Effect of Nanoparticles on Modified Screen Printed Inhibition Superoxide Dismutase Electrodes for Aluminum

senso s A icle E ec o Nanopa icles on Modi ied Sc een P in ed Inhibi ion Supe oxide Dismu ase Elec odes o Aluminum Mi iam Ba que o-Qui ós 1,* and Ma ía Julia A cos-Ma ínez 2 1Depa men o Chemis y, Uni e si y o Cos a Rica, CELEQ, San Ped o de Mon es de Oca, San José 11501-2060, Cos a Rica 2Depa men o Chemis y, Facul y o Sciences, Uni e si y o Bu gos, Plaza Misael Bañuelos s/n, Bu gos 09001, Spain; ja [email p o ec ed] *Co espondence: mi iam.ba que o@uc .ac.c ; Tel.: +506-2511-2442; Fax: +506-2511-2446 Academic Edi o : Robe o Pillo on Recei ed: 26 July 2016; Accep ed: 20 Sep embe 2016; Published: 26 Sep embe 2016 Abs ac : A no el ampe ome ic biosenso o he de e mina ion o Al(III) based on he inhibi ion o he enzyme supe oxide dismu ase has been de eloped. The oxida ion signal o epineph ine subs a e was a ec ed by he p esence o Al(III) ions leading o a dec ease in i s ampe ome ic cu en . The immobiliza ion o he enzyme was pe o med wi h glu a aldehyde on sc een-p in ed ca bon elec odes modi iedwi h e a hio ul alene (TTF) and di e en ypes o nanopa icles. Nanopa icles o gold, pla inum, hodium and palladium we e deposi ed on sc een p in ed ca bon elec odes by means o wo elec ochemical p ocedu es. Nanopa icles we e cha ac e ized ough scanning elec onic mic oscopy, X- ays luo escence, and a omic o ce mic oscopy. Palladium nanopa icles showed lowe a omic o ce mic oscopy pa ame e s and highe slope o aluminum calib a ion cu es and we e selec ed o pe o m senso alida ion. The de eloped biosenso has a de ec ion limi o 2.0 ±0.2 µM o Al(III), wi h a ep oducibili y o 7.9% (n = 5). Reco e y o s anda d e e ence ma e ial spiked o bu e solu ion was 103.8% wi h a ela i e s anda d de ia ion o 4.8% (n = 5). Reco e y o ap wa e spiked wi h he s anda d e e ence ma e ial was 100.5 wi h a ela i e s anda d de ia ion o 3.4% (n = 3). The s udy o in e e ing ions has also been ca ied ou . Keywo ds: supe oxide dismu ase biosenso ; aluminum; e a hio ul alene; sc een-p in ed elec odes; nanopa icles 1. In oduc ion Aluminum oxici y has been shown in i o and in i o , bu complexi y o i s in e ac ions wi h human o ganism makes i e y di icul o assign he esponsibili y in Alzheime ’s disease. I can be conside ed as he combined e ec o oxidan ac ion, pa icipa ion on amyloid cascade, neu onal degene a ion [ 1 ] and accumula ion in neu o ib illa y angles. Aluminum p esence a o s T p o ein link ough phospha e b idge [ 2 ] and al e s homeos a ic ion equilib ium [ 3 ], and i has shown a s ong e ec on eac i e oxygen species (ROS) p oduc ion on li ing o ganisms, due o i on accumula ion in oxida i e s ess [ 4 ]. Ma kedly, ROS impai ed enzymes such as supe oxide dismu ase (SOD) and ca alase eac wi h adical species such as O 2•− , OH •− , and ONNO •− . Al hough O 2•− adical is no so eac i e i sel , in he p esence o Fe 2+ , Fen on eac ion can u n i in o hyd oxyl adical, which is he mos po en adical. These indings show ha O 2•− is in ol ed in cellula damage. Biosenso s ha e employed supe oxide dismu ase (SOD), an enzyme ha sca enges supe oxide o measu e supe oxide anion acco dingly wi h he eac ions [5]: Cu2+ −SOD + O2•−→Cu1+ −SOD + O2 Senso s 2016,16, 1588; doi:10.3390/s16101588 www.mdpi.com/jou nal/senso s Senso s 2016,16, 1588 2 o 19 Cu1+ −SOD + O2•−+ 2H+→Cu2+ −SOD + H2O2 O 2•− a ec s cy och ome c (Cy c) by oxidizing Fe 2+ o Fe 3+ and educing i sel o H 2 O 2 . O2•− adical le els we e ound o be ele a ed in homogenized cance ous b ain issue compa ed o no mal human b ain issue [ 6 ]. O 2•− is o med in li ing biological sys ems by he dona ion o an elec on o molecula oxygen, h ough oxida ion o semiquinone- ype adicals o med in he mi ochond ial elec on anspo chain. In i s p esence, ee adical sca enge s, enzymes such as supe oxide dismu ase and glu a hione pe oxidase (GSH-Px), dec ease hei an ioxidan s a us, and lipid pe oxide le els a e inc eased. Aluminum adminis a ion o labo a o y animals induces SOD dys unc ion and damage on a ge o gans [ 7 – 10 ]. I has been ound ha zinc [ 11 ], selenium [ 12 ] and he apies used agains Alzheime ’s disease [ 13 – 16 ] ha e a p o ec i e ole agains aluminum induced oxici y, imp o ing SOD unc ion. Aluminum p esence in humans is associa ed wi h oxida i e s ess [17,18]. Biosenso s cons uc ed wi h sc een p in ed elec odes (SPCE) o e low de ec ion limi s, easie assembly o me allic nanopa icles (NPs), good ep oducibili y, low con amina ion and excellen biocompa ibili y wi h enzymes and an ibodies [ 18 ]. The me al NPs’ small size, high mechanical s eng h and high chemical and he mal s abili y allow hem, when ac ing as enzyme-ca ie ma e ials, o imp o e he e iciency o immobilized enzymes, acili a ing eac ion kine ics, and supplying a la ge su ace a ea, leading o highe enzyme loading pe uni mass o pa icles. This ac allows achie ing enhanced de ice sensi i i y and educed mass ans e esis ance [ 19 ]. Besides, NPs inc eased elec ic conduc i i y and elec on ans e be ween edox enzyme cen e and elec ode [20,21]. Di e en ypes o NPs such as me al, me al oxides, semiconduc o s, polyme s and composi e-me al NPs ha e been used o assembly minia u ized elec ochemical senso s and biosenso s. Gold NPs (AuNPs), due o hei unique p ope ies, ela i ely low cos and ease o p epa a ion, a e he mos used in many biochemical applica ions [ 22 , 23 ]. AuNPs can be syn hesized by di e en chemical me hods and applied o elec ochemical de ec ion o As(III) [ 24 ]. AuNPs use in ampe ome ic biosenso s and elec ochemical echniques enhances de ec ion sensi i i y [25–27]. Due o hei ine ness, pla inum NPs (P NPs) a e he p incipal me al NPs al e na i e o anodic cu en measu emen and ha e been applied o o maldehyde [ 28 ], neu o ansmi e s [ 29 ], glucose [ 30 ], u ic acid [31], and As(III) de e mina ion [32]. Glassy ca bon elec odes modi ied wi h palladium nanopa icles (PdNPs) ha e been applied o ca echolamines de e mina ion [ 33 ]. P NPs and PdNPs/me hyl hiophene (PMT) senso s ha e been applied o dopamine (DA) and AA de e mina ion [34]. Rhodium NPs’ (RhNPs) main applica ion has been as p ecu so s o he p epa a ion o ca aly ics [ 35 , 36 ] and o ca alysis and sensing o cy och ome c [ 37 ] and H 2 O 2 [ 38 ], as well as o biosensing o α-ke oglu a a e [39]. Due o sensi i i y and speci ici y join bene i s p oduced by modi ied SPCEs wi h me allic NPs and enzymes, his esea ch wo k was conduc ed wi h he goal o compa e he e ec o NPs o Au, P , Pd and Rh deposi ed by dis inc elec ochemical p ocedu es on sensi i i y o ampe ome ic inhibi ion SOD by aluminum, wi h TTF as media o using epineph ine (EPI) as subs a e. Mos de eloped biosenso s ha immobilize SOD enzyme in a ca ageenan gel a e based on H 2 O 2 ampe ome ic de ec ion [ 40 ] and we e success ully applied as a ool o an ioxidan capaci y assessmen o e alua e ed and whi e wines [41], esh he bs and ui s, oli es, ea [42–44], algae [45], phy o e apeu ic p epa a ions [ 46 ], d ugs con aining salicylic and as co bic acid, and β -ca o ene [ 40 , 47 ]. This de eloped biosenso enables measu emen o an ioxidan capaci y o heal hy and diseased issues in i o [40], and was also applied o de e mina ion o o al an ioxidan capaci y o be ies [48]. The o he s SOD based biosenso s ha employ modi ied solid elec odes a e shown in Table 1. Senso s 2016,16, 1588 3 o 19 Table 1. SOD based biosenso s applied o samples o biological in e es . Technique Elec ode Po en ial Modi ica ion Range LOD Sample/Anali e Re e ence CV 1SPCE −0.8–+0.8 V Py ole/SAMs 0.5 ×10−9–5 M 0.5 ×10−9Mcul u ed human ke a inocy es NO2-[49] Ampe ome y CFME 2+0.25 V cys eine/AuNPs (13–104) ×10−9M- O2•−[50] Ampe ome y SPCE −0.1 V po ous P -Pd/na ion (16–1536) ×10−6M 0.13 ×10−6MCell cul u e medium/O2•−[51] CV 1Ch onoampe ome y Gold elec ode −0.2–+0.5 V Au/Cys/SOD 9, Au/GNP/Cys/SOD 10 and Au/GNP/Cys/SOD/Chi 11 0.5–4 Gy 0.03 ×10−6M hallium 201/wa e [52] Ampe ome y GC CFME 20.2 V NTA/HT 710−7–10−4M 21 ×10−9Mb ain issue/O2•−[53] CV 1 Ca bon pas e elec ode Elec ochem 0–0.3 V cy och ome c in solu ion and Fe(III)-p o opo phy in immobilized (1–6) ×10−3M 0.3 ×10−3MXan ine [54] Sys em Ca bon pas e elec ode P o opo phy in and cy och ome bo h immobilized (1–8) ×10−3M 0.2 ×10−3M C onoampe ome y Composi e elec ode −0.3 V P Pd-PDARGO 6(0.016–0.24) ×10−3M 2 ×10−6M DMEM 5/O2•−[55] Ampe ome y GC 8−0.3 V MWCNT 4(0.01–0.3) ×10−3M1×10−6MWines, be y juice/O2•−[56] PEDOT 3 Ampe ome y SPCTTFE12 0.2 V SOD/PdNP 13 (1.0–60) ×10−5M2×10−6M Al(III) This a icle wa e samples 1 Cyclic ol amme y; 2 ca bon ibe mic oelec ode; 3 poly(3,4-e hylenedioxy hiophene); 4 mul iwalled ca bon nano ubes, 5 Dulbecco’s modi ied Eagle’s medium; 6 P Pd poly dopamine educed g aphene oxide; 7 Ni ilo iace ic acid/his idine- ag; 8 glassy ca bon; 9 gold, cys eine, supe oxido dismu ase; 10 gold, gold nanopa icles cys eine, supe oxido dismu ase; 11 gold, gold nanopa icles cys eine, supe oxido dismu ase, chi osane; 12 sc een p in ed ca bon TTF5% elec ode; 13 supe oxide dismu ase, palladium nanopa icles. Senso s 2016,16, 1588 4 o 19 Taking in o accoun ha TTF allows he apid elec on ans e be ween SOD and elec ode su ace can be ca ied ou a lowe po en ial [ 57 ], and ha p o-oxidan ac i i y o aluminum inhibi s SOD ac i i y [ 58 ], his s udy indica es ha Al(III) SOD inhibi ion can be pe o med a lowe po en ial compa ed o o he aluminum enzyma ic de e mina ions. I was shown o he i s ime using SPC TTF Es ha Al(III) inhibi s SOD enzyme linked wi h Alzheime ’s disease a low concen a ions. 2. Ma e ials and Me hods 2.1. Reagen s All solu ions we e p epa ed wi h pu i ied wa e supplied by TKA Gen Pu e, in e se osmosis, wi h a UV lamp i adia ion sys em. SOD enzyme (30 KU), EPI, bo ine se um albumine (BSA), glu a aldehyde (GA) and hyd ogen e achlo oau a e (III) ihyd a e (HAuCl 4 ) we e ob ained om Sigma-Ald ich (Sigma-Ald ich), S einheim, Ge many). Solu ions o pla inum, hodium and palladium 0.1 mM we e p epa ed om ICP solu ions o 1000 mg/L (Me ck, Da ms ad, Ge many). Ti isol solu ions om (Me ck, Da ms ad, Ge many)we e used o p epa e s ock s anda d solu ions o Al(III), Cu(II), Fe(III), Sn(II), Zn(II), Co(II), Ni(II), Se(IV) C (III), Cd(II), Pb(II), W(VI) and V(V). Mo(VI) and Ca(II) solu ions we e ob ained om Ino ganic Ven u es (Lakewood, NJ, USA). As(V) and Hg(II) solu ions we e p epa ed om A omic Spec oscopy S anda ds solu ions (Pe kin Elme Co, Whal ham, MA, USA). Al(III) solu ions used o spike we e p epa ed om High Pu i y S anda ds (Cha les on, SC, USA) con i med agains s anda d e e ence ma e ial SRM 3101. B i on Robinson (BR) suppo ing elec oly e solu ions we e p epa ed as usual wi h bo ic, phospho ic and ace ic acids (Me ck, Da ms ad , Ge many), and he equi ed pH was ob ained by adjus ing wi h NaOH solu ion (Sup apu , Me ck, Da ms ad , Ge many). Se e al inks we e used in he ab ica ion o SPEs, namely Elec odag PF-407 A (ca bon ink), Elec odag 6037 SS (sil e /sil e chlo ide ink) and Elec odag 452 SS (dielec ic ink) supplied by Acheson Colloiden (Acheson Colloiden, Scheemda, The Ne he lands). The wo king elec ode ink was p epa ed by ho oughly mixing ca bon ink wi h e a hio ul alene (CTTF) 5%. TTF was ob ained om Ac os O ganics (Ac os O ganics, Geel, Belgium). 2.2. Equipmen An elec ochemical sys em Au olab PGSTAT Echo Chemie128 N wi h GPS so wa e was used o eco d elec ochemical measu emen s (Echo Chemie, U ech, The Ne he lands). All pH alues we e adjus ed wi h a pHme e (Me le Toledo, Schwe zenbach, Swi ze land). A S-3700 Hi achi was used o pe o m scanning elec onic mic oscopy (SEM) o SPCEs. An IXRF Sys ems model 550iwas used o ob ain spec a o elemen s on he SPCE. A omic o ce mic oscopy (AFM) pa ame e s and images we e ob ained wi h a NanoScopeQuad ex Digi al Ins umen s Veeco Me ology G oup. SPCTTFEs Cons uc ion SPC TTF Es we e homemade buil using a DEK 248 p in ing machine (DEK, Weymou h, UK) using polyes e sc eens wi h app op ia e s encil designs moun ed a 45 ◦ o he p in e s oke. These ansduce s consis ed o h ee sc een-p in ed elec odes deposi ed on o polye hylene e eph hala e ilms (HiFi Indus ial Film, Da dilly, F ance). The di e en inks we e sc een-p in ed and cu ed acco ding o he manu ac u e ’s speci ica ions. The wo king elec ode ink was p epa ed by ho oughly mixing ca bon ink wi h TTF (5% /w) and immedia ely sc een-p in ed. One elec ode is shownin (Figu e 1). Senso s 2016,16, 1588 5 o 19 Senso s 2016, 16, 1588 5 o 18 Figu e 1. Sc een p in ed elec odic sys em used. 2.3. Nanopa icles Elec odeposi ion Me hods SPCTTFEs modi ica ion wi h nanopa icles (NPs/SPCTTFEs) was ca ied ou by bo h con olled po en ialand cyclic ol amme y scan me hods. (A) Me al pla ing was ca ied a wo di e en po en ials namely +0.3 and +0.18 V, in a qua z cell con aining Au(III), P (IV), Rh(IV) o Pd(IV) solu ions (0.1 mM) in H2SO4 (0.5 M) [24]. Following elec odeposi ion p ocess, he NPs/SPCTTFEs was emo ed om pla ing solu ion, insed wi h pu i ied wa e and wiped ca e ully. (B) Cyclic ol amme y deposi ion was pe o med doing a se o se en successi e ol amme ic scans be ween +1.0 and −0.2 Vin a qua z cell con aining Au(III), P (IV), Rh(IV) o Pd(IV) (0.1 mM) in H2SO4 (0.5 M) [59]. Elec odes we e p epa ed by se ing wo cyclic ol amme ic condi ions namely CV1 and CV2. CV1: delay ime 60 s, s ep po en ial 0.0150 V, scan a e 0.050 V/s CV2: delay ime 120 s, s ep po en ial 0.025 V, scan a e 0.100 V/s. A e nanopa icles deposi ion, he elec ode was insed wi h pu i ied wa e and wiped ca e ully. 2.4. SOD Enzyme Immobiliza ion on o AuNPs//SPCTTF Es Enzyme was immobilized by c osslinking polyme iza ion wi h glu a aldehyde [60] on he su aces o AuNPs/SPCTTFEs, P NPs/SPCTTFEs, PdNPs/SPCTTFEs, and RhNPs/SPCTTFEs. To ca y ou he immobiliza ion p ocedu e, supe oxide dismu ase enzyme solu ion was p epa ed by dissol ing enzyme in B i on Robinson bu e a pH 7.0. To a oid loss o enzyma ic ac i i y, BSA was used in a mix u e made o 20 μL o SOD (5.9 mg/mL), 10 μL o BSA (1.69% w/ ) and 10 μL o GA (2.5% / ) [61]. This mix u e was d opped on o he su ace elec ode and s o ed a 4 °C be o e used and be ween measu emen s. The modi ied elec ode was washed wi h pu i ied wa e , be o e and a e use. 3. Resul s 3.1. Op imiza ion o Expe imen al Pa ame e s EPI o igina es an ampe ome ic signal a NPs/SPCTTFE wi h SOD enzyme immobilized (SOD/PdNPs/SPCTTFE), a e which a s eady-s a e cu en is eached. The p esence o Al(III) ions p oduces SOD enzyme inhibi ion which causes a dec ease in he EPI ampe ome ic signal. Al(III) concen a ion in luence inhibi ion p ocess and can be quan i a i ely e alua ed de e mining he di e ence be ween he s eady s a e cu en in absence o Al(III), (I0), and he s eady s a e cu en in he p esence o Al(III), (I) namely Δ(I0-I).Acco dingly, wi h he ollowing wo king p inciple p oposed in Scheme 1, a SOD based biosenso , wi h TTF inco po a ed in elec ode ink, has been de eloped. Figu e 1. Sc een p in ed elec odic sys em used. 2.3. Nanopa icles Elec odeposi ion Me hods SPC TTF Es modi ica ion wi h nanopa icles (NPs/SPC TTF Es) was ca ied ou by bo h con olled po en ialand cyclic ol amme y scan me hods. (A) Me al pla ing was ca ied a wo di e en po en ials namely +0.3 and +0.18 V, in a qua z cell con aining Au(III), P (IV), Rh(IV) o Pd(IV) solu ions (0.1 mM) in H 2 SO 4 (0.5 M) [ 24 ]. Following elec odeposi ion p ocess, he NPs/SPC TTF Es was emo ed om pla ing solu ion, insed wi h pu i ied wa e and wiped ca e ully. (B) Cyclic ol amme y deposi ion was pe o med doing a se o se en successi e ol amme ic scans be ween +1.0 and − 0.2 Vin a qua z cell con aining Au(III), P (IV), Rh(IV) o Pd(IV) (0.1 mM) in H 2 SO 4 (0.5 M) [ 59 ]. Elec odes we e p epa ed by se ing wo cyclic ol amme ic condi ions namely CV1 and CV2. CV1: delay ime 60 s, s ep po en ial 0.0150 V, scan a e 0.050 V/s. CV2: delay ime 120 s, s ep po en ial 0.025 V, scan a e 0.100 V/s. A e nanopa icles deposi ion, he elec ode was insed wi h pu i ied wa e and wiped ca e ully. 2.4. SOD Enzyme Immobiliza ion on o AuNPs//SPCTTF Es Enzyme was immobilized by c osslinking polyme iza ion wi h glu a aldehyde [ 60 ] on he su aces o AuNPs/SPC TTF Es, P NPs/SPC TTF Es, PdNPs/SPC TTF Es, and RhNPs/SPC TTF Es. To ca y ou he immobiliza ion p ocedu e, supe oxide dismu ase enzyme solu ion was p epa ed by dissol ing enzyme in B i on Robinson bu e a pH 7.0. To a oid loss o enzyma ic ac i i y, BSA was used in a mix u e made o 20 µ L o SOD (5.9 mg/mL), 10 µ L o BSA (1.69% w/ ) and 10 µ L o GA (2.5% / ) [ 61 ]. This mix u e was d opped on o he su ace elec ode and s o ed a 4 ◦ C be o e used and be ween measu emen s. The modi ied elec ode was washed wi h pu i ied wa e , be o e and a e use. 3. Resul s 3.1. Op imiza ion o Expe imen al Pa ame e s EPI o igina es an ampe ome ic signal a NPs/SPC TTF E wi h SOD enzyme immobilized (SOD/PdNPs/SPC TTF E), a e which a s eady-s a e cu en is eached. The p esence o Al(III) ions p oduces SOD enzyme inhibi ion which causes a dec ease in he EPI ampe ome ic signal. Al(III) concen a ion in luence inhibi ion p ocess and can be quan i a i ely e alua ed de e mining he di e ence be ween he s eady s a e cu en in absence o Al(III), (I 0 ), and he s eady s a e cu en in he p esence o Al(III), (I) namely ∆ (I 0 -I).Acco dingly, wi h he ollowing wo king p inciple p oposed in Scheme 1, a SOD based biosenso , wi h TTF inco po a ed in elec ode ink, has been de eloped. Senso s 2016,16, 1588 6 o 19 Senso s 2016, 16, 1588 6 o 18 Scheme 1. Oxida ion o EPI on SOD/SPC TTF E. The pa ame e Δ(I 0 -I) depends on EPI concen a ion, applied po en ial (Eap) and pH solu ion. The e o e, an op imiza ion o hese a iables was pe o med in o de o ensu e he quali y o he esul s. Because he dependence be ween Δ(I 0 -I) and Al(III) concen a ion is linea , subs a e esponse was ob ained om pH 5.0 o pH 8.0, and a pH o 5.0 was selec ed ega ding subs a e s abili y o au oxida ion. In he same way, subs a e esponse was ob ained om +0.20 V o +0.60 V, and a po en ial o 0.2 V was selec ed d i ing subs a e oxida ion o epineph inequinone [57]. Then, se e al aluminum inhibi ion calib a ion cu es we e pe o med a di e en po en ial and pH alues and hei slopes we e compa ed, in o de o ob ain Al(III) inhibi ion e ec wi h pH and Eap. Slope calib a ion cu e wi h pH was calcula ed om pH 5.0 o pH 8.0. In he same way, slope calib a ion cu e wi h po en ial was calcula e om +0.20 V o +0.60 V. Highe slope alues we e ob ained a pH 5.0 and Eap o +0.2 V, so hese condi ions we e chosen o pe o m Al(III) inhibi ion calib a ion cu es. Slopes o calib a ion cu es wi h po en ial and pH a e shown in Figu e 2. Figu e 2. (a) Slope alues o Al(III) calib a ion cu es wi h pH; and (b) Slope alue o Al(III) calib a ion cu es wi h applied po en ial. [EPI] = 1.6 × 10 −4 M; B i on Robinson bu e pH 5.0, Eap = +0.2 V s. Ag/AgCl. Findings indica ed ha subs a e s abili y imp o ed a low alues o pH and po en ial; u he mo e, i applied po en ials we e highe han +0.6 V, he elec odes showed e a ic beha io . Since subs a e esponse inc eases wi h concen a ion, a alue o 1.6 × 10 −4 M o EPI was chosen, as his concen a ion gi es a p ope sensibili y, and a e y s able signal wi h e y low noise. Uppe concen a ions p oduced highe noise on ampe ome ic eco ding o calib a ion cu es. Unde he selec ed condi ions, he elec odes showed good pe o mance. Calib a ion cu es o Al(III) using SOD/AuNPs/SPC TTF Es, SOD/P NPs/SPC TTF Es, SOD/PdNPs/SPC TTF Es, and SOD/RhNPs/SPC TTF Es we e ob ained unde he op imized condi ions. Scheme 1. Oxida ion o EPI on SOD/SPCTTFE. The pa ame e ∆ (I 0 -I) depends on EPI concen a ion, applied po en ial (Eap) and pH solu ion. The e o e, an op imiza ion o hese a iables was pe o med in o de o ensu e he quali y o he esul s. Because he dependence be ween ∆ (I 0 -I) and Al(III) concen a ion is linea , subs a e esponse was ob ained om pH 5.0 o pH 8.0, and a pH o 5.0 was selec ed ega ding subs a e s abili y o au oxida ion. In he same way, subs a e esponse was ob ained om +0.20 V o +0.60 V, and a po en ial o 0.2 V was selec ed d i ing subs a e oxida ion o epineph inequinone [ 57 ]. Then, se e al aluminum inhibi ion calib a ion cu es we e pe o med a di e en po en ial and pH alues and hei slopes we e compa ed, in o de o ob ain Al(III) inhibi ion e ec wi h pH and Eap. Slope calib a ion cu e wi h pH was calcula ed om pH 5.0 o pH 8.0. In he same way, slope calib a ion cu e wi h po en ial was calcula e om +0.20 V o +0.60 V. Highe slope alues we e ob ained a pH 5.0 and Eap o +0.2 V, so hese condi ions we e chosen o pe o m Al(III) inhibi ion calib a ion cu es. Slopes o calib a ion cu es wi h po en ial and pH a e shown in Figu e 2. Senso s 2016, 16, 1588 6 o 18 Scheme 1. Oxida ion o EPI on SOD/SPC TTF E. The pa ame e Δ(I 0 -I) depends on EPI concen a ion, applied po en ial (Eap) and pH solu ion. The e o e, an op imiza ion o hese a iables was pe o med in o de o ensu e he quali y o he esul s. Because he dependence be ween Δ(I 0 -I) and Al(III) concen a ion is linea , subs a e esponse was ob ained om pH 5.0 o pH 8.0, and a pH o 5.0 was selec ed ega ding subs a e s abili y o au oxida ion. In he same way, subs a e esponse was ob ained om +0.20 V o +0.60 V, and a po en ial o 0.2 V was selec ed d i ing subs a e oxida ion o epineph inequinone [57]. Then, se e al aluminum inhibi ion calib a ion cu es we e pe o med a di e en po en ial and pH alues and hei slopes we e compa ed, in o de o ob ain Al(III) inhibi ion e ec wi h pH and Eap. Slope calib a ion cu e wi h pH was calcula ed om pH 5.0 o pH 8.0. In he same way, slope calib a ion cu e wi h po en ial was calcula e om +0.20 V o +0.60 V. Highe slope alues we e ob ained a pH 5.0 and Eap o +0.2 V, so hese condi ions we e chosen o pe o m Al(III) inhibi ion calib a ion cu es. Slopes o calib a ion cu es wi h po en ial and pH a e shown in Figu e 2. Figu e 2. (a) Slope alues o Al(III) calib a ion cu es wi h pH; and (b) Slope alue o Al(III) calib a ion cu es wi h applied po en ial. [EPI] = 1.6 × 10 −4 M; B i on Robinson bu e pH 5.0, Eap = +0.2 V s. Ag/AgCl. Findings indica ed ha subs a e s abili y imp o ed a low alues o pH and po en ial; u he mo e, i applied po en ials we e highe han +0.6 V, he elec odes showed e a ic beha io . Since subs a e esponse inc eases wi h concen a ion, a alue o 1.6 × 10 −4 M o EPI was chosen, as his concen a ion gi es a p ope sensibili y, and a e y s able signal wi h e y low noise. Uppe concen a ions p oduced highe noise on ampe ome ic eco ding o calib a ion cu es. Unde he selec ed condi ions, he elec odes showed good pe o mance. Calib a ion cu es o Al(III) using SOD/AuNPs/SPC TTF Es, SOD/P NPs/SPC TTF Es, SOD/PdNPs/SPC TTF Es, and SOD/RhNPs/SPC TTF Es we e ob ained unde he op imized condi ions. Figu e 2. ( a ) Slope alues o Al(III) calib a ion cu es wi h pH; and ( b ) Slope alue o Al(III) calib a ion cu es wi h applied po en ial. [EPI] = 1.6 × 10 −4 M; B i on Robinson bu e pH 5.0, Eap = +0.2 V s. Ag/AgCl. Findings indica ed ha subs a e s abili y imp o ed a low alues o pH and po en ial; u he mo e, i applied po en ials we e highe han +0.6 V, he elec odes showed e a ic beha io . Since subs a e esponse inc eases wi h concen a ion, a alue o 1.6 × 10 −4 M o EPI was chosen, as his concen a ion gi es a p ope sensibili y, and a e y s able signal wi h e y low noise. Uppe concen a ions p oduced highe noise on ampe ome ic eco ding o calib a ion cu es. Unde he selec ed condi ions, he elec odes showed good pe o mance. Calib a ion cu es o Al(III) using SOD/AuNPs/SPC TTF Es, SOD/P NPs/SPC TTF Es, SOD/PdNPs/SPC TTF Es, and SOD/RhNPs/SPCTTFEs we e ob ained unde he op imized condi ions. Senso s 2016,16, 1588 7 o 19 P elimina y expe imen s showed ha modi ica ion o elec ode su ace wi h NPs inc eased he sensi i i y o he biosenso ; he e o e, a ho ough s udy o condi ions o NPs deposi ion was ca ied ou . AuNPs, P NPs, PdNPs and RhNPs we e deposi ed on elec odes su aces acco ding o me hods desc ibed in he Expe imen al Sec ion. 3.2. XRF and SEM o NPs/SPCTTFE S udy METHOD A Two di e en con olled po en ials, +0.18 V and +0.3 V, we e applied o 15 s o SPCEs in o de o deposi NPs o e e y me al. X- ay luo escence emission (XRF) spec a we e ob ained om su aces o SPC TTF Es modi ied wi h he di e en ype o NPs. Table 2shows XRF pe cen age o elemen s deposi ed using indica ed po en ials. The pla ing o me als a +0.18 V o 15 s p oduced a highe pe cen age o Au, Pd and Rh. P deposi ed pe cen age was highe a +0.30 V. Since he Eap o +0.18 V applied o 15 s p oduced a highe pe cen age o Pd, Rh and Au, and he applica ion o a deposi ion po en ial o +0.3 V did no deposi Pd o Au, condi ions o Eap o +0.18 V and 15 s o me hod A we e selec ed o deposi NPs o me als. Table 2. XRF pe cen age o elemen deposi ed on SPCTTFEs by me hod A and B. Me hod A Me hod B Elemen XRF% XRF% XRF% XRF% (+0.18 V, 15 s) (+0.30 V, 15 s) CV1 CV2 Pd 0.136 0.00 0.557 0.632 P 0.223 1.48 2.74 2.71 Rh 0.693 0.380 4.49 2.95 Au 1.42 - 1.87 2.23 The inhibi ion calib a ion cu es o Al(III) a e shown in Figu e 3, whe e he lowes slope alue co esponds o SPC TTF E wi hou NPs deposi ed and he highes co esponds o SOD/AuNPs SPC TTF E. The o he me al NPs modi ied SPC TTF E es ed showed lowe linea adjus men han SOD/AuNPs SPCTTFE. SEM images o AuNPs ob ained by me hod A deposi ed on SPCTTFE a e p esen ed in Figu e 4. Senso s 2016, 16, 1588 7 o 18 P elimina y expe imen s showed ha modi ica ion o elec ode su ace wi h NPs inc eased he sensi i i y o he biosenso ; he e o e, a ho ough s udy o condi ions o NPs deposi ion was ca ied ou . AuNPs, P NPs, PdNPs and RhNPs we e deposi ed on elec odes su aces acco ding o me hods desc ibed in he Expe imen al Sec ion. 3.2. XRF and SEM o NPs/SPC TTF E S udy METHOD A Two di e en con olled po en ials, +0.18 V and +0.3 V, we e applied o 15 s o SPCEs in o de o deposi NPs o e e y me al. X- ay luo escence emission (XRF) spec a we e ob ained om su aces o SPC TTF Es modi ied wi h he di e en ype o NPs. Table 2 shows XRF pe cen age o elemen s deposi ed using indica ed po en ials. The pla ing o me als a +0.18 V o 15 s p oduced a highe pe cen age o Au, Pd and Rh. P deposi ed pe cen age was highe a +0.30 V. Since he Eap o +0.18 V applied o 15 s p oduced a highe pe cen age o Pd, Rh and Au, and he applica ion o a deposi ion po en ial o +0.3 V did no deposi Pd o Au,condi ions o Eap o +0.18 V and 15 s o me hod A we e selec ed o deposi NPs o me als. Table 2. XRF pe cen age o elemen deposi ed on SPC TTF Es by me hod A and B. Me hod A Me hod B Elemen XRF% XRF% XRF% XRF% (+0.18 V, 15 s) (+0.30 V, 15 s) CV1 CV2 Pd 0.136 0.00 0.557 0.632 P 0.223 1.48 2.74 2.71 Rh 0.693 0.380 4.49 2.95 Au 1.42 - 1.87 2.23 The inhibi ion calib a ion cu es o Al(III) a e shown in Figu e 3, whe e he lowes slope alue co esponds o SPC TTF E wi hou NPs deposi ed and he highes co esponds o SOD/AuNPs SPC TTF E. The o he me al NPs modi ied SPC TTF E es ed showed lowe linea adjus men han SOD/AuNPs SPC TTF E. SEM images o AuNPs ob ained by me hod A deposi ed on SPC TTF E a e p esen ed in Figu e 4. Figu e 3. Calib a ion plo s using: ( ) SOD/AuNPs/SPC TTF Es; (■) SOD/PdNPs/SPC TTF Es; (▲) SOD/RhNPs/SPC TTF Es; (  ) SOD/P NPs/SPC TTF Es and (  ) SPC TTF Es pe o med wi h NPs/SPC TTF Es p epa ed by me hod A, [EPI] 1.6 × 10 −4 M, B i on Robinson bu e pH 5.0, Eap = +0.2 V s. Ag/AgCl. Figu e 3. Calib a ion plo s using: ( • ) SOD/AuNPs/SPC TTF Es; (  ) SOD/PdNPs/SPC TTF Es; ( K ) SOD/RhNPs/SPC TTF Es; (  ) SOD/P NPs/SPC TTF Es and ( • ) SPC TTF Es pe o med wi h NPs/SPC TTF Es p epa ed by me hod A, [EPI] 1.6 × 10 −4 M, B i on Robinson bu e pH 5.0, Eap = +0.2 V s. Ag/AgCl. Senso s 2016,16, 1588 8 o 19 Senso s 2016, 16, 1588 8 o 18 Figu e 4. SEM image o AuNPs/SPCTTFE deposi ed a 0.18 V o 15 s. 3.3. XRF and SEM o NPs/SPC TTF Es S udy METHOD B XRF pe cen ages o e e y me al deposi ed wi h me hod B a e shown in Table1. AuNPs, P NPs, PdNPs and RhNPs we e deposi ed on SPC TTF Es acco ding o me hod B and modi ied wi h immobilized SOD. SOD/SPCEs modi ied wi h me allic NPs showed he bes linea adjus edAl(III) calib a ion cu e a CV1 condi ions o P NPs and a CV2 condi ions o PdNPs (Figu es 5 and 6). Reg essions wi h he bes linea i pe o med by me hods A and B showed ha he highes slope co esponds o SOD/PdNPs/SPC TTF Es (Figu e 7). SEM image o PdNPs/SPC TTF Es a CV2 condi ions is shown in Figu e 8, whe e i is obse ed ha PdNPs a e deposi ed in a egula o m on SPC TTF Es o he CV2 condi ions. Figu e 5. Calib a ion cu es o Al(III) inhibi ion o (▲) SOD/RhNPs/SPC TTF Es; (■) SOD/PdNPs/SPC TTF Es; () SOD/P NPs/SPC TTF Es and (  ) SOD/AuNPs/SPC TTF Es pe o med wi h NPs/SPC TTF Es p epa ed unde CV1 condi ion, [EPI] 1.6 × 10 −4 M, B i on Robinson bu e pH 5.0, Eap = +0.2 V s. Ag/AgCl. Figu e 4. SEM image o AuNPs/SPCTTFE deposi ed a 0.18 V o 15 s. 3.3. XRF and SEM o NPs/SPC TTFEs S udy METHOD B XRF pe cen ages o e e y me al deposi ed wi h me hod B a e shown in Table 1. AuNPs, P NPs, PdNPs and RhNPs we e deposi ed on SPC TTF Es acco ding o me hod B and modi ied wi h immobilized SOD. SOD/SPCEs modi ied wi h me allic NPs showed he bes linea adjus edAl(III) calib a ion cu e a CV1 condi ions o P NPs and a CV2 condi ions o PdNPs (Figu es 5and 6). Reg essions wi h he bes linea i pe o med by me hods A and B showed ha he highes slope co esponds o SOD/PdNPs/SPC TTF Es (Figu e 7). SEM image o PdNPs/SPC TTF Es a CV2 condi ions is shown in Figu e 8, whe e i is obse ed ha PdNPs a e deposi ed in a egula o m on SPC TTF Es o he CV2 condi ions. Senso s 2016, 16, 1588 8 o 18 Figu e 4. SEM image o AuNPs/SPCTTFE deposi ed a 0.18 V o 15 s. 3.3. XRF and SEM o NPs/SPC TTF Es S udy METHOD B XRF pe cen ages o e e y me al deposi ed wi h me hod B a e shown in Table1. AuNPs, P NPs, PdNPs and RhNPs we e deposi ed on SPC TTF Es acco ding o me hod B and modi ied wi h immobilized SOD. SOD/SPCEs modi ied wi h me allic NPs showed he bes linea adjus edAl(III) calib a ion cu e a CV1 condi ions o P NPs and a CV2 condi ions o PdNPs (Figu es 5 and 6). Reg essions wi h he bes linea i pe o med by me hods A and B showed ha he highes slope co esponds o SOD/PdNPs/SPC TTF Es (Figu e 7). SEM image o PdNPs/SPC TTF Es a CV2 condi ions is shown in Figu e 8, whe e i is obse ed ha PdNPs a e deposi ed in a egula o m on SPC TTF Es o he CV2 condi ions. Figu e 5. Calib a ion cu es o Al(III) inhibi ion o (▲) SOD/RhNPs/SPC TTF Es; (■) SOD/PdNPs/SPC TTF Es; () SOD/P NPs/SPC TTF Es and (  ) SOD/AuNPs/SPC TTF Es pe o med wi h NPs/SPC TTF Es p epa ed unde CV1 condi ion, [EPI] 1.6 × 10 −4 M, B i on Robinson bu e pH 5.0, Eap = +0.2 V s. Ag/AgCl. Figu e 5. Calib a ion cu es o Al(III) inhibi ion o ( K ) SOD/RhNPs/SPC TTF Es; (  ) SOD/PdNPs/SPC TTF Es; (  ) SOD/P NPs/SPC TTF Es and ( • ) SOD/AuNPs/SPC TTF Es pe o med wi h NPs/SPC TTF Es p epa ed unde CV1 condi ion, [EPI] 1.6 × 10 −4 M, B i on Robinson bu e pH 5.0, Eap = +0.2 V s. Ag/AgCl. Senso s 2016,16, 1588 9 o 19 Senso s 2016, 16, 1588 9 o 18 Figu e 6. Calib a ion cu es o Al(III) inhibi ion o (■) SOD/PdNPs/SPC TTF Es; () SOD/P NPs/SPC TTF Es; (▲) SOD/RhNPs/SPC TTF Es and (  ) SOD/AuNPs/SPC TTF Es pe o med wi h NPs/SPC TTF Es p epa ed unde CV2 condi ions, [EPI] 1.6 × 10 −4 M, B i on Robinson bu e pH 5.0;Eap = 0.2 V s. Ag/AgCl. Figu e 7. Calib a ion cu es o Al(III) o (■) SOD/PdNPs/SPC TTF Es; (▲) SOD/RhNPs/SPC TTF Es p epa ed me hod B; CV2 condi ions; () SOD/P NPs/SPC TTF Es, p epa ed by me hod B and CV1 condi ions and () SOD/AuNPs/SPC TTF Es, p epa ed by me hod A, [EPI] = 1.6 × 10 −4 M, B i on Robinson bu e pH 5.0, Eap = +0.2 V s. Ag/AgCl. Figu e 6. Calib a ion cu es o Al(III) inhibi ion o (  ) SOD/PdNPs/SPC TTF Es; () SOD/P NPs/SPCTTFEs ; ( K ) SOD/RhNPs/SPC TTF Es and ( • ) SOD/AuNPs/SPC TTF Es pe o med wi h NPs/SPC TTF Es p epa ed unde CV2 condi ions, [EPI] 1.6 × 10 −4 M, B i on Robinson bu e pH 5.0; Eap = 0.2 V s. Ag/AgCl. Senso s 2016, 16, 1588 9 o 18 Figu e 6. Calib a ion cu es o Al(III) inhibi ion o (■) SOD/PdNPs/SPC TTF Es; () SOD/P NPs/SPC TTF Es; (▲) SOD/RhNPs/SPC TTF Es and (  ) SOD/AuNPs/SPC TTF Es pe o med wi h NPs/SPC TTF Es p epa ed unde CV2 condi ions, [EPI] 1.6 × 10 −4 M, B i on Robinson bu e pH 5.0;Eap = 0.2 V s. Ag/AgCl. Figu e 7. Calib a ion cu es o Al(III) o (■) SOD/PdNPs/SPC TTF Es; (▲) SOD/RhNPs/SPC TTF Es p epa ed me hod B; CV2 condi ions; () SOD/P NPs/SPC TTF Es, p epa ed by me hod B and CV1 condi ions and () SOD/AuNPs/SPC TTF Es, p epa ed by me hod A, [EPI] = 1.6 × 10 −4 M, B i on Robinson bu e pH 5.0, Eap = +0.2 V s. Ag/AgCl. Figu e 7. 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