Investigation of localized electr ochemi cal interfaces with advanced microscopic techniques: AFM-SECM and FIB/SEM t omography Zur Erlangung des Doktorgrades D r. rer. nat. der Fakultät für Naturwissenschaf ten der Universität Ul m vorgelegt v on Angelika Holzinger aus Heilbronn Ulm, 20 19 Amtierender Dekan: Prof. Dr. Peter Dürre Erstgutachter: apl. Prof. Dr. Ch ristine Kranz Zweitgutachter: Prof. Dr . Jürgen R. Behm Tag der Promotion: 16. Juli 2019 i ii Much to learn you still have . - Yod a iv Co n te n t Content ......................................................................................................................... iv Abbreviations ............................................................................................................... vi Abstract ........................................................................................................................ ix Zusammenfassung ....................................................................................................... xii Experimental background ......................................................................................... - 1 - F UNDAMENTALS IN ELECTR OCHE MISTRY ........................................................................ - 1 - P OTENTIOMETRY ................................................................................................ . - 4 - D IFFUSION - LIMITED CURRENT S AT UME S ................................................................... - 8 - Surface and geometry of UMEs ........................................................................... - 9 - E LECTROCHEMISTRY AT THE I TIES ........................................................................... - 10 - The interface between tw o liqu ids ................................................................... - 12 - Electrochemistry at ITIES ................................................................................... - 13 - E LECTROCHEMISTRY AT NANO - INTERFACES ................................................................ - 14 - S CANNING PROBE TECHNI QUES .................................................................................. - 17 - A TOMIC FORCE MICROSCO PY ................................................................................. - 17 - S CANNING ELECTROCHEMICA L MICROSCOPY ............................................................... - 20 - AFM-SECM ..................................................................................................... - 23 - FIB/SEM TOMOGRAPHY ........................................................................................ - 28 - Results and discussion ............................................................................................. - 32 - H 2 O 2 SENSING WITH AFM TIP - INTEGRATED ELECTRODES ................................................. - 33 - I NTRODUCTION .................................................................................................. - 33 - E XPERIMENTAL .................................................................................................. - 36 - R ESULTS AND DISCUSSION ..................................................................................... - 40 - PB/NiHCF-modified UMEs ................................................................................. - 40 - PB/NiHCF-modified AFM-SECM probes ............................................................ - 54 - Localized detection of H 2 O 2 by AFM-SECM ....................................................... - 63 - C ONCLUSION AND OUTLOOK .................................................................................. - 67 - AFM TIP - INTEGRATED P H SE NSORS ................................ ............................................ - 69 - I NTRODUCTION .................................................................................................. - 69 - E XPERIMENTAL .................................................................................................. - 71 - R ESULTS AND DISCUSSION ..................................................................................... - 75 - AFM tip-integrated pH sensors ......................................................................... - 76 - Investigation of calcite dissolution by AFM-SECM ............................................ - 80 - Electrochemical deposition of iridium oxide film electrodes ........................... - 87 - v C ONCLUSION AND OUTLOOK .................................................................................. - 92 - I NVESTIGATION OF THE ITIES AT NANOP ORE ARRAYS ...................................................... - 94 - I NTRODUCTION .................................................................................................. - 94 - E XPERIMENTAL .................................................................................................. - 98 - R ESULTS AND DISCUSSION ................................................................................... - 103 - Diffusion at nanopore arrays .......................................................................... - 103 - ITIES at nanopore arrays ................................................................................. - 109 - Silica deposits at the ITIES for localization of the i nterface ............................ - 116 - Investigation of the elem ental composition of deposited silica at nanopores- 123 - C ONCLUSION AND OUTLOOK ................................................................................ - 130 - Final remarks ......................................................................................................... - 132 - References ................................ ............................................................................. - 136 - Appendix ............................................................................................................... - 153 - IX. A . U SED CHEMICALS AND MATER IALS ....................................................................... - 153 - IX. B . U SED INSTRUMENTS ........................................................................................ - 155 - IX. C . T ABLE OF T ABL ES ............................................................................................ - 156 - IX. D . T ABLE OF F IGURES .......................................................................................... - 157 - Acknowledgment .................................................................................................. - 173 - Curriculum Vitae .................................................................................................... - 174 - Publications ........................................................................................................... - 176 - Eidesstattliche Erklärung ....................................................................................... - 178 - vi Ab bre vi ati o n s ACT - Aqueous complexation followed by transfer AFM - Atomic force microscopy Ag/AgCl - Silver/silver chloride reference electrode AIROF - Anodic iridium oxide film ATR FTIR - Attenuated total reflection FT IR BSE - Backsc attered electron(s) CE - Counter electrode CV - Cyclic voltammetry DPA - Differential pulse amperometry DRIE - Deep reactive ion etchin g EDX - Energy dispersive X- ray EIROF - Electrochemically deposited iridium oxide film ETD - Everhart-Thornley detector FIB - Focused ion beam FTIR - Fourier-transform infrared spectroscopy HOPG - Highly oriented pyrolytic graphite IBID - Ion beam induced deposition ISE - Ion selective electrode ISFET - Ion sensitive field effect transistor ITIES - Interface between two immiscible electrolyte sol utions Mt/AFM- SECM - Molecule touching/ AFM-SECM NHE - Normal hydrogen electrode NiHCF - Nickel hexacyanoferrate NMR - Nuclear magnetic resonance spectroscopy NPP model - Nernst-Planck, Poisson model OCP - Open circuit potential ORR - Oxygen reduction reaction PECVD - Plasma-enhanced chemi cal vapor deposition vii PB - Prussian blue RE - Reference electrode ROS - Reactive oxygen species SE - Secondary electron(s) SECM - Scanning electrochemical microscopy SEM - Scanning electron microscopy SG/TC mode - Substrate generation / tip collection mode SHE - Standard hydrogen electrode SiN - Silicon nitride SIROF - Sputtered iridium oxide film SNOM - Scanning near-f ield microscopy SPM - Scanning probe microscopy STEM - Scanning transmission electron microscopy STM - Scanning tunneling microscopy SVET - Scanning vibrating electrode technique TARM – AFM-SECM - Tip-attached redox mediator – AFM-SECM TATB - Tetraphenylarsonium tetraphenylborate TEM - Transmission electron microscopy TIC - Transfer by interfacial complexation TID - Transfer by interfacial dec omplexation TLD - Through-the-lens detector TOC - Transfer followed by organic-phase complexation TOIROF - Thermal oxidation of ir id ium oxide film UME - Ultramicroelectrode XRD - X-ray diffraction viii ix Ab st ract Comprehensive knowledge about interfacial processes is essential for the development of innovative miniaturized sensors or the in vestigatio n of biological samples. Miniaturization of sensors with sub-micron and nanoscale dimen sions and sensor arrays i s accompanied by challenges, s uch as changes in the efficiency of the sensor, changes in the general behavior at small se nsor dimen sions, or difficultie s in handlin g of such devices. Th erefore, a detailed eval uation o f possible factors influencing the performance of miniaturized sensors has to be done prior to applications. Studying processes at s urfaces, ranging from e.g., single cells 1 to c orroding ma terials 2 a nd i nvestigations o f nanoscale 3D an d 2D materials, e.g. nanotubes 3 an d graphene 4 , got accessible with high-resolutio n microscopic techniques with improved resolution down to the sub -nanoscale 5 – 7 . Esp ecially comb ined scanning probe techniques (SPM) 2,8 – 10 en able t he investigation of localized surface properties. Amongst other hybrid methods, atomic f orce - scanning electrochemical microscopy (AFM-SECM) is h ighly attractive for stu dying process es at the solid/liquid interface, and also f or in vestigations of liquid/liquid interfaces 11 . In order t o enhance the field of applications, AFM-SECM probes may be modified facilitating the requireme nts for biomedical applications an d for usage with electroanalytical t echniques. Whereas AFM- SECM p rovides predominantly information of surface processes , the combination of focused ion beam (FIB) and scanning electro n microscopy (SEM) 12,13 e nables the detection and reconstruction of the inner st ructure of (nano)porous materials. This was alread y reported for several materials, ranging from nanoparticles in dye-sensitive solar cells 14 to investigations of biological 15,16 and geological 16 samples. Within the f irst part of this t hesis, the focus of researc h is targeted towards modification of AFM-SECM probes fac ilitatin g biomedical ly relevant measurements, such as sen sing hydrogen peroxide (H 2 O 2 ) release or generation in close proximity to a sample surface (chapter 4), and localized pH sensing with associated surface changes (chapter 5 ). In chapter 4, t he surface m odification of AFM tip-integrated electrodes wit h electrocatalytically activ e layers is p resented, namely Prussia n Blue ( PB ) for H 2 O 2 sensing. In contrast to a relative ly high potential (0.5 – 0.8 V vs. normal hydrogen electrode x (NHE) 17 ) for H 2 O 2 oxidation at plat inum electrodes, mo dification with PB pro vides moderate electrochemical reduction p otentials (-0.05 V vs. standard re ference electrode (SHE) 18 ) and improved selectivity 19 . The disadvantage of the limited stability o f PB particular in higher H 2 O 2 concentration can be att enuated by co-d eposition of PB and nickel-hexacyanoferrate (NiHCF) 20 . Prior to AFM- SECM investigations, diffe rent electrode materials and con ditions, e.g. vacuum condition s and drying processes, which are necessary for AFM-SECM probe f abrication, are evaluated on modified ultramicroelectrodes (U ME). Besides a thorough characterizati on, the stability a nd sensitivity of the H 2 O 2 sensor s are investiga ted, an d f irst results t oward s localized H 2 O 2 detection by AFM-SECM are presented in 4.3 .3. In chapter 5, solid-state pH sensors integrated into AFM t ips are evaluated sin ce local p H changes play an important role, fo r example in corrosion scie nce or inves tigation of living cells 21,22 . Antimony/anti mony oxide 23 , 24 , a nodic iridium oxide film (AI ROF) 25,26 and electrochemical iri dium oxide film (EIRO F) 27 wer e inv estigated as AFM tip- integrated pH sensor materials. The most suita ble modif ication with res pect to s tability and s ensitivity in pH calib rations is evaluated (5.3.1 a nd 5. 3.3) and first AF M- SECM meas urements o f t he dissolution of calcite c rystals as a model sample mapping s urface changes along w ith a pH change to more alkaline pH values 28,29 are presen ted in 5.3.2 . In chapter 6, de tailed investigations of FIB- milled nanoporous arrays in silicon nitride (SiN) membranes are presented, which were used as solid- state s upport for el ectrochemistry at the interface b etween two immiscible ele ctroly te solutions (ITIES) 30,31 . FIB can be used for mask-less prototyping of arrays with readily adju stable dimensions 32 – 34 . Sin ce this fabrication process may change t he performan ce of such micro - and nanoporous solid- state materials due to implantation of positively charged Ga ions 35 or due to a varyin g pore shape resulting f rom re-deposition during the FIB milling process 32,36,37 , a comprehensive a nalysis of these devices is essential. The sui tability of AFM -SECM in the investigation of micro- and nanoscale interfaces is shown b y the performance of AFM - SECM with a conductive AFM t ip, which was used to visualize the different diffusion behavior at these na noporo us arrays , as the ra tio between the pore size a nd the dis tance between single pores wit hin an array are affect ing the diffusio n behavior at these devices (6.3.1). Th e det ailed investigations on the location of the nano -interfaces at the xi nanoporous Si N mem branes are shown in 6.3 .3 and 6.3.4 . I n additi on, the localized electrochemical dep ositi on of silica at th e nanoI TIES was u sed t o visualize th e location of the interface within the n anopores . For t he first time, FIB/SEM tomograph y was performed for the investigations of the location of this interface at such silica deposits. xii Zu s am me nf a ss ung Zur Entwicklung in novativer mi niaturisierter Sensoren, sowie zur Untersuchung biologischer Vorgänge, ist ein detailliertes Verständnis der zugrundeliegenden Prozesse notwendig, die a n Grenzflächen a blaufen. Mögliche Einflü sse, die das Ver halten und die Anwendbarkeit solcher M ikrosensoren b eeinträchtigen könn en, m üssen im Vorfel d detailliert untersucht werden. Untersuchungen an Grenzflächen in der Größenordnung weniger Mikro- oder Na no meter profit ieren von den Weiterentw icklungen im Bereich der hochauflösenden Mikroskopie- Technike n 5 – 7 und insbesondere k ombinierter Rastersondentechniken 2,8 – 10 , da hier or tsaufgelö ste Messungen von elektrochemisc he n Oberflächenprozessen ermöglicht werden. D ie K ombinationstechnik von Rasterkraft - und elektrochemischer Ras tersondenmikroskopie ( atomic force - s cannin g electrochemical microscopy , AFM -SECM), welche hoch auflösende Abbildungen einer Oberfläche b ei gleichzeitiger Bestimmung elektrochemischer Ober flächenprozesse vereint, ist ein Beispiel einer solchen Hybridtechnik . Eine weitere Mikroskopie-Technik stellt die Kombination aus fokussiertem Ionenstrahl (FIB) und Raster elektronenmikr oskopie (SEM) dar, die es ermöglicht Proben h insichtlich ihrer mikro - und nanoporösen Strukturen 12,13 zu untersuchen. Dabei erstreckt sich das Anwendungsgebiet d er FIB/SEM Tomographie von der U ntersuchung v on N anopartikel n, die in Solarzellen genutzt werden 14 , bis hin zu Untersuchungen von biologischen 15,16 und geologischen 16 Proben. Der erste Abschnitt der z ugrundeliegenden Arbeit umfasst die Mod ifikat ion von AFM - SECM Sensoren, die biomedizinisch relevante Untersuchungen ermö glic ht, wie zum Beispiel d ie Detektion von Wasserstoffperoxid ( H 2 O 2 , Kapit el 4) od er ortsaufgelöste pH Änderungen (Kapitel 5 ). In Kapitel 4 wird die elektrochemische Modifikation der elektroaktiven Fläche von AFM- SECM Spitzen mit Preußisch Blau (PB) gezeigt. Im Gegensatz z u Platinelektroden, die hohe Oxidationspotentiale (0. 5 – 0.8V vs. NHE 17 ) z ur H 2 O 2 Bestimmung benötigen, kan n mit tels PB -modifizierten Elektroden unter milden Bedingungen (-0.05 V vs. SH E 18 ) gemessen werden. Gleichzei tig wur de eine erhöhte Selektivi tät beobachtet 19 . Ein Nacht eil vo n PB ist die begrenzte Stabilität, besonders b ei hohen H 2 O 2 Konzentrationen, welc he jedoch durch xiii zusätzliche Abscheidung von NiHCF 20 verbessert werden kann. Unterschiedliche Elektrodenmaterialien und M essbedingungen, die während der Herstell ung von AFM - SECM Sensoren erf orderlich sind, wurden a nhand m odifizierter Ultram ikroelektroden (UME) untersucht. Neben einer detaillierten Untersuchung und Charakterisierung der modifizierten Spitzen, wurde die H 2 O 2 Sensitivität und das lineare Ansprec hverhalten der Sensoren untersucht. Er ste AF M -SECM Messungen vo n lokalisiert erzeugtem Per oxid werden in Abschnitt 4.3.3 d iskutier t. In Kapitel 5 wird die Modifikation von AFM -SECM Pr oben mit einem pH Se nsor präsentiert, d a d ie Änderung d es p H -Wertes sowoh l in Korrosion, ab er auch in biologischen Zellen eine wichtige Rolle s pielt, um nur zwei Beispiele zu nennen 21,22 . Unterschiedliche Modifikationen von pH -sensitiven AFM-SECM Spitz en wurden un tersucht, und zwar Antimon/Antimonoxid 23 , 24 , an odischer Iridiu moxid-Film (AIROF) 25,2 6 und elektrochemisch abgeschiedene Iridiumoxid-Filme (EIROF) 27 als p H-sensitives Elektrodenmaterial. Dabei wurden d iese untersch iedlichen pH Sens oren auf ihre Stabilität und Sensitivität in pH Messu ngen untersu cht und verglichen ( 5.3.1 u nd 5.3.3). Erst e Untersuchungen mittels AFM -SECM werden in Abschn itt 5.3 .2 gezeigt. Dabei wurd en Calcit-Kristalle abgebild et, d ie beim Lösen in wäss riger Lösung eine lokale pH Än derung zu basischen pH erzeugen 28,29 . In Kap itel 6 wird d ie genaue Untersuchung von nano porösen Siliziumnitrid M embranen dargestellt, die als Supportmaterial für nanoITIES ( na no-interface bet ween two immiscible electrolyte solu tions ) verwend et wer den 30,31 . U nter anderem w urde A FM -SECM daz u genutzt, u m das Diffusionsverhalten an diesen nanoporösen St rukturen zu u ntersuchen, da dieses maßgeblich von dem Verhältnis zwischen dem Porenradius und d em Abstand zwischen den Poren abhängt ( 6.3.1). Neben dem Einfluss der Herstellungsart dieser nanoporösen Memb ranen durch FIB, wie in Ab schnitt 6.3.2 gezeigt, wird eine gen au e Untersuchung der Lage der Grenzf läche von ITIES in diesen N anoporen beschrieben und anhand der elektrochemisc hen Abschei dung v on Silik apartikeln an der Phasengrenze zweier nichtmischbaren Flüssigkeiten i nnerhalb des nanoporösen Netz werkes gezeigt (Abschnitt 6.3.3 u nd 6.3.4) D es Weiteren ermöglicht die genaue Untersuchung der Nanoporen mittels FIB/SEM Tomographie ei ne R ekonstruktion der por ösen Struktur und der lokalen Abscheidung der Silika partikel . - 1 - Ex pe rim e nt al b a ck gro un d The following section gives a comprehensive overview o f the theoretic al background, which is the basis of th e research presented wit hin this thesis: fundamental principles of electrochemistry at UMEs and at ITIES, AFM, SECM, the comb ination of AFM -SECM and FIB/SEM tomography. Fun d am entals in el ec tr oc hem is try Electrochemistry includes a ll p rocesses related to elect ric re actions that c an be t raced to chemical p rocesses . In case of potentiometry, no measurable current flow is involved . In case that the process induces a current flow, one can dist inguish between galvanic or electrolytic processes . Thereby, galvan ic reactions are sp ontaneous by connecting two systems by a co nductive media, which is im portant for energy storage and production. If a reaction is forced by an applied external potential larger than the eigenpot ential of t he system, it is called electrolytic. In electrolytic cells, different t ypes of rea ctions are p ossible with t he concordance that ever y syst em s trives for the e quilibrium s tate with minimized molar free en ergy Δ r G, resulting in the chemical p otential µ i of component i wi thin an electrochemical process described as 38 : μ i = μ i 0 + RTlna i (1) µ i = ( ∂ G ∂n i ) T,P,n j≠i (2) With t he st andard chemical p otential ( μ i 0 ) of component i, the activity of the component i (a i ), the molar gas constant (R) and the t emperature (T), the pressu re (P) a nd t he amount of substance (n i ). The equilibrium is dynamic, which means that both, oxidat ion and reduction, are takin g place at the same rate i n the e quilibrium state. The chemical po tential leads t o the Nernst equation, which describes t he Galvani pot ential in equilibrium in dependence of the activity of the participating compounds. If the Galvani potential difference Δ ϕ is measure d against the SHE, it can be replaced by the electrod e potential E 39 : - 2 - E = E 0 + RT nF ( ∏ a ox v ox ∏ a red v red ) (3) With the number of participating electrons ( n), t he Faraday constant (F), the potential at standard conditions (E 0 ) and a stoichiometric fact or (v). To reac h the equilibrium state within a certain t ime ( dt ) , a charge ( dQ ) is tran sferred within the system resulting in a faradaic current (i) 38 : 𝑖 = 𝑑𝑄 𝑑𝑡 (4) The Nernst equat ion applies therefore for reversible reactions. Reversible electrochemical processes are governed b y the concentrations at the surface, by fast electron transfer , and lack of additional homogenous reactions in solution of the produced species forming side p roduct when a reduced sp ecies is oxid ized and vice versa . Whereas for irreversible reactions, the back reaction is kin etically hindered or ad ditional side p roducts inhibit the conversion of all redu c ed spec ies back to oxidized species (and vice versa) . Quasi- reversible reactions are govern ed by kinetic and mass tran sport . The Nernst equation describes the electroche mical reaction fro m a thermod ynamic p oint of view. I f kinet ic processes are affecting the elec trochemical reaction, an additional potential, the so-called overpotential ( η ), has to be applied to overcome the activation en ergy a nd to drive th e reaction . The Tafel equation describes this ove rpotential in relation to t he decadic logarithm o f the current and o f the exchang e current, which d escribes th e resulting faradaic ac tivity in t he equilibrium state with a zero current 38 . The kine tics of a redox re action can be exp ressed by the standard rate constant k 0 with high values (1 – 10 cm/s) 38 for reactions reaching the equilib rium state in a s hort time. The rate constant is used to describe the kinetics of the cathodic reaction k catho dic (or anodic as k anodic ) by : 𝑘 𝑐𝑎𝑡ℎ𝑜𝑑𝑖𝑐 = 𝑘 0 ∙ 𝑒 − 𝛼𝑓 ∙(𝐸 −𝐸 0 ′ ) (5) 𝑘 𝑎𝑛𝑜𝑑𝑖𝑐 = 𝑘 0 ∙ 𝑒 (1−𝛼 )𝑓∙(𝐸 − 𝐸 0 ′ ) (6) - 3 - E 0 ’ is the formal potential and E the relative e nergy of the electro n participating in the redox reaction. The Butler-Volmer 40 describes the anodic (i + ) and t he cathodic (i - ) cu rrent depending on the excha nge current(i 0 ) in dependence of the overpotential by : i = i + + i − = i 0 [𝑒 − 𝛼𝑓 η − 𝑒 (1−𝛼 )𝑓η ] (7) Nonfaradaic c urrent contributions t o the overall recorded cu rrent are based especially on the growth of a capacitance layer, namely the el ectrical double layer. The double layer can be d escribed as a capacitor, resu lting in a current flow b y app lying an external voltage. The curren t i C is described by the differential capacitance (C d ), the potential (E) and time (t) and is dependent on the electrodes’ surface and the electrolyte solution 39 : i C = C d ∙ dE dt (8) The do uble laye r co nsists of differe nt regio ns described by the S tern model . This theory combines the f ormer mo dels of Helmhol tz and of Gouy and C hapman. In close proximity to the electrode, an oriented ion layer is formed, w hi c h is termed the inner Helmholtz layer with adsorbed ions on the su rface of the electrodes due to Van der Waals forces , and the outer Helmholtz layer with the ions su rrounded by solvent molecules and described by the Poisson equation 39 : 𝑑 2 𝜑 𝑑 𝑥 2 = − 𝜌 𝜀 𝑟 𝜀 0 (9) The second derivative of the potential ( ϕ ) to the perpendicular direction to t he electrode ( x) is thereb y dependent on the charge density ( ρ ) and the relative (index r) and ab solute (index 0) permittivity ( ε ). The Gouy-Chapman model describes an adjoining diffu se region considering the thermal motion of the ions near th e electrode surface and includes the presence of counter ions within the diffusive layer, This layer is described b y the so-called zeta potential, wh ich drops exponentially with distance 39 . - 4 - Potentiometry Potentiometric mea surements are b ased on t he d etection of changes in th e eigenpotential of a system due to con centration changes with respect to the p otential of a reference electrode. This is t he basic principle of ion -selective electrod es (ISE), in which an inner reference electrode located in an inner electrolyte solution is sepa rated from the analyte solution b y a semipermeable membrane . Diffusion and exchange of se lective ions within this membrane leads to a potential difference at the mem brane. The membrane can be either made of glass, as used in th e p H glass electrode, or by a solid or liquid crystalline membrane. This membrane is d oped with either charged ions en abling the detection of analytes of opposite polarity, or with io nophores enabling the sele ctive interaction of ions. This ionophores with in the membrane can be both, neutral or charged , depending on the an alyte species 41 . The potential gradient according t o the exchange of the analyte ions within this me mbrane is measured via the open circuit potential (OCP) and is described by the Ner nst equation (1-3). Exemplarily, for detecting p H changes, the Nernst equation can be re -written as: 𝐸 = 𝐸 0 + 𝑅𝑇 𝑛𝐹 ∙ log (𝑎 𝐻 3 𝑂 + ) → 𝐸 = 𝐸 0 − 59 .2 𝑚𝑉 𝑛 ∙ 𝑝𝐻 (10) With the gas constant (R), t he temperature (T) , the Faraday constant (F), th e number of electrons (n) and the activity of H 3 O + ion s (a). Hence, this approach just represents the p otential in the case of thermodynamic equilibrium. Diffu sional, kinet ic or migration proce sses are not taken into acc ount . In case, the solution contains a mixture of ion s, interferences have to b e con sidered. A semi - empirical exte nsion of the Nernst eq uation for the potential (E) of ISEs is described b y the Nikolsky-Eisenman equation 41 . 𝐸 = 𝐸 𝑖 0 + 𝑅𝑇 𝑧 𝑖 𝐹 ∙ ln (𝑎 𝑖 + ∑ 𝑘 𝑖𝑗 𝑝𝑜𝑡 ∙ 𝑎 𝑗 𝑧 𝑖 /𝑧 𝑗 𝑖 ≠𝑗 ) (11) With the constant potential ( 𝐸 𝑖 0 ), ion charge (z), gas const ant (R), temperature (T), Far aday constant (F), activity a and the selectivi ty constant ( 𝑘 𝑖𝑗 𝑝𝑜𝑡 ) for two ions i and j. Th e - 5 - interfering ion contributi ng to the measure d potential is represented by the index j. The equation is b ased on the equilibrium state and an y kinetic effects of ion transfer are neglected. An extension to t he general assumption of the equilibrium state is given by the diffusion-layer-model, which takes into account the steady -state and local equilibrium conditions at the mem brane surface. T he diffusi on and the ion concentrati on a t or within the membrane are thereby time dependent. Th e most det ailed description of potentiometric measurements is given by an advanced non - equilibrium model considering both, variations i n time and in distance. Th is mo del is based on the Nernst - Planck equation, describing the ion flux dependent on time and space and the Poisson equation, defining the c urrent density 42 . The graphs in Figure 1 re present the potentials described by the N ernst-Planck and Poisson (NPP) mod el (Figu re 1 A) in comparison to th e phase b oundary model ( Figure 1 B), which is a classical mod el assuming eq uilib rium conditions a nd n eglecting migration and kin etic effects. Th e variation in th e membrane potential changes in the ran ge of mV for th e NPP model com pared to the p hase boundary model (B). Schemes o f the tim e- and sp ace-depend ent p otentials de scribed by the NPP model (A) and compared to the classical phase bo undary model (B). The membrane poten tial in (A ) is defin ed as the integral over th e distance between a point in t he bulk solution x b,L and a p oint in th e internal s olution x b ,R at different times: 4 x 10 -4 s (a), 1.64 s (b), 13.1 s (c), 26.2 s (d), 104.8 s (e), 420 s (f) an d at stead y-stat e co nditions (13440 s, g). 42 Reprint ed with per mission from Boba cka, J., Ivaska, A. L ewenstam, A. Chem. Rev. 2 008 , 108 , 329- 351 . Copyright 20 19 Amer ican Chemical Soci ety. htt ps://doi.org/10.10 21/cr0681 00w . - 6 - ISEs are used for the detection of a large variet y of ions, as a sta ndard an alytical method in water analysis or f ood contro l, and are also us ed f or characterization o f ph armaceutical or biological samples 43 . Th e most used ISE is for the determination of pH using a hydronium- sensi tive glass elect rode developed ove r a century ago and based on the exchange of ion s within the glass as the semipermeable membrane 44 . Next to ion- selective e lectrodes, ion -selective sensing can be performed with soli d -state e lectrodes, such as conductive polymers, metals or ion -sensitive field-effect t ransistors (ISFETs). This is especially favor able for miniaturized sensors. The capab ility of p H sensing using metals as solid electrode materials is based on the equilibrium between the metal and its oxide, or be tween two oxid ation states of the me tal/material. A variation in pH leads to changes in th e equilibrium state and, ther efore, to a change in the eigenp otential of the material. Thereby, the composition of the metal and its oxide (or of two different oxid es) varies leading to a n electron t ransfer towards t h e elec trode and th e d etection of t he cha nged pH value by the electronic setup. Because there i s a limited amount of the metal and its oxide (or of both metaloxides), especially f or miniaturized se nsors, also the capaci tance of t he material is reduced. Th i s results i n a hig her material resistance, which affect s the potential stability in potentiometric measurements 42 . Competing site-reac tions may lead to false interpretations or to p otential d rifts d uring the measurements . Many met al/metal oxides can be used for pH sensing, wherea s stability and/or linearity vary dep endent on the different met al/met al oxid es, conditions, and comp lexity of sample mixtures 45,46 . Suitable me tal/metal oxides have to be stable in solut ion, but also soluble enough that the ratio of metal and corresponding oxide may vary t o achieve equilibrium depen ding on pH changes. The pH response h as to be fast, just depending on the diffu sion of the H 3 O + ions towards the p H sensor, and reproducible over a wide pH range. Response times determined by a n electr ochemical time- of -flight technique were up t o a tenth o f seconds for AIROF 25,26 electrodes, dependent on the film thickness 47 . Dep endent on the underlying redox reaction of the pH -sensitive materials, the sensitivity varies as shown in Table 1 for a selection of metal/metal oxides. The u nderlying red ox pro cess during pH detection is shown exemplarily for the AIROF and the antimony/ antimony oxide electrode 23 , 24 , which were used in experiments p resented within this thesis. Th e potential d etermining equilibrium for the AIROF and the antimony electrode may be written as f ollows: - 7 - 2 Ir O 2 ( 0H ) (2−𝑥) (𝐻 2 𝑂 ) (2+𝑥 ) ( 2−𝑥 ) − + ( 3 − 2x ) H + + 2e − ↔ Ir 2 O 3 ( OH ) 3 ( H 2 0 ) 3 3− + 3 H 2 O Sb 2 O 3 + 6 H + + 6 e − ↔ 2 Sb + 3 H 2 O metal/metal oxide or metal oxides as pH sensor pH response pH range monocrystalline Sb/ SbO 2 52 mV/pH pH 2- 10 Ir/IrO 2 63 mV/pH pH 2-9 Pd/PdO (th ermal) 59.6 mV/pH pH 2.5 – 8.5 Pd/PdO (electro chemical) 71.4 mV/pH pH 3 - 8 48 AIROF (electro chemical) 65 – 80 mV/pH pH 2.5 – 8.5 AIROF (thermal) 59 mV/pH pH 2 - 12 49 AIROF (sputt ered) 59.5 mV/pH pH 2 – 8.5 PtO 2 46.7 mV/pH pH 5 – 10 RuO 2 61.8 mV/pH pH 2 – 12 RhO 2 x H 2 O 62.8 mV/pH pH 2 – 12 OsO 2 x H 2 O 51.2 mV/pH pH 2 – 11 Table 1 pH response and linear p H range for some metal/metal oxi des suitable as p H sensors 45 . Different fabrication strategies have b een reported with respect to crystallograp hic properties 50 – 52 , stability 53 , and efficiency in p H sensing 54 , 55 . Especially, t he response of iridium oxide electrodes varies depending on the f abrication process, namely, the sputtered iridium oxide film (SIROF) 56 , 57 , thermal oxidation of Ir salts (TOIROF) 58 , 59 or - 8 - electrochemically deposited films like EIROF 27 , and AIR OF 60 – 62 . The slo pe for t he pH response for AIROF elect rodes can also be exp ressed b y 59· (3 - 2x)/2 mV/pH 45 . Additionally, the infl uence of oxidation state 27 and of t he substrate f or the pH -sensitive film with respect to stabi lity 63 has been investigated. All pH sensors have to be calibrate d prior to usage. Diffusion-limited currents at UMEs In voltammetric measurements, the faradaic current related to a redox reaction is recorded. The cur rent si gnal is infl uenced by transport processes in the electrolytic cell, namely diffusion, migr ation and convection. In a system w ith n o additional steering and no temperatur e or density gradient, convection c an be neglected. In elect rolyte solution with h igh electrolyte concentrations in the range of 0.1 – 1 M, the migration of charged particles from the bulk so lution to the ele ctrode surface is most ly suppresse d and can also be neglected because the electrolyte ions are carrying 97 % of the current in bulk so lution 38 . In case of fast ele ctron transfer kinetics, the faradaic current can then be described by the diffusion processes towards t he electrode’s surface. The diffusion- limited current I(t) is desc ribed for a spherical el ectrode by a semi-infinite approach via the Cottrell equation 38 : 𝐼 ( 𝑡 ) = 𝑛𝐹𝐴 √ 𝐷 0 𝑐 0 ∗ √ 𝜋𝑡 + 𝑛𝐹𝐴𝐷 0 𝑐 0 ∗ 𝑟 0 (12) Thereby, the current consists of a time -d ependent and a time- independent term, respectively, defined by the elec trode area (A) a nd radius (r 0 ), Faraday constant (F) , the number of ele ctrons (n), t he b ulk con centration (c 0 *) and diffusion coefficient (D 0 ) of the electroactive species (labelled with the index 0). In case, the diffusion layer, which is rather a region than a la yer, is much smaller than the electrode area , the current signal becomes time -dependent. In the case of ult ramicroelectrodes def ine d by t he c ritical radius of 25 µm 38 , t his relation c hanges a nd the d iffusion layer is i n the same d imension or even larger t han the e lectrode size. The c urrent signal becomes time-indep endent an d is termed steady-state current I ss 38 : - 9 - 𝐼 𝑠𝑠 = 𝑛𝐹𝐴𝐷 0 𝑐 0 ∗ 𝑟 0 (13) At short time scales, however, the time-depending term cannot b e neglected. Calculated by Shoup and Szabo 64 , the t ime-dependence can b e expressed f or a planar d isk microelectrode as: 𝑓 ( 𝜏 ) = 0. 7854 + 0. 8862 𝜏 − 1 2 ⁄ + 0 . 2146 𝑒 (−0. 7823 𝜏 − 1 2 ⁄ ) (14) With τ as a dimensionless variable for the time defined as 𝜏 = 4𝐷 0 𝑡 𝑟 0 2 (15) According to equation ( 15 ), the time dependenc e is strongly related to the size of the electrode and results either in a li near or radial di ffusion. For U MEs, the diffusion is linear to the surface within nano- to microseconds, but this merges within milliseconds into a radial diffusion and a resultin g steady-state current. Due to this fast response at UMEs, fast electrochemical processes can be investiga ted under steady- state conditions. Surface and geometry of UMEs The current response dep ends o n the electrode’s geometry an d surface rou ghness , defined by the relati on b etween t he microsco pic area (A m ) and the geo metric area (A g ), which can b e interpreted as t he pro jection of the surface boundary . For polished electrodes, the surface roughness is low, with 38 : 2 − 3 > 𝐴 𝑚 𝐴 𝑔 (16) In case that the diffusion layer is larger than th e actual electrode size , which is th e case for t ime scales above some seconds at UMEs, just the so-called geometric area of the surface is relevant. In case that the roughness is within the dimension of the diffu si on - 10 - field, which is the case for fast time scales (nano or milliseconds) or unpolished surfaces, the ac tual electrode are a is relevant . Clearly apparent from equation (1 2) and (13), the current is related to the area A of the electrode and d ifferent geometr ies, such as f rame , disk, band , cylindrical, or con ical electrodes re sult in different current respon ses 65 . An overview of the steady-state current re sponses with respect t o the geometrical parameter is summarized in Table 2. electrode geometry steady-state current 66 disc microelectrode I ss = 4nFaDc 0 ∗ (17) ring microelectrode I ss = nF l o Dc 0 ∗ with l 0 = [ π 2 ( b + c ) ] ln [ 16 ( b + c ) / ( c − b ) ] , c b < 1. 25 (18) conical microelectrode I ss = 4nFDc 0 a ( 1 + qH p ) with H = h/a, q = 0.3661 and p = 1.14466 (19) Table 2 Overvi ew o f the steady- state current at nano/micro -el ectrodes with respect to differ ent electrode geo metries. For SECM mea surements (see section 2.2), which allows mapping of local heterogeneity of electrochemical properties, the d imensions of the in sulating sh eath of the UME are crucial, b ecause the c urrent flow , when the UME is in close proximity to t he sample surface, is not on ly influenced by the n ature of th e sample but also by th e insulating sheath of the UME . An optimal ratio (RG value) of t he radius of the electrode (r UME ) and the insulating sheath (r insulating sh eath ) was derived from numerical simulations 67 as 10 ≥ 𝑟 𝑖𝑛𝑠 𝑢𝑙𝑎𝑡𝑖𝑛𝑔 𝑠ℎ𝑒𝑎𝑡ℎ 𝑟 𝑈𝑀 𝐸 (20) Electrochemistry at the ITIES Besides electrochemistry at the interface between a solid electrode and a liquid electrolyte solution, a polarized interface between two immiscible electr olyte solutions (ITIES) is highly int erestin g for ele c troanalysis . Th ereby, electrochemical processes within the liquid/liquid system are described by different mechanisms, summariz ed in Figure 2. - 11 - Scheme of different electroch emical ly induced pro cesses at ITIE S : electron transfer by two redox reactions in both phases (I), ion tran sfer (II) an d facili tated ion tran sfer in the presence of a ligand molecul e in one o f the phases (III). The charge transfe r at ITIES can be b ased on elec tron transfer, but als o on ion transfer, which can be either a direct tran sfer of a molecule soluble in b oth phases or a facilitated transfer in the presence of a ligand molecule in o ne of the phases. The fac ilitated tra nsfer can be grouped into different types of tran sfer, as summar ized in Table 3. types of facilitated ion transfer ACT – aqueous complexation followed by transfer TIC – transfer by interfacial complexation TID – transfer by interfacial decomplexation TOC – transfer followed by organic - phase complexatio n schemes Table 3 Overvi ew of facilitated ion t ranspo rt 68 . Additionally, adsorption an d desorption processes at the interface can be analyzed b y impedance measurements and el ectrocapillary curves. Thereby, the k inetics of layer formation can be investigated, and new materials can be formed at a d efect-free interface between two liquids. The ther modynamics of char ge transfer at the li quid/liquid interface are described by the Nernst equation like in the case of a liquid/solid interface (see equations 1-3) a nd t he Galvani potential of the transfer can be describe d by the solvation potentials in the aqueous and the organic phase, respectively . For the electron transfer, two redox reaction s, one in ea ch liquid , are taking place at the same time . In most cases, the charge transfer is coupled with mu ltiple reactions and additional transfer due to charge b alance within t he system has to be taken int o account . The current is expressed by the Butler- Vol m er equation (see equation 7) with the f lux of ch arge J i or J el , whic h is - 12 - described by first -order kinetics in case of ion transfer and b y second -order kinetics for electron t ransfer at ITIES. The kinetics of the transf er can be expresse d by t he rat e constants of the forw ard (k f ) and backward (k b ) transfer of an ion i w ith a c oncentration c i as 69 𝐽 𝑖 = 𝑘 𝑓 𝑐 𝑖 ( 𝑤 ) − 𝑘 𝑏 𝑐 𝑖 (𝑜) (21) 𝐽 𝑒𝑙 = 𝑘 𝑓 𝑐 𝑅1 ( 𝑤 ) 𝑐 𝑂2 ( 𝑜 ) − 𝑘 𝑏 𝑐 𝑂1 (𝑤 )𝑐 𝑅2 ( 𝑜) (22) The indices R and O in equation ( 22 ) re present the reduced and oxidiz ed species in the water and organic phase, respectively . T he c urrent with in t he liquid/liquid system is defined as positive with t he t ransport of a p ositive charge f rom the water to t he organic phase, and vice versa 69 . The interface between tw o liquids Compared t o electrochemistry at a solid/ liquid interface, for which the charge distribution is described by t he electrical double layer at the compact interface of a solid electrode , the interface bet ween two liquids can be interpret ed as two mirrored, diffusive layers, the so -called back- to - back doub le la yer, each of them facing one of the electrolyte solutions. The two diffusive layers can be described b y the Gouy- Chapman model . The charge distribution at the liquid/liquid interface is described by the modified Verwey-Niessen model 70 , whereas in early publications, both d iffusive layers are se parated by a compa ct inner layer similar t o the inner Helmholtz layer with a negligible potential d rop 71 . An extension t o firs t assumpt ions was the description of t his inner laye r as a sandwich structure of alt ernating solvent mol ecules fro m the aqueous and organic phase, respectively 72 , or as a mixtu re of b oth solvents 73 , 74 . Girault and Schiffrin des cribed additionally inter facial io n pairs of both electrolyt es as a k ind of specific ad sorpt ion at the interface 75 . The potentia l d ifference ( ∆ 𝑜 𝑤 𝜙) at the i nterface can b e expressed as the sum of the se th ree layers, t he interfacial potential drop within the inner layer ( Δ 𝑜 𝑤 𝜙 𝑖𝑛 ) and the potential of the diffusive layers 𝜙 2 in both phases as 69 : ∆ 𝑜 𝑤 𝜙 = Δ 𝑜 𝑤 𝜙 𝑖𝑛 + 𝜙 2 𝑜 − 𝜙 2 𝑤 (23) - 13 - Electrochemistry at ITIES For electrochemical experiments at liquid/liquid interfaces, four electrodes are necessary, where one counter (C E) and one refere nce electrode (R E) ar e placed in each elect rolyte solution ( SY as organ ic and RX as aqueous electrolyte), respectively. For miniaturized electrochemical interfaces, the detected current is low (in the ran ge of pA to nA) and a two electrode setup ca n be used, w ith one re ference electr ode in eac h phase. The polarized interface itself acts as the working el ectrode w ithin the non-polarizable syste m. The electrochemical cell is expressed by the following notation 69 : For the detection of standard potentials of a redox couple u sing a conventional three - electrode system, only the potential of the referen ce elect rod e h as t o b e taken into account. In liquid /liqui d interface measurements, t he potentials of b oth reference electrodes, as well as the potential at the second liqu id/liqui d inter face (represented as SX(w´)|SY(o´) in the notation , marked in b lue) necessary for the referenc e electrode of the organic p hase solution, are influencing the overall potenti al and t he standard potential of the ion/electron transfer. The cell potential (E cell ) can be d escribed as the difference between the p otentials at the referen ce electrode in the aqueous phase (E aq ) and in the organic phase (E org ) 69 : 𝐸 𝑐𝑒𝑙𝑙 = 𝐸 𝑜𝑟 𝑔 − 𝐸 𝑎𝑞 = ∆ 𝜙 𝑜 𝑤 − Δ 𝜙 𝑜´ 𝑤´ + 𝐸 𝑗 (24) The term (Δ 𝜙 𝑜´ 𝑤´ + 𝐸 𝑗 ) represents the potential difference a t the interface of the organic electrolyte solution and th e reference electrode of the organic ph ase with an aqueous reference solution. An aqueous (quasi- ) reference electrode is used in most cases, due to the limited numb er of available non-aqueous reference elec trodes . Th e referen ce electrode of the organic ph ase can be considered as an ion-sele ctive-type electrode with the liquid junction (E j ) at the interface between the reference electrode (aq) and the - 14 - organic electrolyte soluti on. The potential window is d efined or limited b y the transfer of the electrolyte ions. In the electrochemical cell given above, the limit at positive potentials is the transfer of the R + ion from the aqueous to the organic electroly te solu tion or the transfer of th e Y - ion f rom the or ganic to the aqueous solution. The limit at negative potentials is vice versa, the posit ive ion (S + ) of t he organic electrolyte solution is transferred to the aqueous phase or the X - (aq) ion to the organ ic electrolyte solution. Similar to the standard hydrogen ele ctrode, the organic ele ctrolyte tetraphenylarsonium tetraphenylborate (TATB) is defined as the zero potential 69 by ∆ 𝑜 𝑤 𝜙 = 0 (25) within the system, which is used to determine the half-wave potential of an analyte. Th e center of th e current-p otential curve is defined as zero, because the ion size, the d iffusion coefficients in water and the potential for the tra nsfer to wards the aqueous solution are equal for TA + and TB - (or at lea st very similar that they can be assumed to be equal). This is also called th e TATB assumption 76 . Hence, adsorption p henomena at the interface , the energy of t he transfer back to the organic solvent or the solvat ion of t he ion s in both phases ar e not taken into account in the TATB assumption 77 . Electrochemistry at nano-interfac es Electrochemistry at micro- and nanometer -sized interfaces using nanopipettes 78,79 and solid-state s upports s uch as membranes 80 – 86 is a promising tool for analy tical applications as the miniaturizatio n of interfaces resu lts in an enhance d signal sho wing increased charge and mass transfer compared to any macroscopic app roach, which is also th e case for electr ochemical rea ctions at the ITIES 11 . Additionally, mi niaturization reduce s the ohmic d rop and leads to a reduced capacitive current 87,88 . With n anoporou s arrays, a la rge number of single interfaces can be achie ved , leading to an enlarged ele ctroc hemical signal by multiple interfaces. A d etailed u nderstanding of the kinetics of ion tra nsport can be investigated at small scale interfaces, which was reported first b y the groups of Girault 89,9 0 and Mirkin 78 , and for ele ctron transport by Bard and co -workers 91 . Th e d iffusional behavior at micro- and nanopores or -pipettes depends on the location of the interface - 15 - (see Figu re 3) resulting i n either a linear diffusion profile within the pore or pipette, or in a rad ial d iffusion at the orifice of th e pore/pipette 92 , 93 . For ITIES, d iffusion occurs in b oth directions fro m the interface f acing the aqueous and the organic electrolyte, respectively 68 . Scheme o f thre e d ifferent po res with interfac es (marke d in red) located a t different position s within the pores, namely inlaid and recessed f or pores with parallel pore walls, and a recessed interface for a tru ncated con e-shaped geo metry. The c urrent I i at micro- and nano- ITIES for the transfer of an ion i is described by the modified Cottrell equation (see equ ation 13) for radial diffusion towards an in laid interface located at a pore/pipette ori fice with radius r o 94 : 𝐼 𝑖 = 4𝑛𝐹𝐷 𝑐 𝑖 𝑟 𝑜 (26) With th e bulk concentration of the ion i (c i ), the Faraday con stant (F), the di ffusion coefficient (D) and the number of participating electrons (n). Fo r a re cess ed in terface a t the p osition (L) withi n the pore (which can b e also seen as t he len gth of the pore/pipe tte), the limiting current is desc ribed by 94 𝐼 𝑖 = 4𝜋𝑛𝐹𝐷 𝑐 𝑖 𝑟 𝑜 2 4𝐿 + 𝜋 𝑟 𝑜 (27) For a truncated cone- shaped pore or pipette, both radii of the orif ices have to b e considered with r o < r L resulting in the following equation 37 . 𝐼 𝑖 = 4𝜋𝑛𝐹𝐷 𝑐 𝑖 𝑟 𝑜 𝑟 𝐿 4𝐿 + 𝜋 𝑟 𝑜 (28) - 16 - Single pores w ithin a soli d material can be arrang ed either ordered w ithin an arra y with defined distances b etween single pores or randomly distributed in an ensemble 82 . The distance or pore- to -pore separation influe nces the diffusion p rofiles of the array /ensemble, resulting in either overlapped d iffusion regions for pore s in close proximity t o each other (see Figure 4 A) , or in i ndividual diffusion at each pore (see Figure 4 B) 30 . Influence of the p ore- to -por e separation ratio within a pore ar ray . For closely arranged pore s as shown in the SEM image in A, the radial di ffusion at a single pore results in an over laid diffusion profile for th e overall array as schematically repre sented be low. Whereas for a large pore- to -pore separation as shown in the SEM image in B , the diffusion profiles are not overlapping an d in dividual hemispherical diffu sion at the pores , as indicated in th e corresponding sch emes, i s governing th e overall r esponse. Numerical simu lations compared to experimentally ob tained cyclic voltammograms at nano-ITIES within pores 95,96 or por e arrays 97 – 99 we re u sed to localize t he interfaces . Also, simulations were performed to identify individual radial diffusion at the pores with determining a separation ratio between the pore radius ( r a ) and the distance b etween single nanopores (r c ) 97,100 with a critical value for individual diffusion of 30 : 𝑟 𝑐 𝑟 𝑎 > 56 . (29) - 17 - Sc ann in g pr ob e te ch niq u es Atomic force microscopy Since the introduction in 1986 by Binnig, Quate , and Gerbe r 101 , AFM d eveloped into an important too l in surface character ization, ran gin g from material science to biology 102 . In AFM , the force interaction between a surface and a sharp tip located at the end of a cantilever is d etected. These for ces can be described by the Lennard -Jones potential and can be divided i nto a ttractive or re pulsive forces. Th e electric doubl e layer in teractions or interactions r esulting from ove rlapping atomic orbitals are repulsive, whe reas cap illary, electrostatic and Van der Waals forces are attractive. Other for ces such as chemical, hydrophobic, s teric or m agnetic forces may b e attractive or repulsive. The tip is l ocated a t the end of a flexible cantilever, typically made of silicon nitride or Si . The AFM tip ca n be additionally modified wit h e.g. nano tubes 103 , nanowires 104 , microneedles 10 5 , coll oids 106 or single mo lecules 107 for imaging high aspect rat io features or for single -molecule force spectroscopy experiments . Critical for AFM pro bes is t heir r esonance f requency ( f 0 ), spring constant ( k ) and the quality factor ( Q ), which should be chosen carefully in respec t to the targeted application and investigated sample, re spectively. The parameters describe the stiff ness of the AFM ca ntilever a nd with this the ability to react on external forces. With high reso nance fr equencies in the ra nge of 1 – 500 k Hz and with small spring constants (0.07 - 100 N/m), forces of 10 -6 to 10 - 12 N can be measure d, without issues that the cantilever is influenced by external vibrations. Different operation modes are available with respect to the ch aracteristics of the substrate under investigation. The contact m ode is preferably used for hard surfaces d ue to strong force interaction between tip and substrate. The surface can be sca nned i n eithe r constant height or constant force mode (also called top ographical mode) . The t ip is either kept in a defined p osition in the z-axis, recording t he variations in force, or a defined force is kept constant and th e height is adjusted when the force in teraction between tip and sam ple varies while scann ing in x ,y -direction. In the d ynamic mod e, the tip oscillates in a defined distance above t he surface. This mode can be either f requ ency-modulated or amplitude -modulated 108 . For the investigation of soft samples, the tapping mode 109 (or intermittent mode) is used that results f rom the amplitude-modulated mode but ad ditionally st rikes the sample surface - 18 - on every oscillation 109 . Th e tip is oscillating close t o its resonance frequency and the change in the amplitude due to tip -sample intera ction is detec ted. Additionally, a phase shift indicates a varyin g adhesio n or viscosity of the surface mat erial. In all operation modes, the interaction results in a bending of the cantilever, which is det ected - in most cases - via a laser focused at the back side o f the ca ntilever and re flected to a split photodiode. The de tected photocurrent signal is the n converted in to an output volt age, which is used via a n electronic feedback sy stem to maintai n a fixed force, or am plitude or resonance frequency of the AFM probe by the z-piezo when the cantilever is b ent due to the force interactions. Du e to these interactions, t he topography of a samp le can be mapped and additional information about mechanical properties of t he sample can be obtained and different mater ials c an b e dist ing uished due to varying adhesion be tween the tip and the sample surf ace. Scheme of an AFM prob e in front and side view (A ) ; th e dete ction of th e d eflection and frict ion signal in AFM conta ct mode with corresponding s ignals shown as p rofile (B-D). Th e AFM probes in (C-D) labeled with 1 -3 sho w the AFM p robe at three d i fferent positions r epresentin g th e movement of the tip in the scan direction. - 19 - AFM measurements can be performed in air, liqui d and under vacu um condition, wherea s the latter is m ost se nsitive an d atomic res olution can b e achieved under optimized conditions 110 . Tip artifacts, as shown in Figure 6, c an be minimized or avoided by suit able experimental co nditions and a careful a djustment of scan parameters. Nevertheless, the results have to be interpreted carefully. So far, a variety of advanced AFM modes were developed e.g., p eak force t apping TM AFM 111 gaining additional information on adhesion, and Young mo dulus, force volume mode 112 , enabling the mapping o f in teractive forces by recording a se t of f orce curves, scanning Kelvin probe microscopy 113 recording the contact potential difference between the tip and a surface, and esp ecially AF M -SECM, which is discussed in detail in 2.3 . Scheme of possible artifacts i n AFM me asure ments. The topograp hy image may reflect the shape of the AFM tip, in case t hat the structure of the sample has smaller dimensions than the tip (A), edges might b e also imaged not correctly (B) . Con taminated AFM p robes may re sult in double or multip le imaging of the actu al s ample structure (C ). The dis played tips in A- C represents one tip at different times moving in the scan d irection. - 20 - Scanning electrochemical microscopy In SECM, which was first report ed in 19 89 by Bard and coworkers 1 14 , a n ano- or microelectrode is scanned across a sample su rface of interest mapp ing local electrochemical processes . SECM c an b e a pplied in many fields res earch f ields i ncluding life sciences 115 or corrosion studies 116 , and investigations of amperometric, potentiometric or conductive sign als at homogeneous and heterogeneous samp les can be monitored 117 . A comprehensive description of SECM instrumentation, SECM tips , and applications in various f ields is given elsewhere 117 . Briefly summarized, the posit ioning and movement of th e SECM tip is re alized - in most cases – by st epper mo tors and piezoelectric d evices. A thre e- or f our-electrode set-up is used consisting of the scann ing UME as working electrode , a counter , and a reference electrode and if req uired, t he substrate as the seco nd working electrode . In a first s tep, t he SECM tip is brought in close proximity to the s ubstrate ’ s surface, which is realized by recording a pproach curves (Figure 7 A, B). In t he presence of a redox active species with fast electron t ransfer characteristic, the c urrent is measured at the ti p, which is b iased at the req uired potential while approaching to the surface. In bulk solution, the conversion of the redox active species results in a steady-state current signal. When the tip is in a distance of several radii of t he electroactive radius of t he SECM tip to the surface, th e current signal is influenced depending on the nature of the substrate. At insulating su bstrates, the current d ecreases due to hindered d iffusion of t he redox mediat or to the SECM tip, which is termed ‘ negative feed back ’ . In t he p resence of a conductive s ubstrate, th e converted red ox species can be regenerated a t the sam ple s urface resulting in an increased current signal, called ‘ positive feedback ’ . To d etermine the distance between the SECM tip and t he surface, the resulting approach curves are compared to theoretical curves obtained via numerical simulation. While imaging, t he distance between tip and substrate plays a crucial role. For relatively flat sam ples, the scan ning can be performed i n c onstant height , whereas for rough surfaces (influence of r oughness corre sponds t o the size of the SECM tip), approaches have been introduced to keep a constant distance between the sample and the SECM tip (Figur e 7 D) . Th is can be realiz ed by applying a hor izontal vibration to the SECM t ip, which results in damping in the oscillation with varying dist ances. This damping can be mea sur ed either by optical 118 or piezoelectric detectors 119 as published by Schuhmann and co -work ers, or by t he integration of a tuning fork, as presented by - 21 - James et al. 120 . The damping is used as the signal for a f eedback l oop to k eep the distance constant – this mode is adapted from scanning ne arfield microscopy (SNOM) and t ermed shear force mode. A constant distance can b e fu rther detected in the constant current 12 1 or con stant impedance mod e 122 . A similar approach k eeping the electrode area in a defined distance is given by SECM with soft - stylus p robes 123 , w hereas the insulating material is in contact with the samp le surface. T he SECM tip is bent due to the f lexible insulating material. Next to different positioning modes, the imaging of a sample can be also divided into di fferent modes. In the generation/collection mode 124 , the electrochemical species is generated a t t he SECM t ip and then detected at the substrate’s surface or vice versa (Figure 7 C). A variat ion of the generatio n/collection mode is th e redox competition mode 125 , in which the same ele ctrochemical reaction is detected by both, SECM tip and substrate. Schemes of different op eration modes u sed in SECM : The feedback mod e (A) with corresponding approach curves (B), tip generation/ substrate collection (TG/SC) and s ubstra te generation/ tip collection (S G/TC) mode (C) and mod es related to the detection in either constant d istance or constant h eight (D). - 22 - In the so-called direct mode 126 , the a pplied potential between the tip and a substrate c an be used for localized modification of the sa mple, by deposition 126 or by generating localized pit corrosion 127 . SECM has also b een combined with a nu mb er of analytical methods, e.g. optical microscopy 128 , IR sp ectroscopy 129 , surface p lasmon resonance 130 , and o ther sc anning probe techniques. For example, b y replacing t he S ECM ele ctrode with a micropipette, which is known as scanning ion conductance microscopy (SICM) , the conductance between a reference electrode located in an ele ctrolyte solution within the pipette an d t he re ference electrode i n the bath solution is d etected 131 . SI CM is frequently used to map the topography of soft samples given a non- invasive approac h. By combining bo th SE CM and SICM, the topographical information detected by SICM can be directly correlated to electroc hemical processes at the sample surface detect ed by SECM. A miniaturized el ectrode can be ei ther integrated into the pipette as a ring electrode surrounding t he ori fice of t he pipette or in a double (or multi ple) cha nnel arrangement, using o ne barrel of the p ipette filled with ele ctroly te sol ution for SICM and another barrel filled with the electrode mater ial for SECM (Figure 8 A ). The comb ina tion with AFM is described in detail in the followi ng section. Scheme of co mbined SECM/ SICM tips w ith varyin g geometries ( A ); th e black area show s th e electrode material, scheme for the diff erent imaging principles (B): the SECM ring-electro de is detecting electroche mical processes, wher eas the cond uctance between two re ference electrodes (ind icated as green dots) is used to d etermine th e top ography. A. Holzinger, C. Steinbach, C. Kranz, „Chapter 4: Scannin g E lectroche mical Microscopy (SECM) : Fun damenta ls and App lications in Life Sciences“, in Electroch emical Str ategies in Detection Science (Ed . Damien W. M. Arri gan), RSC Detection Science, 201 5. Adapte d from Ref. 115 with permission from the Royal Society o f Chemistry. http s://doi.org/1 0.1039/97817826 22529 -00125 . - 23 - AFM-SECM First publish ed by Macpherson and Unwin in 2000 132 , the combination of AFM and SECM enables imaging of high-resolution topography and ele ctrochemical processes by detection of force interaction between a conical tip and a surface ( AFM) and the electrochemical signal related t o surface pro perties (SECM) . Wherea s in this publication, a cantilever-sh aped conical electrode served as the AFM tip and simulta neously as the SECM electrode, the independent detection of an electro c hemical signal by a recessed AFM tip-integrated electrode was f irst realized by Kranz et al. 133 . First imaging of soft biological sam ples w as re ported for the i nvestigations of th e activity of glucose oxidase 134 or horseradish peroxidase 135 by AFM in tapping or contact mo de, respectively, and SECM generation/collection m ode. Modi fication o f AF M -SECM probes by im mobilization o f redox media tors, n amely, tip-attached redox med iator (TARM)- AFM-SECM 136 and investigation of immobilized macromolecules or DNA in the so -called molecule touching (Mt)/AFM-SECM 137 was s hown by D emaille and co-workers. For example, the detection o f proteins by measurements in AF M tapping mode and SECM feedback mode w ith a latera l resolution in t he topography of a few 10 0 nm 138 was re ported. Th e immobilization of DNA sensors to AFM/SECM pro bes was also realized by modification of the tip -integrated electrode with polypyrrole 139 . Further in vestigations of biolo gical samp les were reali ze d by Hirata et al. 140 , mapping glu cose oxidase immobilized on highly oriented pyrolytic graphite (HOPG) in tapping mode, and by Kranz and co -workers with the localiz e d mapping of enzymatic c onsumption of glucose b y det ection of H 2 O 2 wit h a horseradish peroxidase-modified AF M-SECM probe i n AFM contact mode 141 . Besides investigations of biological samples, AFM -SECM can be used for visualizing corro sion processes and the electrochemical behavior of metal compositions. Macpherson a nd co -workers investigated the beh avio r of me tal an odes by AFM -SECM 142 and Davoodi et al. detected active pitting and local corrosion of mixed Al alloys by an L-shaped AFM tip with an integrated Pt wire as SECM electrode 143 – 145 . The localiz ed corrosio n of copper in acidic solution was rep orted by Izquierdo et al. 146,147 . The dissolution of calcite was detected by AFM -SECM 148 , whereby t he tip-integrated Pt electrode w as used for water oxida tion by an applied anodic potential and afterward used for detecting pits w ithin t he calcite in the acidic region by t he generated protons. Inve stigations of diffusional transport at - 24 - nanopores b y AFM-SECM was reported for the t ransport of glucose 149 , of redox-active species 150 , as well as for electrode arrays within c losely-sp aced microdiscs 150,15 1 . Overview on different AFM -S ECM p robe geometries: Schemes of a han d-made AFM-SECM probe with a conical microelectrod e with a sph erical apex 15 2 (A ); batch -fabricated AFM -SECM probe with a triangular, conductive AFM tip 153 (E) and commercial AFM probe modified by a conductive lay er cond uctive BDD layer and insulated, follow ed by the exposure of a recessed frame el ectrode b y FIB -milling 154 (I). The detection p rinciple of the e lectrochemical signal is depicted in B, F and J and corres pondin g results of AFM -SECM mea surements are sho wn for the d etection of Au parti cles i n Mt/AFM -SECM 152 ( C), indivi dual recorded topograp hy (G) an d current signal (H) with a condu ctive AFM tip 153 and simultan eously detected topography (K, M) and current signal (L, N) of an UME with an AFM-SECM prob e with a rece ssed conductive BDD electrode ; magni fied vi ew of the results (M , N) show the b locked current signal of the UME due to a diamond particle at the UME surface 154 . R eprin t with permission from Eifert, A., Kranz, C. Anal. Chem. 2 014 , 86 , 5190−5200. Copy right 201 9 American Chemical Society. https://doi.org/10 .1021/ac50 08128 . - 25 - Different geometries and f abrication procedures of AFM -SECM probes have been published so f ar, for example, hand-made 155,15 6 , ba tch-fabrication 153,157,158 a nd modification of comm ercially available pr obes 140,159 – 16 1 , of w hich some examples are depicted in Figure 9 . Accordin g t o the fabrication process, different electrode shapes an d sizes are p ossible, n amely ring s haped 162 , s quare 163 , f rame 133,159 , disk 164 , cone-shaped 132 or sp herical 165 . Especially, FIB p rocessing enables man ufacturing a wide range of different geometries, electrode shapes, and siz es, or different arrangements of t he electrode area within the AFM-SECM probe 133,16 1,166 . This contribution is b ased on the fabrication of recessed frame electrodes as first reported by Kranz et al. 133 . This method implies the modification of a comme rcially available AFM probe with a thin adhesive l ayer of Ti and a conductive gold layer, followed by insulation with Si x N y , Si x N y /SiO 2 mixed layers or Parylene C. SEM images of an AFM- SECM probe durin g FIB-milling in two different per spectives, either perpendicular to wards the electron o r ion beam, indicated on the left side: an insulated AFM- SECM prob e with mark-ups o f the area, which i s remov ed by FIB (A) and after FIB -milling (B). By mounting the AFM -SECM p robe in 90° to the original position an d removing the marked region in (C ) by FIB -milling, the ele ctrode is expo sed with a th orn located in the center . The SE M im ages depicted in D-F show the Au and the insulation l ayers d uring the single fabrication steps, 90° to wards th e ion b eam and 52° towards the e -beam, re spectively. Ac celeratio n voltage: 3 kV an d beam curre nt: 36 pA. - 26 - The exposure of the recessed electrode area is done by FIB -milling, which is also used f or the preparation of a new AFM tip located in the center of the square for med by the frame - shaped electrode. The individual fabrication FIB -milling steps are summarized in Figure 10 . The resulting Au frame electrode with a frame wid th of about 100 nm, depending o n the thickness of the deposited Au layer, can be varied in size according t o the desired application. Additional mod ification s of the tip-integrated electrode result in advanced probes suit able f or detection of specific analytes . Th e AFM tip p roduced by FIB-milling is located in the center of the Au frame. In this case, the tip is resh aped from the original Si AFM p robe, but variations in position ar e also p ossible and L-sh aped AFM- SECM p robes made of insulating material (Parylene C) and lo cated n ext to the electrode area were reported and used for the mapping of carbon nanotubes 159 . SEM images of the final AFM- SECM probe imaged in differen t perspectives as shown in Fi gu re 10 (A , B) an d top view of the f inal Au frame (C); Acceleratio n voltage: 3 kV an d beam curren t: 36 pA. A p recise characterization of the actual geo metry of AFM -SECM probes is essential and small variations, the RG values, artifacts within the AFM -SECM p robe and t he posit ion of the tip at or within the AFM- SECM probe 167,168 show an imp act on the current signal, which was shown by simu lations for AFM -SECM probe s with the AFM ti p as co nical electrode . Additionally, the effect at the border between insulating and conductive regions of a sample has been shown by simulations f or AFM -SECM probes with a rec essed electrode geometry p redicting an overlapped current signal of the insulating and conductive region, respectively 169 . - 27 - Different electrode materials ca n be used for AFM -SECM. Gold 133,156,170, 171 or platinum 132,159 are most common ly utilized, b ut also bor on -doped diamond 154,172 , carbon nanotubes 164 or c onductive colloids 165 have been reported so far. Additionally, modification of gold el ectro des w ith a P t/C comp osite deposited via IB ID 173,174 enables a n easily tunable electrode area, adjustable to the re quirements of the targeted AFM-SECM measurements. - 28 - FIB /SE M t om og r aph y In scanning elect ron microscopy (SEM ) 175 , a high en ergy ele c tron beam is generated by a thermionic or field emission emitt er, focused and de- magnified by a set of electromagnetic lenses and is finally scanned over a defined area of the sample surface. When the en ergetic electrons penetrate the sample, elastic and in elastic scattering occurs at atoms of the samp le, whereby the t rajectories of t he el ectrons are changed and energy is tran sferred during scattering event s. This interaction with the sample causes th e emission of seco ndary electrons (SE), as well as c haracteristic X- rays a nd bremsstrahlung . The actual trajectories of the primary electrons within t he sample volume can be described by Monte- Carlo simulations 176 . Incident electrons, which are able to leave the sample after sca ttering are called b ackscattered electrons (BSE). Th e i nterac tion between the primary electron beam and the sample t hereby depends on the acceleration voltage of the electron b eam, as a high er acceleration voltage re sults in a higher penetration depth within the sample (Figure 12 ). Additionally, t he yield of detectable electrons is dependent on the composition and de nsity of the sample m aterial and the incide nt angle of the electron beam tow ards the sample surface. Scheme of a model sa mple and the influence of th e accelera tion voltage of the primary e-b eam (represented by th ree arrows for three different accelerati on voltages) in the d etected SEM (top view): the h igher the ap plied voltage, th e more of th e inn er structure of a sample i s visible in the SEM image. - 29 - Therefore, as they originate from a cer tain depth of the sam ple, the amoun t of BSE abl e to leave the sample depend s strongly on den sity and composition, as well as crystal orientation. SE are se nsitive for surface relie f, due to their l ow er energy cont ent (50 eV and less). On ly SE emitted near the sample surface are able to leave the sample and be collected by a detector. The contrast in SEM images results from vary ing amounts of detected electrons per pixel. SEM gives , therefore , information on the localized structure of material s of known composition wit hin the sample and the surface str ucture. In SEM , conductive samples can be easily imaged, whereas imaging of insulating materials may cause distor tions due t o charging effects. The most commonly used detector in SEM is th e Everhart-Thornley detector (ETD) 177 based o n the conversion of electrons i nto photon s by a scintillator, enhancing the signal b y 10 5 – 10 6 times with in a ph otomultiplier and signal collection by a computational read-out system. The ETD is located perpe ndicular to the optical axis of the primary elect ron b eam wit hin the chamber of the SEM. State- of -the-art microscopes provide an enh anced resolution by using an ele ctron detector p laced above the o bjective lens pole pieces wit hin the electron colum n, whic h are referred to as through-the-lens detector (TLD). The use of the so -called immersion mode, in which the magnetic field from the objective lens pole piece is extended to the sample su rface, attracts SE t o travel towards the TL D. SEM is used primarily as an imaging method , but modification and structuring of samples are also possible with a focu sed ele ctron bea m 178 . A b eam of accelerated ions instead of electrons is preferred for nano- and microstructuring of materials. FIB is high ly suitabl e for mask-less tunable fabricat ion steps compared to other microfabrication processes, e.g. e -beam lithography 179 or deep reactive ion etching ( DRIE) 83 . For FIB, mostly a gallium ion source is used 180 . FIB plays a significant role, ranging from microfabrication, investigat ion of materials perpendicular to the surface by cross- sectioning, TEM sample preparation, up to deposit ion of metals and insulator ma terials by ion beam induce d de position (IBID) eit her as p rotection layer or for surface mod ification. Examples for FIB applications relevant to the research presented within this thesis are th e exposure of the electroa ctive area in AF M-SECM p robes 133,160,161 , fabrication of nanop or ous arrays in solid -state mat erials 31 – 34 , cross-sectioning of nanopores f or investigation of electron or ion be am -induced variatio ns i n pore sh apes 36 and fabrication of high-aspect AFM p robes for investigation of such nanopores by FIB - milling con sidering e.g. t he surface tilt or other param eters 181 . With the availability of - 30 - FIB/SEM d ual-beam i nstruments 182 , the inves tigation and reconstruction of materials b y FIB/SEM tomography 183 becomes possible and structu res of several nanometers can be resolved 184,185 . By an automated successive FIB millin g and SEM imaging of the freshly exposed faces, a whole sample volume can be investiga ted with an axial re solution of 3 – 30 nm 10 . T he acq uired image s tack, which represents t he sampled volume, is proc essed by a 3D software, whereby sample drifts or varia tions in co ntrast d uring FIB/SEM image acquisition can be corrected. The 3D reconstruction of the ac tual mo rphology of t he sample gi ve s access t o the p orosity and inner structure wi th nanometer resolution . Fig ure 13 gives an overview of FIB/SEM tomography and 3D reconstruction of a nanoporous structure, w hich is d iscussed in detail in section 6. FIB/SEM tomography can be a pplied to a large variety o f scientific questions , s uch as the invest igations of nanoparticles in d ye- sensitive solar cells 14 and of nanotube arrays 3 . Investigations of the inner structure of micro- and nanoporous mat erials 12,13 or Al-Si alloys 186 , but also the investigation of biological 15,16 and geolog ical 16 samples have been reported. - 31 - FI B/SEM tomograp hy: For 3D tomograp hy, the sample of interest i s stabilized with a Pt/C protection-layer an d a cros s-s ection is milled b y FIB. Th e FIB -induced SE i mage in A show s the perspective of the ion b eam, whereas in B the perspecti ve of the SEM with a tilt corr ection of 38° toward s the ion beam is shown (th e a ctual angle between ion an d e lectron b eam i s 52° and defined by the spatial arrangement of the e-beam and ion-beam columns). The yellow - framed SEM images repr esent single slices after con secu tive FIB milling. After drift and contrast correction, th e slices can be reconstructed as a 3D image (C). The structure can be presented in different p erspe ctives ( D). A. Holzinger, G. Neusser, B. J. J . Aus ten, A. Gam ero - Quijano, G. Herzog, D. W. M. Arrigan, A. Ziegler, P. Walther, and C. Kranz, Faraday Discuss. , 2018, 210 , 113. Adap ted from Ref. 187 . D istribu ted under th e lic en se Creative Commo ns Attribution-N onCommercial 3.0 Unpo rted (CC- BY -NC 3.0), https://creativeco mmons.org/licenses/by -nc /3.0/ . - 32 - Re s ul ts a n d di sc us s io n In the following, th e results ob tained by modifications of AFM -SECM probes are presented. In chapter 4 , t he influence of the indi vidual pretreatment steps necessary for the mountin g of the AFM tip -integrated PB/NIHCF b ilayers as H 2 O 2 - sensitive mater ial into the exp erimental setup is evalu ated in re spect to sensitivity and stability. First results f or the detection of localize d H 2 O 2 genera tion at an UME is presented i n 4.3.3 . In c ontrast, to the mo dification of AFM-SECM p robes with th e active se nsor mat erial, in chap ter 5, th e modification of AFM-SECM probes with an additional metal layer for t ip-integrated pH sensors is evaluated. First measu rements have already been re ported by Jong Seok Moon 188 , who fabrica ted the Ir and Sb integrated A FM tips, respectively, used in chapter 5. Within this thesis, the performance in pH sensi ng of these mo dified AFM -SECM probes is p resented and first AFM -SECM measurements of localized su rface changes together with ongoing pH changes during the dissoluti on of calcite crystals are detec ted (5.3.2). In chapter 6, the diffusion at nanoporous membranes is presented visualizi ng the d iffusion behavior dependent on the pore- to -pore sep aration of individual nanopores with in an array and showing the capability of AFM -SECM with a conductive AFM t ip for t he detection of localized electrochemical p rocesses 181 . Th e nanoporous solid -state materials , which are presented i n this thesis, were fabricated by FIB milling enabling readily tunabl e geometries of nanopore arrays. Although, FIB fabricated nano porous membranes were already i nvestigated for electrochemical process es at the liquid/ liquid interf ace 30,31 , the actual l ocation of the interface b etween both liqu ids within t hese na nopores is not verified yet. The actual pore s hape and implantation of Ga + i ons d uring FIB milling might change t he hydroph ilic ity of these nanopores and with this chang e the behavior of these devices used as support material for electrochemistry at liquid/li quid interfaces. T his was no t considered so far in previous publications and will be evaluated in chapter 6 , amo ng other investigations, by FIB/SEM tomography. - 33 - H 2 O 2 se ns ing wi th A FM tip -in te gra t ed el ec tr od es Introduction Hydrogen p eroxide ( H 2 O 2 ) plays a crucial role in biological systems as reactive oxygen species (ROS) 189 , as a p roduct of the oxygen reduction reaction (ORR) in acidic media 190 , and thus, H 2 O 2 is import ant for develo ping new c atalytic materials in energy storage 191 . It is also a n important species in environmental water and atmospheric samples indicating phototoxic e ffects and environmental pollutions 192 . Next to others, like fluorometric 193 or spectrophotometric 194 methods, H 2 O 2 can be determined by electrochemical approaches 195 . First det ection approaches with solid metal electro des were reported f or the oxi dation of H 2 O 2 at p otentials of + 0.5 – 0.8V vs. N HE 17 in acidic or neutral s olutions depending on the electrode material, or H 2 O 2 reduction at 1.76 vs. NHE 196 . Hence, reduction of O 2 and other electroactive com pounds present in samples, such as ascorbic acid in biological samples, migh t interfere wit h the electrochemical d etection of H 2 O 2 197 . Additionally, the H 2 O 2 conversion shows large overpotential s at metal electrodes 196 . Therefore, ele ctrode modifications h ave been investigated enabling the conversion of H 2 O 2 at moderate potentials with enhanced sensitivity for H 2 O 2 195 . Examples f or suc h sensors are elect rodes mod ified with enzymes emb edded e.g. in redox p olymers 198 or nano-sized materials, suc h as mul ti-walled carbon nanotubes with nanoparticles 199 . Metal hexacyanoferrates and especially Prussian Blue (PB) 19 show also a promising response in H 2 O 2 d etection via the Fenton reaction 200 . PB , wi th KFe(II )[ Fe(III)(CN) 6 ] as the soluble a nd Fe(III) 4 [Fe(II) (CN) 6 ] 3 t he insoluble salt , and analogues met al h exacyanoferrates form a face-center-cubic structu re with iron(II) and iron(III) ions in t he network of the complex 201 . The ele c trocatalytic activity of PB is related t o the FeN x units in the crystal framework determined by x-ray diffraction ( XR D) and Fourier-transform infrared spe ctroscopy ( FT - IR ) 202 . The reduction of H 2 O 2 occurs at a potential of - 0.05V vs. SCE and is catalyzed by Fe(II) ions 18 , whereas O 2 does not interfere, because the reduction of O 2 to H 2 O occurs at a potential of 0.2V vs. S CE 18 . Additionally, PB is insensitive to other reductant s inter fering in H 2 O 2 sensing in biological samples 203 . Fo r meta l h exacyanoferrates other than PB, t he electrocatalytic activity for H 2 O 2 is due to d efects of PB crys tals in the sal t l attice 19 ,204 . The oxidation and reduc tion of iron hexacyanoferrate lead s to the formation of Berlin Green (or Prussian Yellow, Fe (III)[Fe(III )(CN) 6 ]) 201 and the Everitt’s salt (or Prussian White, Berlin White, K 2 Fe(II)[Fe(II)(CN) 6 ]) with structures comparable to PB 205 . - 34 - Fe [ Fe ( CN ) 6 ] + K + + e − ⇌ KFe [ Fe ( CN ) 6 ] + K + + e − ⇌ K 2 Fe [ Fe ( CN ) 6 ] Prussian Yellow ↔ PB ↔ Prussian White Fe 4 [ Fe ( CN ) 6 ] 3 + 3K + ⇌ 3 KFe [ Fe ( CN ) 6 ] + Fe 3+ insoluble ↔ solu ble Thereby, the role of potassium ions is significa nt for the redox reaction , because the electrochemical p rocesses are re lated t o the t ransport of K + rath er than the t ransfer of electrons within the PB lattice 206 . Th e amount of K + with in the PB crystal stru cture also affects the sensitivity for H 2 O 2 detection. T he infl uence of the used el ectrolyte solut ion , containing K + cations during electrochemical PB deposition was shown by Zh ang et al. 2 07 . A potential shift in the open circuit potential of PB was obta ined in dependence to the used electrolyte . K + ions with in t he crystal lat tice also guarantee the electro -neutrality. The ability for K + (de- )ins ertion during oxidation and reducti on, respectively, enables also the u se of PB f or insertio n electrochemistry in e.g. en ergy stor age due to its capability t o store K + ions 208,209 . PB is frequently used for H 2 O 2 sensing, especially in biosensors 203 and even the synonym “artificial peroxidase” was established in literature 210 . The f irst deposition of PB on electrode materials b y dipping the electrodes into a ferric ferricyanide solution was pub lished in 1978 and the redox behavior was shown by single sw eep voltammograms 211 . The first el ectrochemical deposition w as presen ted by I taya e t al. 212 ,213 . Since th en, d ifferent approaches f or th e electrochemical synthesis of PB has been reported resulting in a va riation of the s tructure and the stoichiometric composition of PB depending on the elect rochemical conditions. Ep itaxial growth o f PB on g old was real ized by electrochemical deposition at a constant pot ential 214 . PB was d eposited on gold nanoparticles t o enhance the sensitivity (10.6 µ A/(µM · cm 2 )) 215 toward s H 2 O 2 compare d to b ulk gold electrodes , and even the sim ultaneous formation of gold nanoparticles and PB on gold or glassy carbon electrodes was report ed by Ku mar et al. with H 2 O 2 sensitivities up to 5 nA/nM 216 . Other materials u sed as supporting electrode materials for PB deposition are Pt and carbon paste usi ng a ferri c yanide solu tion for t he e lectrochemical - 35 - deposition by cyclic voltammetry 217 , or Pt/C composite dep osited via IBID sh owing enhanced st ability and H 2 O 2 sensitivity compared to g old as electrode mater ial 218 . A drawback of PB for H 2 O 2 sensing is its reduced lo ng-term stabili ty. Th e stability of PB is influenced by the p H value of the an alyte solution and a loss of stability can be seen in alkaline solutions due to t he p ossible formation of Fe(OH) 3 219 . Because the conversion of H 2 O 2 results in the production of OH - ions in n eutral an d alkaline media, t he lon g-term stability of PB is limit ed to a few minutes in the p resence of higher amounts (tenths of mM) of H 2 O 2 220 . PB can be stabilized by co-deposition of conductive polym ers 221 enabling additionally the embedding of enzymes in biosensors 203 . An alternative modification to overcome th is limitation is the co -deposition with other metal hexacyanoferrates showing no sen sitivity to H 2 O 2 , but exhibit enhanced stability in neutral and alkali ne media 204,222 . Komkova et al. examined an optimal ratio for mixed metal hexacyanoferrates of three bilayers of PB and nickel hexacyanoferrate (NiHCF) with reduced s ensitivity comp ared to PB films but enhan ced long -term stability for several hours 20 . PB -modified electrodes were already u sed for the localize d detection of H 2 O 2 evolution by SECM in t he generation/collection mode 218 and u sed as a lactate b iosensor in soft-stylus SECM 223 . A H 2 O 2 sen sitivity of 1.6 A/(M · cm 2 ) 223 could be achieved f or the lactate biosensor . Additionally, the d issolution and corresponding loss in H 2 O 2 sensitivity for mixed PB/N iHCF bilayers 20,224 were investi gated via SECM . - 36 - Experimental The modi fication of electrodes with H 2 O 2 sensitive PB/N iHCF w as done a ccording to the procedure published by Komkova et al. 20 . Three bilayers of PB/NiHCF were electrochemically deposited by alternating d eposition of PB and NiHCF. All electrochemical measurements were performed with a (bi)potentiostat (660A or 660C, CH Inst ruments). All solutions were d iluted with ultrapure water (18.2 M , ELGA LabWater, Veolia Water Solutions & Technologies). PB as first laye r was deposited in a solution containing 4 m M FeCl 3 · 6 H 2 O (pro analysi, M erck KGaA ) and 4 mM K 3 [Fe(CN) 6 ] (Honeywell, Flu ka) in 0.1 M HCl (f rom 32% HCl Normapu re, V WR Chemicals) and 0.1 M KC l (pro analysi, Merck KGaA ) by cyclic voltammetry within a potential range of 0.4 to 0.75 V vs. Ag/ AgCl re ference electrode (sat. KCl, RE- 1CP, ALS Co ) at a scan rate of 20 mV/s. The electrodes were rinsed a fter deposi tion with ultrapure w ater and the first layer was dr ied at 80°C for 15 min. NiHCF w as deposited at a potential range of 0 – 0.8 V at 100 mV/s i n a mixture of 1 mM NiCl 2 ( pro analysi, Merck KGaA ) and 0.5 mM K 3 [Fe(CN) 6 ] i n 0.1 M HCl and 0.5 M KCl. Subsequent depositions were d one with out additional tempering by alternating deposition of PB and NiHCF u p to 6 depositions of 2 CV cyc les for each compound resulting in three bilayers of mixed films. Electrochemical deposition of PB (A) an d N iHCF (B) by cycl ic voltammetry : P B: 1 st (blue), 3 rd (red) and 5 th (green) layer were deposited at 0.4 - 0.75 V vs. Ag/AgCl refe rence electrode; scan speed: 20 mV/s. NiHCF: 2 nd (bl ue), 4 th (red) and 6 th (gr een) layer were deposited at 0 - 0.8 V vs. Ag/AgCl referenc e electrode ; scan speed: 10 0 mV/ s. - 37 - After deposition, the fil m was activated by cycling at a poten tial range of 0 – 0.85 V vs. Ag/AgCl re ference electrode at 40 mV/s in 0.1 M HCl/ 0.1 M KCl with varying numbers of scans. Af ter ac tivation, the e lectroactive PB/NiHC F film was d ried at 80 °C for at least 3 0 min. Before and after electrochemical modification of the UMEs, the electr ode surface was invest igated by optical microscopy (AXIO Ima ger.M1m, Zeiss ) a nd by SEM (Qua nta 3D FEG, ThermoFisher Scien tific), if no t s tated other wise. The sensitivity and response of the modified electrodes to ward s H 2 O 2 were tested by calibration at a constant pot ential of 0 V vs. Ag/AgCl reference electrode with s ubsequent addition of equimolar al iquots of H 2 O 2 (40 µL (50 µL) of 1 0 mM H 2 O 2 (fr om 30 % H 2 O 2 , EMSURE, Merck)) to 0.05 M phosphate buffer (4 mL (5 mL), pH 6 -7, consisting of Na 2 HPO 4 · 12 H 2 O (NORMAPUR, VWR Chemicals)/NaH 2 PO 4 · H 2 O (pro analysi, Merck KGaA ) in 0.1 M KCl electrolyte solution within a co ncentration range of 99 – 476 µM. For evaluation of the results, the averaged steady-state currents after c urrent leveli ng were used . High conce ntrations of H 2 O 2 ( 1 – 2 mM ) were us ed next to calibrat ions to test the stability of t he sensors. T he activation in 0.1 M HCl/ 0.1 M KCl was u sed to determine the amount of active PB film on the electrodes after being exposed to H 2 O 2 solution. H 2 O 2 calib ration in 0.05 M phosphat e buffer of a PB/NiHCF - modified Pt UME (diameter: 25 µm) with successive ad dition o f equimolar aliquo ts o f 10 mM H 2 O 2 (A) an d corre spondin g CV in 0.1 M HCl/ 0.1 M K Cl recor ded prior to calibration ; scan rate: 0.02 mV/ s (B). - 38 - Additionally, t he dissolution of PB/NiHCF bilayers was investigated in solution b y con tact AFM -SECM (5500 AFM/SPM microscope, Keysight Techno logies). All AFM image s and correlating h eight and current profiles were processed by Pico View (Keysight Technologies) or Gwydd ion 225 . The electrochemical liquid cell consisted of t he AFM-SECM probe as working electrode, an Ag /AgCl quasi-reference elec trode (G oodfellows) and a Pt wire (Goodfellows) as coun ter electrode. Different conditions were evaluated with respect o n the stability a nd res ponse o f t he PB/ NiHCF film f or H 2 O 2 sensing . Details of the AFM -SECM probe fabrication is based on the procedu res described elsew her e 133 . Brie fly summarized, the modification of an AFM tip-integrated electrode is comparable t o the modification o f U MEs. It has to be e nsured that t he sharp AFM tip rema ins u nmodified . Therefore, the AFM- SECM probe was investigated b y SEM after modific ation with PB/NiHCF bilayers and the AFM tip had to be reshaped in case of deposited particles located on t he AFM tip, because N iHCF is known to deposit spontaneousl y, also without applied potentials d uri ng el ectrochemical deposition and t hus, the dep osition of PB/NiHCF bilayers is not just occurrin g at t he electrode surface. Additionally, the electrode area of ti p-integrated electrodes was en larged by IBID (pr ecursor: methyl - cyclopentadienyl-trimethyl platinum, ThermoFisher Scienti fic ) of a Pt/C composite 174 and the PB/NiHCF films deposited on t his electrode m aterial were evaluated for stability, H 2 O 2 response and sensitivity. The modification of t he AFM -SECM p robes with Pt/ C was done by consecutive deposition of rectangular patterns surroun ding the AFM tip (Figure 16). To guarantee that the AFM tip remai ns f ree of the conductive material, a circular pattern was used removing possible Pt /C deposition at or close to t he AF M tip. Thereby, the AFM tip was reshaped after the final deposition of t he PB /NiHCF film to obtain a sharp and clean AFM ti p. Single steps of the Pt/C deposition an d c leaning by FIB milling are summarized i n Figure 16. - 39 - SEM images showing th e mod ification of A FM- SECM probe s with Pt/C co mposite b y IBID: The exposed Au frame electrode (shown in A) is modified by IBID (rectangular blue area), foll owed by circular cle aning ar ound th e AFM tip an d rectangu lar cleanin g patterns n ext to th e actual electrode area (marked in red) by FIB milling prior to electrochemical modification with PB/NiHCF ( B). Sin gle fab rication steps ar e sho wn 54° tilted, before (C) an d aft er (D ) Pt/C deposition and after FIB cleanin g-(E). Acc eleration voltage : 3kV and beam current: 35.5 p A. The AFM-SECM p robes have to be glued onto the AFM mount (nosecone) and the electrical contact of the electrode was insulated aft erward by UV glue (Dymax 9001 -E- V3.1 or DYMAX 425, Dymax Europe GmbH ). The influence of UV light (U V light: = 320 - 395 nm, 9 W/cm 2 , Dymax UV lamp 75 Blue Wave, Dymax Corp .), which is req uired for curing the glue, on the stability of PB/N iHCF films was also examined. Preliminary measurements were done with UMEs being less time consuming to modif y and enabling simplified handling compared to AFM -SECM pro bes. The Pt and A u UMEs used for these measurements were fabricated as described elsewhere 65,226 . - 40 - Results and discussion This first section summarizes and d iscuss es t he resu lts obt ained with PB/NiHCF-mo difie d UMEs, f ollowed b y th e modification and optimization for the electrochemical deposition of PB/NiHCF on to AFM-tip-integrated electrodes. First re sults in AFM-SECM measurements are presented in section 4.3.3. PB/NiHCF-modified UMEs The modifica tion of UMEs with three PB/NiHCF b ilayers resulte d in an inhomogeneous film with differ ent structures and colors as visible in SEM and optical images (Figure 17) , respectively. Thereby, the varying colors indicate different oxidation st ates of the PB and varying stoic hiometry o f the PB structure. No correlation between the c olor or the shape of the deposited film and the electroch emical deposition could be drawn. SEM (A, C) an d op tical images (B, D, E) of UMEs (diameter: 25 µm ) before (A, B) and after (C -E) electrochemical d eposition of P B/NiHCF bilayer s. - 41 - The i nhomogeneous structure might be also relat ed to an inhomogeneous mixtu re of the PB and the NiHCF at the surfaces of the modified electrodes . To exclude ch anges in morphology and correlating loss of H 2 O 2 sensitivi ty during the measurement due to an irregular composition o f the PB/NiHC F -mixed bil ayers, the elem ental composit ion of the film was investigated via EDX mapping as shown in Figure 18. The counts for Ni are homogeneously distributed over the entire film and the Fe map i n Figure 18 shows ad ditionally an enhanced intensity for Fe compared to Ni as a consequence of the presence of Fe ions in both, iron and nickel hexacyanoferrates. The i ntensity distribution of the Pt signal originating from the electrode m aterial b eneath the PB/NiHC F bilaye rs reveals a n i nhomogeneous thick ness o f t he PB/NiHCF film with less d eposit on the areas with high Pt counts ( white labeled i n Figure 18 , Pt ). The excita tion volum e according to the p enet ration depth of t he electron beam into th e sample volume is, of course, larger than the actual thickness of the PB/ NiHCF b ilayers, which is apparent in the detected Pt intensities. SEM (A, B) and EDX map ping (colored images sho wing the distributio n of th e elements : red (Fe), green (Ni) and blue (Pt)) of an UME (diameter: 25 µm) modified with PB/NiHCF -mixed film . Acceleration voltag e: 5 k V. EDX mapping repr esents th e average el emental distributio n of in sum 512 frames (re solution: 256 x 200 pixels). - 42 - A penetration depth of up to at least a µm has to be assumed in PB/NiHCF layers at an acceleration voltage of 5 kV as used in the f ollowing EDX mappings, depe nding on the local material composition and density. This varying thickness can not be related to t he composition of the mixed film and higher Fe and Ni intensities in the EDX map s are likely related t o an increased thickness of the PB/NiHCF bilayers. The ob served cracks may be associat ed with the vacuum conditions. Because the p recise stoichiometry of PB 227 and NiHCF 228 is not known due to varying composition and oxidation states of PB and varying K + content in both salt structures, quanti fi catio n is not possible. The h omogeneous distribution of Ni is clearly visibl e in Figure 18. In case of an irregular composition of th e mixed PB/NiHC F film, the distributio n of the i ntensity for Ni should be i rregular as well. Another appro ach for the i nvestigation of the composition of the mixed film of PB/NiHCF is the dissolution of singl e PB particles in the presence of H 2 O 2 detecta ble by chan ges in the morphology of the f ilm. The dissolution of the PB/NiHCF was measured in- situ by AFM-SECM and the results are depicted in Figure 19 . N ext to the dissolution of these particles, the change in topography can be also explained by mechanical re moval of surface structures due to the scanning AFM tip . Th ereby, resulting art ifacts w ithin the topographical image are visible and marked by white arrows (see Figure 19 ). While imaging of t he modified UME as depicted i n the images in Figure 19 A and B, no H 2 O 2 was present in the solution and n o potential was ap plied to the AFM -S ECM probe. Hen ce, the ob served ch anges in topography cou ld be addressed to mechanical removal of the PB/NiHCF bilayers. The A FM topography shown in Figure 19 C is the last scan recor ded a fter a duration of in t ot al 7.5 h s howing the PB/NiHCF film that was not re moved d uring th is measurement. Between the results shown in B and C, a potential was applied to the AFM - SECM tip sufficient for generating H 2 O 2 ( - 0.5 V vs. quasi-Ag/AgCl r eference electrode) a nd t he surface c hanges during consecutive AFM scans w ere detected. For improved visualization of the morphological changes, just a small section of the PB/NiHC F -modif ied UME was investigated in detail (white square in Figure 19 B). The results f or three con secutive AFM scans are depicted in the AFM topography images in Figure 19 D-F. The morphology of the PB/NiHCF-modified UM E changed w ithin appro ximately 12 mi n in the presence of H 2 O 2 generated a t t he AF M -SECM probe. The dissolution of a large particle is visible, whic h is also partially removed by the scanning AFM tip. - 43 - Investigation of the stability and surface chang es of a modified UME while H 2 O 2 is generated at the AFM -SECM pro be, by contact AFM in liqu id. Du ring imaging the surface depicted in A-C, no p otential was app lied to the AFM - SECM prob e. Origina l s ize: 50 x 5 0 µ m 2 (A, B), 60 x 60 µm 2 (C), scan speed: 0.6 ln/s ( 60 µ m/s) (A,B), 0.8 ln/s (9 6 µ m/s) (C). The timeline (not to scale) depicted in the figure shows the ti mes (min ) when the imag es were recorded durin g a tota l duration of the experiment of 7.5 h. For the small ar ea of the PB/NiHCF- modified UME depicted in D-F, the AFM -SECM probe was b iased at -0.5 V vs. quasi-Ag/Ag Cl ref erence e lectr ode and H 2 O 2 was generated in close proximity to the surface. Scan direction: down (D), up (E) and down (F), original size: 25 x 25 µm 2 , scan speed: 1.5 ln/s (37 µm/s ). The AFM to pograph y in D-F shows single scans r ecorded in between the scans sho wn in B a nd C, after a d uration o f 198, 204 and 210 min, r espectively. But the reduced size of the particle and shrinkage of its structure visible in th e morphological changes in i mage E to F can be associated wit h the dilution in t he pre senc e of H 2 O 2 generated at the AFM- SECM probe. Thereby, the volume of this particle is reduced by approximately 62%. Following scans at different scan areas in the presence of H 2 O 2 , however, showed no further changes i n the AF M topography (resul ts are no t shown). The AFM -S ECM investigation in Figure 19 shows that higher regions of the PB/NiHCF b ilayers or particles locat ed on t op of the ‘ main ’ PB/N iHCF bilayers are predominantly removed in th e presence of H 2 O 2 . In SEM - 44 - images, t hese regions are also visible a nd chang es in surface morphology after ex posure of the PB/NiHCF-modified ele ctrodes t o vary ing c oncentration of H 2 O 2 w ere obs erved. T he sensitivity loss in H 2 O 2 detection observe d in t he record ed electrochemic al i -t curves may b e correlated to the morphology changes visible i n the consecutively recorded SEM images. H 2 O 2 sensitivity and response of th e modif ied electrodes were d etermin ed by ca libration measurements before and after th e exposure of modified electrodes to H 2 O 2 , whereas in between th e single SE M i mages shown in Figure 20, the U ME was exposed t o solu tions of 0.7, 0.8 and 1 mM H 2 O 2 , res pectively, and a n i-t-curve was recorde d over a period of 2 5 min f or each H 2 O 2 con centration. Comparin g the SEM d ata t o the topographical changes ob served in the AFM-SECM investigation shown in Figure 19, a chang e or dissolution of these particles on top of the ‘main’ PB/NiHCF film would be expected. SEM in vestigation of a PB/NiHCF- modified UME after electro chemical d eposition (A) and after exposure to d ifferent H 2 O 2 co ncentration s: 0.7 mM (B), 0.8 mM (C, D) and 1 mM (E, F ). R egions o f the PB/NiHCF bilayers lab eled by red arrows and circles are stable in the presence of H 2 O 2 . Acceleration voltag e : 5 k V; 53 pA (A,B) and 852 pA (C - F ). - 45 - These particles are mark ed b y a red arrow and cir cle in Figure 20 and s howed h owever almo st no changes during the consecutive H 2 O 2 exposures in between recording t he individual SE images. In contrast to AFM investigations, the main characteristic of th e PB/Ni HCF bilayers is a plate-structured deposit on the UME, which showed a reduction in surface coverage with consecutive H 2 O 2 detection. The changes in the surface coverage are more obvious wit h additional H 2 O 2 exposure, especially visible in t he SEM images di s played in E and F showing a larger area of the PB/N iHC F-mo dified UME. The dissolution of the PB/NiHCF film seems to be homogeneous and along the edges of the visible structures, which is energetically favorable. Whereas the particles on top of th ese main characteristic plate- struc tured deposits were dissolved during AFM investigat ions, t hese struc tures observed in the SEM images as bright single particles, appear to be not a ffected by the H 2 O 2 exposure. Hence, t hese particles might consist either of NiHCF a s a stable component of the mixed film insensitive to H 2 O 2 or these particles a nd structures consist of mixed components of PB/NiHCF. The resu lts obtained b y EDX mapping shown in Figure 18 indicate that a homogeneous mixture of PB and NiHCF is most likely. The localized change and dissolution of a pure PB film in the presence of H 2 O 2 were detected in co nsecutive AFM sca ns as sh own in Figure 21 . Thereby, a Pt UME modified only with PB was in vestigated at a constant H 2 O 2 concentration (1 mM) similar to the concentrations used in the SEM studies (as shown in Figure 20). AFM topograp hy of a PB-modified Pt UME (d iameter: 25 µm) in the presence of 1 mM H 2 O 2 in 0.05 M phosphate buffer. The depi cted AFM i mages are recorded at 16, 84 and 153 min, respectively, of in tota l 2.5 h , showing the ch anges in th e surface coverage during this AFM investigation. Original size: 60 x 60 µ m 2 , s can sp eed: 0.5 ln/s. E lectroche mical dep osition of PB wa s done with 20 cycle s according to t he experimental condition s described in 4.2. - 46 - Within approximately 2.5 h, t he whole PB film was re moved. A possib le influence of the vacuum conditions during SEM imaging was also investigated. A H 2 O 2 cal ibration curve and corresponding loss of PB at a modified electrode with consecutive H 2 O 2 exposure is exemplarily shown in Figure 22 . In between the deposition of PB and N iHC F, the electrodes were exposed to vacuum (chamber vacuum of the SEM , in the range of 10 -6 mbar ), namely after t he 1 st (PB), 2 nd (NiHCF) and 3 rd (PB) layer. After t he deposition of in sum 6 layers in accordance with 3 PB/NiHCF-mixed bilayers, the electrode was investigat ed by SEM. 1 st H 2 O 2 calibration in 0.05 M p hosphate buffer of a PB/NiHCF-modifi ed Pt UME (diameter: 25 µm) and correspondin g CV in 0.1 M HCl/KCl (scan rate: 0.02 mV/s) afte r 1 st calib ration (blue) and afte r successive exposure to 1 mM H 2 O 2 for 25 min, respectively (running order: red, gree n, yell ow). SEM images after PB/NiHC F d eposition (C) an d after H 2 O 2 detection (D) of in tota l of 3.5 mM H 2 O 2 over a period o f approximately 2 h; acceleration voltage: 5kV, 7pA (C) an d 8 nA (D). In between the single H 2 O 2 calibrat ions, the PB/NiHCF-modif ied UME was only characterized by CV in 0.1 M HCl/KCl and the ch ange in the morphology was det ected b y optical m icroscopy avoiding any influence of additional vacuum conditions i n between con secutive H 2 O 2 - 47 - exposures. In the optical images depicted in Fig ure 23 , a clear change in the composition of the mixed bilayers is visi ble, i ndicated by a change in color. T hereby, PB is redu ced to Prussian Yellow, visible as a yellow-brown re gion on the modified UME, and Pr ussian White, which is insensitive to H 2 O 2 . Single spots of the PB/NiHCF film were removed during H 2 O 2 exposure that is visib le i n Figure 23 B and C at t he bright r egions within the fi lm showing the underlying Pt electrode surface. Optical images of a PB/NiHCF -modified UME after electroch emical depo sition of PB/NiHC F bilayers (A), after the 1 st H 2 O 2 calibra tion ( B ) and after 2 nd exposure to 1 mM H 2 O 2 for 25 min (C). Comparable results obtained by optical images of a PB/NiHCF - modifie d Pt UME without vacuum conditions in between the sin gle layer deposit ions showed enhanced removal of the active b ilayers without any distinctive color changes. The change in t he surface of the mod ifie d electrode with successive H 2 O 2 calibrations is show n in Figure 24. Optical images of a PB/NiHCF -modified UME after electrochemical deposition of PB/NiHCF bilayers (A), aft er th e 1 st H 2 O 2 calibration (B) and aft er 5 th calibratio n, which equals an exposure to app rox. 2 mM H 2 O 2 (C). - 48 - In Figure 25, the comparison between two H 2 O 2 calibrations with PB/N iHC F- modified UMEs with a nd without vacuu m conditions, resp ectively, shows e nhanced sta bility for t he b ilayers exposed to vacuum, which is in accordance to the observat ion via optical microscopy. H 2 O 2 calibration of PB/NiHCF-modified Pt UMEs (diameter: 25 µm) with (dashed lines; 1 st : ora nge and 2 nd : green) and witho ut exposure to vacuum conditio ns between lay er deposition (so lid lines; 1 st : blue and 2 nd : red) . The enhanced stability of the modified UMEs, which were exposed to vacuum , might be related to the removal of water (‘drying p rocesses ’) in t he crystal lat tice. The locat ion of water molecules in the PB lattic e has been determined by attenuated total re flection FTIR ( ATR- FTIR) 229 and powder neutron diffraction revealing b oth, coordinated and u ncoordinated regions of water molecules within the crystal lattice of PB 230 . Investigations of PB by 1 H nuclear magnetic resonance spectroscopy ( NMR) showed that the water molecules are bo und to the high-spin Fe(III) ions in the crystal lattice influ enc ing t he co nductivity of the ma terial 231 . O n the other h and, coordinated water mo lecules occupy space in the PB lattic e, which might be filled with K + ion s that en able charge t ran sport during electrochemical pro cesses 227 as described in t he introduction. Water mo lecu les after electrochemical deposit ion f rom aqueous solution might also be located in between PB particles and the removal of incorporated wa ter fr om pores within t he PB film by vacuum might lead to enhanced stability - 49 - of the PB layers. A simpl e explanation for the reported enhanced sensitivity might by the fact that rap id drying under vacuu m conditions indu c es fractures w ithin the PB film , which leads to an e nlargement of the surface area. Thereby, the film might be mo re sensitive to H 2 O 2 but , on the other side, PB dilution p referentially occurs at these edges, which reduce s the st abili ty. This was not observed during the course of these investigations. In the n ext s tep, Pt /C composite as elec trode ma terial for t he electrochemical deposition o f PB/NiHCF-mixed films was investigated. The acti vation of the PB/N iHCF-modified UME b y cycling in 0.1 M HCl/KCl shows an enlarged amount of PB deposited on the UME with Pt/C composite as electrode material (Figure 26 ). The ele ctrode area modified by Pt/C deposition was determined by CV using [Ru(NH 3 ) 6 ]Cl 3 as outer sp here redox species resulting in a n act ive electrode area of 755 µm 2 , whereas a bare Pt electrode used for compa rison had an active electrode area of 825 µm 2 . In contrast to the smaller active electro de area, the current o f the PB redox signal is approximately 5 times h igher for Pt /C comp osites as electrode material compared to the PB/NiHCF modification of bare P t UMEs. CV in 0. 1 M H Cl/ 0.1 M KC l of PB/NiHCF -modified Pt UME (d iameter: 25 µm, A) and on a Pt UME (diameter: 25 µm) modified with Pt/C compo site prior to layer deposition (B), scan rate: 0.02 V/s. Blue CVs were r ecorded after d eposition of PB/NiHC F bilaye rs an d red CVs we re recorded after H 2 O 2 calibration, r espectively. After 4 consecutive calibrations (99 - 476 µM H 2 O 2 for each calibration) a t the UME, where data are shown in (A), and after 5 consecu tive calib rations (9 9 - 476 µM H 2 O 2 for each calibration ) for the Pt/C modified UME sho wn in (B). - 50 - In Figure 27, t he optical imag e of an UME is shown after th e deposition of Pt/ C composite by IBID, whereas the optical images in B and C show the electrodes after electrochemical deposition of the PB/ NiHC F bilayers. In Figur e 27 C, a large overspread o f the PB/NiHCF film is visible, which might be also the explanation for the enhanced amount of PB located on the UME. Optical images o f UME s (diam eter: 2 5 µ m) with add itional deposition of a Pt /C compo site b y IBID ; before (A) an d after ( B, C) the electro chemical deposition o f PB/N iHCF bilayer s. The o verspread of deposited PB/NiH CF depict ed in C is r educed by add itional FIB milling prior to lay er depo sition (A) as depicted in (B). This oversprea d s hould b e avoi ded in t he modif ic ation of AFM -SECM probes du e to the loss of the lateral resolution in localized H 2 O 2 detection. It was assumed that this overspread is related to additional deposition of Pt/C n ext to the actual electrode area. To confirm this assumption and to avoid t his overspread, the region surrounding the ac tual electrode area was clean ed b y FIB m illing after d eposition of the Pt/C composite, b ut prior t o the electrochemical deposition of the PB/NiHCF film ( Figure 27 A). Th e resulting PB/NiHCF modification of the UM E is shown in Figure 27 B with a n egligible overspread around the actual electrode su rface. Thereby, it can be con cluded that t his ove rspread was act ually based on the additional deposition of Pt/C composite . A dire ct comparison of the H 2 O 2 sensitivity of PB/NiHCF-modified UMEs with bare Pt or Pt /C composite as supporti ng electrode material is shown in Figure 28 . Additionally, the effect of the FIB clean ing after deposition of t he Pt/C composite to reduce the overspread of PB/NiHCF is taken into account. - 51 - Comparison of H 2 O 2 sensitivity and stability of a PB/NiHCF-modified UME at d ifferent supporting electrode materials: Pt/C composite (blue), Pt/C composite with ad ditional FIB c leaning (green) and bare Pt UME with no ad ditional treatment prior to electrochemical deposition of PB/NiHCF (red ) (A). The stability in con secutive calibratio ns of PB/N iHCF- modified UME s is co mpared in (B) wherea s dashed lines represent the first and solid lines the s econd calibration, respectively (colors are same as in (A)). The measured current respon se at the 5 consecutive H 2 O 2 a ddition s of the comp ared t hree UMEs vary with a factor of 3.5 ± 0.2 (Pt/C without FIB milling ) and 5.0 ± 0.3 (Pt/C with FIB milling), respectively, within the data sets f or Pt supporting electrode mat erial and Pt/C - modified Pt (o r Au ) UMEs with or without additional FIB millin g ( Figure 28 ). A loss of sen sitivity is observable for con secutive c alibrations for all electrodes, but for Pt /C -modified UMEs improved stability compared to bare Pt as supp orting electrode material is ob served. The enhanced H 2 O 2 sensitivity may be related to an inc reased amount of PB within the PB/NiHCF bilayers, whereas this is not necessarily related t o an enl arged electro de su rface , but to the differen t su pporting elec trode material s, namely Pt and P t/C composite. Th e sta bility of th e PB/NiHCF bilayers within con secutive calibrations (up t o 5 calibration s) of H 2 O 2 in phosphate buffer was investigated on different su pporting electrode m aterials and fo r UMEs exposed to vacuum . - 52 - The results are compared in Figure 29. The loss in sensitivity is clearly recognizable for all investigated electrodes, but increased stability for the electrodes exposed to vacuu m between single H 2 O 2 calibrations i s visib le comp ared to U MEs w ith no additional t reatment. Th e Pt/C modification of the UMEs prior t o th e d eposition of PB/NiHC F bilayers resulting in en hanced sensitivity for H 2 O 2 , shows also enhanced stability in H 2 O 2 detection. Comparison of the H 2 O 2 sensitivity for 5 successi ve calibrations of two PB/NiHCF- modified UMEs with no add itional treatmen t (green) , two PB/NiHCF -modified U MEs addition ally exp osed to vacuum condition s (blue) and two PB/NiHCF-mod ified UMEs with Pt /C as supportin g electrode material (red ). - 53 - Investigation of H 2 O 2 stability after the exposure of the PB/NiHCF -modifi ed electrodes to UV light: consecutive H 2 O 2 calibra tions in 0.05 M phosphate buffer of a PB/NiHCF -modified UME (diameter: 25 µm) with Pt/C composite a s suppo rting electrod e materi al (A ), colors: 1 st (blue), 2 nd green) an d 3 rd (red) calibra tion, and corresponding CV s in 0.1 M HCl/ 0.1 M KCl ( B) a fter depo sition (yellow) , exposure to UV light (purple) an d after con secutive H 2 O 2 ca librations (ru nning ord er: b lue, green, red), scan rate: 0.02 mV/s. The sensitivity of three con secutive H 2 O 2 calibratio ns recorded at three individual UMEs modified with Pt/C (red) and additionally exposed to UV lig ht (green), is compared in C. Also, t he in fluence of UV light towards t he p erformance of PB/ NiHCF -mo dified UMEs i n respect t o H 2 O 2 sensitivity and stability was investigated. Th erefore, a PB/NiHCF-mo dified Au UME with Pt/C composite as supporting electrode material was exposed to UV light for 120 s after electrochemical deposition of PB/NiHCF bilayers and after thei r act ivation by cycling in 0.1 M HCl/0.1 M KCl ( Figure 30 ). The H 2 O 2 calib ration in phosphate buffer showed good linearity wit hin the con centration range of 99 – 909 µM H 2 O 2 and a sensitivity o f 0.82 µA/(cm 2 ·µM) for the first calibrat ion ( Figure 30 A), which is comparable to the results obtained - 54 - by modified Pt/C-UM Es without UV light exposure. Between recording the calibrations, the PB/NiHCF bilayers were characterized b y CV in HCl/KCl ( Figu re 30 B). Additionally, the influence of the UV light in respect to the H 2 O 2 response was compared i n the CVs recorded prior and after the exposure of the modified U ME to UV l ight (see yellow and purple CVs in Figure 3 0 B). No significant change in the amount of PB could be detected and no negative effect within the following consecutive calibrations was observed . Both treatments, namely the vacuum and the UV exposure of the modifie d electrodes, might res ult in a loss of hydrate water and an e nhanced drying of the H 2 O 2 -active film. In lit erature, the exposure of PB to ligh t of different wavelength has been reported a nd an increase in the formation of PB could be associated t o the photo-reduction of the iron(III) in the [Fe(III)(CN) 6 ] 3- ani on 232 . The transfer of ferricyanide to ferrocyan ide was shown for UV lig ht ( = 365 m) 232,233 . Th e photo -reduction is not linked to the wavelength of the used light source, but to t he en ergy transferred by irradiation. T herefore, i t is p ossible that residues o f FeCl 3 an d [Fe(III)( CN) 6 ] 3- within the freshly prepared PB films are reduced t o Fe(II) and PB is add itionally formed. H owever, the redox signal of PB shown in Figure 30 B doesn’t show signif icant differences b etween the CVs recorded prior and after th e exp osure of the modifie d UMEs to UV lig ht. Also, IR investigations indicated no change of t he vibrat ion bands within the film (data n ot shown). Th ereby, a chan ge of the PB structure can be excluded and the enhanced stability of the PB/NiHC F - modified films is probably re lated t o the fast drying of t he film and additional PB formatio n for both, vacuum and UV c onditions. In co nclusion, vacuum conditions and UV light, which are necessary for the preparation of modified AFM -SECM probes, seem to h ave a positive effect on the performance of the ele c trodes . Impro ved stability is observed under both conditions. Pt /C composite as supporting electrode material is also suit able and show ed enhanced stability and sensitivity compared to bare Pt UMEs as supporting electrode material . PB/NiHCF-modified AFM-SECM probes Modification of AFM tip -integrated ele ctrodes with PB/NiHCF bilayers, which h as been investigated within this thesis for the first t ime, was obtained u sing the s ame conditions as described for UMEs with concent rations of t he solutions for PB and NiHCF dep osition adopted from Komkova et al. 20 (4 mM FeCl 3 /K 3 [Fe(CN) 6 ] and 1 mM NiCl 2 / 0.5 mM K 3 [ Fe(CN) 6 ]) . Thereby, the el ectrode area was exposed by FIB milling and increased with a Pt/C composite by IB ID 174 . First measurements with these modified AFM-SECM probes wer e performed in H 2 O 2 - 55 - calibration experiments to investigate their H 2 O 2 sen sitivity and stability (Figure 31 C). In Figure 31 A and B, an AFM -SECM p robe after the d eposition of 3 PB/NiHCF bilayers is shown. The SEM images reveal a complete coverage of the whole pyramidal AFM tip. Because the deposition of NiHCF is known to be spontaneous, this might be reasonable for the o bserved deposition of the H 2 O 2 -sensitive PB/NiHCF layer onto the whole AFM tip. This should be avoided to ensure localized measurements of H 2 O 2 . SEM image o f a PB/NiHCF -modified, tip -integrated: top view (A) and side view (B) of th e py ramidal AFM tip. Linearity an d H 2 O 2 sensitivity of 5 successive calibratio ns (C; ru nning order: blue, red, green, orange, purple). Because o f th e huge ove rspread of the PB/NiHCF bilayers , the electrode area was not tak en into account an d the absolute current signal is correlated to the H 2 O 2 concentration (C). Therefore, the deposition of PB/NiHCF bil ayers has been optimi zed with tip -less AFM-SEC M probes and tested in H 2 O 2 calibrat ion experimen ts. In Figure 32, the deposit ion of PB/NiHCF following the procedure of K omkova et al. 20 is depicted i n A, whereas the de p osition of two PB/NiHCF bilayers using a reduced concentration of the original deposition solutions by 66 % is sh own in B. It is ob vio us that the optimization of the PB/NiHCF layer deposit ion resulted in - 56 - the localized deposition of H 2 O 2 -sensitive material located at the or iginal electrode area wit h an overspread d ue to a radial diffusion at the microelectrode during the electrochemical deposition. In Figure 32 C, the PB/NiHCF d eposition occurred additionally at the area surrounding the actual microelectrode as a result of t he Pt/C deposition by IBID and/or the spontaneous deposition of NiHCF . SEM images of PB/NiHCF -mo dified, tip-integrated electrod es, with 4 mM FeCl 3 /K 3 [Fe(CN ) 6 ] an d 1 mM NiCl 2 / 0.5 mM K 3 [Fe(CN) 6 ] con centrations for the ele ctrochemical depositio n of 3 PB/NiHCF bilayers (A ) and reduced concentra tion s of the used soluti ons of 1.33 mM FeCl 3 /K 3 [Fe(CN) 6 ] an d 0.33 mM NiCl 2 / 0. 17 mM K 3 [F e(CN) 6 ]) du ring electrochemic al depo sition of 2 PB/NiHCF bi layers (B - D); without (C) an d with (D) addition al FIB cleaning aft er IBID of th e Pt/ C composite. Acc eleration voltage: 5 kV (A) and 3 kV (B- D) / 24 pA (A), 43 pA (B) an d 86 pA (C, D) . Th e SE M images i n ( B – D ) are recorded in the im mersion mode. This increased d eposition of a thin Pt/C layer next to the actual microelectrode area was reduced by s ubsequent FIB milling as d epicted in Figure 32 D. Th e sensitivity and s tability o f the PB/N iHCF-modified AFM -SECM p robes with a reduced concentration of the deposition solutions were tested in consecutive H 2 O 2 calibrations. In the following graph (Figure 33), the H 2 O 2 sensitivit y of a PB/NiHCF-mo dified, tip-integrated electrode (in blue) is compared to results obtained with m odified UMEs. The reduction of t he amount o f PB and NiHCF d uring - 57 - electrochemical deposition didn’t show a reduced sensitivity , in contrast even an enhanced sensitivity for the f irst 3 H 2 O 2 calibrations with t he PB/NiHCF-mod if ied, tip-integra ted electrode was ob served. The i nfluence of UV light exp osure on the behavior of the PB/NiHCF bilayers was also investigated as the mounting of the AFM-SECM probes requires the exposure to UV light. Comparison of the H 2 O 2 se nsitivity for 5 successive calibra tions of a PB/NiHCF -modified tip - integrated electrod e (blue) to P B/NiHCF -modified UME s mo dified with Pt/C (light an d dark red). The investigation of the i nfluence of the UV light was already tested w ith modified UMEs (s ee 4.3.1), resu lting in enha nced stabili ty of PB/NiHCF b ilayers. The AFM- SECM probes were exposed to UV light after the deposition of the 1 st and the 3 rd layer corresponding to the deposition of PB, whereas the 2 nd and 4 th layer correspond to the deposition of NiHCF resulting in two PB/N iHCF bilayers. In the following section, individ ual AFM -SECM probes are compared showing good H 2 O 2 sensitivity in consecutive cali brations. Using a re duced conce ntration of the deposition solutions, for the modification of AFM -SECM probes with PB/NiHCF bilayers resulted in ra ther limited rep roducibility. In f act, only the 4 (13 %) presented probes show ed a response t o H 2 O 2 and 27 (87 %) tested AFM -SECM probes sh owed either n o sign al in the - 58 - ‘activation’ CV i n HCl/KCl or no H 2 O 2 se nsitivity at all. In t he following, the different responses of th e presented probes are evaluated in d etail t o identify the re ason for th is good H 2 O 2 sensitivity. The H 2 O 2 sensitivity of different AFM- SECM p robes with slightly different electrode sizes and geometries are compare d in Figure 34 A. The compared probes are named according to the colors in the graphs, namely purpl e, blue, red and black. Comparison of H 2 O 2 sen sitivity and linearity o f PB/NiHCF-modified AFM-SECM probes with UV exposure (purp le, blue, red) and w itho ut (black) UV ex posure d uring electrochemic al layer deposition (A). CV in 0. 1 M H Cl/ 0.1 M KCl of PB /NiHCF -m odified AFM -SECM p robes (B) recorded prior to ca librations depicted in (A), scan rate: 0.02 V/ s. Corr esponding dat a are of the sa me color. A summary of the sensitivities and elec trode area is given i n Ta ble 4. The 1 st H 2 O 2 calibrations obtained wit h PB/NiHC F-modified AFM -SECM p robes are shown in Fig ure 34 A, being ex posed to UV lig ht (pu rple, blue and red dashed lines ; results correspond to three individual AFM - SECM probes), an d wit hout being exposed t o UV light (black dashed li ne). It is clearly visible that the se nsitivity of the probes exp osed to UV light is s trongly enhanced by a factor of 4.9 up to 16.3 in relation to the resu lts labeled in black. Ju st one PB/NiHCF -modified AFM -SECM probe with additional UV exposure (red dashed line in Fig ure 34 A) shows a red uced sensitivity by a factor of 0.5. Although the same conditio ns for th e electrochemical deposition of PB/NiHCF bilayers were app lied t o all samples, the deviation in the resul ting sensitivities is huge. A comparison of the ‘activation‘ CVs o f the H 2 O 2 - active bilayers in 0.1 M HCl/ 0.1 M KCl - 59 - black red blue purple exposed to UV light No Yes FIB cleaning after PB/NiHCF bilayer deposition No No Yes H 2 O 2 sensitivity [µA/(cm 2 · µM)] 1.24 0.65 6.10 20.17 ratio - 0.5 4.9 16.3 electrode geometry square, no AFM tip Pt/C frame electrode + AFM tip electrode surface [µm 2 ] 13.8 12.7 1.1 1.8 Table 4 Comparison of H 2 O 2 sensitivity and corres pondin g ratio o f AFM- SECM probes exposed t o UV light in respect to the AFM-S ECM p robe not expose d to UV light (black), electrode surf ace as determined by CV in 1,1’ -ferrocenedimethan ole and H 2 O 2 se nsitivity regardl ess of the a ctive electrode surfac e of PB/NiHC F-modi fied AFM-SE CM prob es as depicted in Figure 3 4. (the amount of PB can be estimated by its redox signal at appro ximatel y 0.2 V vs. Ag/AgCl reference electrode (Figure 34 B)) doe sn’t explai n these enhanced sensitivities. Only the data obtained for one AFM-SECM probe ex posed to UV light during ele ctrochemical layer deposition (displayed in r ed colors in Figure 34 a nd Table 4) sh ows a re duced se nsitivity an d a low con centration of PB, accordin g to t he redox signal for PB in the CV displayed in Figure 34 B. Indeed, the electrode revealing t he highest signal (black CV in Figure 34 B) shows for the 1 st calibration the worst sensitivity (not exposed to UV ligh t). The comp arison of the active electrode surface as determined by CV in 5 mM 1,1’ -ferro cenedimethano le shows deviations for the respective AFM-SECM p robes as summarized in Table 4. The differ ences in electrode size and H 2 O 2 se nsitivity are shown in Figure 34 B and in Table 4, respectively. The individual samples are named according to the colors in Fig ure 34 . Th e data in Table 4 sh ow sig nificant deviations in the H 2 O 2 sensitivities, which does not correlate with the actual e lectrode surface. Whereas f or the probe ‘black’ (no UV exposu re), a square electrode was used without an AF M tip (as depicted in Figure 32 B), the A FM -SECM probes with an electrode area (named as bl ue and purple) reflect Pt/C frame electrodes s urrounding the AFM tip (SEM image of the ‘ blue’ - 60 - AFM -SECM probe is shown in Figure 32 D). The AFM-SECM probe labeled as ‘red’ is shown in the SEM image in Figure 32 C, without additional FIB cleaning after Pt/C deposition, b ut also with an AFM tip surrounded by the active Pt/C - modified electrode area. Because t he ‘re d’ AFM -SECM probe shows c omplete coverage of the pyrami dal AFM p robe i n the SE M image in Figure 32 C, the actual electrode area before the deposition of PB/NiHCF -bilayers is larger (see values listed in Table 4). The detection of H 2 O 2 correlates with the amount of PB as the H 2 O 2 - sensitive material. The amou nt of PB is d ependent on t he concentration of FeCl 3 and K 3 [Fe(CN) 6 ] during elec trochemical deposition an d the actual elec trode area, on which the PB is deposited. Because the concentration during the electrochemical deposition for all AFM - SECM p robes in the compared results was kept the same, the d ifferenc es in sensitivity are either related to the electrode s urface or to the p ositive influence of the e xposure to UV ligh t in between the deposition of the individual layers. In deed, the AFM - SECM probe (‘red ’), exposed to UV light and w ith the high est electrode area (see CV recorded in 1,1’ - ferrocenedimethanole) an d a PB/NiHCF coverage of t he whole pyramidal AFM p robe visibl e in the SEM image in Figure 32 C, shows the worst sen sitivity with the lo west amo unt of PB according to the results shown in Figure 34 . The difference b etween t his AFM -SECM probe and the other probes is th e additio nal FIB cleaning next to the act ual electrode area, which guarantees a localized d eposition of PB/N iHCF b ilayers. This add itional t reat ment doesn’t h ave a direct influence on the H 2 O 2 sen sitivity but on the amount of deposited material. The amount of P B as est imated by the redox signal in the CVs shown in Figure 34 B is comp arable for the ‘purple’ a nd ‘blue’ AFM p robes, b ut lower for the ‘red’ AFM probe. Up to this p oint, no correlation could be f ound between the amount o f PB/NiHCF bilayers , detected by the ‘activation’ of the b ilayers by cycling in HCl/KCl solut ion and the coverage area observed in the SEM images, and the resulting H 2 O 2 sensitivity of the modified AFM -SECM probes. All PB/NiHCF-modified AFM- SECM probes showed a sensitivity loss with cons ecutive H 2 O 2 calibrations, w hich is in accordance with a diminishing concentratio n of PB during H 2 O 2 detection due to more alk aline p H values of the s olution induced by the H 2 O 2 reduction. - 61 - The ‘blue’ AFM -SECM p robe t ested by two succe ssive H 2 O 2 calibrations showed a sensitivity loss of approximately 58 %, t he ‘purple’ probe sh owed a sensitivity loss of 45 %, red probe of 31 % an d black probe of 25 %. The PB/NiHCF elect rochemical deposition of two bilayers with reduced concentration compared t o the origi n al deposition p rotocol was repeated. It would be f avorable to reactivate used PB/NiHCF -modified AFM- SECM probes for further investigations. H 2 O 2 sensitivities for two consecuti ve calibrations of the PB/NiHCF -modified AFM -SECM prob es. The colors are in a ccordan ce with the modified AFM -SECM prob es summarized in Table 4 . Additional deposition of PB and NiHCF shou ld also result in a n enlarged amount of PB on the AFM -SECM p robe. Beca use t he sam e p robe is used, there are no d iffere nces in preliminary preparation s teps (electrode siz e, additional FIB c leaning , etc.) of the AFM-SECM probe that showed already good behavior in H 2 O 2 sensing. With the assum ption that the electrochemical deposition results in PB/NiHCF- mixed b ilayers as postulated by Karyakin and coworkers 222 , t he amount of PB/ NiHCF-mixed material s hould be increased wit h additional deposition. Because the modified A FM -SECM p robe was u sed in H 2 O 2 calibration and according t o the loss of sensitivity of the H 2 O 2 -active film during the 2 nd calibration, the amount of PB and NiHCF still left on the electrode area is unknown. Neverthel ess, an enhan ced H 2 O 2 s ensitivity after the - 62 - 2 nd electrochemical d ep osition of mixed b ilayers is expected. In Figure 36 A, the H 2 O 2 calibration (gree n d ashe d line) actually shows an en han ced sensitivity, which is three times higher than the sensitivit y of the 1 st calibration after the 1 st ele ctrochemical deposition of Comparison of H 2 O 2 sensitivity and linearity of one PB/N iHCF-modified AFM-SECM prob e with UV exposure du ring electrochemical layer deposition (A): 1 st (bl ue) and 2 nd (red) H 2 O 2 calibrat ion after 1 st electroche mical lay er d eposition an d H 2 O 2 calibration ( green) after 2 nd e lectroch emical layer deposition. CV in 0.1 M H Cl/ 0.1 M KCl o f the PB/NiHCF-m odified A FM -SECM prob e (B) recorded after 1 st (blue) an d 2 nd (green ) electrochemi cal layer depo sition an d prior to calibratio ns d epicted in (A), scan rate: 0. 02 V/s. PB/NiHCF (blue), b ut the amount of PB sho ws just a small d eviation in the CVs r ecorded in 0.1M HCl/ 0.1 M KCl (Figure 36 B). In both electrochemical depositions, the AFM -SECM probe was exposed to UV light after the 1 st and the 3 rd layer deposition. The difference in sensitivity is t herefore not associat ed with the exp osure of the PB/NiHCF -modified AFM-SECM probe to UV ligh t. T he enhanced st ability can be addresse d neither to t he exp osure of th e PB/NiHCF- modified AFM- SECM probes to UV light nor to the amount of PB. Because the same AFM - SECM probe was use d in this comparison, t here w ere no additional diffe rences, namely the electrode area or geo metry of the original Pt/C - modified electrode. Add itionally, the hypothesis that alternating electrochemical deposition of PB and N iH CF results in the formation of bilayers is questionable. The presented re sults are reflecting individual AFM - SECM probes, at th is st age, no quantitative conclusion could b e drawn. The investiga tion o f - 63 - 27 of 31 PB/N iHCF-modified AFM- SECM probes showed no H 2 O 2 sensitivity or any st able behavior in H 2 O 2 detection if this is related to h andling issues (e.g., electrical discharge , etc.) or insufficient stability of the deposited layers cou ld not be clarified . Localized detection of H 2 O 2 by AFM-SECM For t he first AFM-SECM meas urements, t he ori ginal deposition pr otocol as p ublished by Komkova et al. 20 was u se d resulting in a PB/NiHCF- modified AFM- SECM probe with complete coverage of the pyramidal AFM ti p . In t his investigation, no localized signal was exp ected t o reflect the si ze of an electrode as a model sample during H 2 O 2 generation by an applied potential. There fore, the PB/NiHCF-mod ifie d AFM-SECM p robe was used for imaging the H 2 O 2 evolution at a Pt/C-modified Pt UME biased at – 0.5 V vs. quasi-Ag/ AgCl reference electrode, which is sufficient to generate H 2 O 2 234 . The mo rphology was detected i n AFM contact mode and th e generated H 2 O 2 in SECM generation/collection mode (Figure 37 B, D , and F). Control experiments were performed at a sample bias of 0 V vs. Ag/AgCl quasi - reference electrode, which is not sufficient for H 2 O 2 gen eration (Figu re 37 A, C and E). The current signal in Figu re 37 F recor ded b y the PB/NiHCF-mo dified, tip-integrated electrode clearly sh ows a respo nse to H 2 O 2 generated at the UME. Even a difference between the original electrode area and the overlapping Pt/C composite could be detected. - 64 - AFM -SECM image s record ed at an UME with a PB/NiHCF- modified AFM -SECM pro be ; setup according to the schemes depicted in (A) and (B): topograp hy (C, D) and corre sponding tip current (E, F) recorded at th e AFM -SECM probe with a s ample b ias of 0 V (E) and – 0.5 V (F) v s. Ag/AgCl quasi-reference electrode, res pectively, and a tip bias at 0 V vs. Ag/AgCl quasi -reference e lectrode in both measurements. Origin al size: 54 x 54 µm 2 , scan rate: 0.2 ln/s (10 .9 µm/s). - 65 - Although the used AFM- SECM probe was completely covere d with H 2 O 2 -active PB/NiHCF bilayers (as sh own in Fig ure 31 A, B ), still localized information could b e obtained, as the signal of the H 2 O 2 red uction ma inly results fro m t he film at or close to the AFM -integrated electrode surface. This b ecomes more obvious in the 3D representation of the AFM topography overlaid with the current signal as shown in Figure 38. 3D representa tion of the re sults sho wn in Figu re 37. The cu rrent signal o f H 2 O 2 reduction detected at a PB/NiHCF- modified AFM -SECM p robe (A) and AFM to p ography (B). In addition, the H 2 O 2 -sensitive PB/NiHCF bilayers may also h ave been remo ved during previous scan s or calibrations, which would also explain that t here are no obvious artifact s in the topography visible. Th e H 2 O 2 conversion is predominantly located at PB/NiHCF bilayers deposited onto the AFM tip- integrated microe lectrode. Nevertheless, for investigations of unknown sample m orphology and/or s patially he terogenous H 2 O 2 release , the H 2 O 2 se nsing layer should b e well defined and confined to the electrode surface. Although the current signal was stable during 5 consecutive AFM scans, partial dissolution of the PB/NiHCF film du ring measurements might lead to a f alse interpretation of the det ected current signals for unknown samples. - 66 - To exclude electrochemical activity in the absenc e of the PB/NiHCF bilayers an d any crosstalk between the c urrent signal and the topography det ected simu ltaneously by AFM -SECM, an unmodified AFM-SECM probe was us ed addition ally as shown in Figure 39. As expected, n o current response related to H 2 O 2 consumption could be detected b y the unmodified AFM - SECM probe. AFM -SECM image of an UME recorded with an unmodified AFM- SECM pro be; s et-up acco rding to the scheme depicted in (A ): Topography (B) and correspon ding tip current (C) recor ded at a n unmodified AFM -SECM probe with the s ample bias ed at – 0.5 V v s. Ag/AgCl quasi-reference electrode and with a n AFM -SECM p robe bias of 0 V vs. Ag/AgCl qu asi -reference electro de. Original size: 46 x 46 µm 2 , scan rate: 0. 25 ln/s (11.4 µ m/s). - 67 - Conclusion and outlook Within this section, PB/NiH CF-mod ified UMEs and AFM -SECM probes for H 2 O 2 d etection have been investigated. Thereby, significant influenc es of parameters used for the fabrication of AFM -SECM probes have been identified, namely the influe nce of vacuum c ondit ions, UV light and Pt/C composite as supporting electrode material. In general, no negative effect of all these parameters co uld b e detected . In contrast, a significantly improve d st ability an d sensitivi ty for the detection of H 2 O 2 was achieved. Preliminary experiments for mapping localized H 2 O 2 evolution at an UME using a PB/NiHCF-modified AFM -SECM probe are presented. Althou gh the used AFM -SECM probe was completely covered with PB/NiHCF bilayers, a localized detection of H 2 O 2 could be shown. This can be explained that only the PB/NiHCF-covered tip- integrated el ectrode area close to the sample surface contribu ted predominantly to the recorded signal. Addit io nally, a change in the morphology of the PB/NiHCF film, as shown in 4.3.1 for the modificatio n of UMEs, might resul t in that the non - electrod e areas cover ed by PB/NiHCF film are not significantly c ontributing to the overall signal. To avoid the coverage of the c omplete tip, AFM-SECM investigations h ave t o be repeat ed w ith o ptimized parameters for the electro chemical d eposition of PB/N iHCF b ilayers reported within this section . The bilayer-modified probes, either UMEs or AFM tip -integrated ele ctrodes, showed a good response t o H 2 O 2 with enhanced stability compared to PB as electrocatalytic layer for H 2 O 2 reduction . A reduced concentration and number of d eposited PB/NiHCF bilayers u sed for the modification of AFM tip -integrated ele ctro des showed a reduce d amount of PB/NiHCF locat ed on the AFM- SECM p robe as detected by SEM, but H 2 O 2 calibrations showed enh anced sensitivity for H 2 O 2 . However, the reproducibility with respect to AFM -SECM probe modification needs to be improved i n future experiments . C haracterization of individ ual PB/NiHCF-modified AFM-SECM probes s howed additionally n o comparable behavior and n o correlation between the redox signals with in cycli ng in 0.1 M HCl/ 0.1 M KCl for ac tivation of the PB/NiHCF bilayers an d the actual sensitivity toward s H 2 O 2 . All investigations of the PB/NiHCF bilayers by AFM -SECM, SEM, EDX, optical microscopy and electrochemical measurements led to t he con clusion that the f ormation of bilayers by alternating electrochemical deposition of PB and N iHCF c ould not be confirmed, b ut rather the f ormation of a homoge neous PB/NiHC F film w ith vary ing thicknesses in rela tion to the am ount of deposited PB and NiHCF is mos t likely. Fu rther investigati ons w ith r espect to the actual composition of PB/N iHCF-mixed layers are needed in future. With a detailed investigation of - 68 - the mixed f ilms, the modification of AFM-SECM pr obes can be optimized and a h igher yi eld of PB/NiHCF-modified sensors with appropriate p erformance in H 2 O 2 sensing can be gained. - 69 - AF M tip -in tegr a ted pH s ens ors Introduction The miniaturiza tion of pH sensors is essential for localiz ed pH m easurements and enables research of metabolic or signaling pr ocesses i n biomedical related researc h f or examp le mapping changes of p H at t he cell level 21 , as well as the detection of loca lized corrosion processes 22 . Th erefore, the sensor material ha s to be biocompat ible, mechanically resistant and th e pH sensor has to be miniaturize d 45 . The most common pH sen sor is the pH -sensitive glass electrode . C arter et al. 235 used a glass microelec trode for t he stud y of corrosion processes. For th e investigation of biologi cal samp les, P. C. Caldwell used a glass microelectrode for intracellular pH sensing at muscle fibers of crabs 236 and at a giant squi d axon 237 . Next to pH- sensitive glass electrodes, m etal/metal oxides were w idely used for pH detection d urin g the l ast century 45 , 238 . A range of suitable me tal/metal oxides, such a s RuO 2 239 , WO 3 240 , and most important irid ium oxide 62 and the antimony/antimony oxide 23 , 24 electrode, h ave been investigated so f ar and su ccessfully applied for pH monitoring in biological resear ch. For example, intracellular investigation of the giant squid axon wit h a 1 µm antimony electrode 241 and in ex tracellular investigations o f endothelial cells 242 and carbonic anhydrase located at t he brain 243 have been demonstrated . SECM is highly suitable for the localized detection of changes in electrochemical signals at a surface and potentiometric measurements combined with SECM were also s uccessfully sh own 244 – 246 . Mirkin and co-work ers investigated pH chan ges around human b reast epithelial cells with a 7 µm Sb UME 247 . The investigation of corrosion processes by SECM has bee n reviewed by Niu et al. 248 . For the detection of the oxygen reduction reaction and corresponding pH changes due t o corrosion of metal or metal alloy surfaces, Sb 22 , Ir oxide particles 249 or h y drogen ionophore liquid membranes 250 in combination with SECM have been used so far. Although SECM is highly su itable for mapping local changes in electrochemical signals, typically it is challeng ing t o obtain simultaneously topogr aphical information of t he investigated sample. This gap can be closed by the combination of SECM with other surface sen sitive techniques, e.g. with scanning vibrating electrode technique (SV ET) 251,252 an d SICM 253 for pH mappin g. pH measurements in close vicinity to the area of interest with h igh spatial resolution may - 70 - further provide in formation on t he reactio n mechanism, the r ate of r eaction or th e involved species itself. The combination of a pH -sensitive electrode inte grated in to an AFM p robe may enable the local detection of pH chan ges sim ultaneou s w ith chan ges in topography. Although the metal/metal oxid e pH sensors have b een used in a broad variety of applications, i nsufficient stability was reported, e.g. for the iridium oxide electrodes, and different oxidation states of the iridi um oxide or different fabrication s chemes of the active films showed a distribut ion of p H sensitivities of 82 – 92 mV/pH 27 , 62 – 74 mV/pH 61 , or 59 - 77 mV/pH 45 . Additionally, decreasing p H sensitivi ty in consecutive calibrat ions w as also observed for i ridium oxide electrodes 49 . How ever, n o statistical d ata are available in literature and most of the publications present investigations in respect to individual sensors, which will be al so discussed in the f ollowing sections. Within this thesis, Ir and Sb – modified AFM- SECM p robes are investigat ed as pH sensors and the performance of the modified AFM-SECM probes is investigated by monitoring the dissolution of calcite crystals. Unwin and co -workers investigat ed the d issolution of calcite using a p latinized Si x N y AFM cantilever to reduce the pH value t o more acidic pH values , whi ch accelerates the crystal dissolution 148 . Fu rthermore, the electrochemical deposition of EIROF on to AFM tip – integrated Au and Pt/C – modified Au microelectrodes is evaluated within the follo wing chapter and investigated at Au and P t/C – modified Au UM Es. - 71 - Experimental Ultrapure water (18.2 M , ELGA LabWater, Veolia Water Solutions & Te chnologies) was used for the preparation of all solutions. For Ir -modified AFM probes and iridium oxide modified U MEs, CV s in de-aerated 0.5 M H 2 SO 4 (from 98 %, VWR Chemicals) in a po tential range of - 0.45 V to 0.75 vs. Hg/HgSO 4 reference electrode (sat. K 2 SO 4 , CH Instruments) a t a scan rat e of 1 V/ s was used to activate t he iridium oxide film ( Figure 40 A) p rior to pH calibration. CV (A ) in 0.5 M H 2 SO 4 vs. Hg /HgSO 4 referenc e electrode, showing the 1 st (black) and the last (blue) CV of in sum 500 cycles. Scan rate: 1 V/s . OCP m easurement (B) in 0.05 M TRI S buffer with corre spondin g pH valu es determined by a pH gla ss electro de at ro om t emperature after addition of HCl and N aOH, respectively . OCP measure ments (Figure 40 B) vs. Ag/ AgCl reference electrode (sat. KCl, RE -1CP, ALS Co) were done in 0.05 M TRIS buffered solution (pH 6.96, tris(hydroxymethyl)- aminomethane, Mer ck KGaA) within a pH range of 2 – 11 u sing a (bi)potentiostat (CHI660A and CHI660C, CH Instruments). The pH values w ere adjusted by addition of 5 M HCl (from 30 % HCl, Normapure, VWR Internatio nal GmbH) or 0.5 M NaOH (Merck KGaA), respectively. A comp lete exchan ge of different buffered sol utio ns took s everal minutes, which imp edes proper d etection of the response time o f the pH sensor towards p H changes. Therefore, the pH calibration was done by con secutive addi tion of aliquots of HCl or NaOH, re spectivel y. Af ter mixing the s olution, a pH glass electrode (InLab ® Expe rt pro, Mettler-Toledo Intl. Inc.) with an int egrated temperature sen sor was used t o determine the p H values of the buffered solutions. Th e pH calibrations were done at room temperature with small f luctuations of 1 – 2 °C du ring meas urements. A simultaneous detection of the pH with the pH glass el ectrode during OCP measureme nts in the same - 72 - solution was not p ossible due t o strong i nterfere nces in the d etected OC P signal b y t he glass electrode (see Figure 41 ). Influence of the p H glass elect rode towards the OCP signal o f an Ir /IrOx – modified AFM pr obe . OCP me asure ment in 0.05 M TRIS buffer at pH 2.5. The pH glass ele ctrod e w as kept within the liquid cell in th e Faraday ca ge and was turned o n/off as labeled with in the graph . Different AF M tip- integrated p H se nsors w ere inv estigated within this thesis, w hich w ere fabricated by former co- workers of Mizaikoff an d Kranz. AFM probes modified with a pH - sensitive Sb layer were fabricated by Jong Seok Moon 188 . Briefly, a commercial Si x N y AFM cantilever was mod ifie d with a t hin Ti adhesion layer and an Au layer (100 n m) with a DC sputter coater, followed by the deposition of Sb at the AFM tip with varying thicknesses (35 - 225 nm) by an RF sputter system. Afterward, t he AFM-SECM probes were in sulated by PECVD with Si x N y and SiO 2 . An active pH -sensitive electrode and a defined AFM tip were exposed by FIB milling (Quanta 3D FEG, ThermoFisher Scientific) as reported in section 4.2. AFM-SECM probes were batch-fab ricated by Heungjoo Shin 254 und modified with Ir. Silicon cantilevers were mod ified with a th in P t layer and an Ir layer with a thickness of 300 - 40 0 n m. After the re lease of the cantilevers, ad ditional deposition is re quired t o compensate stress in order to use such probes for AFM measurements. - 73 - SEM of Ir (A , C) and Sb ( B, D) modified AFM probes. The whole AFM tip shown in (A) was cut by FIB milling to expos e all components of the Ir – mod ified AF M tip (labeled in C). The electrode area of an insulate d Sb – mod ified AFM probe ( B) was also e xposed by FIB milling. The Sb layer deposited on Au is labeled in (D). Acc eleration voltage: 3 kV,10 pA (Ir)/ 20 pA ( Sb). The different geometries of the A FM tip -integrated pH sensors are summarized in Figure 42 . Prior to the cali bration of the AF M tip - i ntegrated p H sensors, the AFM -SECM p robes were mounted onto an AFM n osecone or an i nsert usin g UV glue (Dyma x 9001- E-V3.1 or DYMAX 42 5, Dymax Eu rope Gmb H). Electroc hemical deposition of iridium oxide film electrodes was done according to Yamanaka et al. 255 . 71.3 mg/50mL iridium(I V) oxide (Alfa Aesar GmbH & Co. KG) was stirred for 30 min, followed by the a ddition of 0.5 mL H 2 O 2 (30%, Merck KGaA). After 10 min stirring, 208.2 mg anhydrous oxalic aci d (Fluka Chemie GmbH) w as a dd ed to the sol ution. T he pH was adjusted t o 10.5 by the addition of KCO 3 (Merck KGaA). After storing the solution for t hr ee d ays, th e solut ion was purged with argon f or 30 min prior to electrochemical deposition . Au wires (diameter: 10 µm, Goodfellows) were used as electrodes fabricated as described elsewhere 65,226 . Some Au electrodes were additionally mo dified with a Pt/C composite by IBID (precursor: methyl - cyclopentadienyl-trimethyl platinum, ThermoFisher Scien tific) with respect to th e EIROF - 74 - modification of AFM tip-int egrated pH sensors. AFM-SECM probes were prepared based on the procedures as already reported 1 33 and described in section 4.2. Th e AFM tip – integrated Au electrode was modified with Pt/C via IBID . 5 mM [Ru (NH 3 ) 6 ]Cl 3 (98 %, Aldrich) in 0.1 M KCl (Merck KGaA) and 5 mM 1,1’ -ferrocenedimethanole (98 %, Acros Organics) in 10% ethanol ( from 96 %, VWR Chemicals) and 0.1 M KCl (Merck KGaA) were used to determine the electroactive ele ctrode surface of t he UMEs and the AFM -SECM probes prior to electroc hemical modification with EIROF. pH -active iridi um oxid e films were deposited u sing a (bi)p otentiostat. D ifferent depositio n techniques were evaluated as summarized in Table 5. deposition parameters constant current 0.2 nA for 300 s constant potential 0.75 V vs. Ag/AgCl for 300 s CV - 0.45 V - + 0.7 vs. Ag/Ag Cl, scan rates: 1- 10 V/s, 20 - 500 cycles pulsed potential E 1 = 0.75 V, E 2 = -0.45 V for 0.25 s, respective ly; number of cycles: 25 -250 Table 5 Summary of deposition techniques with corresponding d eposition parameter s u sed for the EIROF formation , by CV , at a constant current, and a t constant or pulsed po tentials. The m odified UMEs were investigated after electrochemical deposition b y optical microscopy (AXIO Imager .M1m, Zeiss), SEM (Q uanta 3D FEG, Thermo Fisher Scientific) and contact mode AFM (55 00 AFM/SPM microsco pe, Keysight Tech nologies) with silicon nitride tip s (OTR-P, Olympus). All AFM images and correlating height and current p rofiles were processed by Pico View (Keysight Technologies) or Gw yddion 225 . AFM-SECM measurements were done in contact mode A FM detecting the dissolution of calcite and the local associated pH changes by OCP measurements using a bipotentiostat (CHI842B, CH Instru ment). The OCP outp ut signal of the bipotentiostat was f ed to an AD channel of the AFM controller for correlating the potentiometric data with the topographical image. - 75 - Prior to AFM-SECM investigations in TRIS buffered solution, the calcite sample was imaged in air in AFM contact mod e to locate single calcite crystals. Calcite crystals were synthesized by the ammonia diffusion method fr om dissolved CaCl 2 (95 %, Carl Roth) and (NH 4 ) 2 CO 3 (Me rck KGaA ) under N H 3 – saturated atmosphere 256 . T he calcite crystals were embedded in Crystalbond ( 509, Plano) a nd parti ally polished with Al 2 O 3 susp ension (5 0 nm, MasterPrep, Buehler ). Results and discussion In the following section, resu lts of AFM -SECM probes modified with either an Ir or Sb metal layer are p resente d by p H calibrations i n 5. 3.1 . Due to a limited n umber of available AFM -SECM p robes modified with an Ir or Sb metal layer, respectively, the following results might b e interpreted as first investigations sh owing the suitability of these AFM tip - integrated pH s ensor s, but also in detection of localized pH c hanges, exemplarily shown by AFM-SECM investigations of the d issolution of calcite crystals in 5.3.2 . The fabrication of such modified AF M-SECM probes resu lted i n st rong be nding of the AF M c antilever due to intrinsic stress du ring sputteri ng of the e.g. Ir metal layer 254 . A limited number of Sb or Ir coated probes were available for co mbined measurements in the time frame of this thesis showing a can tilever bending o f 1 – 2°. AFM cantilevers with more bending were used i n OCP measurements f or pH calibrations in bulk experiments. Additionally, EIROF - modified microe lectro des were t ested in consecutive pH calibrations at UMEs (diameter : 10 µm), as presented in 5 .3.3, as alternative f abric ation s trategy of a pH sensor integrated into AFM probes. - 76 - AFM tip-integrated pH sensors After insulation of the AFM probes and exposure of an ac tive electrode area b y FIB milling as described in section 4.2, the pH se nsitivity and linearity was investigated performing calibrations. The AFM p robes with an integrated pH – sensitive AIROF electro de showe d a sta ble pH response in long-term measurement s (Figure 43 ) wit hin consec utive calibrations. The pH response for th e exemplarily pre sented pH sensor was 54.3 mV/pH, whereas a p H re sponse of 65.4 mV/pH - also referred to as super-Nernstian b ehavior reported f or AIROF electrodes 45 - could be determined in the buffered r egion s hown in the zoomed graph in Figure 43. The linearity of the calibration graphs is in an acceptabl e range (inlaid table in Figure 43) t o investigate qualitative p H changes, localized at a sample surface in following AFM-SECM investigations. OCP vs. Ag/ AgCl referenc e electrode of an AIRO F-modified AFM prob e in 0.05 M TRIS b uffer detecting pH changes b y ad dition of either 5 M HCl or 0.5 M NaO H, respectively. The tab le summarize s the sensitivity and lin earity of the whole calibrat ion experiment an d the b uffered region shown in th e zoomed extra ct. - 77 - Exemplarily, the results f or 4 consecutive calibrations of one Ir -modified AFM -SECM probe are shown i n Figure 44, pH sensitivity and linearity are give n in the imp lemente d table for the different calibrations. The result s reveal that th e OCP signal show ed a more linear behavior with a h igh r ecovery within con secutive calibrations for neu tral to alkal ine pH values, whereas the d istribution of the detected p otentials was increased in the acidi c pH ran ge. This h as been alre ady reported for AIROF ele ctrodes showing a varying p H response with different pH sensitivities in correla tion to the measured pH val ues 257 . Calibration of an AIROF -modified AFM -SECM probe: Lin ear regre ssions of 4 consecutive pH calibrations with sensiti vity and linearity given in the implemented table labeled by the respective colors. Th e 3 rd calib ration is n ot shown due to i nsufficient stability (see also F igure 45 ). The OCP measu rement for th e third and the fourth calibration of this AIROF -modified AFM -SECM prob e is p resented in Figure 45 . In contrast to Sb – modified electrodes, the iridium oxide f ilm can b e regenerated a nd pH se nsitivity and lineari ty can be recove red by cycling in H 2 SO 4 . In Figure 4 5, t he OCP meas urements recorded wit h an AIROF -mo dified AFM probe ar e shown bef ore and after the regeneration of the active ir idium oxid e film, demonstrating the great ad vantage of Ir over S b. The p resented results here correspo nd - 78 - to o ne AF M-SECM pro be tested in consecutive pH calibrations due t o a limi ted number o f available AFM-SECM probes. Limitatio ns accordin g to available AFM-SECM probes were also a problem for Sb-modified AFM- SECM pro bes. Regeneration of AIROF electrodes: O CP vs. Ag/AgCl r eferen ce electrod e in 0.05 M TRIS b uffer detecting pH changes b y addition of either 5 M HCl or 0.5 M NaOH, re spective ly, bef ore (A) and after (B ) cycling in 0.5 M H 2 SO 4 . Th e linear regression of t he re sults shown in (B) is depic ted in Figure 44 as ca libration 4 (red). - 79 - Individual probes could be tested i n pH calibrations, as exem plarily depicted in Figure 46 , showing two OCP mea surements with correlati ng pH values of the buffered solution detected with a 10 0 nm Sb-mo dified AFM pr obe in a short (A) and a long-t erm (B ) calibration, The results sh ow a fast an d stable pH respon se up to 300s (A) a nd being linear over severa l minutes at the same time (B). Th e pH sen sitivity was 50.3 (A) and 55.9 (B) mV/pH, respectively, whereas the linearity i n the OC P measurements was 99 % (A) an d 97 % (B). OCP vs. Ag/AgCl re ference el ectrode in 0.05 M TRIS b uffer detecting p H changes by addition of either 5 M H Cl or 0.5 M NaOH, respectively. The change in short time s cales (A) and stab ility within long time measuremen ts (B) were investigat ed. However, on ly a small percentage of available potentiometric Sb AFM - SECM sensors showed a measurable pH response, whic h may b e associated with the long storage time (approx. 4 years) under ambient conditions . Additionally, some sensors showed a strong surface ch ange after OCP measurements as depicted in Figu re 47 B an d C. It seems that the Sb layer u ndergoes s ome dissolution process and sprea d as small p arti cles around the actual electrode area, which may be associated with the complete oxidation of t he Sb layer during the long sto rage time. The Au layer is still visible and the in -plane area of the Au can be identified as the f rame in the zoomed SE image in C, whereas the pyramidal shape of th e Au modified AFM tip became visible in the surroundin g region formerly covered by t he Sb layer. In case of Sb – modified A FM-SECM probes, b oth met al layers (Au and S b) are exposed and connected within an electrolyte s olution resulting in a ga lvanic couple and, therefore, in th e dissolution of S b as the less nob le metal. Loca lized corrosion and the adjoin ing r emoval of the Sb film because of impurities on the Sb electrodes was - 80 - SEM image s of a Sb-modi fied AFM - SECM probe before ( A) an d after (B) calibration. T he zoomed region (C) s ho ws th e removal of th e Sb layer. Acceleration voltage: 5 kV, 27 pA (A) /47 pA (B, C). already reported, whereas these impurities were not specified 52 . However, prior studies using these Sb-modified AFM - SECM probes didn’ t show t h is behavior 188 . T o elimi nate a ny possible dissolution of the Sb metal layer b y forming a galvan ic cou ple, t he deposition of Sb should be limited to the AFM tip and p art of the cant ilever, whereas Au as an elec tronic connection layer might be just deposited onto the AFM chi p and cantilever 188 . Another possibility would be the exposu re of an electrode area next to the pyramidal AFM tip, exposed by FIB milling in a top-down a pproach. Thereby, ju st the Sb meta l deposited on top of Au is exposed. The SECM signal is t hen sh ifted towards the topograp hical information with respect on the distance between the AFM tip and the electrode area located close to the AFM tip. These investigations were not the focus of this t hesis. Investigation of calcite dissolution by AFM-SECM First AFM-SECM measurements were c onducted with S b- and AIR OF-modified AF M- SECM probes. Changes in the OCP detected b y the AFM t ip -integrated pH sensor were interpreted just qualit ati vely according to the obtained results in section 5.3.1. Prior t o AFM -SECM investigations, t he p H sensors were test ed towards their pH sensitiv i ty to guarantee a change in the OCP signal to more negative potenti als f or more alkaline pH values. As a model system, t he dissolution of calc ite was imaged with the AFM- SECM pH - 81 - sensors. Calcite is dissolved in aqueous solution by characteristic surface changes showing rhombohedral e tch pits due to d issolution 28 . Add itionally, the dissolution process results in a pH change to more alkaline p H values 29 . The reby both, a surface change an d a p H change sh ould b e detectable by the AFM-SECM probes . Similar in vestigations were published by Unwin and coworkers 253 , detecting the dissolution of a calci te crystal with an edge length of approximately 25 µm by SECM-SICM revealing limited t opographical information. In Figure 4 8, the r esults for the AFM- SECM in vestigations with an AFM tip - integrated Sb electrode are shown. The electrode area is located next to t he actual AFM tip as depicted in the SE images i n A - C. In the AFM deflection and topography images shown in D and E, the change in morphology of the calcite crystal is clearly vi sible revealing AFM -SECM measure ment of the dis solution of calcite u sing an AFM tip -integrat ed Sb electrode: SEM images o f the used AFM -SECM pro be in sid e view (A) and 52° t ilted (B), wi th a zoomed view to the a ctual electrod e area with an edge l ength of 189 nm (C). Accelera tion - 82 - voltage: 3 kV, 43 pA. AFM deflection (D) and topography ( E) recorded in AFM con tact mode and correlating poten tial chan ges d etected b y OCP v s. Ag/AgCl quasi- reference el ectrod e (F). Original size: 80 x 80 µm 2 , scan rate: 0.1 ln/s (16 µ m/s). the characteristic pit s tructures. Th e potential c hanges during AFM sc annin g are depicted in Figure 48 F showing low er potential values compared to regions without calcite, which correlate to a p H change toward s more alkaline pH values. The lateral r esolution in Figure 48 F is insufficie nt within the single li ne scans ( x-direction ), w hereas a change in signals is localized with respect to t he sca n directio n (marked b y an arrow in Fig ure 48 , y-direction). Same investigations were repeated with AFM- SE CM probes with tip-integrated AIROF pH sensors. One AFM -SECM investigation is exem plarily shown in Fig ure 49. Thereby, a smaller calcite crystal (approx. 2-8 µm in length), compared to the one depicted in Figure 48 , was mapped u sing th e maximum sca n range o f the piezoe l ectric positioner in case the missing lateral resolution is correlated to convection effects of the moving tip. Althou g h the Ir e lectrode ar ea in tegrated i nto t he AF M pr obe is large with an oute r diameter of the ring electrode o f 3.6 µm, the resol ution in th e scan direct ion (y-direction) w as su fficient to detect localized pH c hanges re lated to the dissolution of calcite. However, no laterally resolved information on pH change in x-direction w as observed. The height and potential profiles at the marke d position s in E and D of Figure 49 are clearly showing a potential change towards lower potentials correlating with a higher pH value at the position of t he calcite crystal with an elevated topography visible comp ared to the surro unding su rface, in which the calci te crys tal was e mbedded. Nevertheless, the change in the potential is still s pread over t he wh ole dis tance i n x-direction l acking late ral informatio n in the r egion of the calci te crystal. This broadening of the OCP signal seems to be ab sent in the y - direction, which indicat es that the lack of resolution is not associated with the actual electrode size . - 83 - AFM -SECM ima ging of the calc ite dissolution with a tip-integrated AIROF el ectrode: SEM image of the u sed AFM -SECM prob e with a ring electrode with an inn er diameter of 2.9 7 µm (o uter diameter = 3.64 µ m, A). Acceleration voltage: 3 kV, 10 pA. AFM deflection (B) and topography (C) dete cted in AFM conta ct mode with corre lating poten tial changes detected b y OCP vs. Ag/AgCl quasi-refer ence electrode (D). The actu al, expose d calcite crystal is marked by a white dotted circle in ( B) embedded in crystal b ond. The pro files of the top ography and O CP at th e marked region in (C) an d (D ), respectively, are given belo w. Original size : 90 x 79 µm 2 , scan rate: 0.14 ln/s (25 µ m/s). The AFM-SECM measurement shown in Figure 49 is analyzed in d etail in the following figures. I n Figure 50 , t he chan ge in the detect ed OCP signal extracted as single line scans (x -direction) is evalu ated in de tail. There by, the onset of a decreasing p otential correlated - 84 - to the more alkaline pH value in close pro ximity to the calcite crys tal can b e seen i n the first potential profile depicted in B and labeled with 1. This data corresponds to t he d ashed white line (1) in the AFM t opography imag e shown in A. Line (2) corresponds t o the las t extracted profile as marked in Figure 50 A, indicating the actual location of t he calcite crystal. This position is also marked in the corres ponding heightprofile (labeled with an arrow, 2 in B). Thereby, the onset of the detectable p otential change is shifted by approximately 7.3 µm, re presenting t he distance between t he dashed w hite li nes 1 an d 2 in (A). The minimum value for t he OCP signal represents a change of a pprox. 3 mV in an area of 20 µm in t he x- direction (dashed lines) shown in the profile in (C) corresponding to the OCP signal in the 3D represe ntation in (B) as indicated by a blue arrow . Response tim e o f the OCP sig nal correlated to the loca tion of the calcite crystal: Single height profiles as labeled by white d ashed line s in the AFM topog raphy (A) for th e fir st (1) and the last (2) extracted profiles ar e comp ared to correspondin g changes of th e OCP at same p ositions (B). The scan direction (y-directio n) is labeled by a black d ashed arrow (in A and B) and the location of the crystal is indicated b y an arrow in (B). Dat a correspond to the results shown in Figure 49. - 85 - The detailed evaluation of the response time w ith re spect to the sca n dir ection (y- direction) is shown in Figure 51 . At the position m arked by a white dotted line in the OCP image in A, t he h eightprofile in B and the OCP profile vs. d istance shown in D are extracted. Additionally, the det ecte d OCP signal vs. t ime as recorde d b y t he external bipotentiostat is shown in C . Indeed, the profiles in C a nd D are almost identical, i ndicating no resol ution in x- di rection during the detected resu lts. The onset of the changing potential towards more negative potentials correlating with a mo re alkaline pH is shi fted towards the location of the calcite crystal by 7 .3 µm. This larg est potential change recorded du ring 2- 3 µm (in y -direction) is approximately 10 mV followed by an i ncreasing potential leveling to the background response in betwee n a distance of 10 µm in the y-direction. Evaluation of the response time in AFM scan direction (y -direction): Extracted pro file s at the location marked b y a dashed white line in the OCP signal (A) showing th e potential chan ges in scan d irection (ind icated by a black, dashed arrow). The height profile (B) with the loca tion of the act ual calcite crystal marked by an arro w is compare d to th e OCP signal vs. time dete cted by the external b ipotentio stat (C) and the OCP change vs. distance (D) as extracted from the OCP image in (A). Data corr espond to the results shown in Figure 49. - 86 - In comparison, the obtained results reported by Unwin a nd coworkers 253 for SECM -SICM investigations us ing an AIROF-modified nanoelectrode, show that the detected OCP signal is increased compared to the actual size of the calcite crystal by the factor 2 -3 or by a factor of 1.5 – 2 comp ared t o a simulated potential shift according to the pH distribution above the calcite crys tal. Whereas in this pub lication, the p H sensor had a dim ension of 100 nm and additionally, the calcite dissolutio n was recorded in the SECM hopping mode recording single positions of the SECM-SICM probe along t he x,y -direction without la teral scanning. The det ected potential changes were record ed in a distance of approximately 100 nm toward s th e calcite crystal. Compared to this investigation, the obt ained result shown in Figure 49 to Figure 51 is in a comparable range. The distance between the AIROF- modified el ectrode and the sample surface was in the range of 1 µm as defined b y the length of the reshaped AFM tip, which also in dicates a broadened signal due to enhanced diffusion. Indeed, the enlarged AIRO F sensor used in the presen ted results in com parison to the nanoelectrode used by Unwin and coworkers 253 showed an enhanced resolution with respect to the scan directi on (y -direction). This can be definitively interpre ted as a positive trend for f urther investigations with AIROF -mod ified AFM-SECM probes. However, t he resolu tion in the x -direction or the direction of single line sc ans has t o be improved for future st udies. In the AFM-SECM measurement shown in the previous figures, the resolution was 512 x 51 2 p ixel, representing a line scan every 0.17 µm. With the scan speed of 0.14 ln/s (25 µm/s) one line wa s recorded in approximat ely 7 s an d the area m arked by t he white dashed lines 1 a n d 2 in Figure 50 wit h the distance of 7.3 µ m represent 43 lines, which were recorded in 5 min. This also correla tes to the OC P sig nal detected by the external bipotentiostat showing a c hange in th e potential i n a durat ion of 7 min. Wipf and cowor kers reported an optimal re sponse t ime of < 10 s for AIROF microelectrodes 249 , wher eas for EIR OF-mod ified electrodes, detected i n a potentiometric time- of -flight exper iment, the response t ime was det ermined as s everal t enths of seconds in relation t o t he thickness of deposited EIROF 258 . Thereby, t he detected insuf ficient pH response in the x-direction of the single line scans migh t be indeed re late d to a scan speed, which is too fas t for detec ting an a dequate pH ch ange . Similar AFM-SECM measurements with comparable Ir and Sb - modified AFM- SECM probes with only small d eviation in electrode size were used with varying AFM scan s peed and scan si ze, resulting always in insufficient resolution in the x-direction (results are not shown). One exp lanation for t he - 87 - lack in resolution may be additionally t he mentioned convective e ffects of t he scannin g AFM probe. This effect should b e strong for high scan speeds, whereas , during slow scanning, convection should be negligible. Electrochemical depositio n of iridium oxide film electrodes Both, Ir- and Sb-derived p H active layers showed a broad distribution in pH sensitivity . I n addition, bending of the cantilever might be an i ssue for the fabrication of such p robes due to intrinsic st ress during sputtering of Ir t hat requires additional microfabrication steps. Hence, the electrochemical deposition of pH -sen sitive material onto the AFM tip integrated Au electrode was in vestigated. EIROF was deposited by d ifferent electrochemical techniques (see Tab le 5 in 5.2). The evaluation of optimized d eposition parameters was tested with Au UMEs (dia meter: 10 µm) p rior t o the m odification of AFM- SECM probes. Ad ditionally, the pH res ponse of EIROF el ectrodes deposited to Pt/C - modified Au UMEs was evaluated . Pt/C composite was tested as the sub strate material for EIROF pH sensors as the AFM tip -integrated Au frame ele ctro des ca n be incr eased by an ad ditional Pt/C composite layer. This addi tional step results in an enh anced d eposition of pH active iridium oxide, which is localized at a small electrode area bu t contains a sufficient amount of ac tive pH ma terial f or stable p H response giv en the e nlarged sur face area by Pt/C deposition compared to the 1 00 nm thin Au frame electrode . EIROF deposition o n Au UMEs (diameter: 10 µm) modified with a 150 nm thin Pt/C co mposite by IBID: AFM topography of t he modified UME (A) and corresponding height profiles (B) of the bare Au UME (blu e), after modification via IBID (green) an d after EIROF d eposition by 100 pulse cycles (red), record ed in AFM contact mode. Origina l size: 1 7 x 17 µm², scan speed: 0. 34 l n/s. - 88 - The first i nvestigations of the mo dification o f Au and Pt/C - modified Au UM Es were evaluated by imagi ng the el ectrode surface by optical microscopy, SEM an d AFM to guarantee a homogeneous deposition on the whole electrode area by a thin EIROF layer. Figure 52 A shows the AFM topography of an A u UME (diameter: 1 0 µm) mo dified with 150 nm Pt/C layer via IBID, which is additionally mo dified with EIROF by applying 100 potential pulse cycles. The corresponding height profiles of the UME are shown in Figure 52 B after ea ch modification of the surface with the final height p rofile showin g the surface after deposition of a t hin iridium oxid e f ilm (red). The EIROF deposition s b y CV and multiple pulses showed the mos t satisfactory results and both t echniques were evaluated in more detail b y chang ing deposition parameters. The pH response of the EIROF pH sensors was tested in consecutive p H calibrations ( 2 – 4 calibrations depend ing on the stability of t he individual sensors) comp aring d ifferent deposition c ycles for both techniques . The r esults obtained for EIROF-modif ied UMEs with respect t o t he p H response in consecutive calib rations and t he linea rity o f the obtained sig nals during these calibrations are presented in Figu re 53 . In A, the sensitivity is compared for sensors deposited via CV (shown in squares) and via multiple pulses (m arked b y crosses). The grey bar represe nts t he s ensitivity rang e for neutral to alkaline pH values (> p H 6) for EIR OF- modified sensors as re ported by Wipf et al. 257 . Only two ou t of 8 sensors, which are compared in Figure 53, showed a respo nse to p H changes wit hin 4 consecutive calibrations, whereas m ost of the o thers gave a stable response in two calibrations. Indeed, t he li nearity for all presented pH sensors as shown in Figure 53 B , was insufficient for q uantitative pH de tection. T he b lack dotted li ne i n B labels linearity of 90 % a nd the majority of the results are below this linearity. - 89 - Sensitivity (A) an d linearity (B) of pH calibrations for 8 EIROF -modified UMEs (diam eter: 1 0 µm): The graphs r epresent results obta ined with Au and Pt/C - modi fied UMEs by EIROF deposition via CV (square s in A an d solid b ars in B) an d via MP (crosses in A and dashed b ars in B) with varying nu mbers of cy cles or p ulses. The dashed bar in (A) shows the s ensitivity range reported by Wipf e t al. 257 , the dashed line in (B) marks linear ity of 90 %. Correspondin g data is depicted in the same colors . Similar t o the results presented in section 5.3.1 , the EIROF ele ctrodes showed a broad distribution in pH sen sitivity. The stability and the linearity in bet we en consecutive calibrations, but also for different tested se nsors, was unsatisfactory. The res ults of consecutive pH calib rati on of exemplarily two EIROF- modified Au UMEs are presented in Figure 54 . These results correspond to the t wo p H sensors sh own in Figure 53 with 4 consecutive calibrations. The quite large distribution measured with a singl e EIROF- modified sensor is for the obtained OCP values approximately 200 mV at pH values of 6 – 8 in co nsecutive pH calibrations, for both, EIROF deposited via CV (A ) and multiple potential pulses (B). - 90 - Calibration of EIROF-modifi ed Au U MEs (diameter: 10 µm), electrochemically depo sited by CV (A) or by multiple potential puls es (B): Linear re gressions of 4 consecutive pH calibrations with sensitivity and lin earity summ arized in th e tables below lab eled in correspond ing colors. Similar results were also obt ained by other rese archers, e.g. by Wipf et al. with EIROF- modified carbon fiber microe lectrodes 257 , by Marz ouk using d ifferent mm -sized substrates as supporting material f or AEIROF sensors 63 , or by Elsen et al., who investigated different deposition protocols for EIROF -modified gold microelectrodes 258 . In all of these reported investigatio ns, ind ividual pH sensors were reported also s howing broad distributions in pH sensi tivity betwee n individual sensors and no informa tion is given in respect to long-term stability of th ese pH sensors. Alt hough d ifferent suppo rting electrode materials or different sizes of t he modified electrodes a re presented in the described examples above, a clear trend is recognizable. T he results obtained in this thesis with the EIROF modif ication of either UMEs or AFM -SECM probes are in agreement with the reported findings . The electrochemical deposition of EIROF was init ially investigated to overcome limitations of AFM - SECM p robes with int egrated Sb or Ir metal layers as pH sensor. But the res ults for EIROF-modified A u and Pt/C UMEs also show ed a broad response in the range of 17 to 96 mV/pH wit h ins ufficient li nearity (Figure 53Figure 5 3 B) . Nonetheless, first EIROF modif ication of AFM -SECM probes with gold frame el ectrodes - 91 - and electrodes additiona lly modified with P t/C c omposite via IBID w ere ele ctrochemically modified by either CV or multiple poten tial pulses and characterized via calibration experiments. Thereby, the re sponse t ime of the EIROF -mod ified AFM-SECM probes towards pH changes i n TRI S buffered solution was investigated. The p H calibration c urves obtained for 4 individual pH -sensitive AFM-SECM probes are summarized in the table shown in Figure 55 . Again, a bro ad distributio n ranging f rom 37 to 91 mV/pH in pH sensitivity f or d ifferent probes, similar to the results of EIROF – modified UMEs was observed. Calibration of EIROF -modif ied AFM-SECM probes, electro chemically deposited b y mul tiple pulses (MP ) or CV on Au or Pt/C - modi fied Au AFM -SECM probes: Lin ear r egressions o f 4 pH calibration graphs with sensitivity and linearity given in th e tab le in respect to electrochemical deposition and substrate, are labeled in corre sponding colors. No differences between either the electroch emical deposition technique nor the underlying ele ctrode material for the iridium oxide f ilm showed prefera ble behavior in stability, pH sen sitivity or linearity, in concordance w it h the r esults published elsewhere by Wipf et al. 257 , Marzouk 63 or Elsen et al. 258 . Similar investigations for EIROF sensors at a macroscopic rotating ring-d isk electrode as reported by Steegstra et al. 27 reported also a broad distribution in pH sen sitivity according to the oxidation state of t he iridium oxides. Indeed, they also address the results of in dividual EIROF ele ctrodes and again no statistical data were presen ted. As the f abrication p rocess is more t ime- consuming i n respect to t he modification of AFM- SECM probes, as every single AFM -SECM probe has to be modified - 92 - individually, further op timization of EIROF -modified AFM tip-integrated pH sen sors was not further pursued within this thesis . Conclusion and outlook Within this section, AFM t ip-integrated Sb and Ir pH sensors were investigated towards their pH re sponse. The experiments performed within this thesis also show a limited reproducibility of the miniaturized pH electrodes as already reported in severa l publications (see introduction) . For all different modification strategies, a b roa d distribution of pH sensitivity was ob served. pH sen sors prepared under the same conditions showed varying p H sensitivity f or d ifferent pH electrodes but also for consecutive pH calibrations . The stability of the pH sensors was examined for approximately 1 h showing good pH sensitivity and recovery after sever al minutes. In case of EIROF pH sensors electrochemically deposi te d on Pt/C-modified Au electrodes, either in AFM-SECM probes or as UMEs, the pH sensitivity and linearity was i nsuf ficient, and the electrochemical deposition of iridium oxide see ms t o be un suitable for tip-integrated p H sensors. Although the successful mod ification of car bon nanoelectrodes w ith electrochemical deposit ed iridium oxide used in combi ned SECM-SICM investigations of the calcite d issolution was reported b y Unwin and co- workers 253 , no information was provided for long-term stability of the p resented pH sensors , nor multiple pH measurements were shown. Within this thesis, no clear improvement of the electrochemical modification in respect t o reproducib ility of the p H sensors cou ld be observed. In section 5.3.2, Sb and Ir-modified A FM -SECM probes were us ed to detect t he localized dissolution of calcite crystals with simultaneously record ed pH ch anges. No lateral resolution of pH ch anges cou ld be observed. Variations in sca n speed, scan size, the original pH of the electrolyte solutio ns or the used pH sensor, either Sb or Ir, alw ays resulted in a change of the detected potential close to calcite particles , but with out a clear correlation t o the imaged morphology. However, compared to already reported investigations, e.g. by SEC M-SICM 253 , the resoluti on in the prese nted resul ts of this t hesis obtained for AIROF-modified AFM- SECM probes are in an acceptable range , esp ecially in the sc an d irection (y - direction, see 5.3.2) of the measurements. Whereas a broad ran ge in sensitivity was observed for both, Sb and Ir-modif ied AFM-SECM p robes, the great - 93 - advantage of Ir-based pH sensors ove r Sb is its possib ility t o be ‘ reactiv ated ’ b y anodic oxidation in H 2 SO 4 . AIROF electrodes were also in vestigated by Wipf and coworkers 249 towards their long-term stability in pH sensing. The size of the reported el ectrode was in the range of 1 0 µm i n d iameter consisting of Ir microparticles located in a microcap illary with an active electrode surf ace significantly larger than the h ere reported AFM-SECM sensors . Nevertheless, t hey re ported the s tability of individual s ensors f or over 2 month s , also showing a r educed s ensitivity in pH calibrations, but with linear beh avior over severa l calibration experiments du ring these 2 months. Indeed, the results presented in this publication refer to one individual AIROF sen sor showing good linearity d uring this period an d just sm all deviations in sensi tivity ra nging from 66 – 74 mV/pH 249 . T hey also reporte d a yield of 30 – 50 % of in total 510 inves tigated AIROF sensors. Studies involving such a large number of miniaturized pH sensors is beyond the scope of this thesis. However, the reported long-term stability o f t he AIROF p H se nsor seems t o b e promising also for AFM tip-integrated AIROF electrodes and further investigati ons should be f ocused on the optimization of the fabric ation of these pH sensors. - 94 - In ves tig ation of the I TIE S a t n an op ore arr a ys Introduction The detailed characterization of nanoporous array s used for electrochemis try at the ITIES is crucial for a compre hensive understanding and interpretation of transport ph enomena taking p lace at t he liquid/liquid interface. Especially, when changing the f abricatio n processes of such nanosized devices, the behavior , e.g. in diffusion or the location of the interface in these d evices, might change dramatically. The direct visualization o f diffusio n phenomena a t FIB-mille d nano porous ar rays w as shown by localized deposition of silica at the liquid/liquid inte rface in collaboration with Damien Arrigan’s group at Curtin University (Li et al. 181 ) . The localized d eposition showe d different diffusional behavior for nanoporous arrays with varying pore- to -pore separation . However, it has to be noted that the final silica formation is realiz ed by the h ydrolyzation initialized by the e lectrochemical ion transfer of a p recursor at the ITIES, followed by rinsing and d rying pr ocesses, which may change or influence the actual or localized silic a format ion. For the in -situ observation of d iffusion processes at nanoporous arrays, AFM- SECM is highly suitable. ITIES at nanoporous ar rays profit from an en hanced ch arge transfer and are a p ro mising in terface for sensing applications 259 . The diffusion b ehavior at these micro - and nanoporous membranes are not just dependent on the dimension s (length and diamet ers) of the actual micro- or nanopo res, but on the pore- to -pore sep aration resulting in an overlaid diffusion p rofile for sho rt p ore d istances w ithin the array (s ee also 1.4). Micro- and nanoporous membranes can b e fabricated by standard microfabrication processes . For example, DRIE has been applied for membranes used in ITIES investigations 83 , as well as e-beam lithography 98 . Especially, FI B e nables the f abrication of arrays, without the need for shadow masks and with varyin g an d easily adjustable p ore- to - pore sep arations, as already reported for various materials 32 – 34 . The f abrication of nanosized geometries by FIB processing is favorable due to an eas ily tunab le geometry within on e f abr ication ste p i n varying materials, e.g. thin SiN membranes 31 or p orous alumina 260 . Tong e t al. 261 reported the fabrication of n anopores with diameters do wn to 10 nm by addit ional silica deposition using low-pressure chemical vapor deposition to FIB-milled nanopores within SiN membranes. Additional (c old) ion b eam sculpti ng results in nanopores of j ust a f ew nanometer s in diameter as repor ted by Li et al. 32 and Kua n et al. 34 . F or investigations of the ITIES at nanopo rous arrays, theo retical simulat ions and cyclic voltammetry showed - 95 - additionally that the separation of the individual interfaces p lays a crucial role in the current signal and results in an overlapped d iffusion profile for closely- spaced nanopores 30,97 . Local di ffusion processes can be detected by sc anning probe t echniques as alrea dy re ported f or the diffusion at nanop orous membranes by SECM 262 or SICM 263 – 266 . AFM-SECM offers the ad vantage of simultaneous detection of the topography of nanoporous ar ray by AFM and the l ocalized detection of the electrochemical processes. The diffusional tran sport through pores by AFM -SECM has been shown by Kueng et al. for the transport of glucose 149 , of redox-active species by Macpherson et al. 150 and for the diffusion at electrode arrays with closely-spaced microdiscs 150,151 . For the used nanoporous arrays in SiN membranes fabricated by e-beam lithography, an inlaid interface was p redicted with the organic phase located within the nanopores due to t he resulting electrochemical signals of the transfer of a model analyte at ITIES 26 7 . Investigations at similar nanostructured samples pred ic ted the aqueous phase within the nanopores as shown by deposited nan oparticles located at the inter face at n anoporous alumina membra nes 86 or size exclusion of a lig and molec ule at nanoporous silic alite membranes 268 . T he h ydrophilicity of t he SiN membranes w as characterized by contact angle 98,267 resul ting in a hydrophobic back an d a h ydrophilic front side of the membrane . The p ore walls were also assumed t o be hydrophobic and therefore, the pores should be filled with the organic ph ase, w hich was suppor ted by calculatio ns 267 . The same ge ometry and location of t he interface were assumed for FIB-milled nanoporous arrays and calculations of electroc hemical transport at ITIES at these nanoporous arrays were base d on an inlaid interface 31,267 . Scheme of th e used SiN m embranes with correspon ding dimensions; top view (A) and side view (B). - 96 - FIB fabrication results in the implantation of Ga + ions 35 and results i n a truncated cone- shaped geometry of the nanopores due to re-deposition of FIB-milled material 32,36 ,37 . Indeed, t he impla ntation of charged ions in the SiN membranes sh ould show an influence on the hydrophobicity of the surface. Contact angle measurements are not possible for the inner wall of nanosc ale pores, hen ce the location of the interface has to be d etected otherwise. With the t runcated cone- shaped ge ometry resulting from FIB-millin g, the nanopores have two var ying orifices located at either the organic or aqueous f acing side of the SiN m embrane , respectively. This effect has not been ta ken into account yet f or nanopore arrays used for ITIES i nvestigations, only for solid-state electrodes recessed at th e bott om of truncated pores 37 . Therefore, in the following section of this thesis, t he nanopore arrays were fabricated by FIB -milling from either the front or the back of the SiN membrane resulting in varying pore diameters at each side of the membrane. Scheme of front (left) and back (right) side FIB-milled n anopores and the varying pore diameters at on e side of the membrane marked in red and green. With the comparison of the re sulting currents for the tran sfer of a model analyte , e.g. tetrapropylammonium chloride at the ITIES, the location of t he interface at or within the nanopores can be predicted. Th is prediction is sti ll based on theoretical assumption and the actual loca tion o f t he i nterface ca n dramatically vary from t hese predictions, in particular when the inner side walls of the pore might be altered due to FIB millin g . The interface betwee n two liquids may be also of dynamic nature and a s tatic interface, which is used for theoretical calculations, may not be suit able to characterize t he locat ion of these int erfaces in detail . Different approaches for the in vestigation of the liquid/liquid - 97 - interface have b een reported so far b y spa tial scannin g sp ectroelec trochemistry a nd localized d eposition of nanoparticl es 269 , by UV -VIS spectroscopy 270 , and R aman c onfocal spectroscopy 271 with a spat ial resolution in z-direction in the µm- range. Hence, the investigation of t he interface between two immiscible liquids by spectroscopic approaches is lacking the required resolution essential to investigate nano-sized por es within membranes. T he parallel displacement of t he excitation lig ht s ource as used in the mentioned techniques is n ot suitable f or solid-st ate membranes. The visualization of the interior and shape of nanopore s in SiN membranes has been demonstrated by high- resolution TEM tomography 272,273 . The first part of this section will show the visualization of different diffusi on al behavior in relation t o th e p ore- to -pore separation by mapping th e diffusion of a redox species at ITIES by AF M-SECM 181 . T hese measurements c omplete the investigations of the influence of t he pore- to -pore separation by CV and corresponding theoretical calculations . Next to AFM -SECM, the localized d eposition o f silica deposits at ITIES w as used for visualization of the diffusion p rofiles sh owing overlapped or individual diffusion in dependence of the pore- to -pore s eparation 181 . T he fo rmation o f a solid phase directly at the i nterface due to an ion t ransfer at t he ITIES may be used t o identify the location o f this interface within th e nanopores. In section 6.3.2 , different current responses of the transfer of tetrapropylammonium chloride (TPrACl) as a model analyte at the ITIES with in FIB -milled nanopore arrays, fab ricated either f rom the front or the back, are compared. Electrochemical silica deposit ion at the interface at the t wo differen t na nop orous array s is used t o visualize the differences between both approaches. Addition ally, such silica deposits at ITIES were investigate d b y FIB/SEM tomography enabling the recon struction of the pore shape, the mo rphology of the deposited silica d irectly located at th e nanopore s and especiall y within the nan opores. Ad ditional E DX and STEM measurements were conducted to characterize the silica deposits. The following results have been published partly by Liu et al. 181 and by Holzinger et al. 187 . - 98 - Experimental SiN membranes (SIMPore Inc. and Du raSiN Films, Protochips) with 50 and 100 nm thickness, respectively, were used as supporting material for nanoporous arrays fabricated by FIB milli ng (Zeiss Neon 40EsB, Carl Zeiss N ano Technology Systems for the arrays used for visualization of diffu sion p rocesses and FEI Helios Nanolab 600, ThermoFisher Scientific for electrochemical measurements and FIB/SEM tomography) . The AFM-SECM p robes used in p art 6.3.2 of th is chapter were p roduced as described elsewhere 133,160 . In brief, n on -metalized silicon nit ride probes ( OTR-P, Olympus) were modified with a 5 nm Ti adhesio n layer and a 100 nm gold layer p rior to insulation by silicon nitride (PECVD) . Then, the probes were additionally modif ied with a conical conductive tip of Pt/C composite by IBID (precursor: methyl -cyclopentadien yl-trimethyl platinum, Therm oFisher Scientific). The fabrication steps by FIB milling and IBID a re displayed in Figure 58. SEM images of th e u sed AFM -SECM probe with a conductive Pt/C tip : the Au electrode fr ame (approximately 600 nm in diameter) was exposed by FIB mil ling (30 kV, A ), Pt /C comp osite was deposited by IBID on top of the Au frame (square -shaped pattern with an edge le ngth of 1 µm and an ap proximate height of 466 n m, B) an d reshaped by FIB milling r esulting in a curva ture radii of 20-25 nm (C). Adap ted with p ermission from Y. Liu , A. Holzinger, P. Knittel, L. Poltorak, A. Gamero-Qu ijano, W. D.A Rickard , A. Walcarius, G. H erzog, C. Kran z, and D.W.M. Arrigan . Visualization of d iffu sion with in nan oarrays. Anal. Chem. 88, 6689 – 6695 (2 016). Copy right 2019 American Chemic al Soc iety . http s://pubs.acs.org/doi/10.102 1/ac s.analchem .6b00513 . - 99 - The AFM-SECM probe was characterized by CV i n d e -aerated 5 mM Ru( NH 3 ) 6 Cl 3 / 0.1 M KCl (98 %, Aldrich/ pro analysi, Merck KGaA) a t a scan rate of 0.1 V/s ( Figure 59 B). For AFM -SECM mea surements, AFM (5500 AFM/SPM microscope, Keysigh t Technologies) was used i n contact mode and SECM was d one in the generation - collection mode in 0.1 M KCl (pro analysi, Merck) d etecting the reduction of Ru(NH 3 ) 6 3+ at - 0.3 V vs. Ag/AgCl in a three-electrode setup with the AFM-SECM probe as working elect rode, a Ag/AgCl qu asi- reference electrode (Goodfellows) and a Pt counter electrode (Goodfellows). All AFM images a nd correlati ng h eight a nd current p rofiles were processed by Pico View (Key sight Technologies). All solutions were prepared with ultrapure water (18.2 M , ELGA LabWater, Veolia Water Solu tions & Technologies). Dynamic mode AFM was addit ionally used to characterize the p ore shapes of t he nanopores used in section 6.3 .2 and 6.3.3 with a FIB-sharpened NCL probe (k = 48 N/m, 190 kHz, Nano World). The electr ochemical Scheme o f the AF M- SECM setup used for detection of different diffusion beha vio r at nanop orous arrays within SiN membranes (A) , with the upper reservoir filled with 0.1 M KC l (light b lue) an d the low er re servoir b eneath the membrane filled with 20 m M Ru (NH 3 ) 6 Cl 3 / 0.1 M KCl (grey-blue). CV of the conductive AFM tip recorded in 5 mM Ru(NH 3 ) 6 Cl 3 / 0.1 M KC l, scan rate: 0.1 V/s (B). Ada pted with permission f rom Y. Liu, A. Holzinger, P. Knittel, L. Poltorak, A. Gamero-Qu ijano, W. D.A Rickard , A. Walcarius, G. H erzog, C. Kran z, and D.W.M. Arrigan . Visualization of d iffu sion with in nan oarrays. Anal. Chem. 88, 6689 – 6695 (2 016). Copy right 2019 American Chem ical Soc iety . https://pub s.acs.org/doi/10.102 1/acs.analchem .6b00513 . - 100 - investigations in 6.3.2 of ITIES at nanoporous arrays are based on the f ollowing electrochemical cell: Ag/AgCl | x µM TPrACl, 10 mM LiCl aq || 10 mM BTPPATPBCl DCH | Ag using T PrACl ( Aldrich) as t he model analyte in lithium chloride solu tion (LiC l, ≥ 99%, Sigma Life Scie nce) as the aqu eous ele ctrolyte. Aliquots of TPrACl were add ed t o the aqueous phase and gently mixed with a pipette for a homogeneous concentration during calibrations. The organic ele ctrolyte was synth esized as d escribed elsewhere 274 by metathesis of potassiumtetrakis (4 -chlorophenyl) borate (K + TPBCl - , Alfa Aesar) and bis(triphenylphosphoranylidene )ammoniumchloride (BTPPA + Cl - , Alfa Aesar). Bis(triphenylphosphoranylidene )ammoniumtetrak is (4 -chlor ophenyl)borate (BTPPATPBCl) was used as organic electrolyte in 1,6-dich lorohexane (98%, Aldrich). All electrochemical measurements were done by using a (bi)potentiostat (CompactSt at, Ivium Technologies) with Ag/AgCl quasi-reference electrod es . The electrochemical deposition of sil ica at t he interface b etween water and 1,2 -dichloroethane was obtained according to Poltorak et al. 275 and Herzog and co-workers 187 . A sol of 50 mM TE OS in 5 mM N aCl was adjusted to pH 3 b y addition of aliquots of 1 M HCl, stirred for 90 minutes at room temp erature to allow hydrolysis to occur. The ethan ol p roduced by the hydr olysis was removed by evap oration and the pH w as raised to pH 9 by t he additio n of aliquots of 1 M NaOH solution (unless stated otherw ise). The used e lectrochemic al c ell can be described by: Ag/ AgCl ǀ 50 mM TEOS hydrol yzed + 5 mM NaCl aq ǀǀ 14 mM CTA + TPBCl - D CE ǀ Ag The template salt in the organic phase was prepared by metathesis f rom K + TPBCl - and cetyltrimethylammonium bro mide ( CTAB) as d escribed els ewhere 276 . A potential of - 0.1 V or 0 V was a pplied for 30 - 60 s enabling th e t ransfer of CTA + f rom the organic to t he aqueous phase, triggerin g t he conden sation reaction bet ween silanol groups to for m Si - O-Si bonds. After the electrochemical deposition step, the membrane was carefully removed from the sol ution a nd ri nsed with a flow of ultrapure w ater to avoid any formation of silica through evaporation. The me mbranes were the n p laced in an oven a t 130 °C for 16 h to ensure cross-lin king. - 101 - Automated data acqui sition for FIB/SEM tomography (Helios Nanolab 600, FEI, ThermoFisher Scien tific) was performed with the ‘slice and view’ so ftware p ackage (FEI, ThermoFisher Scientif ic) with autom ated FIB milling (30 kV, 1.5 - 48 pA) providing a lateral resolution of 5 or 10 nm for slicing, respectively, and SEM imaging (5 kV, 86 pA) i n immersion mode after each milling step. Stabilization of the silica deposits while FIB milling or TEM f oil prepa ration w as achieved b y additional d eposition of a thin P t layer (3 - 4 n m) by sp utter coating (SCD 005, BAL-TEC) and by Pt/C deposi tion via IBID. Th e 3D slices were processed by Fiji 277 using the ‘linea r s tack ali gnment wi th SIFT’ 278 a nd applying a FFT bandpass filter. 3D graphs were processed by Aviz o 9.1.0 Lite (FEI, Thermo Fisher Scientific). An overview of the d ifferent fabrication steps in FIB/SEM tomography is shown in Figure 60. SEM imag es (5 kV/ 8 6pA) of the prepar ation steps for FIB/SEM tomography : A singl e silica deposit (A) was covered with a Pt /C protection layer by IBID (30kV, 48pA) (B) and th e sample was exposed by a cross-secti on to loca lize the SiN membrane below the silica d eposit ( C). A different number of slices (8 0 – 400) with th icknesses of 5 and 10 nm, respectively, was recorded du ring ‘ Slice an d View ’ . The slices showin g the p ore were then used to generate 3D projections of the sampl e (SEM in 2D and correspondin g 3 D representation are gi ven in th e - 102 - lower pane l of the figure ). A. Ho lzinger, G. Neu sser, B. J. J. Austen, A. Gamero -Quijano , G. Herzog, D. W. M. Arrigan, A. Zi egler, P. Walther, and C. Kran z , Faraday Discu ss. , 201 8, 210 , 113. Adapted from Ref. 187 . Dis tributed under the licen se Creative Commons Attribution - NonComm ercial 3.0 Unpo rt ed (CC - BY - NC 3.0 ), https:// creativecom mons.org/licenses/by - nc/3.0/ . TEM lamellae were pr epared by several successive FIB milling steps as described in detail elsewhere 279 and summarized in Figure 61 . TEM (EM 912 TEM, Zeiss) images were recorded with an acceleration voltage of 1 20 kV . STEM a nd EDX measurements were done with an FE -SEM (30 kV, S-5200, Hitachi), equipped with a X- ray detector ( Pheonix, E DAX) and a STEM detector (Hitachi). The images were also processed by Fiji 277 . Overview of th e pr eparation of TEM lam ella: app rox. 1 µm (in thickn ess) la mella is isolated from th e membran e conta ining the silica -modified nan opore by FIB milling step s . The la mella is then attached via IBID (30 kV) to a micromanipu lator needle (Omniprobe) (A ), removed from the sample and transferred onto a Cu TEM grid (Om niprobe) (B, b lue square marks the micromanipu lation needle wit h the TEM sample ) and fixed via IBID to th e grid ( C ). Aft erward , the micro manipulat ion n eedle is re moved (D ) an d the lamella i s thin ned b y FIB to a thickn ess of ap proximately 150 - 200 nm (E). A. Holzinger, G. Neusser, B. J. J. Austen, A. Gamero-Qui jano, G. Herzog, D. W. M. Arrigan , A. Ziegler, P. Walthe r, and C. Kran z, Faraday Discus s. , 201 8, 210 , 113. Adapted fr om Ref. 187 . Distribute d under the license Creative Comm ons Attribution - NonComm ercial 3.0 Unpo rt ed (CC - BY - NC 3.0 ) , https://creative commo ns.org/ licenses/by- nc/3.0/ . - 103 - Results and discussion Diffusion at nanopore arrays The diffusion of a redo x med iator through nanoporous arrays , driv en by a concentratio n gradient of two reservoirs located below and above the nanoporous array (see Figure 59 ), is detected . The AFM-SECM probe was located in t he upper comp artment containing solely KCl ele ctrolyte solution and detected the d iffusion of Ru(NH 3 ) 6 3+ from the lower reservoir sep arated by the SiN membrane containing the n anopore array toward s the AFM -SECM p robe. In Figure 62, the results for the diffusion at nanopores with small pore - to -pore spacing are pres ented. AFM -SECM images of a nanoporous array with 21 times se par ation of the individual pores in a hexagonal arrangement. Contact mode AFM topography (A) and GC mode SECM current signal (B) due to th e diffusion of [Ru(NH 3 ) 6 ] 3+ throu gh the nan opores is depicted sho wing an overlapped diffusion. The co rresponding height and current pro files are shown below according to the marked lin e in (A) and (B). Original size: 35 x 35 µm 2 , scan speed: 32.0 µm/s (0.5 ln/s), scan an gle: - 14.4 °. Adapte d with permission fr o m Y. Liu, A. H olzinger, P. Knittel, L. Poltorak, A. Game ro- Quijano , W. D.A Ric kard, A. Walcarius, G. Herzog, C. Kranz, and D.W.M. Arrigan . Visualization of diffusion within nanoarrays. Anal. Chem. 88, 6689 – 6695 (2016). Copyright 2019 Am erican Chemic al Soc iety . https://pubs.acs.or g/do i/10.1 021/acs.analche m.6b00513 . - 104 - The ratio of th e distance between single pores and t he rad ius of the pores ( r c /r a , see equation 29) is 21 and s maller than the cri tical v alue f or i ndividual d iffusi on o f 5 6 as p ore - pore sepa ration defined by Liu et al. 30 a nd re sulting in a n overlapped diffusion behavior at t he nanoporous array. The AFM topography and the cor responding height prof ile at the marked line in the AF M i mage are shown in A, w hereas the correlating current respo nse with the current p rofile at the same location is s hown in B. A clear overlapped current signal was detected with a c urrent d ensity of 0. 64 µA/cm² above the na noporous a rray. Single current p eaks are visible at t he location of t he individual nanopor es. These observations can be explained by the conductive AFM tip pe netrating the nanopores and detecting the current response , which reflects t he concentration in the lower compartment of [Ru(NH 3 ) 6 ] 3+ . Calculations of the expected theoretical current res ponse (based on the electrode area, diffusion coefficient, concentration , et c.) f or these additional current p eaks with a conical AFM tip results in a current response in the range of 0.3 – 1 nA (Ta ble 6 ) . Th ereby, the theoretical currents I dif are calculated b y equ ation (19) (see Table 2 ). measure d; peak curr ent signal, N = 50 calculated; for a 1 = 100 nm , h 1 = 155 nm calculated; for a 2 = 40 nm , h 2 = 40 nm calculated; for a 2 = 30 nm , h 2 = 30 nm curr ent [nA] 0.08 ± 0.02 1.04 0.36 0.27 Table 6 Measur ed and calculated current for the additional current peaks as illustrated in Figure 63 . The calc ulations w ere obtain ed with equation (19). Only th e part of the electroactive area of the AFM -SECM p robe within the pore and reaching the other side of the membrane (with height h 1 and radius a 1 ) containing the high conce ntrated [Ru(NH 3 ) 6 ] 3+ solution (20 mM) is use d t o calcul ate t he theoretical current response (see scheme in Figure 63). - 105 - Current respon se (A) accordi ng to the AFM-SECM image s sho wn in Figure 62 and scheme of the AFM tip penetratin g a single nan opore (B). The h eight h an d radiu s a, u sed for calculation of the theoretical current va lues for th e ob served curr ent peak as shown in the over laid current response (red cu rve), ar e labeled in white and y e llo w, respectively. Additionally, the curren t is calculated for t he AFM tip jus t immersing into the high concentrated [Ru(NH 3 ) 6 ] 3+ solution with the h eight h 2 and the radius a 2 according to th e actual dimensions of the used AFM -SECM probe (in Table 6, t wo different heights and radii are comp ared based on the SEM images of the actual AFM -SECM probe, which are measured from two different sides of the AF M tip). Th e calculated currents are up to 10 times higher than the actual measu red c urrent response. Ind eed, a perfect cone sh ap e was used for th e calculations, which is n ot the case for the actual shape of th e FIB -milled AFM tip, a nd a maxim um conce ntration of Ru(NH 3 ) 6 3+ w as used as c 0 . Bec ause diffusion close to and within th e pores changes the actual concen tration of the redox mediator in the s olution below the nanoporous array and also t he penetration of the AFM tip into t he pores results in ad ditional convection in opposite direction (tip moves towards the si de of the membrane facing th e concentrated solution, whereas d iffusion is dir ected from the high to less co ncentrated solut ion). Hence, the actual concentration for c 0 may be lower than the original bulk concentration of Ru(NH 3 ) 6 3+ . Ad ditionally, the t ilt of the AFM tip towards the surface is not t aken into account , which changes th e actual size of the AF M tip placed within the nanopore . Fo r an estimation of the d etected current signal, the calculations used in Table 6 are sufficient and these sin gle p eaks visible for individual nanopores can be addressed b y the electroac tive area of t he AFM- SECM actu ally - 106 - penetrating the nanopores. Additionally, the over all current signals are ov erlapped in the surrounding of the pore s due to t he overlap ping hemispherical diffusion profiles at the individual pores . Same mea surements at nanoporous arrays with a por e- to - pore separation sufficient for i ndividual diffusion (w ith r c /r a = 91, see equation 29) as predicted by electrochemical in vestigations and theoretical calculations 30,31,97 are presented in Figure 64 and show clearly h emispherical diffusio n above the single nanopores. The AFM topo grap hy with corresponding height profile is depicted i n A and the correlating curren t image and profile at the marked line in the current image is shown in B. Th e current density obtained from these re sults is l ower (0.11 µA/cm²) compared to the results sh own in Figure 62 for the overlapped diffusion, but just a small sectio n of 8 nanopores is visibl e within the d epicted imag es in Figure 64 co mpared to the w hole array with 100 nanopores in Figure 62 . By calculation of the current response f or solely one single nanopore within both arrays, a current density of approximately 13.3 nA/cm² f or la rge p ore/pore separation compared to 6.4 nA/cm² for small distances between single pores show th e enhanced current response for single nanopores w ith individual hemispherical diffusion profile. Th e current profile in Figure 64 B also shows the current peaks with enhan ced current signals at the nanopores, which is again related to the p enetration of the conductive AFM tip into the pore filled with th e solution of high concentration of Ru(NH 3 ) 6 3+ . - 107 - AFM -SEC M images of a nanoporous array with 91 time s se par ation of the individual pores in a hexagonal arrangement. Contact mode AFM topography (A) and GC mode SECM current signal (B) du e to th e diffusion of [Ru (NH 3 ) 6 ] 3+ thro ugh th e nanop ores is d epicted sho wing ind ividual diffusion p rofiles at th e po r es. The cor respondin g heigh t and current p rofiles are shown beneath according to the marked line in (A) and (B). Original s ize: 25.3 x 25.3 µm 2 , scan speed: 47.9 µm/s (1.0 ln/s), scan angle: - 34.9 °. Ada pted with pe rmission f rom Y. Liu , A. H olzinger, P. Knittel, L. Poltorak, A. Gamero -Quijano, W. D.A Rickard, A. Walcarius, G. Herzog, C. Kranz, and D.W.M. Arri gan . Vi sualization of d iffusion within nan oarrays. Anal. Chem. 88 , 6 689 – 6695 (2016). Copyrigh t 2019 Americ an Chemic al Society . https://pubs.acs.or g/doi/10.1 021/ac s.analchem.6b005 13 . The AFM-SECM investigations were repeated with an AFM tip -integrated recessed Au frame electrode to detect the diffu sion without a dditional current peaks due to the penetration of a conductive AF M tip into the nanop ores. Thereby, a h in dered diffusion could b e detected b esides the pore as shown in Figure 6 5, with some pores seem t o be blocked, indicated by an absent current signal. The results s how a nanoporous array with a por e- to -pore sep aration of 21 in the AFM t opography with the corresponding height profile (A) and t he curr ent response in B, which sh ows not the exp ect ed overlapped diffusion. In deed, the current response is t oo l ow compared to calculations of the - 108 - predicted current for the diffusion of Ru(NH 3 ) 6 3+ and the current density (0.04 µ A/cm²) is just 6 % of the current density for t he results sh own in Figure 62, leading to t he assumption that the whole nanoporous arra y was blocked and not just some individual pores of the array. AFM - SE CM images of a nanoporous array with 21 time s s eparat ion of the individual pores in a hexagonal arrangement. Contact mode AFM topograph y (A) and GC mod e SECM current signal (B) d ue to th e dif fusion o f [Ru(NH 3 ) 6 ] 3+ through th e nan opores i s d epicted. Some of the p ores are blocked and no diffusion is detectable in (B ) repres ented by dark region above the nanop ore array. The correspo nding height and current profile s are sho wn beneath according to the marked lin e in (A) an d (B). Original size: 4 0 x 40 µm², scan spe ed: 47.7 µ m/s (0.6 ln/ s). The observed small change in current may be ex plained th a n by the cha nge in distance between the surface a nd the tip integrate d electrode when the re -shaped AFM tip penetrates the p ore s. This behavior of blocked pores could be also observed in ITIES measurements at the nanoporous arrays. The reason for bl ocking has not been fully investigated and might be re lated eithe r to impurities of the used solu tions or to redeposited ma terial durin g FIB-mil ling o f t he a rrays. Additionally, small air bu bbles, which may b e loca ted at the SiN membrane and may be possibly introduced during the filling of the glass tubes with one of the electrolyte solut ions, might also block single p ores. - 109 - For a de tailed interpretation of the ob served pore blocking, a detailed understanding of the l ocation o f the ITIES within t he nanopores is essen tial. In t he following section , electrochemistry at the ITIES and theoretical calculations are compared to verify the prediction of the i nterface being located at the aqueous facing side of the nanopores as reported for arrays produced by e-beam li thography 98,267 and also assumed for FIB-mille d nanoporous arrays 31 . ITIES at nanopore arrays For the identification of the interface within nanoporous arrays, the electrochemical ly driven t ransport of TPrA + as a model analyte at the ITIES is investigated. This was already used for characterization of porou s SiN membranes and led to t he con clusion that the interface a t FIB-mil led nanopores is com parable to na nopores fabricated by e-beam lithography 31 . Thereby, an inlaid i nterface facing th e aqueous electrolyte solution was assumed and the calculations are b ased on equation ( 26 ) (see section 1.4). Because FIB- milling results in the implantation of positively charged Ga ions , which may result in a hydrophilic surface, the b ehavior of FIB-milled nanopores might be different for ITIES compared t o nanopores obtained by e- beam lithography. Additionally, the t runcated pore shape due to redeposited ma terial during th e milling process, which was already r eported by several groups 32,36,37 , w as so far n ot considere d. C V recorded at ITIES (A) : black curve re pr esents the backgroun d and color ed curves represent the CV a fter cons ecutive addit ion of TPrACl to t he aqueou s phase. The curr ent re sponse after backgroun d sub traction is de picted in (B). Colors: 20 µM (purp le), 40 µM (yello w), 60 µM (green), 80 µM (blu e) and 100 µM (brown) TPrA Cl. Scan sp eed: 5 mV /s. - 110 - In Figure 66 , the CV for th e t ransport of TPrA + from the aqueous to the or ganic ph ase during the forward scans are shown wi thin a concentration range of 20 – 100 µM TP rA + , with the black CV as the background response p rior to the addition o f t he an alyte. In B, the background subtracted current response of the f orward scan (TPrA + tran sfers from the aqueous t o the organic phase) is depicted. A limit ing current at 0.6 – 0.65 V is visible for the pu rple and the yellow curve in Figu re 66 B. The current at this potential was compared t o theoretical cu rrents calculated by the different equations summarize d in 1.4 . Prior to the electroche mical investigations, the nanoporous array was characterized by AFM u sing a high- as pect ratio AFM tip, showing the p ore shape a nd the pore orifices o f the FIB-milled nanopores (see Figure 67). Whereas in prior publications, the diameters of the nanopores were evaluated by SEM 30,31 , AFM provides an accur ate geometry and size of the pore. Dynamic mode AF M topograp hy (A, original s ize: 2.75 x 2. 75 µm²) of a nano pore ar ray with height p rofile (B) accord ing to the marked l ine in A. Th e re sults sho wn in Figur e 66 an d depicted as black points in the calibration curve in C are co mpared to theoretical calcu lations for an inlaid geometry of the int erface (black line , at h = 0 n m) and a recessed interface at different locations within the nan opore as marked in th e depth profile in (B). The d iameter s used for calculations of the th eoretical current s are summariz ed in (D). - 111 - The measurement of the pore diameter f rom SEM images is suitable for p ores with parallel in ner walls and the same p ore diameters at both sides of the membran e, whereas for a truncated p ore shape, th e invest igation of the por e shape of nanoporous memb ranes with thicknesses within 50 to 100 n m via SEM will always result in mixed pore size of t he truncated cone-shaped pore due t o th e p enetration d epth of the electron beam. Th ereby , the calculatio n of th e c ur rent respo nse based on an inlaid interface, which re presents the large orifice of the t runc ated nanopores facing the aqueous side within the reported investigations 30,31 , whereas for the reported calculation s the pore dimensions determined by SEM were used. These pore dimensions represent indeed an intermediate pore diameter, in b etween both orifices of a truncated cone-shaped pore. In Figure 67, the AFM image obta ined with an ultra -sharp AF M ti p (A) an d th e corresponding depth profile of the nanopores is depicted in B. The dashed lines within the profile repres ent the posit ion of the i nterface used for t he calculations of the theoretical c urrent by equat ion (28) fo r truncated cone-shaped n anopores. The diameter of the nano pores at the orifice (at 0 nm in the depth p rofile i n Figure 6 7 B, black dashed line) is 187 nm ± 3 nm ( N = 3) a nd wa s used as r L in t he calculation of the theoretical current (bas ed on equation ( 28) in 1.4) at different p osition of the interface within t he n anopores marked by different colors in Figure 67 B. The diameters for t he assumed r ecessed inter faces at diffe rent depths are shown in Fig ure 67 D and were used for r 0 in t he calculations. Th e experimental data for the t ransfer of TPrA + as shown in Figure 66 are presented as black p oints in the c alibration shown in Figure 67 C. Thereb y, the data points fit well with the theoretical calibration for an interf ace located in the p ores at a depth (or length) of L = 50 n m. The nanop orous array had a pore- to -pore separation of 10.2 ± 0.2 (N = 3) for the results shown in Figu re 66 and Figure 67 . Th is indicates an overlapped diffusio n p rofile a t t his n anopore arra y as shown in the AFM-SECM investigations in chapter 6.3.1 (see Figure 62). Liu et al. 30,97 reported that th e current re sponse for a n overlap ped diffu sion is u p to 54 % 30 reduced to individual diffusion at the n anopor es in dependence to the pore- to -pore separat ion. In Figure 68 , two different assumptions for a n overlapped diffusion are c ompared t o the experimental data and to the theoretical current with th e interface locat ed within t he trun cated cone - shaped nanopores at a depth of L = 50 nm. - 112 - Schemes (A) showing th e a s sumptions u sed for theor etical calculations accordin g to an overlapped d iffusion comp ared to the calibration curve (B, black points) of the results in Figure 66 . The red and b lue lines in B correspond to the theoretical a ssumption of one UME with the same size as the activ e area (red) or an UME of the same size as the w hole array (blue). The green line in B corresponds to the theoretica l assumptio n o f a rece ssed inter face loc ated towards the organic el ectrolyt e. The schemes in Figure 68 A are illustra ting the t heoretical assu mptio n used for t he theoretical values depicted i n the calibration curves in B in corresponding colors. Fo r the approach colored in red, the active area of the single nanopores is summarized and replaced by an interface of the same area, whereas for the calculation of the whole area, the size of the array was used a nd replaced by a d isk-shaped interface with th e same area. Both approaches can be con sidered as t he u pper and lower limit o f current r esponse that can b e expected for an overlapped diffusion. An other approach for the investigation of the location of t he ITIES within FIB-milled nanopores is shown in Figure 69 . Th e tr uncated cone shape of FIB -milled nanopores is used in t he comparison of n anoporous arra ys fabricated from t he front or the back, so that either the small orifice is facing the aq ueous electrolyte solution and vic e versa, as illustrated i n Figure 69 A. The used SiN membrane s had a thickness of 100 nm and same parameters were u sed for the fabrication of the nanopore arrays by FIB millin g. A similar approach was alread y reported by Alvarez de Eulate et al. 84 for microporous glass membranes fabricated by laser ablation with truncated cone-shaped pores. The hydr ophobicity of such inner pore walls was determined by conta ct angle measurements an d corresponding experimental and simulated data of t he diffusion at the microporous array with pores oriented t owards - 113 - either t he aqueous or the organ ic electrolyte solu tion results in t he assumption of a hemispherical shape of the i nterface. Fo r the arrays rep orted in t his p ublic ation , the pores were filled with the organic electrolyte solution 84 . For characterization of the n anopores in the experiments presented within this thesis, an ultra - sharp AFM t ip was used. Th e sharp AFM probes were obtained by FIB milling t aking into account the instrumental mounting an gle (9°) of the AFM p robe towards the sample surface. The AFM topography of a front and back side milled nanopore are show n in C with corresponding height profiles displayed in D. Comparison o f front an d back side m illed po res: the scheme in (A) represents the or ientation of th e truncated con e-shape d pores towards the aqu eous and or ganic phase, respectively, depending on the orientation o f the membrane d uring FIB milling. AF M to pography recorded in dyn amic mode (B) of a fron t side (l eft) and back side (righ t) milled pore with co rrespon ding height profiles as marked in (B) and depicted in (D). A high-aspect-ratio AFM tip is repre sented in the SEM image in (C) ob tained b y FIB milling. AFM par ameters: 152 kHz (left)/ 169 kHz (right), scan speed: 1 ln/s ( 6 µm/s), original size: 3 x 3 µm ². A. Holzinger, G. Neusser, B. J. J. Austen, A. Gamero- Quijano, G. Herzog, D. W. M. A rrigan, A. Ziegler, P. Wa lther , an d C. Kran z, Faraday Discuss. , 201 8, 210 , 113. Adapted from Ref. 187 . Distributed u nder the license Creative Commons Attribu tion -NonCommercial 3.0 Unported (CC -B Y-NC 3.0), https://creativeco mmons.org/licenses/by -nc /3.0/ . - 114 - Due to the geometry, only the pore diameter of the small orifice is accessible via AFM measurements f or the back sid e milled nanopo res. Th e dimen sions of t he nanopores are represented by t he height profiles of the back side milled pore (light blue line) and the front side milled pore (dark blue line) in Figure 69 D corre sponding to the topography marked by dashed lines in B in corresponding col ors. Th e profil e of the back sid e milled pore represents thereby th e shape of th e AFM t ip and solely the dime nsion of the small orifice can be gained fro m these investigations. Th e fabricated nan oporous arrays, FIB- milled either from the front or the back of the SiN membrane , were examined in electrochemical investig ations via CV fo r the transport of TPrA + . In case that the i nterface is located towards the aqueous phase as assumed i n previou s publicat io ns 30,31 , a lower current response for the b ack side milled nanopores is expected due to t he smaller orifice facing the aqueous phase. In Figure 70 A , the b ack ground c orrected forward sweep of the current response in dependence of the concentration of TPrA + in the ran ge of 20 – 100 µM is shown and the corresponding calibration graph is represented by red points in B. Current response after background subtraction (A) of consecutive addition of TPrACl at a back side mill ed nanopore array. Colors: 20 µM (pu rple), 40 µM (yellow), 60 µ M (green), 80 µ M (blue) and 100 µM (brown) TPrACl. Scan speed: 5 mV/s. Th e calibration curve (B) comp ares the results of A (red data) with the data obtained at ITIES at a front side milled nanopore array (black d ata) and the th eoretical assumption (green line) of an inlaid interface f acing the aqueous electrolyt e. - 115 - The green line in B represents t he calculate d d ata for an inlaid interface facing the aqueous electrolyte solution and the black p oints rep resent the experimental results for a front si de mill ed n anoporous ar ray as already sh own in Fig ure 6 6 . Alt hough the lin earity for the experimental data for the nanopores FIB-milled from the back is poor, the detected current is up to 5 times higher than for the front side milled nanoporous array. The pore- to -pore s eparation for this array with r espect t o the radius of th e sm all ori fice is 17.6 ± 0.8 ( N = 9) compare d to 10.2 ± 0.2 ( N = 3) for the results shown in Figure 66 and represented by the black data points in Figure 70 B. According to the results obtained so far 30 , the diffusion at this p ore array is overlapped. Either for individual or overlapped diffusion profiles, the current response has to b e reduced for smaller pore d iameters re presented by the back side milled n anoporous array i n case of an i nlaid in terface facing the aqueous electrolyte solution du ring electrochemical investigations. Of course, the influence of implanted Ga + ions has to b e considered re sulting in c hanges in the hydrophilicity of t he membrane d ependent on the orientation of the SiN memb rane towards the ion b eam during FIB milling. This parameter was neglected so far an d h as to b e t aken into account in future studies. Similar results showing a variation in electrochemical invest igations performed at conical pores in two different orientation was already re ported by Arrigan and coworkers, b ut for µm-sized pores in a glass memb rane fabricated by laser ablation 84 . However, the hydroph ili city of these micropores w as modified by an additional surface modification, which en sures the same hydrophilicity for the d ifferent orientation s of th ese micropores. This is not the case for the FIB- milled nanopores re ported wi thin this thesis. Thereby, this is the first time that t his direct comparison of n anoporou s arra ys in SiN membranes u sed for nan oITIES (see Fig ure 69 a nd Figure 70 ) are investigated t owards the orientation of the membranes du ring FIB -milling. The different behavior in electrochemical investigations at nanoITIES with respect to the orientation of cone- shaped nan opores will be also considered in th e following sections. The lo calization of the interface b y AFM-SECM is d ifficult d ue t o the p enetration depth o f t he AFM t ip into th e nanopores a nd an accompanying mixture of the solutions. The d iffusi on be havior at nanoporous arrays was also visualized by silica depositi on at the ITIES within nanoporous arrays. The siz e of the silica deposits for med at t he interface between an aqueous ph ase and DCE was evaluate d 181 . Within the n ext sec tion, the size variation of t he silica deposits is compared with respect to the actual pore diameters. - 116 - Silica deposits at the ITIES for localization of the interface The localized deposition of silica at the ITIES is used within the following investigations to distinguish between nanopores FIB -milled either from the front or t he back as shown in Figure 69. Th e pore- to -pore separation in both a rrays is sufficient for independent por e diffusion and the diameters determined by AFM f or the or ifice facing t he aqueous electrolyte solution a re 18 3 nm ± 29 nm (N=3) f or front sid e milled a nd 72 n m 12 n m (N=3) for the b ack side milled arrays, respectively. Th e SEM images of the arra ys after silica d eposition are depicted in Figure 71 A and the diameter of the single silica deposits for both arrays are com pared in B. Thereby, a wide distribution of different deposit si zes can be seen for both approaches with larger diameters for the back side milled nanoporous array that i ndicates an enhanced io n transport . The siz e dis tribution within the 100 nanopores of o ne array is even larger t han the difference betw een both arrays fabricated by front or back side FIB milling. A paire d two-tailed t-test w ith a 95 % confidence level showed that the variation of the silica deposit s in each array is significant and th e standard deviation of the silica deposits (43 % an d 57% of the mea n value for diameters, respectively) within one array, which has on ly a small standard deviat ion in the original pore size, corresponding t o 16 – 17 % of the mean value, is larger, than the difference between the silica depositions at both membranes. These results confirm the interpretation gained by the comparison of electrochemical results of the t ransfer of TPrA + at t he nan oITIES as d iscussed in 6.3.2, showing t he same trend of an enhance d diffusion for FIB-milled nanoporous ar rays fabricated from the back of the S iN membrane. However, d ue to the broad size distribution of the results presented in Figure 71 , additional investigations h ave been done at these modifi ed nanopore arrays. Therefore, the silica deposits of t he n anoporous array fa bricated from the back are investigated in detail by FIB cross-sections and FIB/SEM tomography. Du ring electrochemical deposition of silica at the ITIES, the template CTA + is transferred from the organic to the aqueous phase enabling the formation of silica by hydrolysis and condensation of TEOS. 275 Si, either as TEOS or silica, is ju st p resent in the aqueous phase, hence the locati on of the silica deposits is an indication for the location of the aqueous phase. - 117 - SEM images of the silica depositions at front (A , left) and back (A, right) side milled pore arrays, respectively. Th e size d istributio n of the array s in A i s given in B. yello w: front side, blue: b ack side approach . The electroch emical deposition was don e a t 0 V for 90 s (aqueous phase : pH 9). A. H olzinger, G . Neusser, B. J. J. Austen, A. Gamero -Quija no, G. Herzog, D. W. M. Arrigan , A. Ziegler, P. Walther, and C. Kranz, Faraday Discuss. , 2018, 210 , 113. Reprodu ced from Ref. 18 7 . Dist ributed un der the license Creative Com mons Att ribution -NonCommercial 3.0 Unported (CC- BY -NC 3.0), https://creati vec ommons.org /licen ses/by-nc/3.0/ . In Figure 72 A, the FIB-induced secondary electron image is shown of one silica deposit covered with a layer of Pt/ C, which prevents ch arging effec ts while imaging and milling, and additionally prevents beam -induced damage of the silica deposit. In the SEM images in B-E , the cross-sections of two silica deposi ts with varyi ng diameters are depicted . As shown in Figure 71, the siz e distribution of the silica deposits is large and both , small - 118 - (< 5 µm, in B, C) and large (> 9 µm, in D, E) deposits were investigated in detail by FIB/SEM tomography. A close-up of t he silica deposits close to the nanopores is shown in C and E. Whereas for small deposits, the silica is d irectly attached at the SiN membrane, a gap between the silica an d the SiN membrane is visible for large ones ( Figure 72 E). This gap might be related to the drying step after silica formation, which might cau se shrinkage of the silica dep osit due to solve nt l oss. This e ffect is lik ely stronger for large deposits resulting in the separation of the deposit from the SiN membrane. Differences in silica morpholo gy : Cross-sections of silica depo sits are compared for large (> 9 µm in diameter) and small ( < 5 µ m in diameter) deposits. The FIB-induced SE image in A shows the silica deposit partially covered with a Pt/C protection layer, whereas the SE images in B - E show the deposit s in a 38° tilted perspective. The SE imag es shown in C and E represent a zoomed view of the samples shown in B and D, respectively, during consecutive FIB s ectionin g. The b lue arro ws in E point out an inner radial region sho wing different density o f the depos ited silica. A. Holzinger, G . Neusser, B. J. J. Austen, A. Gamero-Quijano , G. Herzog, D. W. M. Arri gan, A. Ziegler, P. Walther, and C. Kranz, Faraday Discuss. , 2018, 210 , 113. Reprodu ced from Ref. 187 . Dist ributed u nder the lic ense Creative Commons Att ribution -NonComm ercial 3.0 Unported (CC- BY -NC 3.0), https://creati vec ommons.org/licen ses/by -nc/3.0/ . - 119 - Any other p rocesses following the silica formation, suc h as removing the elec trolyte solutions, drying, and cle aning might also have an influence on the silica deposits and with this o n the investigated interface. In both examples shown in Fig ure 72 , a dense structure of t he silica deposit is visible, wherea s for the l arge deposit in Figure 72 D and E, the dense structure shows a hemispher ical region in close proximity to the nanopore (marked by arrows in E) with higher density acc ording to t he contrast i n t he SE imag e, compared t o the adjacent region showing a slightly different contrast. This variation in the silica deposit can b e addressed to an en hanced h emispherical diffusion near the n anopore du ring electrochemical deposition resulting in an enhanced f ormation of silica and a denser structure comp ared to the adjacent re gion. Next to the dense str ucture located in close proximity to the Si N membra ne visible in the SE images, which correl ates with the side facing the aqueous ele ctrolyte solution during electrochemical deposition, there is an inhomogeneous st ructure visible below the me mbrane f acing t he organic electro lyte solution. Th is is recognizable for small an d large silica d eposits ( Figure 72 C, E). This structure w ill be referre d to as “residue” with in the following discussion . In the SE image shown in C, som ething appearing as a ‘ single particle ’ is l ocated within the nanopore. In Figure 73, several SE images of th is si lica deposit are sh own in the FIB/SEM tomography , where the lateral distance between single SE images was 10 nm. The f ocus in these images was laid on the nanopore and t he residue i n close proximity to the nanopore. The conic al shape of the nanopore is clearly visible in these images. - 120 - Processed SEM imag es showing a nano pore (marked area in (A)) within a series o f image s (A- I) with 10 nm d istance between single SE images. SEM: 5 kV/ 86 pA, FIB: 30 kV/ 48 pA, 10 nm/ slice. Holzinger, G. Neusser, B. J . J. Au sten, A. Gamero -Quija no, G . Herzog, D. W. M. Arrigan, A. Ziegler, P. Walther, an d C. Kranz, Faraday Discu ss. , 20 18, 210 , 113. Based on Ref. 187 . Distributed und er th e license Creative Common s Att ri bution -Non Commercial 3.0 Unp orted (CC - BY -NC 3.0), htt ps://creativeco mmons.org/licen ses/by -nc /3.0/ . Additionally, the bright particle in th e pore (see Fi gure 73 F) has a dimension of 10 – 20nm as a sm all variation of the contrast in t he SE image in E might be p art of t his p article, but the particle is already no longer vis ible in im age G. Additio nally, the particle is not completely visible in image E, which might indicate that the pore is also filled with some other residue. In case that th e pore is empty (ex cept this particle) a nd th e contrast just indicates t he SiN membrane behind th e n anopore, this single particle has to be also visible in th e images C-E. I t is d if ficult to discrimin ate b etw een t he in -plane region of the exposed area and t he sam ple vol ume because the co ntrasts in the SE images are al ways a result of overlapped signals due to t he p enetration d epth of the electron b eam 184 . Th is is also called - 121 - ‘the sh ine through artifac t’ 280 . The structure of the silica deposit is homogeneous except small variation i n co ntrast in close proximity to the nanopore, w hich migh t indica te differences in the silica formation rate due t o enhan ced concentrations directly at the nanopore. A detailed interpretation giving the variation in the SEM contrast is at the current state not p ossible . The residue at the si de of the Si N membrane facing the organic electrolyte sol ution du ring ITIES meas urements shows different regio ns given t he varying, observed contrasts. The black regions are holes within t his residue, whereas b right regions might be eithe r single particles of unknown content or edges indicating the in-plan e area sectioned by the ion beam. Again, it is difficult t o interpret t he differen t contrast in SE images with unknown samples and to distinguish between th e in - plane area and the sample volume. S tructures, which a re visib le i n more than one S E im age d uring FIB sectioning can be associated wit h the sample vol ume. Focusing on the nanopore, there is some connection b etwe en re sidue and the nanopore visible, especially in th e SE images shown in Figure 73 C - G, with a single p article s how n in F. A cle ar differentiation between the SiN membrane an d this residue within the na nopore is still challe nging and d ata have to be interpreted with ca re to avoid false results. The residue can be clearl y addressed to the diffusion p rocesses in close pro ximity t o the n anopore because the residue is just located at this nanopore. These observat ions are more o bvious in an other silica d eposit investigated by SEM/FIB tomography , which is presented in Figure 74 . Single slices of the 3D stack are presented in A, I-IV s howing the resi due on the organic facing side of t he SiN membrane and located close t o the orifice of the nanopore. Three differen t regions migh t be differentiated within this residue. The f irst p art seems to be some kind of encapsulati ng layer, followed by a poro us region a nd a diffuse i nner structure of the residue. The diffuse inner structure seems also to be within th e nanopore. In tot al 24 SE images of the slice & view p rocedure a re su mmarized in a 3D representation of the n anopore , pr esented by a voxel siz e o f1.92 µm x 1. 92 µm x 10 nm . T he resid ue is shown in Figure 74 B with the inner diffuse structure marke d in green an d the outer layer sh own in yellow. The borders o f the SiN membrane are i ndicated in blue. T he truncate d cone shape of the nanopore is clea rly visible in t he 3D reconstruction. The d rop -like re sidue in this example is l ocated around the n anopore, whereas the thin layer marked i n y ellow seems to cover the SiN mem brane over a larger region. - 122 - Processed SEM image sequen ce of a b ack side milled n anopore after silica deposition with a large residue stru cture locate d at the nanopore facing th e organic electrolyte (A, I -IV). The distance between singl e slices is 30 nm. 3 D reconstruction of th e slices and v iew stack i s sho wn in B. SEM: 5 kV/ 86 pA, TLD a t 38° (corrected tilt), FIB: 30 kV/ 48 pA, 10 n m/slice; in to tal 80 slices are imaged . A. Holzin ger, G. N eusser, B. J. J. Austen, A. Gamero -Quijan o, G. Herzo g, D. W. M. Arrigan, A. Ziegl er, P. Walther , and C. Kran z, Farad ay Discuss. , 2 018, 210 , 113. Reproduce d from Ref. 1 87 . Distributed un der th e lic ense Creative Commons Attributio n - NonComm ercial 3.0 Unpo rt ed (CC - BY - NC 3.0), https:// creativecom mons.org/licenses/b y- nc/3.0/ . The results s hown i n Figure 73 and Fig ure 74 indicate that the pores are filled with some residue. Th ere is a connection between this residue and the silica deposit above th e membrane facing the aqueous electroly te solution during electrochemical investigations, however, the structure of t he par ticles or structures within the na nopores show different contrast in the SE images compared to the silica deposits. A detailed in t erpretation is still difficult due t o the ‘ shine t hrough effect ’ of porous structures 280 . Additional in formation is gained by the investigation of the elemental composition as presented in the f ollowing section. - 123 - Investigation of the elemental composition of deposited silica at nanopores For the d etermination of t he el emental c omposition of th e silic a deposits a nd t he residue at the sid e of t he membrane facing the organic electrolyte solution during electrochemical deposition, a thin TE M lamella was p repared fr om a single silic a deposit. T he TE M lamella with a thickness smaller th an 150 nm was investigated by EDX resulting in the elemental maps shown in Figure 7 5 (color- coded SE images in res pect to the pr esent elements o n the right). The fabrication of the TE M lamella by FIB milling resu lted in re-deposition of material, which is indicated by the EDX signal of Pt (light blue image). The signal has a maximum valu e at t he edge of the diffuse residue. An enhanced signal at energies for X- rays charact eristic for the other elements at this border can be also seen in the other images and is therefore negligible . TEM im age of a nan opore with a d iameter of 80 nm (A) with a zoomed vie w o f the pore (B) and SEM image of th e same loca tion, but vertically flipped by 180°. EDX mapping of th is area is given on the right side marked by false colo r images according to the elemental composition given in the single imag es. Acceleration voltage: 10 kV. A. Holzinger, G. Neusser, B. J. J. Austen, A. Gamero-Qu ijano, G. H erzog, D. W. M. Arrigan, A. Zi egler, P. Walther , and C. Kran z, Fa ra day Discuss. , 2018, 210 , 113. Ad apted from Ref. 187 . Distrib uted u nder the license Cre ative Commons Attribu tion -NonCommercial 3.0 Unported (CC - BY -NC 3.0), https://creativeco mmons.org/licenses/by -nc /3.0/ . - 124 - The residue located at the nanopore shows high intensities for chloride (red), carb o n (green), but also silic on ( pink). Thereby, the Si sig nal is more pronounced and it is unlikely that t his signal originates ju st due to rede posited material. Therefore, t his resi due mig ht consist of Si, either as silica or as TEOS indicating the l ocation of the aqueous phase during electrochemical silica deposition. For a deta iled interpretation of the elemental composition of this residue, additional investigations w ill b e necessary. Th e interior of t he nanopore cannot be evaluated h ere becau se the signal is overlaid with the signal originating from t he SiN membrane. The STEM image depicted in A shows the TEM lamella from one side, whereas the SE image in C and t he c orresponding E DX mapping (right side) show a vertically flipped view of the same p osition. From t his perspective, the nanopore is not visibl e and the SiN memb rane in this image is entirely covering the pore. For b oth, SEM and EDX, the penetration de pth of the exc itation e-beam is larger tha n the thickness of the TEM lamella itself 184 . For clear identification of the content within the nanopores, the pore has to be larger than the TEM lamella. Because t he p reparation of th in lamellas below 100 nm is challenging due to limits in the fabrication pro cess 281,282 , the n anopores prepared by FIB milling were in creased to a range of 25 0 – 350 nm in diameter. As visualized in the schemes in Figure 76, the larger pores are accessible from both sides in lateral dimension that the contrast in SEM, TEM and th e signal detected by EDX of the lamellas solely represents the inner struct ure wit hin the pores avoiding t h e convolution of the signals with the signals originati ng from the SiN membrane. Scheme o f two TEM lam ellas with varying po re dia meters , in sufficient (left) and suffici ent (right) for investigation of the content within the n anopores. A. Holzinger, G. Neusser, B. J. J. Austen, A. Gamero- Quijano, G. Herzog, D. W. M. A rrigan, A. Ziegler, P. Wa lther , an d C. Kran z, Faraday Discu ss. , 201 8, 210 , 113. Reproduc ed from Re f. 187 . Distributed under the license Creative Commons Attribution -NonCommercial 3.0 U nported (CC - BY -NC 3.0) , https://creativeco mmons.org/licenses/by -nc /3.0/ . - 125 - For the nanoporous arrays with larger diameters ranging from 250 to 350 nm, Si N membranes with a th ickness of 50 nm were u sed. All arrays were FIB milled fro m the f ront side facing the aqueous electrolyte solution duri ng electrochemical silica deposition. To distinguish between Si particles located in the pore and associated with the silica deposit process, which migh t be moved into the pore during drying or cleaning steps after silica formation, the pH value for one silica deposition was adju sted t o suppress t he silica formation. Additionally, the deposition time was altered, and its influence is analyze d towards the silica deposits and the remaining residue in or below the nanopore f acing the organic ele ctrolyte solution during electroc hemical d eposition. An overview of silica deposits fabricated at different de position conditions are shown in Figu re 77 . SEM images of different silica deposits with v arying depositi on parameters: array with 9 (A, B) and 4 (C) pores and silica d eposits (D -F) corr esponding to the arrays above. Parameters : applied p otential: - 0.1 V for all array s; for 60 s at pH 9 (A, D), for 5 s at pH 9 (B,E), for 10 s (3 times) at pH 3 (C,F). The pH value corresponds to the aqueo us phase. 5 kV, 86 pA, tilt: 0° (A-C) and 52° (D- F) . Adapte d from Ref. 187 . Dist ributed under the lice nse Creative Commo ns Attribution-No nCommercial 3.0 Unp orted (CC - BY -NC 3.0), https://creativeco mmons.org/licenses/by -nc /3.0/ . - 126 - Thereby, the SE images in A and B show a varying size of t he silica d eposit s with changes in deposit ion time at pH 9. In C, the results f or the deposition at pH 3, insufficient for the silica formation, is show n. At acidic pH values , the mononuclear specie s of TEOS is dominant in the aqueous phase, whic h doesn’t facilitate the transfer of the template from the organic to the aqueous phase and no condensation reaction of the TEOS occurs 276 . It is clearly visible that no silica d eposits were formed above t he individual p ores at the aqueous facing side durin g electrochemical deposition. However, some p articulat e material was located with in the nanop ores in absen ce of silica d eposits, which ar e embedded within the residue that was already ob served for nan oporo us arrays with smaller p ore diameters (see Figure 72 to Figure 75). With in creasing deposition times, the diameters of the silica deposit s i ncreased with 1.4 ± 0.3 µm ( N = 27) for 5 s compared to 3.7 ± 0.5 µm ( N = 27) for 60 s, whereas in both examples shown in Figure 77 A and B, slightly different sizes f or single deposits in one arra y can be seen in accordance to the results presented in section 6.3 .3 . For short deposition times of 5 s, the silica deposits were jus t for med at some pores and 5 o f 27 nanopores of t his investigated n anoporous array showed no silica formation. Fo r t he n anoporous array used in ITIES measu rements with insu fficient pH values of the aqueous phase, no silic a deposits were obtained (Figur e 77 C, F), but some nanop ores showe d re sidue wit hin t he pores as shown in F. In Figu re 78 , the cross -sections of a silica deposit formed at pH 9 (A and B) are compared to the cross - section of a nanopore w ithout silica for mation due to an insufficient pH valu e (pH 3, C). The cross-sections shown in A and B, as well as the 3D reconstruction in D, show a dense silica d eposit as already reported for th e n anopores with smaller dimensions and a diffuse residue (marked yellow) within the p ores with a few sin gle particles (marked in gree n) and some holes (black ). In the SE image in C, a large particle is visible within the nanopore embedded in the so-called residue. - 127 - Influence of the p H in the aqu eous p hase on silica formation : A processed SEM image of a s ilica deposit formed at pH 9 shown at two different magnificatio ns (A, B) and no silica f ormatio n at pH 3 (C), 38° tilt ed view. The residue formed at large nan opores is shown in detail in th e 3D representation (D, accord ing to the nanopore sho wn in A , B) with: SiN me mbrane (b lue), si ngle particles (gr een), holes (black) and the diffuse residu e (yell ow). SEM: 5 kV/ 86 pA, TLD at 38° (corrected tilt), FIB: 30 kV/ 48 pA, 5 nm/slice and in sum 300 slices. A. Ho lzinger, G. Neusser, B. J. J. Aust en, A. Gamero-Qui jano, G. Herzog, D. W. M. Arrigan, A. Ziegler, P. Walther , and C. Kranz, Farad ay Discuss. , 2018, 210 , 113. Rep roduced from Re f. 187 . Dist ributed un der the license Creative Commons Attribution -NonCommercial 3.0 U nported (CC - BY -NC 3.0), https://creativeco mmons.org/licenses/by -nc /3.0/ . Because t here is no silic a con densation at the acidic aqu eous solution i n accord ance to literature 276 , detailed investigations of these p articles or re sidues might give further information on the residues located on the side of the SiN membra ne f acing the organic electrolyte solution during electrochemical silica deposi tion. Therefore, a TEM lamella was prepared b y FIB milling and the elemental composition of t he residue withi n t he nanopore was investigated. I n Figure 79, SE images of a nanopore (A), the c ross-sectio n of a nanopore (B) and the preparation of a T EM lamella (C) of the nanoporous array without - 128 - silica formation is sh own. The EDX s pectra of two regions within the nanopore, located i n a TE M lamella thinne r than the actual pore dia meter (STEM image in D), are compared showing a clear differe nce between the regio n of a bright par ticle and the residue surrounding this particle. The bright particle consists of Si as mar ke d i n blue in the EDX spectra, wher eas th e other residue shows almo st no Si signal (marked in y ellow). The Cu signal i n both E DX sp ectr a is associat ed with the copper T EM grid. No silica is f ormed at the nanopore shown in Figure 79 according to insufficient pH val ue. SE images of n anopores after electroch emical investigation s with no silica d eposits d ue t o pH 3 insufficient for th e sil ica formation at th e ITIES (A - C). SE image a fter cro ss-sectioning of a nanop ore filled with a bright particle (B) and SE image during the preparation of a TEM lamella by FIB milling with additional Pt/C as a protection layer (C) and STEM image of a TEM lamella (D). EDX spectra represent the marked regions in (D) in corre sponding colors. SEM (A-C ): 5 kV/ 86 pA, 38° tilted, STEM (D): 30 kV. A. Holzinger, G. Neusser, B. J. J . Austen, A. Gamero -Quij ano, G. Herzog, D. W. M. Arrigan , A. Ziegler, P. Walther , and C. Kran z, Faraday Discus s. , 2018, 210 , 113. Reproduced from Ref. 187 . Distribute d under the license Creative Commons Attribu tion - NonComm ercial 3.0 Unpo rt ed (CC - BY - NC 3.0), https://creativec ommon s.org/licenses/by - nc/3.0/ . - 129 - The observed particulate material within the nanopore might b e the result of a pre - concentration of n on-condensed Si(OH) 4 of the aqu eous ph ase during removal of the electrolyte solutions while cleaning the samp le. Because Si is just located in th e aqueous phase, this silica- rich p article in the nanopore was either due to the presence of the aqueous phase within the nanopore during ITIES invest igations or had to b e moved into the nanopore during cleaning and drying steps . Both effe cts may be also a ssociated with the res ults o btained at nanoporous arrays u nder electrochemical c onditions sufficient for silica formation. In Figure 80, different cross-sections of silica deposits eit her formed at small n anopores with pore diameters of 72 nm ± 12 nm (N=3) (A, B) an d for large with nanopore diameters of 322 nm ± 8 4 nm (C ). In all SE images shown in Figure 80, there are particles located in the r esidue at t he side of the mem brane facing the organic elec trolyte solution during ITIES me asurements . In case of a large pore as shown in C, these particle s may have be en moved to this side of the membrane due to cleaning an d drying steps, whereas for the examples shown in A a nd B for small nanopores, the d imensions of the particles are too large to be accident al ly moved to this side of the SiN membrane. Differences in the residue : silica formation at nanoporous a rrays with s mall nanopores (72 nm 12 nm, A, B) with p articles within the residue for large (A) and small (B) silica depo sits, compared to silica formation and th e characteristic re sidue at the organic electrolyte facin g side of the mem brane at nanop ores with large pore diamet ers (322 nm 84 nm, C) . SEM: 5 kV / 86 pA, 38° tilted view. A. Holzin ger, G. Neusser, B. J. J. Austen, A. Ga mero -Quijano, G. Herzog, D. W. M. Arrigan, A. Zie gler, P. Walth er, and C. Kranz, F araday Discuss. , 201 8, 210 , 113. Reproduce d from Ref. 1 87 . Distributed un der th e lic ense Creative Commons Attributio n - NonComm ercial 3.0 Unpo rt ed (CC - BY - NC 3.0), https://creativec ommon s.org/licenses/by - nc/3.0/ . - 130 - Therefore, it is most likely that t hese particles indicating the location of the aqu eous p hase and wi th this the interface between both electrolyte solutions. All SE images and EDX mappings shown with in this section reveal th at t hese p articles visib le within single nanopores or in the residue at t he side of the SiN membrane, which face the organic phase contained Si- related s pecies . At this point, it is not possible to distinguish between silica or TEOS. Based on the results shown in Figure 80, it is h ighly unlikely that these large particles are part of th e silica formed at the sid e of the membrane facing the aqueous electrolyte solutio n duri ng deposition. Either t hese particles i n dicate the location of the aqueous ph ase durin g ele ctrochemical silica deposit ion at the ITIES, or the residue represents some kind of mixed phase close to the nanopores. Indeed, e thanol is formed during t he hydrolysis of TEOS to silica 283 , which is soluble in the aqueous electrolyte solution, but also in DCE. Although ethanol was removed p rior to electrochemical deposition at the ITIES, the r esidue may still be related to mixed layer when ethanol was not completely removed . It has been shown th at the ion transfer at trunc ated pores is influenced by osm otic pr essures or do uble layers within the nanopores, ev en for miscibl e solutions. This leads to an ion current recti fication with the electroosmotic flow dependin g on the ion f low directio n with in the truncated cone- shaped geometry 284 . This mig ht also be a reason for some kind of mixed phase forming the ob served residue. Hence, the observed particles may be the result of a combina tion of the discussed effects. Conclusion and outlook The advantages of FIB milling for prototyping nanop orous arrays wit hin SiN mem branes were shown in detail as already reported in previous publica tions 30,31,97 . The importance of the p ore- to - pore separation with respect to th e current response for nanoporous arrays at the liquid/liquid interface cou ld b e visualized by AFM -SECM. Additionally, the position of t he liquid/li quid interface within the n anopores plays a crucial role in the application of such nano sized interfaces in electroanalysis, which was investigated by the transport of TPrA + between both electrolyte solut ions and by electrochemical silica formation d irectly at the water/DCE interfac e. Fo r the first time, 3D FIB/SEM investigations were used to visualize the silica formation in detail, enabli ng a close look into th e nanopores and i nto th e silica deposition at the side of t he SiN membran e facing - 131 - the aqueous electrolyte solu tion during ele ctrochemical d eposition. Additionally, some kind of r esidue was observed at th e side facing the or ganic electrolyte solution, which was characterized by SEM and by EDX measurements performed at TEM lamell as. The results obtained h ere indicate that the nanop ores are filled with t he aqueous phase, whereas additional cleaning or heating procedures during samp le preparation might change the actual situation/location du ring electrochemical ITIES measurements dramatically. Equally reasonable and con ceivable explanations would be some kind of mixed phase within the nan opores, which changes during the ele ctrochemical investigation a t the ITIES and results in a mixed, diffuse residue in close proximity to the nanopores. Hence, the assumption that the interface is located inlaid towards the aqueous ele ct rolyte sol ution and that the nanopores are f illed with the organic electrolyte solutio n as reported previously 31,267 , mig ht be doubted i n case of F IB-milled arrays based on the s tudies presented here . T he direct characterization of ITI ES w ithin nanoporous membranes is s till challenging, and the ap proach presented here is highly interesti ng accordin g to the obtained results. In future st udies, different analytes shou ld be investigat ed b y FIB/SEM tomography, forming a solid phase directly at the interface between two liquids an d showing a sufficient contrast in SE images, such as metal ions or co nductive materials. To prevent changes of the deposition due to additional cleaning and d rying steps and to avoid art ifacts due to th e rem oval of the elec trolyte solutions, cryogenic FIB/SEM tomography might be a valu able approach for t he investigations of the interface b etween two liquids 285 . - 132 - Fi na l re m ar ks In this thesis, the modification of AFM tip-integrated electrodes is presented eval uating the suitability of integr ated electrochemical sensors for localized detection of H 2 O 2 release (see chapter 4 ) or p H changes (see chapter 5 ) in close proximity to a sample surface. The results presented with in this thesis are f acing the essential challenges of integrated min iaturized sen sors . The fabrication of AFM -SECM probes involves several additional st eps be fore being used i n an AFM-SECM meas urement, namely FIB/SEM (vacuum conditions) to exp ose the ele ctrode area, to reshape a sharp AFM tip and to image the modifications, or heating and UV light exposure d uring mounting of AFM -SECM probes an d ins ulation of the electrical c ontact. These additional tre atments might change or influence the performance of integrated sensors. In the case of PB/NiHCF as H 2 O 2 - active material, these additional influences were investigated showing improved stability of the sensing layers in consecutive H 2 O 2 calibrations (see 4.3.1 ). For AFM-SECM investigations, it is essential t o use ele ctrodes with fast response t imes in order to observe local changes in t he elec trochemical signal during scans of the sam ple surface. The H 2 O 2 sensors, as well as the pH s ensors i ntegrated in to AFM - SECM probes, have been t ested towa rds their sta bility in long- term measurements showing stable behavior in external calibrations. For PB/NiHCF- modified sen sors, a change in morphology was detected by co nsecutive SEM imagi ng o f modified UMEs af ter bei ng exposed to H 2 O 2 (Figure 20 ), by AFM in the pre sence of relatively high H 2 O 2 concentrations of 1 mM (Fig ure 21 ), and by AFM-SECM g ener ating H 2 O 2 at the AFM tip-integrated microelectrode (Figure 19 ). Th ese morpholo gy changes might resul t in some kind of dist ortion of the detected signals, either by a change of the detected current correlated to the reduced a mount of H 2 O 2 -sensitive PB mat erial or b y AFM tip artifacts d ue t o t he d issolution of the sensor material during scanning. Althou gh, changes of electrochemically deposited PB/NiHCF can be investigated b y SEM prior and after AFM/SECM mea surements, changes occu rring during ongoi ng measurements may remain undetected b ut might play a cr ucial limi tation of these integrated H 2 O 2 sensors. In section 4.3.2, modifications of AFM tip -integrated microelectrodes with optimi zed d eposition parameters for localize d deposition using - 133 - diluted solu tions were presented, w hich sh owed, however, a poor reproducibili ty in several consecutive calibrations. No clear correlation between the amount of PB as indicated by the redox sign al o f PB i n th e f ilm activat ion via CV i n H Cl/KCl an d t he sensitivity in H 2 O 2 d etection could be drawn. T his might be also related to the separa tion of parts of the PB/NiHCF f ilm resulting in a reduced amount of active PB actually in contact with the underlying microelectrode and , thus, being detected in electrochemical measurements. Especially f or PB/NiHCF- modified sensors, the modification of an AFM tip - integrated electrode is challenging becau se the e xact composition of these mixed layers is unknown. The ED X mapp ing pre sented in Figure 18 indica tes a homogeneous distribution of PB and NiHCF, though, a laterally resolved determination of the distribution between both salts is dif ficult due to the given resolution of t he method and the penetration d epth of the acceleration e -beam, whic h is larger than the actu al t hickness of the b ilayers. The dissolution of the PB/NiHCF bilayers dep osited on U MEs, det ected either by AFM or by consecutive SEM imaging, showed individual regi ons of th e material being predominantly removed under H 2 O 2 exposure. Th is indicates a variation in t he local composition of these mixed layers. A detailed investigation of the mixed P B/NiHCF layers with respect to th e composition of the material see ms to b e essential for these miniaturized H 2 O 2 sen sor s, enabling a targeted optimization of deposition p arameters for the modification of AFM-SECM probes. For the results p resented in ch apter 5 , just a limited number of A FM -SECM probes with integrated Sb or Ir meta l layer suitable f or imaging experiments have b een available as these p robes h ave been fabricated b y f ormer co-workers of Kranz an d M izaikoff . The presented investigations are focused on the evaluation o f the most suitable material for AFM tip-integrated pH sensors facilitating future optimization of the fabrication procedure of these p H sen sors. Th ereby, Sb/Sb O 2 , AIROF, an d EIROF-modified AFM -SECM probes were investigated in terms of p H sensitivity and stability. Althou gh the number of available sensors was limited, t he presented resu lts re veal trends about the most promising material for m odification of AFM-SECM probes. For all investigated materials, a broad dis tribution in pH sensiti vity was detected ra nging f rom 37 to 91 mV/p H for EIROF (see 5.3.3), 31 to 54 mV/pH detected in con secuti ve calibrations of one individual AIROF - modified AFM-SECM probe (see Figure 44 ), and 50 and 56 mV/pH for two investigated - 134 - AFM tip-integrated Sb/SbO 2 sensor s (see Figure 46). A significant advantage of AIROF electrodes in comparison to Sb is the p ossibility of re generatio n of the s ensor by anodic oxidation in H 2 SO 4 . In first AFM -SECM investiga tions w ith tip-integrated pH sensors, t he dissolution of calcite crys tals was map ped resulting in a changed morpholo gy of the crystals with adjoining changes in pH to more alkaline pH values. However, all investigations p resented in section 5.3.2 were lacking a lateral resolution for t he detected OCP signal of the Sb/SbO 2 or AIROF sensors in x-direction, but a good resolution in the scan direct ion (y-direction), which is also comparable t o results detected by SECM- SICM 253 . Changes in the OCP sig nal during me asurement could be correlated t o t he location of t he actual calc ite crystal but lacking suf ficient lateral resolution . Therefore , further investigations should focus on optimization of response t imes of AFM tip - integrated pH sensors. In chapter 6 , arrays of nanopores fabricated by FIB milling in soli d-state SiN membranes were presented and in vestigated in terms o f diffusion behavior and location of the interface of two immisc ible solutions within nan opores. AFM -SECM mea surements revealed the chan ge in diffusional profiles with respect to the pore - to -por e separation of individual por es in an array an d showed an overlapped diffusion for closely spaced p ores and individual diffusion for pores being separated by a ratio of r c /r a = 91 (see equation (29)). Furt her investigations of the potential -driven ion transport of TPrA + at ITIES and localized deposition o f silica p articles at the ITES within nanoporous arrays, ad dresses the question of t he location of the i nterface. In previous publications, it was postulated t hat the na nopores are filled wit h the organic elec trolyte solut ion, which i s t he case for nanoporous arrays f abri cated b y e-b eam lithography. The assumption that FIB -milled nanoporous arrays show t he same b ehavior may b e questionable due to the known implantation of positively charged Ga ions and a conical - shaped ge omet ry of the nanopores. Further investigations were presented in c hapter 6 a nd FIB/SEM tomogra phy, used f or th e first t ime for the investigation of micro- and nano interfaces located in solid - state materials, is presented in section 6.3.3 and 6.3 .4. The nanopores investiga ted throughout this thesis are mo st likely filled wi th the aqueous phase forming some kind o f mixed p hase w ithin the nanopores during t he presented electroc hemical sil ica deposition at the nanoITIES. It sh oul d be n oted that the presented results obtained for the localized - 135 - silica d eposition at t he in terface between two immiscible liquid s requires post - treatments, n amely rinsing and drying, prior to bein g investigated b y 3D FIB/SEM , which may influence the location of the interface. To circumvent this, investigations of the liquid/liquid interface c ould be performed under cryogenic conditions. 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