!
!
!
Phthalocyanine - Fullerene0Dyads0and0 0
DNA0In terst rand 0Cros s - Linking0on0Surface 0
!
!
!
vorgelegt!von!
Cinthya!Yamila! Vé liz!Montes!
geb.!in!Guayaquil,!Ecuador!
!
!
!
von!der!Fakultät!III!–!Prozesswissenschaften!
!der!Technischen!Universität!Berlin!
und!der !
Autonome! Universi tät!Madrid,!Spanien!
(im!Rahmen!des!Doppel-Promotionsabkommens)!
zur!Erlangung!des!akademischen!Grades!
!
!
Doktor!der!Naturwissenschaften!
-! Dr.!rer .! nat.-!
!
!
genehmigte!Dissertation!
!
!
!
Promotionsausschuss:!
!
Vorsitzender in: ! Prof.!Dr.! Annemieke!Madder!(Universität! Gent )!
Gutachter:!! Dr. !habil.!Rudolf!J.!Schneider! (TU!Berlin) !
Gutachter:!! Prof.! Dr. !Felix!Zamora!Abanades!(Autonome!Universität!Madrid)!
Gutachter:!! Prof.! Dr. !João!Paulo!Costa!Tomé!(Universität! Aveiro )!
Gutachterin:!! Prof.!Dr.! Maria!Salomé!Rodríguez!Morgade!(Autonome!Universität!Madrid)!
!
!
Tag!der!wissenschaftlichen!Aussprache:!15 .! März!2017!
a n!der! Autonome!Universität!Ma drid ,!Spanien!
!
!
Berlin!2018 !

Universidad Autónom a de Madrid
Faculta d de Ciencias
Departamento de Qu í mica Org á nica

Technische Universität Berl in
Faculty I II Process S ciences
Department of Applied Biochemistry

PHTHA LOCYANI NE - FULLE RENE DYADS AN D
DNA INTER STRAND CROS S - LINKI NG
ON SURFACE

CINTHYA YAMILA V ÉLIZ MONTES
Doctoral Thesis

Madrid 2017

“You never fail until y ou stop trying.”
Albert Einst ein

This work was developed in t he Department of
Organic Chemistr y of The Autonoma University
of Madrid, under the supervision of Prof. Dr.
Tomás Torres Cebad a, Dr. Olga Trukhina a nd
in the Fachbereich 1.8 of Bundesanst alt für Ma-
terialforschung un d - prüfung ( BAM) tutored by
Dr. habil. Rudolf J. Schneider under the super -
vision of Prof. Dr. Jens Kurreck whom I express
all my thankfulness.

Parts of t his work was presented in the following art icle s:
• “ Photo - induced cross- linking of short furan - modified DNA on s urfaces ”. C. Véliz
Montes, H. Memc zak, E. Gyssels, T. Torres, A. Madder and R. J. Schneider. Langmuir ,
2017 , in press ; DOI : 10.1021/acs.langmuir. 6b03855 .
• “Unsubstituted, Covalently - linked Pht halocyanine - C60 Fullere ne Dyads”. C. Véliz
Montes, O. Trukhina, G . Bottari and T. Torres, 2017. Manuscr ipt in preparat ion .

This work ha s been develop ed in large pa rts within the EU Marie Curie Ini tial Trainin g
Network FP7 - PEOPLE - 2012 - I TN “SO2S” , grant no. 316975.

• The first part of my doctoral resear ch was perf ormed in the group direc ted by Dr.
Rudolf J. Schneider at Bundesan stalt für Materialfor schung und - prüfung (BAM ), Berli n,
Germany, from June 2013 unt il August 2015, in close collabor ation with Fr aunhofer
Institute for Cell Therapy and Immunology (IZI - BB) , Branch Bioanalytics and Biopr o-
cesses, Pot sdam, Germany.
• Experience in t he chemistry of nucleic aci ds and DNA interstrand cr oss - linking
methodologies was gained durin g a research stay in the group of Prof. Annemieke
Madder, Organic and Biomimet ic Chemistry Research Gr oup, Ghent University, Bel-
gium, from August to September 2013, with the help of her experienced P hD students
Ellen Gyssels and Nat halie De Laet.
• The last step was done at Universidad Aut ónoma de Madrid in the gr oup of Prof.
Tomás Torres in the Depar tment of Or ganic Chemistry from August 2015 to August
2016, under t he kind guidance of Dr. Olga Trukhina a nd Dr. Giovanni Bottari.
I would like to thank all of them warmly .

Acknowledge ments
First of all, I would like to express my gratitude to “The Singlet Oxygen Strategy ” Marie
Curie Initial Training N etwork (ITN) under t he European Union S eventh Framework pro-
gramme FP7 - PEOPLE - 2012 - I TN for the financial sup port.
I would also like to express my most sincere gratitude to my supervisors Prof. Tom ás
Torres and Prof. Jens Kurr eck for their time and guidance. I am very gratef ul and indebted
to my research tut or Dr. habil. Rudolf Schneider for his continuo us encourag ement , sup-
port, and freedom for developing my work.
I express my pr ofound gratitude t o the whole Fachbe reich 1.8 of Bundesanstalt f ür
Materialfors chung und - prüfung (BAM) for scientific discussion, a dvice and direct assis-
tance in the lab and help with the German language. I’d like to extend my t hanks for scien-
tific discussions an d ideas to m y friends and PhD co lleagues : H olger , Peter, Stephan, M ar-
tin, Mafalda, Luc í a, Ana, Lidia and Rob ert. Special tha nks to Krist in Hoffman and Sabin e
Flemig for their honest friendship and assistance. Thanks to Christin Heinrich for the big
help with the manage ment.
I would also like to thank Prof. Dr. Dirk M. Guldi and staff of the Department of Chem-
istry and Pharmacy at Friedrich - Ale xander - Universität Er langen/Nür nberg for th e spectral
characterizat ion of my compounds , as well as the Surface Science Laborator y of Madrid
Autonoma University, in particular PhD st udent Luigi Terracciano for his attempts of UHV
STM deposition of pr epared dyads ont o surfaces.
Thanks to Dr. Olga Trukhina for her friendship, pat ience and excelle nt guidance in
teaching me or ganic chemistr y. I extend my sincere tha nks to all the other members at the
Nanoscience and M olecular Materials Res earch Group of the Departm ent of Organic
Chemi stry of The Autonoma Univ ersity of Madrid , for their help and suggest ions. I dedicat e
a special acknowledg ement to the senior scientists : Gianni, Andres, Salom e, Gema, David
and Marivi for being good examples of e xpertise and commitm ent to science. Special
thanks to Giovanni Bottar i for his scientif ic contributions and guidance. Thanks Irene for
becoming a good fr iend , keeping the contact .

My sincere t hanks to Henr y Mem cz ak and Walter Stöcklein from Fr aunhofer I nstitute
for Cell Therapy and Imm unology, Branch Bioanalyt ics and Bioproces ses (IZI - BB) for col-
laboration on the SP R measurements . Thanks also to t he members of the group of Bi-
omarker Validati on and Assay Development , wit hout their assistanc e I could not have man-
aged the experimental work .
I also want to thank for the opportunity that Prof. Annemieke Madder gave me to ex-
perience the chemistry of nuclei c acids during my research st ay as well to her group of
Organic and Biom imet ic Chemist ry . Special t hanks to Ellen Gy ssels and Nath alie De Laet.
I’m ver y thankful to all my friends that during t his period making me laugh and for their
constant encouragem ent. M y sincere thanks to my Rock Berlin er’s fr iends who directly
suppo rted me along this jour ney. I am deeply grateful to Nahla, Benita, Rafael, Margar ida
and Ana for hearing me, and sharing m y daily frustrat ions and successes .
Thanks to my parents, who taug ht me fr om an early age to alwa ys see ligh t on the
difficulties and to help without expecting anything back, and to my dear littles Michelle and
Kristel, for m otivating me being b etter. I thank my whole family f or their support and be ing
by my side. My aunts Nubia and Tanya are specially acknowledg ed for their love and mo-
tivating words.
I cannot express all my respect and grat itude I feel for all of you . O nce again, I thank
you.

“There is no other G od who can rescue like this.”
Daniel 3:29

Abbreviations

AFM

Atomic force micros cope

CHCA

α - cyano -4- hydroxycinnam ic acid

CR

C harge recombination time

CRP

C- reactive protein

CS

Charge separat ion time

cTnI

Cardiac troponin I

CVD

Cardiovascular D iseas e

D-A

donor - acceptor

DDI

DNA - directed imm obilization

EADS

E volution - a ssociated d ifferential spect ra

ELISA

Enzyme- linked immunosorbent assay

hFABP

Heart - type fatty acid binding pr otein

HRP

H orseradish peroxida se

ICL

Chemical i nterstr and crosslinkin g

MALDI - TOF

matrix - assisted laser des orption / ionization t ime - of - flight

MB

M ethylene blue

MS

mass spectroscopy

NA

Neutravidin

OD

O ptical density

ODN

DNA oligon ucleot ide

OPV

Organic P hotovoltaics

OSCs

Organic Solar C ells

PA

P hotoinduced absor ption

Pc

Phthalocyanine

PCE

Power Conversion E ff iciencies

Pc -C 60

Phthalocyanine - C 60 fullerene

PET

photoinduced elect ron tr ansfer

PMMA

P olymethyl - met hacrylate

Por

Porphyrin

PPV

poly(p - phenylenev inylene)

PS I

Photosensit izer

ROS

R eactive oxygen spec ies

RU

Resonance Units

SA

Sinapin ic Acid

SAM

Self - assembled monol ayer

SE

S timulated emis sion

SPR

Surface Plasmon Resonanc e

STM

Scanning Tunnellin g M icroscope

TA

T ransient absorption spect ra

THF

Tetrahydrofur an

TLC

thin - layer chrom atography

TMB

3,3 ’ ,5,5 ’- tetramethylbenzidine

TPB

1,1,4,4 - tetrapheny l - 1,3 - butadiene

ZnPc

Zinc Pht h alocyanine

Contents
Abstract ........................................................................................................................ 1
Resumen ...................................................................................................................... 5
Zusammenfassung ....................................................................................................... 9
Thesis Outline ............................................................................................................ 13
1. Introduct ion ......................................................................................................... 17
1.1 Phthalocya nines ................................................................................................ . 19
1.2 Phthalocya nines - fullerene dyads ........................................................................ 21
1.2.1 Nanotechnology and self - assembly ................................................................ 21
1.2.2 Donor - acceptor ensembles ............................................................................. 23
1.2.3 Covalently - lin ked Pc -C 60 systems ................................................................... 26
1.3 DNA interstrand cr oss - linking on surf ace ............................................................ 35
1.3.1 Biosensor s in cardiovascula r diseases detection ............................................ 35
1.3.2 Biosensor s and DNA in nanotechnolo gy ........................................................ 43
1.3.3 Furan mediat ed DNA - ICL cross - linkin g .......................................................... 54
2. Object ives ........................................................................................................... 59
2.1 Phthalocya nine - fullerene dyads .......................................................................... 61
2.2 DNA interstrand cr oss - linking on surf ace ............................................................ 63
3. Materials and M ethods ....................................................................................... 65
3.1 Phthalocya nine - fullerene dyads .......................................................................... 67
3.1.1 Chemicals and instr umentation ....................................................................... 67
3.1.2 Synthesis of dy ad precursors .......................................................................... 68
3.1.3 Synthesis of Zn( II)Pc -C 60 fuller ene dyads ....................................................... 75
3.1.4 Experiment al techniques ................................................................................ 79
3.2 DNA interstrand cr oss - linking on surf ace ............................................................ 85
3.2.1 Chemicals and instr umentation ....................................................................... 85
3.2.2 Experiment al procedures ................................................................................ 87
3.2.2.1 Surface Plasmon Resona nce ...................................................................... 87

3.2.2.2 ELISA .......................................................................................................... 90
3.2.2.3 M ALD I - TOF sample pr eparation proc edures ............................................... 90
3.2.2.4 Cr oss - linking in solut ion ............................................................................... 91
3.2.3 Antibody - oligon ucleotide conj ugation ............................................................. 93
3.2.4 Cross - reactivity experiment s of the multiplex immunoass ay .......................... 94
3.2.5 Experiment al techniques ................................................................................ 95
4. Results and Discussion .................................................................................... 101
4.1 Phthalocya nine - fullerene dyads ........................................................................ 103
4.1.1 Synthesis and ch aracterizat ion ..................................................................... 103
4.1.2 Photophysica l studies of the dyads ............................................................... 113
4.1.2.1 Photophysics in THF solution .................................................................... 1 19
4.1.2.2 Photophysics in PMMA films on glass ....................................................... 121
4.1.2.3 Photophysics in drop casted films on glass ............................................... 124
4.1.3 Microscopic charac terization ......................................................................... 126
4.2 DNA interstrand cr oss - linking on surf ace .......................................................... 129
4.2.1 DNA - directed im mobilization on SPR b iosensor chips ................................ . 130
4.2.2 Light - induced im mobilizatio n on microplate surf aces .................................... 136
4.2.3 Multiple x immunoassay cros s - reactivi ty ........................................................ 137
4.2.4 Implementat ion of ICL in immunoassays ...................................................... 138
5. Conclusions ...................................................................................................... 139
5.1 Phthalocya nine -C 60 fullerene dya ds ................................................................ . 141
5.2 DNA Interstr and cross - linking on surfac e ......................................................... 142
6. Conclusiones .................................................................................................... 143
6.1 Diadas ftalocian ina - ful l ereno ............................................................................ 145
6.2 Enlaces quí micos intercatenarios de DNA en superficie .................................. 146

1

A bstract

Abstract

3
Nanotechnology deals with structur es and components of nanosc ale size in order to
create new propert ies or to improve signif icantly their physical, chemical an d biological
properties, pr ocesses and pheno mena. Many ef forts of molecular nanot echnology are d i-
rected to design artific ial nanostr uctures on surf aces due to their pot ential applications in
nanodevices and bios ensors. This t hesis includes two areas of application, phthalocya -
nine - based photov oltaic dyads an d using phthalocya nine - based photos ensitation in creat -
ing a stable biosensor chip sur face.
To date, phthalocyani ne (Pc) -C 60 fullerene arr ays have shown interest ing electro-
chemical, photophysic al and photovoltaic pr operties. In this case the sys tems are consti -
tuted by an unsubstit uted phthalocyanine ( donor) and a f ullerene (acceptor) unit. As a re-
sult of the absence of any substituent at the Pc peripheral positions , these building blocks
show a better organization at supramolecular level. This results in important changes in
some physical propert ies of these system s with respect t o their individual counte rparts.
Assembling of Pc -C 60 nanostructur es on substrate s urfaces and their photo - i nduced en-
ergy/electron t ransfer propert ies were also studied.
The fabricat ion of biosensor chip s for clinical dia gnosis is bec oming an increa singly
important topic. A larg e number of biosens ors are aimed to t he diagnosis of cardiovascu lar
diseases, which represent 45% of all deaths in Europe. DNA - direct ed immobiliz ation (DDI )
is a technique that allows the preparat ion of protein immunoarray s by forming a self -
assembled monolaye r (SAM) of DNA target m olecules with DNA probes pr eviously
immobilized on a s urface. One of the drawbacks of DDI is t hat it requires long oligonucle-
otides (ODNs) f or recognition and s table duplex form ation. Shorte r ODNs would be suffi -
cient for antibody addressing but they are not st able enough during storage and handl ing
(e.g. rinsin g) st eps.
Chemical interst rand crosslinking ( ICL) of short ODNs in solution has been studied
before by several gr oups due to its important clinical applicat ions. The fur an - based ICL
strategy has been widely st udied in solution and pres ents the advantage of selective oxi -
dation of the fur an - modified ODN which leads to the fast f ormation of a covalent bond with
the complementar y ODN sequence. This very stable DNA duplex showed ex traordinary
resistance towards enzymatic digestion and higher melting temper atures in compar ison
with the non -cross- linked DNA duple x. One studied oxidant is singl et oxygen, produced in
a photosensitised r eaction involving a pht halocyanine.

Abstract
4
On this basis t his thesis also desc ribes as an applicat ion the developm ent of a novel
platform based on DDI to detect three cardiac biomark ers in a multiplex immunoarray, re-
quiring only short dodecamer (12mer ) ODNs for addressing the antibody, as a conse-
quence of the increas e d DNA duple x stabilit y after cross - linki ng . Met hylene blue and a
pht h alocyanine we re u s ed as photosensiti s ers f or the ICL formation.
This thesis repor ts on recent exper iments on both Pc -C 60 and immobil ized DNA ICL.
O pen problems and f uture applications are also disc ussed.
Key words: Nanotechnology, bio sensor, phthalocya nine, fullerene, nanostructures,
cardiac bio mark er, DNA, DNA - direct ed immobilization, interstra nd cross- linking , furan , s in-
glet oxygen .

5

Resumen

Resumen

7
La nanotecnología t rata con estr ucturas y componentes de t amaño nanométrico par a
crear o mejorar sus propiedades , pr ocesos y fenómenos fís ic o s, químic os o biológi cos. E n
este campo, una gr an parte de la investigac ión está dirigid a a diseñar nanoes tructuras
artificiales en super ficies, debido a su pot encial aplicaci ó n en nanodispos itivos y biosen-
sores. Esta tesis abarca dos areas de aplicac ión, diadas fotov oltaicas basadas en ftalo-
cianinas y fotosensibili zacion basada en ft alocianinas para cr ear un chip con una superficie
estable para un biosensor. A lo largo de la misma se describen los resultados obtenidos
en ambos temas , diadas Pc -C 60 y ADN inmovilizado, adem ás de discutir los p roblemas
observados y las f uturas aplicaciones.
En las últimas décadas , el estudio de diadas ftalocianina (Pc) - fu l ler eno C 60 ha r esul-
tado en la demostración de int eresantes propied ades electroquí micas, fot ofísicas y foto-
voltaicas. En esta tes is , se presenta diadas f ormadas por una ftalocianina sin sustituy entes
periféricos (dador ) y un fu ll ereno (aceptor ). Como resultado de la ausencia de sust ituyen-
tes en las posiciones periféricas, nuest ras diadas mostrar on una ma y or capacidad de or-
ganización a nivel sup ramolecular . E st o repercute en algunas de sus propiedades f ísicas
de estos sist emas con respecto a s us homólogos individ uales. En est e trabajo se presenta
un estudio del ordenamient o de las nanoestructur as Pc -C 60 en superficies , y sus propie-
dades de transfer encia de energía o elect rones fotoinducida s.
Por otro lado, la fabr icación de chip s bios ensores par a aplicaciones diagn ósticas se
está convirtiendo en un tem a de creciente importancia y atractivo cientí fico . El principal
objetivo de estos biosensor es es el diagnóstico de e n fer medades car diovasculares, las
cuales representan el 45% de todas las muert es en Europa. La i n movilizac ión dirigida p or
ADN (DDI) es una técnica que p ermite la preparación de matrices mediante la formación
una monocapa auto - ensamblada (SAM) de moléculas diana de ADN . Una de las desven -
tajas del DDI es la necesidad de e mplear oligonulcleótidos de alto peso molecular (ODNs)
para el reconocimiento y la formación est able de la doble hélice. ODNs m á s cortos podrían
ser suficient es para interaccionar con el anticuerpo per o no son lo suficiente mente esta-
bles para su conservación y paso s de manipulación (ej . lavado).
La formación de e nlaces químicos intercatenarios ( ICL) de oligonucleóti dos cort os
han sido estudiados en di solución por var ios grupos debido a sus importantes aplicaciones
clínicas. La estr atégia de ICL basada en el furano ha sido utilizada ampliamente en diso-
lución y presenta com o principal ventaja la oxidaci ón selectiva de l ODN modificado con e l

Resumen
8
furano, la cual produce la f ormación de un enlace covalente con el ODN de la sec uencia
complement aria. Esta doble hélice resultó ser extraor dinariamente resistent e a la digestión
enzimática , además de presentar un punto de fusi ón bastante elevado en compar ación
con la doble hélice de ADN no enlazada. Para la oxidaci ón de los oligonucleótidos pr epa-
rados en esta tesis se utilizó el oxígeno singlete, pr oducido en una reacción fotosensi bl e
implicando una f talocianina.
A partir de los resultados obteni dos, esta tesis tambi é n describe com o una aplicación
el desarrollo de una nueva plataf orma basada en DDI para detectar tres biomarcadores
cardiacos en un dispositivo que incluye sólo ODNs cortos dodecámer os, 12 mer ) para
direccionar el anticuerpo. Esto ha sido posible gracias a l increm ento de la estabilidad de
la doble hélice de AD N después de enlace int ercatenario . P ara la f ormación de dicho en-
lace, se han utilizado a zul de m etileno y ftalocianina co mo fotosensibilizad ores.

Esta tesis report a ambos experi mentos, Pc -C 60 e inmovil izado ADN ICL. Problemas
observados y f uturas aplicaciónes t ambién se comentan.

Palabras clave: Nanotecnolo gía, biosensor , ftalocianina , fu l lereno, nanoes truct uras,
biomarcador es car diacos, ADN , inmovilización dirigida por ADN, enlace intercat enario, fu-
rano, oxígeno singl ete.

9

Zusammenfas sung

Zusammenfassung
11
Nanotechnologie unt ersucht Bestandt eile und Strukturen von Mater ialien im
Nanobereich, um Prozesse und Phänomene bess er zu vers tehen beziehungs weise neue
oder verbessert e physikalische, chemische sowie bi ologische Eige nschaften zu er zeugen.
Viel Forschungsarbeit im Bereich der m olekularen Nanotechnolo gie wurde gel eistet , um
künstliche Ober flächennanostruk turen zu erz eugen um diese als Bi osensoren ode r
Nanoröhren zu verwende n. Diese Arbeit beinhaltet zwei Anwendungsbereiche,
Phthalocyanin - basi erte Photovolt aik - Dyaden und Phthalocyan in - basiert e
Photosensibil isatoren , um eine stabile Chipoberf läche zu generieren un d diese als
Biosensor zu verwend en.
Phthalocyanin - Ful lerene (Pc ) -C 60 haben interessant e elektrochemische,
photophysikalisch e und photovoltaische Eige nschaften. In diesem Fall besteht das System
aus einer unsubstituier ten Phthalocyanin - (Donor) und einer Fulleren - Einheit (Akzeptor).
Durch Abwesenheit einer peripheren Substitut ion am Pc, zeigen diese Gebilde eine höher e
Ordnung auf supramoleku larer Ebene. Daraus r esultiert ein entscheidender Wechsel
einiger physikal ischer Eigensc haft en. Der Aufbau der Pc -C 60 N anostrukturen und ihr e
photoinduzier ten Energie - beziehungsweise Elektr onentransfer eigenschaften wur den
ebenfalls unters ucht.
Die Herstellung von Biosensor chips zur Anwendung in der klinischen Diagnos tik wird
ein immer wichtigeres Them a. Eine Großzahl von Biosensoren werden angewendet zur
Diagnostizier ung von Herz - Kreisl auf - Erkrankungen, welche verant wortlich für 45 % aller
Todes fälle in Europa s ind. Die DNA - vermitt elte Immobilisierung ( DDI) ist eine Technik,
welche die Herstellung von Protein - Im munoassays durch Bildung einer selbstorganisierte n
Monoschicht v on spezifischer DNA mit zuvor auf einer O ber fläch e immobilis ierte n,
komplement ären DNA , ermöglicht. Ein Nachteil von DDI ist, dass lange Oli gonukleotide
(ODNs) für die Bildun g von stabilen Duple xformen notwend ig sind. Kurze ODNs wären
ausreichend stabil zur Immobilisierung der Ant ikörper, j edoch nicht für die Lagerung und
Wasch schritte .
Chemische intra - St rang Kreuzvernetzungen (ICL) von kurzen ODNs in Lösung wurden
aufgrund ihrer Wicht igkeit für die klinische Diagnosti k bereits häufiger unter sucht. Die
Furan - basiert e ICL - Strategie stellt den Vort eil der selektiven Oxidation von Furan -
modifizierte n ODNs dar, welche eine schnelle k ovalente Verknüpf ung mit dem
komplement ären ODN - Strang erm öglicht. Dieser sehr stabile DNA - Duple x wei s t
außergewöhnlic he Robustheit gegenü ber enzymat ische m Verdau und eine höher e

Zusammenfassung
12
Schmelztem peratur im Verglei ch zum nicht ver knüpften DNA - Kompl ex auf. Ein
untersuchtes O xidationsmit tel ist Singule tt - Sauerst off, welcher dur ch eine Photoreakt ion
vom Phthalocyanin produzier t wird.
Auf dieser Gr undlage beschreibt diese Arbei t auch die Anwendun g einer neuart igen
Plattfo rm , basierend auf DDI , um drei Herz infar kt - Biomarker in einem M ultiplex -
Immunoarray zu m essen. Dabei sind, aufgrund der hohen Stabil ität des DNA - Duplex nach
Verknüpfung, nur Dodecamere (12mer e ) von ODNs zur Immobilisierung de r Ant ikörper
notwendig. Met hylenblau und ein Phthalocyanin wurde n als Photosensibilis atoren für die
ICL benutzt.
Diese Arbeit bericht et über neue Exper imente sowohl an Pc -C 60 als auch a n
Oberflächen - DNA - ICL . Offene Fragen und zukünftige Anwendungen wer den ebenfalls
diskutiert.
Schlüsselbegrif fe: Nanotechnolog ie, Biosensor, Pht halocyanin, Fullere ne,
Nanostrukturen, Herz infarkt - Biomarker , DNA - vermitte lte Immo bilisi erung , intra- Stra ng
Kreuzvernet zung, Furan, Singulet t - Sauerstof f .

13

Thesis Ou tline

Thesis outline
15
Each chapter of this thesis has been div ided into two section s. The first par t is
centered around the donor - acceptor Pc -C 60 dy ads and the second par t discusses the DNA
interstrand cr oss - linking (ICL) on surface . This dissertation is ar ranged as follows:

1. Introduct ion
This section provides t he motivation of the work, back ground informat ion, and a litera-
ture review. The f irst part introduces the self - assem bly system s in nanoscience and mo-
lecular nanotechnolo gy, also some basic concepts about phthalocyanine s ( Pc) and Pc -C 60
based conjugates.
In the second part the role of biosens ors in cardiovasc ular disease s detection , DNA in
nanotechnology and biosensors are int roduced, as well as basic c oncepts about DNA a nd
ICL format ion in solution.
2. Objecti ves
States the major questions that the research intends to answer. In summ ary, the aim
of the first section is the synthes is of four novel coval ently - linked Zn Pc -C 60 conj ugates and
to deposit them on surfaces t o study their organization by TEM, STM, AFM, and to study
the potential applicab ility of t he novel dyad s for energy convers ion.
The main aim of the second secti on is the establishment of a new method for DNA - ICL
on the surface via singlet oxyg en, using methylene bl ue or a ZnPc derivative (“TT1”) as
photosensitizer s, and to study the possible applic ations in a multiplex address able immu-
noassay for the detection of t hree cardiac biomarkers using short ODN for addressing the
antibody (C - reac tive protein, h eart - t ype fatty acid binding prot ein and Troponin I).

Thesis outline
16
3. Materi als and Met hods
T he first part of this section gives experiment al details about the s ynthesis of unsubsti-
tuted Pc -C 60 dyads and their precur sors as the experimental techniqu es used to perform
the surface and photoexcitat ion dynamics st udies.
The second section is about the steps f ollowed during the development of the method-
ology for ICL of immobilized oligo nucleotide. SPR, ELI SA and microarr ay experiments are
described.
4. Results and Discussion
The results are pres ented, analysed and discussed. Synt hesis of the dyads ’ precursor s
and the Pc -C 60 dyads’ conjugat es with its photophy sical study in sol ution as well as o n the
surface and the diff erent microscopic charac terizations ar e provided.
Within the second sec tion, a novel ICL met hodology on surface - immobil ized sh ort DNA
oligonucleot ides is det ailed and t he problems found for the implem entation of the new DNA
platform in a multiplex address able immunoassay ar e also described.
5. Conclusion s
Provides a summary of key findings on both topics. Briefl y, the results of the first section
suggest the necessar y use of na nocomposite polym ers as a possible t emplate f or enhanc-
ing the supram olecular ordering and cons equent organi c photovoltaics performance of the
ZnPc -C 60 dyads.
The main conclusion of this section is that ICL form ation of immobilized DNA is a very
useful method for increasing the stability of shor t DNA oligonucleot ide on a surface. Com -
paring both photos ensitizers, methylene blue was m ore efficient t han the ZnPc. The deve l-
oped methodology has t he drawback that it cannot be implemented in an addressable im -
munoassay due t o the loss of the recogniti on activity of the capture antibody fo r the prot ein
of analysis.
Summarizing it can be said that for pht halocyanines another t wo applications could be
developed which sho w the high versatility of this diverse group of compounds.

17

1. Introducti on

Introduction
19
1.1 Phtha locy anines

Phthalocyanines ( PCs) enjoy a privileged position within the large family of
porphyrinoid syst ems. These chromophores, which have a two - dimensional (2 - D) and 18 -
π - electron aromat ic system, hav e been commended as ther mally stable excellent light -
harvesters with maj or absorption at the maximum of the solar photon flux – at around 700
nm. More importantly, the optical and electronic proper ties as well as the redox charact er
of these macrocycles can be easily modulat ed by the careful choice of t he metal center
and/or the peripheral s ubstituents, m aking them perfect building blocks f or light harvest ing,
photovoltaic, and molecular photonic app lications. 1
Their 18 π - electron system generates absorption spectr a with two major intense bands :
The Q and the Soret band. The Soret band i s centered in the rang e of 350 nm and, and is
usually a broad and has low intensity. The Q band lies in the visible region around 620 -
720nm. The position and t he shape of the Q band det ermines the color of the Pc, and
depends on several factor s such the central metal, type of substitution ( peripher al, non -
peripheral and axial s ubstitution, Figure 1) 2 , the solvent in which the compound is dis-
solved, its aggregatio n behavior, and the extent of π - conjugat ion.
The nature of the centr al atom of the Pcs has a strong impact in their photop hysical
properties, inf luencing their fluorescence elect ronic absorption, fluorescence and phos pho-
rescence spectr a, and the triplet excited state generation. 3 Trans ition metal ions produce
short triplet lifetim es, in the nanosecond range, while close d- shell and diamagnet ic metals,

1 a) G. Bottari; G. de la Torre; T. Torres, Acc. Chem. Res., 2015 , 48 , 900−910; b) N. L. Bill.,
O.Trukhina, J. L. Sessler, T. Torres, Chem. Commun., 2015 , 51 , 7781 – 7794; c) G. Bottari.;
O.Trukhina, M. Ince; T. Torres, Coord. Chem. Rev., 2012 , 256 , 2453−2477; d ) F. D'Souza; O. Ito,
Coord. Chem. Rev., 2005 , 249 , 1410 – 1422 ; e) G. de la Torre, G. Bottari, M. Sekita, A. Hausmann,
D.M. Guldi, T. Torres, Chem. Soc. Rev ., 2013 , 42 , 8049 - 8105; f) H. Imahori , T. Umeyama, K. Ku-
rotobi, Y. Takano, Chem. Commu n ., 2012 , 48 , 4032 - 4045; g) Handbo ok of Porphyrin Science ,
Academic Press, San Diego, 2010 , Vols. 1– 15.
2 L. Martin - Gomis, F. Fernandez - Lazaro and A. Sas tre - Santos, J. Mater. Chem. A., 2014 , 2, 15672.
3 a) A. Morandeira, I. López - Duarte, M. V. Martínez - Díaz, B. O’Regan, C. Shuttle, N. A. Haji - Zai-
nulabidin, T. Torres, E. Palomares, J. R. Durran t, J. A m. Ch e m. Soc ., 2007 , 129 , 9250 - 9251; b) Y.
Rio, M. S. Rodríguez Morgade, T. T orres, Org. Biomol. Chem., 2008 , 6 , 1877 - 1894; c) A. Li sto rti ,
I. López - Duarte, M. V. Martínez - Díaz, T. Torres, T. Dos S antos, P. R. F. Barnes, J. R. Durrant,
Energ. Environ. Sci., 2010 , 3 , 1573 - 1579; d) X. F. Zhang, X. Shao, H . Tian, X. Sun, K. H an, Dyes
Pigm., 2013 , 99 , 480 - 488.

Introduction
20
such as zinc, alum inum and galli um, leads to high tr iplet state quantum yields, short life-
times and high singlet oxygen quantum yield s, Φ Δ ≥ 0 . 7. 4

Figure 1 . Typical structure of a Pc showing the non - peripheral, peripheral and axial positions
which can be functionalized, together with a typical UV - vis spectrum of a metallophthalocyanine.
A dopted from ref 16 with permission from The Roy al Society of Chemistry (RSC).
Zinc phthalocy anines (ZnPc) sho w high absor ption in the red r egion of solar spectrum
(Q- band) and good singlet oxygen gener ation yield, 5 r endering them promising candidates
as photosensitizers f or DNA cross - linking. However, the formation of dimer ic aggreg ates,
which is promoted in aqueous solutions, reduces its photoactivit y 6 and singlet oxygen
quantum yields comp ared to the corresponding m onomer. 7 TT1 (Scheme 1) is a well -
known ZnPc photosensitizer bearing a carboxylic moiety and three bulky tert - butyl gr oups
that help to m inimize th e format ion of molecular aggr egates. 8 The photophysical properties
in aqueous media of TT1 functionalized with cholest eryl oleate have been st udied before,
pointing out that this functiona lized TT1 derivative can still effectively photosensit ize 1 O 2
formation. 9

4 G. Jori. In CRC Handbook of Organic Photochemistry and Photobiology, F. Lenci, W. Horspool,
(Eds.), CRC Press , 2004 . 146 – 141.
5 C. M. Allen, W. M. Sharman and J. E. Van Lier, J. Porphyr. Phthalocya. , 2001 , 5, 161 - 169.
6 J. R. Darwent, P. Douglas, A. Harriman, G. Porter and M. C. Richoux, Coord. Che m. Rev. , 1982 ,
44, 83 - 126.
7 X. F. Zhang and H. J. Xu, Faraday Trans. , 1993 , 89, 3347 - 3351.
8 J- J. Cid, J. - H. Yum, S.- R. Jang, M. K. Nazeeruddin, E. M. Ferrero, E. Palomares, J. Ko, M.
Graetzel and T. Torres, Angew. Chem. Int. Edit. , 2007 , 46, 8358 - 8362.
9 L. E. S. Contreras, J. Zirzlmeier, S. V. Kirner, F. Setaro, F. Martinez, S. Lozada, P. Escobar, U.
Hahn, D. M. Guldi and T. Torres, J Porphyr. Phthalocya. , 2015 , 19, 320 - 328.

Introduction
21
Scheme 1 . Synthesis of carboxyphtalocyanice TT1. Reproduced with copyrights permisions
from John WILEY ‐ VCH Verlag GmbH & Co.
1.2 Pht h al ocya nines -f uller ene dyads
1.2.1 Nanot echnology a nd self - assembly
The first time that nanotechnology was for mally recognize d as a viabl e field of research
was during the annual meet ing of the American Physical Society on December 29 th 1959.
During the lecture delivered by the Nobel Prize Winner Richard P. Feynman entitled
“There’s plenty of room at the bottom - an invitation to enter a new field of physics“ 10 it was
described how the nat ural laws should not limit the ability t o work at the molecular level,
atom by atom. He challe nged the resear chers to develop ap propriate techniques a nd
equipment for doing so. The age of nanotechnology s tarted relatively recently, 30 years
ago, once scientists had develop ed the scanning tunn eling microscope 11 (STM) and the
atomic force micr oscope 12 (AFM) whi ch posses s three - dimensional resolut ion down to the
atomic scale.
The general and co mplete defi nition of nanot echnology is t his statem ent: “The es-
sence of nanotechno logy is the ability to work at the molecular l evel, atom by at om, to
create large str uctur es with f undamentally new molecular organiza tion. Nanotechnology is
concerned with mater ials and systems whose st ructures and components exhi bit novel and
significantl y improved physical, c hemical, and bio logical pr operties, phenomena, and pr o-
cesses due to t heir nanoscale siz e. The aim is t o exploit these propert ies by gaining control
of structures and devic es at atomic, molecular, and supramolec ular levels and to learn to

10 R. Feynman , “There’s Plenty of Room at the Bottom” at the American Physical Society meeting
at Caltech, 1959 .
11 G. Binning, H. Rohrer, Ch. Gerber and E. Weibel , Phys. Rev. Lett. , 1982 , 49, 57.
12 G. Binning, C. F. Quate, Ch. Gerber, Phys. Rev. Lett ., 1986 , 56, 930.

Introduction
22
efficiently manuf acture and use these devices”. 13 In order to produce nanoscale materi als
in reproducible and contr ollable manners, top - down and bott om - up approaches were de-
veloped. 14 T he top - down approach is t he assembly by manipulating components with
larger devices t o give smaller features, for example by lithograp hic techniques. Howe ver,
this technique has l imitations whe n the size of the desired feat ure is below 100 nm . Thus,
the bottom - up approach is an alter native way for the creation of nanostructur es because it
can easily go far beyond t he previous size li mitation. The bot tom - up approach cons ists in
the self - assembly of machines from basic c hemical buildings blocks, and this is considere d
to be an ideal through which nanot echnology is im plementing its elf . Nanosc ience and nan-
otechnology have become prom ising approaches f or many applications including m edical,
pharmaceut ical, microbiology, energy conversion, etc. 15

13 Interagency Working Group on Nanoscience, Engineering and Technolog y "National Nanotech-
nology Initiative: Leading to the Next Industrial R evolution" US National Science and Technology
Council, 2000 .
14 G. A. Mansoori and T. A. Fauzi So elaiman , “Nanotechnology–An Introduction for the Standards
Community” , J. ASTM Int., 2005 , 2, 6.
15 a) G. A. Mansoori, “Principles of Na notechnology: Molecular Based Stud y of Condensed Matter
in Small Systems”, WCPC, 2005; b) T. Rajagopalan, K. Ve numadhav, G. Arkasubhra, C. Nrip en,
G. Keshab and G. Shubhra, Rep. Prog. Phys. , 2013 , 76 , 066501; c) E. J. Chung and M. Tirrell,
Adv. Healthc. Mater. , 2015 , 4 , 2408 - 2422; d) K. Avijit, B. Kaustuv and L. Peter, Nanotechnology ,
2017 .

Introduction
23
1.2.2 Donor - acceptor ensembles
The rational des ign and success ful fabrication of functional mater ials received m uch
attention over t he last decade, extensively em ploying the vers atile tools of supra molecular
chemistry . 16 Thus, with well - defined pr inciples of molecular recognition 17 in hands, mult iple
supramolecular species have been generat ed by research society for sensing and cat alytic
applications, resulting from the intermolecul ar association of the correspondi ng molecular
building blocks . 18 However, the fabrication of larger entities by spontaneous ass ociation of
undefined number of species into a specific phase with well - defined m acroscopic charac-
teristics remains a challe nge. Curr ently, many research gr oups keep exploring t he basic
principles and conc epts to assemble building blocks into organized st ates exhibiting spe-
cific functions inclu ding optical, elec trical, and magnet ic propert ies. 19 Compoun ds with an
extended π - conjugation are cons idered promising candi dates in this respec t, step - by - step
finding their applicatio ns in orga nic thin - film electr onics such as organic light - emitting di-
odes, 20 or ganic field - effect transi stors , 21 and organic phot ovoltaics (OPV). 22
OPV devices have been intensely invest igated in the last decades owing to their po-
tential to open up new m arkets where classical si licon t echnology cannot com pete. Their
exceptional pr operties such as transparency, light weight, flexibilit y and tunabilit y of colors
and others, rem ain very attr active for integrat ion of OPV devices i nto consumer products
and buildings, yielding environmentally fr iendly electricity generat ion at zero additional foot-
print. However, bot h power conversion eff iciencies (PCE) and operat ional stability of OPV

16 a) A. Harada, R. Kobayashi , Y. Takashima, A. Hashidzume, H. Yamaguchi , Na t . Che m. , 2011 ,
3, 34 - 37; b) O . Ikkala, G . ten Brinke, Science , 2002 , 295,5564, 2407 - 2409; c) Q. Zhang, J. He, H.
Zhuang, H. Li, N. Li, Q. Xu, D. Chen and J. Lu, Adv. Funct. Mater ., 2016 , 26, 146 - 154.
17 A. D. Buckingham, A. C. Legon, S. M. Roberts, Principles of Molecular Recognition , Springer
Netherlands, 1993.
18 a) D. Sun, et al., J. A m. Chem. Soc. 2002 , 124, 6604 - 6612 ; b) Y. Liu, T. Pauloehrl, S. Presolski,
I. Albertazzi, L. Palmans, J. Am. Chem. Soc . 2015 , 137, 13096 - 105; c) Schreiner, P. R. Chem.
Soc. Rev . 2003 , 32, 289 - 96; d ) E. Morales - Narváez, L. Baptista - Pires, A. Zamor a - Gálvez and A.
Merkoçi, Adv. Mater ., 2016 , DOI: 10.1002/adma.201604905.
19 a) Herr, D. J.C., J. Mater . Res ., 2011 , 122 - 139; b) Bottom - up Nanofabrication , e d. K. Ariga and
H. S. Nalwa, American Scientific Publishers, CA, 2009.
20 a) Hong, M.; Ravva, M. K.; Winget, P.; Bredas, J. - L. Chem. Mater ., 2016 , 28, 5791 - 5798; b) L. -
S. Cui, et al., Angew.Chem. Int. Ed ., 2016 , 55, 6864 - 6868.
21 a) Ford, M. J.; Wang, M.; Patel, Sh. N.; Phan, H.; Segalman, R. A.; Nguyen, T. - Q.; Bazan, G. C.
Chem. Mater. 2016 , 28, 1256 - 1260; b) Ji ang, H.; Ye, J.; Hu, P.; Wei, F.; D u, K.; Wang, N.; Ba, T.;
Feng, Sh.; Kloc, Ch., Sci. Rep . 2014 , 4, 7573.
22 a) S. Holliday, R. S. Ashraf, A. Wadsworth, D. Baran, S. A. Yousaf, C. B. Nielsen, C. - H. Tan, S.
D. Dimitrov, Z. Shang, N. Gasparini, M.a Alam oudi, F. Laquai, C. J. Brabec, A. Salleo, J. R. Durrant,
I. McCulloch, Nat. Commun ., 2016 , 7, 11585; b) R. C. Masters, et al., Nat. Commun ., 2015 , 6,
6928; c) Y. Liu, et al., Nat. Comm un ., 2014 , 5 , 5293.

Introduction
24
devices are st ill significantly low er than those of inorganic com pounds. 23 In recent years,
progress has been made by r educing typical loss pr ocesses for charge generation and
extraction, by using lower bandgap polymers 24 and by controlling the nanomor phology of
the photoactive layers using additives, 25 ternary blends, 26 and cont rolling the processin g
conditions. Howev er, the fundam ental prim ary proces s of OPV, namely char ge generation
and separat ion across the donor - accept or interfac e, is still not fully underst ood.
The presence of ultrafast direct charge generat ion has been shown to depend on size
of the donor and accept or - rich phases and on t he excitation wavele ngth, howev er ambigu-
ities remained with respect to the extension of the primary char ge transfer state, as well as
to the influence of specific don or and acce ptor conform ations at the inter face. For thes e
reasons, donor - accep tor dyads have been int roduced as synthet ic model systems for the
donor - acceptor interface.Owing to t heir biological r elevance and us eful propert ies, porphy-
rinoids has been extensively incor porated into light - to- energy conv ersion schemes as both,
photosynthetic ant enna and reaction center m odule . 27
Up to date, phthalocyanines (Pcs) has been c ommended as t hermally stable excelle nt
light - harve sters with major absorption at the maximum of the solar photon flux – at around
700 nm, Pc also posses s in fact unique p hysicochemical pr operties whic h render these
macrocycles valuable building blocks in nanomaterials science . To this end, f ullerenes rep-
resent another class of widely explored molecular materials that have generated an enor -
mous interest in t he field of nanoelectr onics. On one hand, t heir rigid aromat ic structure
evokes low reorganiza tion energies in elect ron transf er reactions. On t he other hand, their
extended π - c onjugation affor ds efficient charge stabilizat ion. 28 Unique archit ectural flexi-
bility and chemical versatility of fullerenes allow the f ine - tuning of their properties. Covalent

23 S. Karuthedath, T. Saue rmann, H. - J. Egelhaaf, R. Wannem acher, C. J. B rabe, L. Lüer, J. Mater.
Chem. A., 2015 , 3, 3399 - 3408.
24 C. Wang, et al, Adv. Energy Mater ., 2016 , doi: 10.1002/aenm.201600148.
25 Y.-S . Jung, J.-S.k Yeo , N.-K . Kim , S . Lee, D.-Y . Kim , AC S Appl. Mater. Interfaces ., 2016 , 8,
30372 - 30378.
26 M . Koppe, H.-J . Egelhaaf, E . Clodic, M . Morana, L. Lüer, A . Troeger, V . Sgobba, D . M . G uldi, T.
Ameri , C . J . Brabec, Adv . Energy Mater ., 2013 , 3, 949 - 958.
27 a) A. Satake, Y. Kobuke, Org. Biomol. Chem. , 2007 , 5 , 1679 - 1691; b) H. Imahori, J. Phys. Chem.,
2004 , 108 , 6130 - 6143; c) D. M.G uldi, Chem. Soc. Rev., 2002 , 31 , 22 - 36.
28 a) M. Lederer, U. Hahn, J. - M. S trub, S. Cianférani, A. Va n Dorsselaer, J. - F. Nierengarten, T.
Torres, D. M. Guldi, Chem. Eur. J., 2016 , 22, 2051 - 2059; b) S. Kirner, M. Sekita, D. M. Guldi, Adv .
Mat., 2014 , 26 , 1482 - 1493; c) A. J. Ferguson, e t al., Mat. Le tters 2013 , 90 , 115 - 125; d) Ch. - Z, et
al., Jen, J. Mat. Chem. 2012 , 22 , 4161 - 4177.

Introduction
25
modification of their exterior 29 and incorpor ation of m olecular guest into t heir interior 30 con-
stitutes extrem ely efficient strat egies in this connection. In light of the aforementioned, a
myriad of electron donor - ac cepto r conjugates/ hybrids have been designe d featuring por-
phyrins , 31 phthalocyanin es, 32 subpht halocyanines 33 and other chromophors as light - har -
vesting electr on donors and fullerenes as ele ctron acceptor s. All mentioned provide a ver -
satile toolbox for t uning the photophysica l properties in ter ms of the type of process (pho-
toinduced energy/ electron trans fer), the nature of the interactions bet ween the electroac-
tive units (through bond or s pace), and the kinetics of the formation/decay of the photo-
generated species. N oteworthy, the pr eparation of multicompone nt system s with tunable
photophysical proper ties and highly order ed nanoarchit ectures may assist in enhancing
high charge mobilities . In this context, t he use of Pcs and fullerenes may result in the prep-
aration of int riguing one - and two - dimensio nal materia ls with improved s elf - assemblying
and conducting proper ties. In this chapter, recent progress in t he construction of covalent
and supramolecular syst ems comprising Pcs will be s ummarized, with a par ticular empha-
s is on their photoinduc ed and aggregat ion behaviors. I t is believed that the high degr ee of
control achieved in t he preparation of Pc -C 60 sys tems, together with the increasing
knowledge of the factors gover ning their photophysics, will allow for the design of next -
generation light - fueled elect roactiv e systems. Poss ible implement ation of these st ructures
in high performance devices is env isioned, finally tur ning into reality much of the expecta-
tions generat ed by these mater ials.

29 a) E. E. Maroto, M. Izquierdo, S. Reboredo, J. Marco - Martinez, S. Filippone, N. Martin, Acc.
Chem. Res., 2014 , 47 , 2660 - 2670; b) M. Garcia - Borras, et a l., Chem . Soc. Rev., 2014 , 43 , 5089 -
5105; c) J. L. Delgado, et al., Chem. Commun., 2010 , 46 , 4853 - 4865; d) A. Hirsch in The Chemistry
of the Fullerenes , John Wiley & Sons, 2008 , pp. 1 - 215.
30 a) M. Rudolf, S. V. Kirner, D. M. Guldi, Chem. Soc. Rev ., 2016 ,45, 612 - 630; b) J. Zhang, S.
Stevenson, H. C. Dorn, Acc. Chem . Res., 2013 , 46 , 1548 - 1557; c) M. Rudolf, et al. , Chem. Eur. J.,
2012 , 18, 5136 - 5148.
31 a) T. Hasobe in Handbook of Carbo n Nano Materials (Eds.: F. D'Souza, K. M. K adish, 2012 , 4 ,
95 - 130; b) O. Ito, F. D'Souza, Molecules , 2012 , 17 , 5816 - 5835; c) S. Fukuzumi, T. Kojima, J. Mat.
Ch e m. , 200 8 , 18 , 1427 - 1439.
32 a) M. Lederer, U. H ahn, J. Fernandez - Ariza, O. Trukhina, M . S. Rodriguez - Morgade, C. Dam-
mann, T. Drewello, T. Torres, D. M . Guldi, Chem. Eur.J., 2015 , 21,5916 - 5925; b) G. Bottari, M.
Urbani, T. Torres in Organic Nanom aterials: Synthesis, Characterization, and Device Applications
(Eds.: T. Torres, G. Bott ari), John Wiley & Sons, Inc., Hoboken, New Jer sey, 2013 , pp. 163 - 187;
c) G. Bottari, J. A. Suanzes, O. Trukhina, T. Torres. J. Phys. Chem. Lett., 2011 , 2, 905 - 913.
33 a) H. M. Rhoda, M. P. Kayser, Y. Wang, A. Y. Nazarenko, R. V. Belosludov, P. Kiprof, D. A.
Blank, V. N. Nemykin, Inorg. Chem., 2016 , 55, 9549 - 9563; b) M. Rudolf, O. Trukhina, J. Perles,L.
Feng, T. Akasaka, T. Torres, D. M. Guldi. Chem. Sci., 2015 ,6, 4141 -4 147; c) D. Gonzalez - Rodr i-
guez, et al., J. Am. Chem. Soc. 2010 , 132, 16488 - 16500.

Introduction
26
1.2.3 Cova lently - link ed Pc -C 60 systems

Organic chemistry of fers a wide range of synthetic strategies for the preparation of covalently -
connected Pc -C 60 systems. The first report on covalently connected Pc -C 60 molecular system ap-
peared in 1997 by Hanack, Hirsch and coworkers, who prepared P c-C 60 dyad through a Diels -Alder
reaction between C 60 fullerene ( i.e. , dienophile) and Ni(II)Pc bearing two terminal double bonds
( i.e. , diene). This latter compound obtained by cyclotetramerization reaction of the corresponding
phthalonitriles (Scheme 2). 34 The [4+2] cycloaddition occurs at a [6,6] - r ing junction of the C 60 full-
erene cage, giving rise to the formation of Pc -C 60 dyad, named by the authors “ green fullerene ” due
to the intense green color in solution impar ted by the Pc chromophore.

N
N
N
N
N
N
N N
Ni
R R
R
R
R R
R
R
CN
CN
CN
CN
O
+ O N
N
N
N
N
N
N N
Ni
R R
R
R
R R
O
i) ii)
R =

Scheme 2 . Synthesis of the first Pc -C 60 fullerene dyad. C onditions: i) Ni(OAc) 2 , DBU, 1 - pen-
tanol, reflux. ii) C 60 fullerene, toluene, reflux.
Since that first report, a large number of covalently - linked Pc -C 60 systems have been
designed. Compound s 1a - c became one of the most examined examples of covalently -
connected system s constituted by a Pc and C 60 and wer e prepared by 1,3 - dipolar cycload-
dition reaction of azo methine ylides, generated in sit u fr om formy l - Pcs and N - methylg ly-
cine, to C 60 fullerene, allowed to obt ain Pc -C 60 dyads 1 in reasonable yields ( i.e., 40% ( 1a ),
43% ( 1b ), 41% ( 1c )) (Schem e 3). 35 This reaction (also known as “ Prat o reaction ”) consists
in the formation of a pyr rolidine macroc ycle attached to a fulleren e moiety and repres ents,
nowadays, one of t he mostly used synt hetic str ategies for the f unctionalization of fuller-
enes. An alternative s ynthetic route f or the prepar ation of Pc -C 60 dyad 1a has also b een
involved the prepar ation of 4 - vinylphthalonit rile from 4 - iodophthalonitrile, foll owed by the
oxidative cleavage of t he vinyl functional ity to obtain 4 - form ylphthalonitrile (Sc heme 3). The
latter compound was then reac ted with C 60 fullerene in the presence of N - methylglycine to

34 T. G. Linssen, K. Duerr, M. Hanack, A. Hirsch, J . Chem. Soc., Chem. Commun., 1995 , 103 - 104 .
35 a) A. Gouloumis, S. G. Liu, A. Sastr e, P. Vazquez, L. Echegoyen, T. Torres, Chem . Eur. J. 2000 ,
6 , 3600 - 3607; b) D. M Guldi, I. Zilbermann, A. Gouloumis, P. Vazquez, T. Torres, J. Phys. Chem.
B., 2004 , 108, 18485 - 18494.

Introduction
27
af ford the fullerene - cont aining phthalonitr ile, which was subseque ntly condens ed with 4 -
tert - butyl phthalonitrile in the presence of zinc (II) chloride, af fording Pc -C 60 dyad 1a in low
yield .

N N
N
N
N
N
N
N
M
N
1
N N
N
N
N
N
N
N
M
I
a M = Zn
b M = H
2
N N
N
N
N
N
N
N
M
R
R = CH=CH
2
R = CHO
ii
) iii)
iv)
N
vi)
c M = Cu
NC
NC I
NC
NC
i)
NC
NC CHO
v)
CN
CN

Scheme 3. General reaction conditions for the preparation of Pc -C 60 dyads 1a - c . i) ZnCl 2 or
Cu(AcO) 2 , dimethylaminomethanol (DMAE), reflux, argon. For H 2 Pc: lithium, 1 - penthanol, amy l al-
cohol, reflux, argon. ii) t ributyl(vynil)tin, Pd(PPh 3 ) 4 , tolue ne, 100 o C. iii) OsO 4 , NaIO 4 , THF , room
temperature. iv) C 60 fullerene, N - methylglycine, toluene, reflux. v) C 60 fullerene, N - methylglycine,
toluene, reflux. vi) only for 1a : 4 - tert - butylphthalonitrile, ZnCl 2 , DMAE/ o - DCB, reflux .
A detailed analysis o n the photophysica l properties ( i.e., steady - state and time - r e-
solved fluorescenc e measuremen ts and transient absorpt ion measurement s) of dyads 1a -
c in solution was carried out, confir ming the occurrence of phot o - induced electron t ransfer
(PET) events . 36 Exper im ental evidence of long - lived CS in t he solid state with l ifetime sev-
eral orders of magnitude higher than in solution and the first demons tration of working solar
cell with dyad 1a as a ctive layer have been also reported, showing that the spin coatin g

36 D. M. Guldi, A. Gouloumis, P. Vazquez, T. Torres, Chem. Comm un., 2002 , 2056 - 2057.

Introduction
28
tec hnique is efficient tool for preparation dyad - containing thin f ilms, and that the morphol-
ogy of the condensed phase is an important paramet er to consider for photovoltaic appli-
cations. 37
An amphiphil ic Pc -C 60 conjugat e 2 have also been prepared with t he aim of st imulating
the supramolecular or ganization of t his dyad through a combination of π - π stacking and
hydrophilic/ hydrophobi c interactions (Figur e 2a). The system comprised of a tert - butyl sub-
stituted Pc covalently linked t o a fulleropirrolidine m oiety and beared a polyet hylene glyco l
unit terminated wit h an ammoniu m function. 38 Interestingly , such an amphiphilic system is
able to form aggr egated species when d ispersed in wat er , as demonstrat ed by UV - vis (Fig-
ure 2c) and light - scatt ering studies. Fur ther insights into t he morphology of these aggre-
gates in water wer e gathered by transmiss ion electron microscopy (TEM) studies in whic h
the format ion of uniform, micr ometer long 1 - D nanorods was be o bserved (Fi gure 2b). In-
teres tingly , these nano tubules are rem iniscent of those observed for a D - A, porphyr in -C 60
dyad. 39
F igure 2. a) Molecular s tructure of Pc -C 60 dyad 2 . b) TEM image of the nanotubules formed
by dyad 2 in water . c) Ground - state absorption spectra of Zn(II )Pc -C 60 Boc- protected (dashed spec-
trum) in THF and ZnPc -C 60 (solid spectrum) in H 2 O. Adopted with permission from J. Am. Chem.
Soc., 2005, 127, 581 1, copyright 2005, American Chemical Society (ACS).

37 a) M. A. Loi, P. Denk, H. Hoppe, H. Neugebauer, C . Winder, D. Meissner, C.J Brabec, N. S.
Sariciftci, A. Gouloumis, P. Vazquez, T. Torres, J Mater. Chem., 2003 , 13, 700 - 704; b) M. A. Loi,
M. A. Loi, P. Denk, H. Hoppe, H. Neugebauer, D. Meissner, C. Winder, C. J. Brabec, N. S. Sariciftci,
A. Gouloumis, P. Vazquez, T. Torres, Synth. M et. 2003, 137, 1491 - 1492.
38 D. M. Guldi, A. Gouloumis, P. Vazquez, T. Torres, V. Georgakilas, M. Prato J. Am. Chem. Soc.
2005 , 127 , 5811 - 5813.
39 a) V. Georgakilas, F. Pellarini, M. Prato, D.M. Guldi, M. Melle - Franco, F. Zerbetto, Proc. Na tl.
Acad. Sci. U.S.A. 2002 , 99, 5075 - 5080; b) D. M. Guldi, G. M. A. Rahman, F. Zerbetto, M. P rato,
Acc. Chem . Res. 2005 , 38 , 871 - 878.
a) b) c)

N N
N
N
N
N
N
N
Zn
N
O
O
NH
3
13
TFA

2

Introduction
29
Steady - state and tr ansient absor ption studies showed that t he self - organizatio n ability
of the amphiphilic ensemble 2 in water has a profound influence on the photophysica l prop-
erties of these 1 - D na no - objects. Part icularly , trans ient absorption m easurements on t hese
nanotubules r evealed the formation of a long - lived photoinduced charge - transf er product
as inferred by the decay analysi s of the radical pair species of this D - A supramolecular
ensemble at 850 ( i.e., Pc •+ ) and 1050 nm ( i.e., C 60 •‒ ). For such a system , an impressive
stabilizatio n of more than 6 orders of magnitude was observed for the CS lifetime of self -
assembled dyad 2 (i.e., 1 .4 ms) with re spe ct to a stru ctural ly rel ated Pc -C 60 dyad which
lacks the term inal ammonium unit and which is not able to form nanotubules (i.e., ∼ 3 ns).
The use of supramolecular int eractions for the “ bottom - up ” fabr ication of Pc -C 60 na-
noscale functional sy stems on solid surfac es has been recently explo ited. 40 A structu rall y
rigid, covalently - linke d 2.8 nm long Pc -C 60 conj ugate 3 (Figure 3) has been prepar ed, and
its organizat ion ability on highly ordered pyrolytic gr aphite (HOPG) and graphite - like sur -
faces has been investi gated by using atom ic force m icroscopy (AFM) and conductive AFM
(C- AFM). The latter technique resulted to be a power ful tool for m easuring electr ical prop-
erties in nanostruct ured architectures. AFM studies revealed the f ormation of supram olec-
ular fibers and films a s a result of a combination of molecul e - to molecule as wel l as mole-
cule - to- substrate int eractions, whereas C - AFM studies showed electr ical conductivity v al-
ues as high as 30 μ A f or bias voltages ranging from 0.30 to 0.55 V (Figure 4). Control
experiments rev ealed that the high electric al conductivit y values recor ded for the solid -
supported, self - as sembled Pc -C 60 conjugate are st rongly related to t he supramolecular or-
der of the dyad within the nanostr uctures.
An idea about possibl e composition of these fibers could be t aken from anot her recent
work on a similar sy stem (Figure 3c) . Thus, a covalent Pc - C 60 dyad with a shor t and sem-
iflexible bridge gave rise t o a liquid crystalline material that exhibits efficient photocurrent
generation and good short - r ange and long - range ambip olar charge transport properties. 41
Interestingly , pr eheated samples of this dyad showed a 5 - fold inc rease in the charge m o-
bility with respect to the unheated mat erial, a phenome non attributed t o a better alignme nt
of the Pc -C 60 units in columns upon thermal t reatment, in tur n, facilitating the c harge
transport .

40 G. B ottari, D. Olea, C. Gomez - Na va rro, F. Zamora, J. Gomez - Herrero, T. Torres, Angew. Chem.
Int. Ed. 2008 , 47 , 2026 - 2031.
41 H. Ha yashi, W. Nihashi, T. U meyama, Y. Matano, S. Seki, Y. Shim izu, H. Imaho ri, J. A m. Chem.
Soc., 2011 , 133 , 10736 - 10739.

Introduction
30

Figure 3 . a) Molecular structure of Pc -C 60 conjugate 3, b) MM1 optimized structure of 3, c)
Schematic representation of the colum nar arrangement of liquid crystalli ne Pc - C60 dyad. Repro-
duced with permission from Acc. Chem. Res., 2015, 48, 900, copyright 2015, ACS.
Figure 4 . C onductive - AFM studies on HOPG. A region of the substrate is scanned with an
AFM tip, when bias voltage is applied an electr ical current flows through the dyad film. The I-V
values obtained for the f iber and the layer are very close to that of HO PG (an excellent electr ical
condu ctor with a very low resistivity), which is ver y remarkable for a supr amolecularly - organized
system. Adopted from Reference 120, by permission of John Wiley & Sons Ltd.
3

Pc -C 60 d ya d drop casted on HOPG

Pristine H OPG

Introduction
31
A dif fer ent synthetic procedure toward t he preparation of highly - ordered Pc - bas ed sys-
te ms was also utilized pr eviously by Gr eets and co - workers. 42 A mesogenic Pc -C 60 dyads
4a -d were prepared b y an esterifi cation react ion between unsymm etrically subs tituted Pcs
bearing a term inal alcoxy group and a fullerene derivat ive beari ng a term inal acid moiety
(Figure 5). Howev er , UV - vis and electrochem ical studies on these conjugates did not show
any sign of ground - state electr onic communication be tween the acceptor and the donor
moieties.
The thermotr opic properties of these dyad s were studied by po larized opti cal micros-
copy and differential scanning calorimet ry , revealing the for mation of liquid - cry stalline
mesophases in the c ase of Pc -C 60 ensemble 4d . These results suggest t hat in this series,
a long linker is necessary in order to allow the bulky C 60 moiety to be accommodated in
the columnar liquid - crystalline m esophase for med by the Pc macr ocycles. No infor mation
on their char ge - transport c haracteristics was given. Following the same str ategy , a m eso-
genic Pc -C 60 dyad 5 has been reported by T or res and co- workers, which consisted of a
hexadodecyl - subs tituted Zn(I I)Pc covalently connected t hrough a flexibl e spacer to a C 60
fullerene via a Bingel - Hirsch cyclopr opanation react ion (Figure 5). 43 Polarized optical mi-
croscopy and differential scanning calorimet ry studies on this dyad r evealed its liquid - crys-
talline behavior bet ween 80 and 180 o C. Complement ary XRD studies showed t hat 5
adopts a rectangular s ymmetry . Similarly , no studies on t heir charge - tr ansport behavior
were performed .

N
N
N
N
N
N
N N
Zn
O
O
O
4 a-d
O O
O
n
C 10 H 21
C 10 H 21
C 10 H 21
C 10 H 21
C 10 H 21
C 10 H 21
n = 3 - 6
O
O O
N
N
N
N
N
N
N N
O
5
Zn
C 12 H 25
C 12 H 25
H 25 C 12
H 25 C 12
H 25 C 12
H 25 C 12
3
O

Figure 5 . Molecular structures of mesogenic Pc -C 60 dyads 4 and 5 .

42 Y. H Geerts, O. Debever, C. Amato, S. Sergeyev, Beilstein J. Org. Chem. 2009 , 5, 49.
43 M. Ince, M. V. Martinez - Diaz, J. Barbera, T. Torres, J. Mater. Chem . 2011 , 21, 1531 - 1536.

Introduction
32
Optimization of the intermolecular π – π stacking ordering and directions relative to the
substrate represent s a key issue towards enhanci ng charge trans porting propert ies of or-
ganic semiconductor s and, consequently, defines t heir performance in organic elect ronic
materials and devices. It has been shown t hat π – π stacking ar rangement s significantly
improve the charge transport ing propert ies of the active layer in organic solar cells
(OSCs), 44 organic light emitting diodes (O LEDs), 45 and phot odetectors. 46
A straightforward sol ution - proces sed approach towar ds large - scale, ultra - dens e and
vertically st anding π – π stacks on indium tin oxide (ITO) substrate has been repor ted by Lu
et al . 47 ZnPc with four terminal carboxylic functions ar med through four amide groups has
been used f or the preparation of a face - on monolayer by anchorin g ZnPc on I TO substrat e
(Figure 6 a). Synergy of the π – π stacking int eractions and H - bonds involving amides a nd
carboxyls caused the form ation of coherent perpendicular π – π stacks on the singl e mo-
lecular layer (Scheme 6a). AFM and SEM images su ggested the typical d iameter of the
nanorods ranging f rom 30 to 45 nm, whereas TEM studies revealed the fac e - to face pack-
ing of ZnPcs s een as a 1 nm - long horizont al lines within the r ods (Figure 6 b) . The conduc-
tivity of the ZnPc nanor ods was found to be of t he order of 10 − 3 S cm − 1 , as it is shown in
the Figure 5c, approxim ately 100 times greater than that of an amorphous ZnPc film (av.
2.0 × 10 − 5 S cm − 1 ). The hole mobiliti es were est imated from the s pace charge limited cur-
rent region ( I–V 2 ) of the I–V curves, with the average va lue of 2.9 × 10 − 3 cm 2 V − 1 s − 1 , that
is smaller than the r eported values of 10 − 2 to 10 1 cm 2 V − 1 s − 1 , typical for discot ic liquid
crystals and bulk crystals. 48 The linear dependence bet ween conductivity /mobility value s
and the length of t he nanorods indicated un ifor mity and reproducib ility of their st ructures.
Finally, ZnPc nanorod arr ays were used as hole - transport ing materials in OSCs with con-
figuration of with t he configurations of I TO|ZnPc|P3HT:PC 61 BM |Ca|Al, obtainin g powe r
conversion eff iciencies compar able to those of the reference cell based on PEDOT:PSS

44 a) C. Y. Chang, C. E. Wu, S. Y. Chen, C. H. Cui, Y. J. Cheng, C. S. Hsu, Y. L. Wang and Y . F.
Li, Angew. Chem. Int. Ed ., 2011 , 50, 9386 – 9390; b) H. Imahori, T. Umeyama and S. Ito , Acc. Chem.
Res. , 2009 , 42, 1809 – 1818.
45 a) J. E. Anthony, Chem. Rev ., 2006 , 106, 5028 – 5048; b) A. P. Kulkarni, C. J. Tonzola, A. Babel
and S. A. Jenekhe, Chem. Mater ., 2004 , 16, 4556 – 4573.
46 a) H. L. Dong, H. F. Zh u, Q. Meng, X. G ong and W. P. Hu, Chem. Soc. Rev ., 2012 , 41, 1754 –
1808; b) B. Mukherjee, M. Mukherjee, Org. Electron . , 2011 , 12, 1980 – 1987.
47 Z. Lu , Ch. Zha n, X. Yu, W. He, H. Jia, L. Chen, A.g Tang, J. Huang, J. Yao, J. Mater. Chem .,
2012 , 22, 23492 - 23496.
48 a) S. Laschat, A. Baro, N. Steinke, F. Giesselmann, C. Hägele, G. Scalia, R. Judele, E.
Kapatsina, S. Sauer, A. Schreivogel, M. Tosoni, Angew. Chem., Int. Ed ., 2007 , 46, 4832 – 4887 ; b)
Y. Shirota, J. Mater. Chem ., 2000 , 10, 1 – 25.

Introduction
33
(Figure 6b), and exceeding t hose of the c ells fabricated with amorphous Z nPc powder ,
strongly suggestin g the impact of oriented π – π st acks of ZnPC on photovo ltaic perfor-
mance .

Figure 6 . a) (T op) Molecular structure of ZnPc and (down) schematic representation of H -
bonds and the π – π stacks, b) (T op) TEM of a typical ZnPc nanorod, (middle) AFM and (down) SEM
images of a dense nanorod - array ed ZnPc film. c) (T op) Plots of the calculated conductivity and hole
mobility vs . nanorod length, (middle) the J-V curves and (down) E QE characteristics of OSCs based
on the hole - transporting layer of a d ense nanorod - arrayed ZnPc film. D ashed line shows the ab-
sorption spectrum of the film of a dense nanorod - ZnPc array (av . 36 n m) and P3HT :PCBM ( av .
200 nm)on the IT O. Adopted from Reference 4 7 with permission from RSC .
Considering st udy of on - surface events for the Pc - based systems, the last r ecent ex-
ample is worthy of mentioning. Thus, t unable photovolt age responses of Pc -C 60 dyads wit h
modulated polarit y have been desc ribed by Lemmetyinen et al . 49 The synthesized dy ads
had polar OH - tails either on the Pc electr on donor side or on the fulleren e electron accep-
tor side of the dyad (Figure 7a) . The Pc -C 60 dyads were deposite d successfully onto s olid
substrates as highly or iented phases using th e Langm uir − Sch ä fer method ( Figure 7b). For-

49 J.Ranta, K. Kaunisto, M. Niskanen, A. Efimov, T. I. Hukka, H. Lemmetyinen, J. Phys. Chem. C ,
2014 , 118, 2754−2765.

Introduction
34
mation of a vertically orient ed monolayer and t he following electron transf er from the pho-
toexcited phthalocyan ine to fullerene was demonst rated by the time - resolved Maxwell dis-
placement charge met hod. The electr on transfer direct ion was found to be reversed for the
dyads with reversed polarity (Figure 7c) , demonstrating t he ability to control charge transf er
direction in the f ilm.

Figure 7. a) Molecular structures of ZnPc - based C 60 conjugates, b) Signal polarity in electron
transfer experiments , c) Photovoltage responses of the dyad monolayers, λ e xc. = 720 nm (U bias =
0.5 V). Adopted with permission from J. Phys. Chem. C 2014, 118, 2754, copy right 2014, ACS.

Introduction
35
1.3 DNA int erstr and cross - l inkin g on s urfac e
1.3.1 Biosensors in cardi ovascular diseases d etection

According to The Wor ld Health O rganization ( WH0), cardiovascula r disease (CVD)
remains the leading cause of death in the world and Europe. By 2030 it is estimated that
almost 23.6 million people in t he world will die from CVDs, mainly from heart disease and
stroke. 50 The lat est data showed t hat CVD causes m ore than 4 million deaths each year
in Europe, accounting for 45% of all deaths. Figure 8 shows the distribution of all woman
deaths in Europe, t he number of deaths from CVD is higher in wom en than men in Europe,
with CVD accounting f or 49% ( 2.2 million) of all deat hs in women and 40% (1.8 million) of
all deaths in m en. Although many adv ances have been m ade in through ea rly detection
and treatment of CVD , it still remains the le ading cause of death worldwide. The WHO
informed that out of the 16 million deaths under the age of 70 are due to non - communica ble
diseases, 82% are in middle an d low - incom e countries and 37% ar e caused by CVDs.
During the World Economic For um it was shown that in 2010 CVD repres ented 50% of
non - communicable d isease deat hs and the est imated global cost of CVD wa s $ 863 billion,
and it is estimated t o rise to $ 1044 billion by 2030. 51

Figure 8 . Representation of the proportion corr esponding to women deaths in Europe.

50 WHO, Cardiovascular Disease (CVDs) Fact Sheet, World Health Organisation, Mediacentre,
2011 .
51 a) Bloom, D.E., Cafiero, E .T., Jané - Llopis, E., A brahams - Gessel, S., Bloom, L.R., Fathim a, S.,
Feigl, A.B., Gaziano, T., M owafi, M., Pandya, A., Prettner, K. , Rosenberg, L., Seligm an, B., Stein,
A.Z., & Weinstein, C. “The Global Economic Burden of Noncomm unicable Diseases”, World Eco-
nomic Forum, 2011 ; b) N. Townsend, L. Wilson, P. B hatnagar, K. Wickramasinghe, M. Rayner and
M. Nichols, “Cardiovascular disease in Europe: epidem iological update 2016”, Eur. Heart J ., 2016 .
A ll other causes
22%
Injuries and
poisoning
4%
Respiratory
disease
6%
Other cancer
11%
Stomach cancer
1%
Breast cancer
3%
Colo- rectal cancer
2%
Lung cancer
2%
Other
CVD
15%
Stroke
14%
Coronary
heart
disease
20%
Cardiov ascular
disease
49%

Introduction
36
Thus, for many reasons CVD represent s a considerable impor tant social and clinica l
issue and effective preventive measur es are necessary. By screening patient s during the
admission to t he hospitals , the m edical cost could be d ecreased by f ocussing the res ources
to those patients who have t he special risk. Therefore, a rapid and mor e sensitive platform
to fulfill the rapid dia gnosis of CVD and t he measurement of cardiac marker s would play a
key role in the diagnosi s of CVD. Biosensors and biomar kers have a very important r ole in
the diagnostic r evolution of CVD diseases. The rapid advances in nanot echnology indust ry
over the last year s have resulted in the development of biosensors for diagnost ics and
many research groups ar e focussing on the development of new diagnostic devices as well
as trying to enhance th e existing immunologi cal methods for the detection of cardiac mark -
ers , 52 which are biological analytes that can be detect ed in t he blood upon the progres sion
of a CVD disease.
The ideal cardiac biomarker s should be highly specific f or cardiac tissue and absent
from non - myocardial t issue as well as the need to be easily accessible to achieve high
diagnostic sensitivit y. For early diagnos is, small solubl e molecules with rapi d clearanc e
from injur ed can be useful as suit able biomar kers. Nevert heless, a highly stabl e biomarker
with a long plasma ha lf - life is esse ntial in the c ase of late diagn osis. As a res ult, peak level s
should be reached relat ively quickly and the biomarker should persist in circulation for a
few hours.
Troponins ar e cardiac prot eins. The Troponin comple x has three subunits on the t hin
filament combined wit h myocardial contr actile muscle to control t he calcium ions bindings:
cTnC (responsible f or calcium bin ding), cTnT ( the tropomyosin - binding) and cTnI (inhibit s
the ATPase activity of actomyosin). Car diac muscle and skeletal muscle shar e troponin C
isoforms, rendering th is protein unsuitabl e for diagnostic use. Whereas, cTnI is f ound only
in heart tissue and is not express ed in any type of skelet al muscle. Troponins T and I have
unique cardiac isofor ms and are consider ed the gold standar d for the detection of
myocardial inj ury (MI) . 53 The com plex for m ed by cTnI and cTnC is t he predominant form

52 a) B. McDonnell, S. Hearty, P. Leonard and R. O'Kennedy , Clin. Biochem ., 2009 , 42, 549 - 561;
b) G. - J. Zhang, Z. H. H. Luo, M. J. Huang, J. A. J. Ang, T. G. Kang and H. Ji, Biosens. Bioelectron . ,
2011 , 28, 459 - 463; c) Z. Altintas, W. M. Fakanya and I. E . Tothill, Talanta , 2014 , 128, 177 - 186; d)
Q. Wang, F. Liu, X. Y ang, K. Wang, H. Wang and X . Deng, Biosens. Bioelectron ., 2015 , 64, 161 -
164; e) B. Rezaei, M. Ghani, A. M. Shoushtari and M. Ra biee, Biosens. Bioelectron ., 2016 , 78,
513 - 523; f) D. Bhatnagar, I. Kaur and A. Kumar, Int. J. Biol. Macromol ., 2017 , 95, 505 - 510.
53 a) P.G. Steg, S.K. James, D. Atar, L.P. Badano, et al., Eur. Heart J ., 2012 , 33, 2569–619; b) P.T.
O'Gara, F.G. Kushner, D.D. Ascheim, et al., J. Am. Coll. Cardiol., 2013 , 61, 78 – 140.

Introduction
37
which comprises 95% of cTnI in human blood. The det ection of unusual cTn levels occ urs
generally 4 – 6 h after the myocardial injury and r emains in the blood for at least 7 days .
Heart - type fatty acid binding pr otein (hFABP) is a small heart protein located in t he
cytopl asm. It i s also being expressed at low levels in e xtra - cardiac tissues including kidney s
and skeletal muscle . 54 Because of its location and low mol ecular weig ht hFABP is released
rapidly into the cir culation right aft er (1 - 3 h) myocardial injury and is helpful in t he early
diagnosis of M I. 55 It is not usef ul for monitor ing patients mor e than 6 h after first symtoms
onset because its plasma clear ance is relatively short , 56 usually the hFABP level can return
to be normal within 24 hour s.
The C - reactive protein ( CRP) is an acute - phase protein reactant, which is released in
response to acute inj ury, infect ion or inflammatory st imulation. CRP is the m ost widely used
infla mmatory biomarker. Because CRP induces t he expression of adhesion m olecules and
other inflammat ory cells, CRP also has pro - inflammat ory effects. It has been shown tha t
CRP can be useful to predict fut ure cardiovascular events, including stroke, development
of peripheral arter ial disease and first - ever acute myocardial infar ction (AMI) . 57 Figure 9
shows the struct ure of the cardiac biomarkers described above.
Figure 9 . Cr ystal structure of heart fatty acidic binding protein , 58 human C - reactive Protein , 59
and cardiac troponin C - troponin I complex. 60

54 A. Colli, M. Josa, J. L. Pomar, C. A. Mestres and T. Gherli, Cardiology , 2007 , 108, 4 - 10.
55 a) C. Carroll, M . Al Khalaf, J. W. Ste vens, J. Leaviss, S. Goodacre, P. O. Collinson and J. Wang,
Emerg. Med. J. , 2013 , 30, 280 - 286; b) R. Janko vic, et al., BioMed Res. In.t , 2015 , 8.
56 G. Haltern, S. Peiniger, A. Bufe, G. Reiss, H. G ülker and T. Scheffold, Am. J. Cardiol. , 2010 , 105,
1- 9.
57 a) S. De Servi, M. Mariani, G.M ariani, A. Mazzone , J. Am. Col.l Cardiol ., 2005 , 46,1496 – 502; b)
S. J. Aldous, Int. J. Cardiol ., 2013 , 164, 282 - 294; c) D. del V al Martin, M. Sanmartín Fernández
and J. L. Zamorano Gómez, IJC Metab. Endocr ., 2015 , 8, 20 - 23.
58 C. Lücke, M. Radem acher, A. W. Zimmerman, H. T. v an Moerkerk, J. H. V eerkamp and H.
Rüterjans, Biochem. J. , 2001 , 354, 259 - 266 (RCSB PDB: 1 G5W).
59 A. K. Shrive, G. M. T. Gheetham, D. Holden, D. A. A. Myles, W. G. T urnell, J. E. Volanakis, M.
B. Pepys, A. C. Bloomer and T. J. Greenhough, Nat. Struct . Mol. Biol ., 1996 , 3, 346 - 354 (R CSB
PDB: 1GNH).
60 M. X. Li, L. Spyracopoulos and B. D. Sykes, Biochemistry , 1999 , 38, 8289 - 8298 (RCSB PDB:
1MXL).
C- reactiv e Protein

Troponin I – Troponin C

Heart fa tt y acidic bindi ng Protein

Introduction
38
T able 1. Summary of clinically utilized cardiac biomarkers (MI: myocardial inf arction). Adapted
from Reference 51b with permission fro m RSC.
Cardiac
biomarker

MW
(kDa)

Clinical
cut- off
levels

Initial
elevati on

above
clinical
cut- off (h )

Duration
till peak
elevati on
(h)

Duration
of
e levation

CVD
indicator
t yp e

Biomarker
Specificit y

Troponin I

23.5

0.01 – 0.1

ng/ml
4–6

18 – 24

4– 7 days

Detection of
MI and tool for

risk
stratificatio n
High: specifically

increased
Following
m yocardial
necrosis

h- FABP

15

6 ng/ml

1–3

6–8

24 – 36 h

Early detectio n

of MI
Low: release d

following s keletal
muscle injury , rapid
clearance foll owing

necrosis and
perceived
increases du e to
compromise d renal

function

C- reac tive

Protein
125

< 1 μg/ml low

risk,
1– 3 μg/ml inter-
mediate ris k,
>3 – 15 μg/ml
high risk. Sti ll no

defini tive
consensus

No

clinical
consensus

No

clinical
consensus

No

clinical
consensus

CVD - related

inflammatory
response
based
biomarker
High: a large body

of evidence t o
suggest a dir ect
link between
elevations ab ove
clinical cut -
offs and

recurrent isch emic
events

Biosensors are one type of small devices for detecting target analytes that are usually
biomolecules such as proteins, pept ides and nucleic acids. Biosensors use biologica l mol-
ecules to recognize t he target and employ output elements (so- called tr ansducers) which
are able to translat e the biorecognition pro cess into mass - sensit ive, optical or electrical
signals.
Biosensors ar e probably one of the mos t promising too ls for a fast, co st eff ective and
sensitive measur ement which ar e used as a rapid screening to ol to detect CVD at the
earliest stage . 61 Immunos ensors are biosens ors with i mmunoreagents as sensing ele-
ments. These are principally antibodies im mobilized in close contact with physicochemical
transducers, for example, optical fiber s and electrodes or f ree antibodies that bind t o im-
mobilized antigens. O ptical, Electrochem ical, piezoelectr ic, magnetic and ther mometric
transduc ers ar e the common types. Wher e the measurement of the analytes is achieved

61 a) M. Mascini and S. Tombelli, Biomarkers , 2008 , 13, 637 - 657 ; b) E. B. Bahadır and M. K.
Sezgintürk, Anal. Biochem ., 2015 , 478, 107 - 120 ; c ) M. - I. Mohammed and M. P. Y. Desmulliez, Lab.
Chip , 2011 , 11, 569 - 595 ; d) S. R. Sh in, Y. S. Zhang, D. - J. Kim, A. Manbohi, H. Avci, A. Silvestri, J.
Aleman, N. Hu, T. Kilic, W. Keung, M. R ighi, P. Assawes, H. A. Alhadrami, R. A. Li, M. R. Dokmeci
and A. Khademhosseini, Anal. Chem ., 2016 , 88, 10019 - 10027.

Introduction
39
by the selective tr ansduction of the receptor – tar get binding , obtaining a quant ifiable elec-
trical or optical binding signal as result. I mmunosensors can achieve continuo us detectio n
of a broad variety of analytes. El ectrochemical im munosensor are appr opriate when o n -
site monitoring capab ilities are re quired and t hey offer high sensitiv ity and select ivity due
to the use of imm unochemical int eractions. O ptical transducer s include meas uring fluores-
cence, optical density, surface plasmon r esonance (SPR), or luminescence.
In colorimet ric and fluor escence - based detec tion, either target or biorecogn ition mole-
cule is labe lled with a c hromogenic or fluorescent tag, such as dyes. An enzyme can also
be the label for t he conversion of a colourless t o a chromogenic substrat e. The change of
the colour/fluorescenc e intensity signa l indicates the presence of the t arget molecules,
which can be extremely sensit ive and detect even a s ingle molecule . There are also label -
free detection methods , in those the target molecules are detect ed in their native forms
and do not need a label, these methods allow quantit ative/kinetic meas urement of molec-
ular interactions such as surface plasma resonance bi osensors . One of the limitations of
the immunochemical sensors is the cross - r eactivity or interference, which depends on the
type of the target analytes as well as the applic ation of the immunosensor . Figure 10 shows
some possible biosens ors applicat ions for the d etection of biomark ers .

Bacteria
Meal ions
Neutravidin
Biotinylated
protein
Antibody
Antigen
Enzyme Glucose DNA
probe
1. Unmodified sensor surface
2. Surface modification
3. Ligand immobilization
4. Analyte detection

Figure 10 . Examples of biosensor applications and possible surface modification.

Introduction
40
The analysis of cardiac marker s can be perform ed using antibody - based met hods
such immunoassays. Originally, pr otein assays were developed i n microtit er plate format
for enzyme - linked immunosor bent assay ( ELISA). ELISA is a method for quantification and
detection of a specific analyte i n a comple x mixture. Within t his method, the v isualization
of the t arget molecule is realized through a colour - generat ing enzyme which is covalently
linked to a specific det ection mol ecule such as an anti body. The colour for mation is indi-
rectly proport ional to the concent ration of the analyte a nd can be used for quantif ication by
measuring the absorbanc e.
The m ajor role in imm unoassays is playe d by the antibodies. Those are produc ed by
B- cells as part of the immune r esponse that identifies and neutralizes f oreign species such
as pathogens. Ant ibodies have been used a s recognition element s in immunoassay and
subsequent ly immunosensor development due t o their high specificity, affinity and versa-
tility as well as their c ommercial availability. The antibodies which are der ived from sepa-
rate cell li n es t hat recognize various r egions on the immunogen a re known as polyc lonal
antibodies, and those derived from sin gle cell line are known as monoclonal antibodies.
There are five major classes or isoty pes of antibodies - IgG, IgD, I gE, IgA , an d IgM, the y
are classed accor ding to the heavy c hain they contain – alpha, delta, epsil on, gamma or
mu respectively. The IgG is the most abundant class in ser um (70 - 80%), it is monomeric
with a molecular we ight of appr oximately 150 kDa. It is the major class of antibody of the
secondary immune r esponse and has the longest half - life (20 - 24 days). For the detect ion
of proteins, ther e are four main types of immunoassay protocols: direct, indir ect, competi-
tive, and sandwich imm unoassays.
Figure 11 shows t he schemat ic illustrat ions of direct, indirec t and sandwich im muno-
assays. The basic prin ciples for the assays are similar and inclu de the capture of the ana-
lyte of interest, block ing of the non - reac ted surface, and addition of the det ection compo-
nent. In the case of competitive - di rect assay, the analyte and an anal yte - enzyme conjugate
compete for the bi n ding sites of the primary antibody. The indirec t immunoassay format
has the ability to im prove the sensitivity of the det ect ion. In comp etitive - indirect immuno-
assay the competit ion for the biding sit es of the primary antibody i s between the immobi-
lized analyte - protein conj ugate and the analy te in solution. In this met hod, the analyte of
interest is bound to a specific antibody and after a label l ed secondary ant ibody against this
primary antibody is then added f or detection. I t is very important that the secondary ant i-
body be raised in anot her species t han the primary antibody t o avoid non - specif ic binding.

Introduction
41
In the sandwich immunoassay the unlabe led protein is sandwiche d between two an-
tibodies and then label l ed specif ic detection antibod ies are used to detect bound proteins.

Direct Assay Indirect Assay Sandwich Assay
Secondary
Antibody
Primary
Antibody
Analyte-enzyme
conjugate
Analyte-protein
conjugate
Secondary
antibody
conjugate
Capture
antibody
Analyte
Secondary
antibody
conjugate
Primary
Antibody

Figure 11 . Schematic illustration of direct, indirect, and sandwich immunoassays.
Even though ELISA has been since long the standar d for quantitative bioanalys is, this
technique off ers not enough throughput , due to lack of low mult iplexing abi lity, and has
high sample and reagents consumption. Theref ore, the develop ment of fast multipl ex
methods for t he quantificat ion of proteins is very important. Antibody microar rays may be
a very useful tool for m olecular diagnosis d ue to its ability to m easure multiple prot eins in
complex mixtures usin g a small amount of s ample. This multiple analysis can r educe false
positive and negative results relat ive to the tests based on sing le markers. In Figure 12 are
compared ELISA and m icroarray immunoass ays.
In the m icroarray platfo rms, the antibody is immobilized o n a chemically modifie d solid
support in an array f ormat for the following q uantification of proteins in biologic al samples.
Mostly, glass m icroscope slides are us ed as platforms and ther e are many comm ercially
available diffe r ent kinds of surf ace modification of gl ass slides. G enerally, fluoresc ence
signal can be achi eved by using f luorophore - conjugat ed with t he detect ion antibody or wit h
streptavidin molecules, which bin ds to biotin - labeled d et ection antibodies t o generate a
fluorescent signal .

Introduction
42
[hapten]
[hapten]
hapten
Primary
antibody
HRP secondary
antibody
CY5 secondary
antibody
ELISA Microarray

Figure 1 2 . ELISA vs. protein microarray.

Introduction
43
1.3.2 Biose nsors and D NA in na notechnology

During the last decad e, nanotec hnology has s hown to have a cle ar impact on t he de-
velopment of biosens ors. Biosensors have ac hieved great succ ess both in the academic
and commercial ar eas due to the need of mult iplex applicat ions that this versat ile technol-
ogy can have. Ther anostics, defined as di agnostic tes t directly linked t o the application of
specific therapies, inf luence the developmen t of biosensors since the pharmaceut ical com-
panies seek t o deliver an efficacio us therapeutic. The dem and for more analytical dat a and
facilitating t hat the patients can add dat a themselves is prom oting the develop ment of bio-
sensors as well. The world market for biosensors est imated from sever al commercia l
sources over the last years and t he prediction for the future is shown in the followin g Figure
13 . 62
Figure 13 . Biosensors world market in million US dollars.
The boom in persona l diagnostic s is expanding the fie ld to the developmen t of new
and inexpensive sensor platforms that are able to sat isfy costumers' needs. Theref ore,
next generation diagnos tics manufact uring is heading for creat ing complete sensing sys-
tems that can interface s eamlessly with modern t elecommunications . New technologies
are focussing on fusion electronic printed syst ems and the future mobile technology such
as smartphones. The research is becoming mor e and more interest ing in the developm ent
of new sensors for the delivery of molecular i nformat ion with high sensit ivity and specificit y
avoiding the instability and cross - react ivity with other nat ural molecules prese nt in the sys-
tem to analyze . Currently, deoxyr ibonucleic acid ( DNA ) , peptide, antibody, aptamers
arrays, as well as molecular ly imprinted pol ymers, are par ticularly prom ising tools with in
this research area. The opportunit ies for success are enhanced by the potential utility of
some of these nanomaterials for novel diagnostic platf orms. Synthetic biology analo gs can

62 A. P. F. Turner, Chem. Soc. Rev., 2013 , 42, 3184 - 3196.
0
5000
10000
15000
20000
1995 2000 200 5 2010 2015 202 0

Introduction
44
facilitate biochem ical reactions in or der to improve diagnostic proc esses by using self - as-
sembled systems t o build supramolecular s tructur es.
It has been m ore than sixty years since the st ructure of DNA was first r evealed in 1953
by Watson and Crick. 63 In 1962 they were awarded the Nobel Prize since their work e st ab-
lished the c hemical basis for discerning genetics. DNA is a long polymer of nucleotides
and is form ed by three differ ent component s: a heterocyclic b ase, a deo xyribose sugar ,
and a phosphate group. There are t wo purine bases, adenine (A) and guanin e (G), and
two pyrim idines, thymine (T) and cytosine (C) (Figure 14 ).
OH
O
Sugar
(2'-deoxyribose)
N
N
NH
2
H
H H
H
O
CH
2
H
O
P
O
O
O
_
_
Base
(cytosine)
phosphate
1'
2' 3'
5'
4'
N
N
N
N
H
N
NH
N
N
H
N
H
N
NH
2
NH
2
O
O
NH
2
N
H
NH
O
NH
2
Adenine
Guanine
Cytosine
Thymine
a) b)

Figure 14 . a) Nucleotide structure, the repeat unit of DNA. b) Purine and pyrim idine DNA
bases.
To form the polynucleot ide chain, the sugars are connecting by a 3´ - 5´ phosphodiester
linkage, which is a covalent bond for med between the 3´ - hydroxyl group of one sugar and
the 5´ - hydro xyl of the next sugar (Figure 15a). The direc tion of the polynucleotide chain is
given by the chemist ry of the phosphodiest er bond, from t he 5´ - to the free 3´, the two
strands of the duplex DNA are antipar allel, meaning that t he strands are oriented in oppo-
site directions . Figure 14b shows t he base - pairing in DNA, the purine bases pair with the
pyrimidine bases by h ydrogen bonds, in this way the two units of backbone s are pulled
together and form the double helix struct ure. 64 The structure of the DNA double heli x is
shown in Figure 15c. The diamet er of the molecule is 2 nm and one total rotat ion takes
place every 3.4 nm , consisting o f 10 - 10.5 base pair s (bp) which ar e separated by 0. 34
nm. This classical for m of the double helix is known as the B - form and r epresents the form
found in cells. B and A - for m are the most common type duplexes.

63 J. D. Watson and F. H. C. Crick, Nature , 1953 , 4356, 737 - 738.
64 J. D. Watson, The double helix , 1968 .

Introduction
45
The A type (11 bp/helical turn, 2.6 nm molecule diameter) of the helix is observed
under conditions of low humidity or in solutions t hat contain organic solvent s or high salt
concentrations.
N
N
N
N
H
N
N
N
N
H
H
N
O
N
HN
N
H
O NH
O
O
N NH
Adenine
Guanine
Cytosine
Thymine
H
H
H
H
H
O
O
P
O O
O
O
O
OH
O
P
O O
O
O
O
P
O O
O
5' END
3' END
T
A
C
G
O
P
O O
O
_ _
_
_
_
3'-5'
phosphodiester
linkage
3'
5'
a) b) c)

Figure 15 . a) DNA polynucleotide chain . b) Base- pairing of DNA bases, dotted lines represent
hydrogen bonds . c) DNA double helix structure.
The double helix is a r elative ly stable struct ure due to its non - covalent intera ctions, the
hydrogen bonds of the base pairs and t he π - π interactions of the stacked base pairs. This
provides the therm odynamic stability of the double helix. Because o f these relatively weak
interactions , the two strands coul d separate easily duri ng the DNA replicati on and tran-
scription. When the double helix molecule is exposed to high temperature or pH conditions,
the strands can be separated because t he hydrogen bonds that hold both strands are bro-
ken, a process known as denaturat ion. Denaturat ion is reversible when the denatur ed DNA
is slowly c ooled, as single - st randed , complem entary strands can r eform double helixes.
This process is called renatur ation, hybridization or annealing. The capacity of the DNA
molecule to renat ure/denature allows t he formation of artificial hybr id DNA and is the basis
for several fundamental techniqu es in molecular biology like DNA addressable arr ays and
Southern blot. The DNA denaturation and hybr idization can be m onitored by measur ing
the absorbance of ultraviolet light of a DNA solut ion. DNA absorbs ultraviolet light m ax i-
mally at a wavelength of 260 nm, t he bases are responsible for this absorpt ion. Upon de-
naturation the absor ption of light at 260 nm by DNA incr eases, this is called hyper chromic

Introduction
46
effect. When com plementary DNA refor ms the duplex struct ure its absorbance at 260nm
decreases, the so- c alled hypochrom ic effect. The tem perature at which half of the DNA
molecule is denatured or unwound is the melting tem perature Tm. 65 DNA goes through a
transition fr om a very well order ed double - h elical struct ure to a much less or dered s in g le -
stranded str ucture. The sharpnes s of the increase in ab sorbance at the Tm indicates that
the denaturation and r enaturation pr ocesses are highly cooperat ive, a zippering - like pro-
cess. Renatur ation probably occ urs as a slow nuc leation pr ocess, a relatively s mall stret ch
of DNA bases of one st rand pair with its complement on the corr esponding com plementary
strand. After this, the remainder of t he two strands is very quickly zipped up from the nu-
cleation site to r eform the double helix.
The base composition of each DNA molecule affects the Tm and is most ly determined
by the Guanine - Cytos ine (GC ) content and the ionic strength of the solution. The higher
the percent of G C base pairs in the DNA, t he higher the Tm . The reason of this is beca use
it takes more heat energy t o break three hydr ogen bonds of a GC base pa ir than the t wo
hydrogens bonds of an AT base pair. An increase of the Tm also occurs at a higher salt
concentration (high ionic st rength) of the solution; The phosphoryl groups of the DNA ba ck-
bone carry negative c harges. If these charges are not shiel ded, they can in duce that the
strands repel each other when t he two DNA strands are close and f acilitate their separa-
tion. At high ionic strength, these negat ive charges are shielded by cations and by that,
they are stabilizing t he double heli x. Automated DNA synt hesis allows the in troduction of
chemical modificatio n in oligonucleot ides in a site - specific and ef ficient way. 66
DNA has become a popular mat erial in nanotechnolo gy due to it s previously descr ibed
specific properties of structur al stability, self - direction and t he possibility t o modify DNA
sequences . 67 The advantage of the DNA for being easily assembled makes this molecule
a useful tool for t he design and fabricat ion of nanostr uc tures . 68 DNA has been em ployed
to build structur es in different dim ensions with severa l geometries. To construct dynamic
nanoscale struc tures with different features most of the scientists have used the bott om -

65 Burton E. Tropp, “Molecular Biology, genes to proteins”, 4th edition, Jones & Bartlett Learning ,
2012 .
66 S. L. Beaucage and R. P. Iyer, Tetrahedron , 1993 , 49 , 1925 - 1963 .
67 a) M. Zahid, B. Kim, R. Hussain, R. Amin and S. H. Park, Nanoscale Res. Lett ., 2013 , 8, 1 - 13;
b) Y. Krishnan and F. C. Simmel, Angew. Chem. Int. Ed ., 2011 , 50, 3124 - 3156; c ) J. J. Thiaville, et
al., Proc. Natl. Acad. Sci. USA , 2016 , 113 , E1452 - E1459.
68 a) N. C. Seeman, A nnu. Rev. Biochem ., 2010 , 79, 65 – 87; b) D. Han, S. Pal, Y. Yang, S . Jiang,
J. Nangreave, Y. Liu and H. Yan, Science , 2013 , 339, 1412 - 1415 ; c) V. Linko , et al., Sci. Rep .,
2015 , 5, 15634; d) S. Krishnan, et al. , Nat. Comm un . , 2016 , 7, 12787.

Introduction
47
up approach of self - assembly . 69 DNA - bas ed nanost ructur es allow to carry or organize pep-
tides, proteins, viral capsids, c arbon nanotubes as well as nanoparticles in a c ontrollable
manner, obtaining a nanomat erial with nov el properties and function . 70 Several research
disciplin es in t he field of DNA nanotechnol ogy are iden tifie d in Figure 1 6.
Figure 1 6 . Representation of different areas bases on DNA nanotechnol ogy.
DNA biose nsors/ genosensors an d DNA chips/ biochips/m icroarrays can allo w sensi-
tive and fast detec tion of DNA hybridizatio n. In the DNA biosen sors, the ODN probe is
immobilized ont o a transducer surface and allows single - shot measurement s. On the con-
trary, DNA chips allow a m ultiple parallel det ection in a s ingle experim ent on the surface
which can be of s ilicon, plast ic or glass. DNA ba sed chips are a n important tool for the
high - throughput analys is on surf aces . 71
A high sensitive DNA chip devel opment depends on t wo factors: the meth od used for
the immobilization of the probes (shor t ODN, 20 - 30mer; long O DN, 60 - 70mer), whic h
should provide repr oducibility of the attachment chemistr y, and the good accessibility of
the probes to the t argets DNA . 72 The probes are pr e - synthesized and then spott ed on the
surface chip by contac t or non - contact print ing.

69 a) R.N. Kallenbach, et al. , Nature , 1983 , 305, 8 29 – 831; b) A.V Pinheiro, D. Han D , W. M. Shih,
H. Yan , Nat. Nanotechnol ., 2011 , 6, 12, 763 – 772 ; c) F. Wang, et al. , Chem. Rev ., 2014 , 114, 2881 -
2941.
70 a) N. Stephanopoulos, M. Liu, G.J. Tong, Z. Li, Y. Liu, H. Y an, M. B. Francis, Nano. Lett ., 2010 ,
10, 2714 – 2720; b) R. Chhabr a, J. Sharma, Y. Ke, Y. Liu, S. Rinker, S. Lindsay , H. Yan, J. Am.
Chem. Soc ., 2007 , 129, 10304 – 10305; c) W. - W. Zhao, J. - J. Xu and H. - Y. Chen, Chem. Rev ., 2014 ,
114, 7421 - 7441; d) W. Wang, et al., Nucleic Acids. Res ., 2016 , 670.
71 a) A. S. Krylov, O. A. Zasedateleva, D. V. P rokopenko, J. Rouviere - Yaniv, A. D. Mirzabekov,
Nucleic Acids R es ., 2001 , 29, 2654 – 2660; b) H. Ravan, et.al, Anal. Bioche m ., 2014 , 4 44, 41 - 46; c)
B. S. Nimse, et al., Int. J. Mol. Sci. , 2013 , 14, 5723 - 5733.
72 a) P. Kumar, S. K. Agrawal, A. M isra, K. C. Gupta, P. Kumar, Bioorg. Med. Chem. Lett. , 2004 ,
14, 1097 -1 099; b) A. Sassolas, et al. , Chem. Rev ., 2008 , 108, 109 - 139.
DNA
Nanotechnology
Functional Enzyme/DNA
Structure
DNA Hydrogels
Nanostructures/
machines
Sensors

Introduction
48
Many immobilization techniques for fabricat ion of DNA chips have been developed
and optimized to obt ain high reactivit y, stability and accessibi lity of the probes in order to
increase the sensitivit y of the detection m ethods . 73 The main techniques to imm obilize
ODNs are presented in Figure 17 .

Physical
adsorption
Covalent
immobilization
Avidin-Biotin
immobilization

Figure 17 . DNA immobilization techniques.

Physical adsor ption is based on t he ionic interact ion between the negative ly charged
ODN probe and the positive charges from the surface , 74 the technique has been used t o
develop DNA microar rays . 75 By using this te chnique, t he ODN can for m many contacts
with different orientat ions, is randomly oriented and weakly att ached to the surface. Due to
the electrostat ic interactions, the ODN can be easily rem oved during the assay conditions
by changes in pH, tem perature or by the dif ferent ionic s trength of the buffer solutions
used.
The immobilization by covalent att achment provides the necess ary high stability. The
silanol gro ups (SiOH) at the glass surf ace are modifie d to obtain nucleo philic (c arboxyl,
epoxy, aldehyde, isot hiocynate ) or electr ophilic (maleim ide, me rcaptosilane ) funct ionali-
ties, which then react with the amino or thiol groups of ODNs, r espectively (Table 2).

73 a) A. N. Ra o, C. K. Rodesch, D. W. Grainger, Anal. Chem ., 2012 , 84, 9379 - 9387; b) K. - S. Song,
S. Balasaheb Nimse, J. Kim, J. Kim, V. - T. Nguyen, V. - T. Ta, T. K im, Chem. Comm ., 2011 , 47,
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4261; d) S. Nimse, K. Song, M . Sonawane, D. Sayyed T. Kim, Sensors , 2014 , 14, 22208; e) A.
Chiadò, C. Novara, A. Lamberti, F. Geobaldo, F. Giorgis and P. Riv ol o, Anal. Chem ., 2016 , 88,
9554 - 9563.
74 S. V. Lemeshko, T. Powdrill, Y. Y. Belosludtsev and M. H ogan, Nucl. Acids Res. , 2001 , 29, 3051 -
3058.
75 S. D. Conzone and C. G. Pantano, Materials Today , 2004 , 7, 20 - 26 .

Introduction
49
Table 2 . Functional groups for DNA immobilization with the corresponding O DN.

The interact ion between biotin an d all proteins f rom the avidin fami ly (avidin, st reptav-
idin, neutravidin) are usef ul in a wide variety of applications. Avidin pr oteins are composed
of four identical subunits , each one being able to form a strong complex with biotin (vitamin
H or B7) as it can be observed in Figur e 18 .
Figure 18 . a) Biotin structure . b) Avidin- biotin complex formed with four biotin guest s. 76

Table 3 shows the c omparison o f the avidin pr oteins. Their associ ation const ant can
be up to K a = 10 15 M − 1 and is comparable with cov alent bonds. Avidin has a basic isoelectric
point, theref ore, at physiologica l pH, it is posit ively charged and can bind non - specif ically
to negat ively charged molecules. St reptavidin and neut ravidin are used more than avidi n
because their isoelect ric points around 6 makes them more specific. Another good ad-
vantage of this syst em is that the carboxylic acid group of the biotin can be m odified v ia es-
ter or amide formation. This allows t he easy biotiny lation of ant ibodies, enzymes or amino -
modified DNA strands .

76 M. Holzinger, A. Le Goff, S. Cosnier, New J. Chem. , 2014 , 38 , 5173 - 5180 .
Surface

modificatio n
Group

structure
ODN probe

modificatio n
Aldehyde

CHO

- NH 2
Carboxyl

COOH

Epoxy

CHCH

2
O

Isothiocyanat e

N=C=S

Maleimide

HC

2
(CO)

2
NH

- SH
Mecaptosilan e

Si -R- SH

a)

b)

HN NH
S
O
H
H
COOH

Introduction
50
All previo us outstanding proper ties result in the biotin – st reptavidin/neutr avidin system,
an important t ool for immobilization via af finity int eractions on any kind of sur faces . 77
Table 3 . Comparison of biotin binding proteins.

Avidin Streptavidin Neutravidin
Molecular Weight 67 K 53 K 60 K
Isoelectric point 10 6.8 - 7.5 6.3
Specificity Low High Highest

The utilization of novel nanom aterials has promot ed the study of DNA chips and bio-
sensors towards t he development of simple, inexpensive and sm art detection of targets.
But there are still some issues to impr ove the utilization of new m agnification methods t o
decrease the limit of detection, new output signa ls to improve the existing detec tion meth-
ods, the enhancement of the assembly production of the DNA probes and targe ts to in-
crease the s ensitivity. In this concept, DNA nanotechno logy provid es new opportunities by
allowing the modif ication of the properties of nucleic acids.
Protein micr oarrays are frequent ly used in sev eral applicat ions such as prot ein - protein
interaction analys is, drug scr eening, and biomar ker detection . 78 Even if t he protein
microarra y has the ability to beco me an import ant tool for many diagnostics applications,
its production is limite d by the technical cha llenges relat ed to microarray pr oduction, being
the printing of the proteins on the micr oarray surface one of t his challenges. The intensity
of the signal obtained i s related to the amount of analyte that is captur ed by the immobilized
antibody, It is im portant that the antibody is attac hed to the surface with its bind ing sites
oriented towards t he solution . 79

77 a) T. Liebermann, W. K noll, P. Sluka, R. Herrm ann, Colloids Surf. A., 2 000 , 169, 337 - 350; b) C.
Larsson, M. Rodahl, F. Höök, Anal. Chem ., 2003 , 75, 5080 - 5087; c) J. Ladd, C. Boozer, Q. Yu, S.
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78 a) G. Mac Beath and S. L. Schreiber, Science , 2000 , 289, 1760 - 1763; b) H. Sun, Grace Y. J .
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79 a) P. Peluso, D. S. Wilson, D. Do, H. Tran, M. Venkatasubbaiah, D. Quincy, B . Heidecker, K.
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Introduction
51
Figure 1 9 shows the possibles o rientation of antibodie s on solid surfaces and DNA -
directed immobiliz atio n (DDI) s trategy to enhance the immobilizati on . 80

Figure 1 9 . The structure of antibody (Fc: fragment, crystallizable, Fab: fragment, antigen
binding, disulfide bridges and antige n binding sites are indicated) and so me strategies for antibod y
immobilization.

Amine - reac tive traditional sur face chemist ry, such as p r oducing carboxy l, epoxy, al-
dehyde or isothiocyn ate gr oups on surface can m ask the antibody bind ing site due t o
multiple covalent int eractions t hat are possible betwe en the antib ody and the sur face
groups. 81 Additiona lly, var iable protein immobi lization y ields within a nd across m icroarrays,
as well as inconsistenc y in the morphology and uniformit y of printed microspots ar e
parameters that affect the robustness and accuracy of the assay . 82

80 Y. Jung, J. Y. Jeong and B. H. Chung, Analyst , 2008 , 133, 697 - 701.
81 H. Sun, Grace Y. J. Chen and Shao Q. Yao, Chem. Biol , 2013 , 20, 685 - 699.
82 a) U. B. Nielsen and B. H. Geierstanger, J. Immunol. Methods , 2004 , 290, 107 - 120; b) S . L.
Seurynck - Servoss, A. M. White, C. L. Baird, K. D. Rodland and R . C. Zangar, Anal. Biochem .,
2007 , 371, 105 - 115; c) V. R omanov, S. N. Davidoff, A. R. Miles, D. W. Grainger, B. K. Gale and B.
D. Brooks, Analyst , 2014 , 139, 1303 - 1326.

Introduction
52
Researchers hav e explored alternat ive methods of pr otein immobilizat ion to address
the mentioned issues and enhance t he activity of t he antibody to impr ove the sensitivity of
the assay. The development s of bioconjugation t echniques made possib le the modifi cation
of the molecules to obtain vers atile surface chemistr ies to immobilize proteins. DDI is one
of those techniques which combines D NA microarrays with pr oteins for diagnost ic
purposes by using pr otein - DNA conjugates ( Figure 20a ) , being the prot eins cov alently
attached to a specific DNA sequence . 83 The antibodies are dir ected and immobilized on
the surface via sequence s pecific DNA - DNA hybridizati on. Due to t he DNA spacer between
the immobilized molec ule and the surface the ant igen binding capacity of ant ibod ies is
enhanced by allo wing a bett er orientation an d decreasing t he steric hindrance . 79 Moreover,
DNA microarr ay production is less labor ious than protein microarray fabr ication due to easy
optimizat ion of DNA printing . 84
The use of nucle ic acid hybridiz ation for di rected immobilizat ion of component s on
surfaces is not limit ed to prot eins; with the aim to detect protein – l igand interac tions it can
also be applied to small molecules such as steroids , 85 for direct ing steroid - oli gonucleotides
on surface plasmon r esonance (SPR) gold chip as is shown in Figure 20 b . Colloidal
compounds, s uch as metal nanopar ticles can also be d irected t o a sensor surfac e by DDI . 86
Figure 20 c ) . As well, DDI has proven to be a powerful tool for nucleic acid analyses and
a versatile technique within the research an d biomedical diagnost ics fields . 87

83 a) C. M. Niemeyer, L. Boldt, B. Ceyhan, D. Blohm, Anal. Biochem ., 1999 , 268, 54 - 63; b) R. C.
Bailey, et al., J. Am. Chem. Soc ., 2 007 , 129, 1959 - 1967; c) A. L. Washburn, et al., Anal. Chem .,
2011 , 83, 3572 - 3580; d) C. Boozer, et al., Anal. Chem ., 2006 , 78, 1515 - 1519.
84 a) H. H. Cao, N. Nakatsuk a, A. C. Serino, W. - S. Liao, S. Cheunkar, H. Yang, P. S. Weiss and A.
M. Andrews, ACS Nano , 201 5 , 9, 11439 - 11454; b) J. Fredonnet, et al., Microarra ys , 2016 , 5, 25;
c) A. H. Loo, C. K. Chua and M. Pumera, Analyst , 2017 , 8, 15 - 28; d) R. Castagna, et al, Langmuir ,
2016 , 32, 3308 - 3313.
85 N. Tort, J. P. Salvador, A. Aviñó, R. Eritja, J. Comelles, E. Martínez, J. Samitier and M. P. Marco,
Bioconjugate Chem. , 2012 , 23 , 2183 - 2191.
86 a) T. A. Taton, C. A. Mirkin and R. L. Letsinger, Science , 2000 , 289, 1757 - 1760; b) C. M. Nie-
meyer and U. Simon, Eur. J. Inorg. Chem ., 2005 , 3641 - 3655.
87 a) N. L. Rosi and C. A. Mirkin, Chem . Rev . , 20 05 , 105, 1547 - 1562; b) S. Nimse, K. - S. Song, J.
Kim, D. Sayyed and T. Kim , Int. J. Mol. Sci. , 2013 , 14, 5723; c) R. Meyer, et al., Curr. Opin. Chem.
Biol ., 2014 , 18, 8 - 15; d) M. - X. Chen, et al., PLoS. Negl. Trop. Dis ., 2016 , 10, 12; e) S P Flynn, et
al., Nanotechnology , 2016 , 27, 465501.

Introduction
53

Figure 20 . a) Scheme of DNA microarra ys using protein - DNA conjugates , b) Schematic rep-
resentation of functionalization of an SPR chip with steroid – oligonucleotide bioconjugates, c ) DNA
arrays using oligonucleotide - modified gold nanoparticle probes. With copyrights permissions of the
ACS ( b) and The American Association for the Advancement of Science ( c).

b)

c)

a)

Introduction
54
1.3.3 Furan mediated DNA - ICL cross - linking

Many ty pes of research are inspired by the toxicity of furan, which is classifi ed by the
International Age ncy for Research on Can cer as a possible hu man carcinogenic co m-
pound . 88 Furan is oxidized by cyt ochrome P450 to cis -2- butene - 1,4 - dialdehyde, which re-
acts with proteins 89 (amino acid side chains) and nucleosides 90 (exocyclic amines) (Figu re
21 ).
O
O Oligo 2
N
N
NH2
Oligo 1
O N
N
HN
Oligo 1
O
OH
Oligo 2
N
N
N
Oligo 1
O
N
N
N
Oligo 1
O
O
OH
OH O
Oligo 2
Oligo 2
O
O
O
O
P450
cis-2-butene-1,4-dialdehyde

Figure 21 . Cis -2- butene - 1,4 - dialdehyde product and the reaction mechanism with nucleo-
sides.
A new cross - linking methodolo gy has been develop ed by Halila and co - workers 91 (Fig-
ure 22). They observed a select ive in situ oxidation of a furan unit incorporat ed in an oligo-
nucleotide nucleoside, resulting in a f ast site - specific cross- link formation between com-
plementary oligonucl eotide st rands. Oxidation was ac hieved wit h N - bromosuccinimi de
( NBS) .
Figure 22 . Furan - oxidation triggered DNA cross - linking. After hybridization with the right tar-
get, furan is selectively ox idized to a keto - butenal functionality which forms a covalent bond with
the nucleophiles (exocyclic amines) of the complementary oligonucleotide sequence .

88 International Agency for Research on Cancer. Dry - Cleaning, some Chlorinated Solvents and
Other Industrial Chemicals. IARC Monographs on the E valuation of Carcinogenic Risk s to Humans ,
1995 , 393 - 407.
89 M. B. Phillips, M. M . Sullivan, P. W. Villalta, L. A. Peterson, Chem. Res. Toxicol. , 2014 , 27, 129 -
135.
90 a) L. A. Peterson, Drug Metab. Rev ., 2006 , 38, 615 - 626; b) D. Verzele, L. Carrette, A. Madder,
Drug. Discov. Today Technol ., 2010 , 7, 115 - 123.
91 S. Halila, T. Velasco, P. D. Clercq, A. M adder , Chem. Commun ., 2005 , 936 - 938.
Hybridis ation

ICL- formatio n

O

O

O O

Oxidation

Introduction
55
Figure 2 3 shows the results of the furan oxidation using NBS and t he ICL formation
with a cytosine nucleos ide. There is a c ompetitive brom ination reaction on the fur an moiety,
which occurs in the case of an extended ar omatic system. 92

O
R O
R
Br O
R
Br O
R
Br
O
R
Br
O
R
Br
R OH
H 2 O
-HBr
O O
-H
+
undesired side
product
desire reactive
intermediate
NBS
N
N
O
R'
N
HO
OH
R ICL

Figure 2 3 . Oxidation mechanism of furan with NBS and the undesired side reactio n product .
DNA interstrand cros s - link (ICL) formation posses s es im portant clinical applicat ions. 93
Therefore, the develo pment of new ICL methodologies is an area of significant int erest. A
series of furan - modified build ing blocks were design ed by the group of A. Madder in order
to improve the furan cross - linking m ethodology by favouring the reaction of the ket o - bu-
tenal with the nucleop hiles of the complement ary oligonucle otide sequence. Figure 24
shows the str uctures of some of thes e furan building blocks, which can be selec ted
depending on the desir ed cross - link outcome, site selectivity or high cross - linking yie ld.
Furan - modified nucle osides 1, 2, 3, and 4 formed c ross - links both to c omplementary A or
C bases. Building bloc ks 5 and 6 allowed a mor e selective cross - linking and only yielded
a cross - linked product if a complementary C base was pr esent.
O
OH
OH
O
O
HO
HO
HO
HO
O
O
OH
OH N
NH
O
O
O
O
OH
OH N
NH
O
O
HN O
O
O
OH
OH N
NH
O
O
HN O
H
N
O
1 3
2
4 5 6

Figure 24 . Furan - modifie d building blocks .

92 a) K. Stevens, D. D. Claey s, S. Catak, S. Figaroli, M. Hocek, J. M. Tromp, S. Schürch, V. Van
Speybroeck and A. M a dder, Chem. Eur. J ., 2011 , 17, 6940 – 6953; b) L. L. G. Carrette, E. Gy ssels,
J. Loncke and A. Madder, Org. Biomol. Chem. , 2014 , 12, 931 - 935.
93 a) S. R. Rajski and R. M. Williams, Chem. Rev. , 1998 , 98, 2723 - 2795; b) M. L. Dronkert and R.
Kanaar, Mutat. Res . , 2001 , 486, 217 - 247; c) D. L. Boger, J. Desharnais, K. Capps, Angew. Chem .,
2003 , 42, 4138 - 4176; d )Y. Huang and L. Li, Transl. Cancer. Res. , 2013 , 2, 144 - 154.

Introduction
56
The acyclic phenyl extended nucl eoside 2 can be synthesized ac cording to a method
optimized in the labor atory of Professor A. Madder. 92 The compo unds obtained alon g the
synthesis are shown in Schem e 7 .
O
O
OH
+
Br
Br
O
O Br
O
O
O
HO
HO
O
HO
DMTO
O
O
DMTO
O
P N
O
CN
a) b) c)
d) e)
I II
III IV V

Scheme 7 . S ynthesis of the furan modified phosphoramidite V: a) KOH, toluene, T= 135°C,
24h. b) furan boronic acid, Pd(PPh 3 ) 4 , Na 2 CO 3 , toluene, MeOH, T= 48°C . c) HCl (4M), THF, 2h . d)
DMTrCl, pyridine, T= 0°C, 3h . e) (iPrN) 2 (NCCH 2 CH 2 O)P, N,N- diisopropylammonium tetrazolide ,
DCM, 0° - kT, 48h.

The synthesis of the furan modified phos phoramidite V invo lves the follow ing steps:
first , SN2 substitution react ion where hydroxyl group of the (S) - Solketal attacks the
electrophilic 4- bromobenzylbrom ide to for m compound I. Suzuki - Miyaura cr oss - coupling of
the aromatic bromide I with 2 - furan boron ic acid and catalyz ed by palladium t riphenyl
phosphine ( Pd(PPh 3 ) 4 ) generat es II. After, hydrogen chloride completes t he deprotection
of the diol skeleton III. B y addition of DMTrCl as a limiting reagent in the next step, select ive
protection of t he primary alcohol is guarant eed because ditrit ylation is avoided. Subse-
quent protection of the primary alcohol results in the formation of the dimethox ytrityl ated
protected nucleos ide IV . 2- Cyanoethyl - diisopr opylphosphor amidite in the pr esence of
diisopropylam moniumtetr azolide as an activating spec ies is used in the phos phorylation
reaction to obtain t he building block V ready for incorporat ion into the desired olig onucleo-
tide sequences.

Introduction
57
To open up the pathway t o biological app lications, a more bioco mpatible oxid ation
method based on the use of singlet oxygen for ICL format ion was developed inspired by
the studies of singlet oxygen - mediat ed furan oxidation in t he aqueous solution shown in
Figure 25. 94 Comparison of the cross - linki ng yields trigger ed by singlet oxygen upon red
light irradiation in the presence of methy lene blue (MB), and NBS, showed that for all furan -
modified nucleos ides building block s except 2, NBS oxidation led to hig her cros slink
yields . 95
Figure 25 . Oxidation of furan by the traditional path a versus the singlet oxygen mediated path
b . Reproduced with copyright permission from RSC .
Other photosensitizer s (PS) , e.g. phthalocyanines t hat show high a bsorption and pr o-
duce singlet oxygen in good quantum yields 96 were also proposed as PSs for DNA cross -
linking.
Mechanism of 1 O 2 generation through elect ron or charge t ransfer from the excited
state of a photosensitizer to the molecular oxyg en (O 2 ) present in the media is shown in
Figure 26. 97 Herein, the PS is first excited from the ground singlet st ate to the ex cited
singlet state by absor ption of a photon upon ir radiation with light. The PS can relax to the

94 D. Noutsias, I. Alexopoulou, T. Montagnon, G. Vassilikogian nakis, Green. Chem. , 2012, 14,
601 - 604.
95 M. Op de Beeck and A. Madder, J. Am. Chem. Soc. , 2012 , 134, 10737 - 10740.
96
C. M. Allen, W. M. Sharman and J. E. Van Lier, J. Porphyr. Phthalocya. ,
2001
, 5, 161 - 169.

97 T. Dai, B. B. Fuchs, J. J. Coleman, R. A. P rates, C. Astrakas, T. G. St. Denis, M. S. Ribeiro, E.
Mylonakis, M. R. Hamblin, G. P. Tegos, Front. Microbiol ., 2012 , 3, 120.

Introduction
58
longer lived triplet stat e, microseconds compared to the nanosecond s of the excited s inglet
state, and can inter act with m olecular oxyg en in two pathways , type 1, which leads t o the
formation of reactive oxygen species ( ROS) and type 2, which leads to the formation of
singlet oxygen.

Figure 2 6 . Schematic illustration of singlet oxygen ge neration including the Jablonski diagram
to describe the states of the photosensitizer molecule (PS). Credits to the aut h ors of Reference 97.

59

2. Objective s

Objectives
61
2.1 Pht h al ocya nine -fu l lerene dyads
Considering t he above - men tione d examples, the objective of this work is to synt he-
size four novel covalent ly - linked Pc -C 60 conjugat es 6 - 9 with bett er sel f- organization ability
(Figure 27). To achieve t his goal, we will employ peripher ally non - substitut ed ZnPcs to
maximize the π − π s tacking int eractions between planar conjugated poliaromat ic species,
and, in the same time, to avoid the presence of r egioisomeric products which ar e obtained
when monosubstit uted phthaloni triles are used as prec ursors in the Pc synthesis. Worth
notable, the prepar ation and further synt hetic modification of non - substit uted Pcs repre-
sents a non - trivial task due to their poor solubility, and, t hus, has been scarcely expl ored
in the literature, being repr esented by carboxy - 98 and amido - 99 monosubstituted ZnPcs as
the only examples. In this work , we will establish a st raightforwar d route for the preparat ion
of mono - substituted asym metric ZnPc suitable f or its easy purification and f urther incorpo-
ration in Pc -C 60 dyads.
In their tur n, for the preparation o f these covalent conj ugates t wo main functionaliza-
tion strategies will be us ed, the 1,3 - dipolar cycloaddiction reac tion between Pc moieties
bearing an azomet hyne ylide function and C 60 (Prato r eaction, 100 dyads 6 - 7 ) or the cyclo-
propanation reac tion (Bingel - Hirs ch reaction, 101 dyads 8 - 9 ). Moreover, t he distance be-
tween ZnPcs and C 60 f ullerene will be mo dulated as a func tion of the m olecular spacer
connecting both spec ies. Its variable flexibilit y will additionally ass ist in possible improv e-
ment of supramolecular pac king of the dyads. In order to see if these Pc -C 60 conjugates
will be able t o give rise to order ed structures being depo sited on surf aces, AFM, STM and
TEM studies will be perform ed. Their electron transfer reactivity will be invest igated to es-
timate the potential a pplicability of the hybrids for the energy conver sion schem es.

98 Chen, J.; Chen, N.; Huang, J.; Wang, J.; Huang, M., Inorg. Chem . Commun . 2006 , 9, 313.
99 Li, L.; Luo, Z.; Chen, Z.; C hen, J.; Zhou, Sh.; X u, P.; Hu, P.; Wang, J.; Chen, N.; H uang, J.,
Bioconjugate Chem ., 2012 , 23, 2168.
100 M. Maggini, G. Scorrano, M. Prato, J. Am . Chem. Soc., 1993 , 115 , 9798 - 9799.
101 C. Bingel, Eur. J. Inorg. Chem ., 1993 , 126, 8, 1957 – 1959.

Objectives
62

N
N
N
N
N
N
N
N
Zn
N
N
N
N
N
N
N
N
N
Zn
N
O
O
H
3
N
TFA
N
N
N
N
N
N
N
N
Zn O
O
O
O
N
N
N
N
N
N
N
N
Zn
O
O
6 7
8 9

Figure 27. Molecular structures of ZnPc - based C 60 conjugates.

Objectives
63
2.2 DNA int erst rand cr oss - linkin g on sur face
T he main objective of this wor k wa s to e stablish a met hod for ICL on surface (u sing
methylen e blue and TT1 phthalocyanine as photosensitizer s) towards a DDI based
platform which posses es a c onsiderable enhance ment of its DNA duplex st ability . This new
platform , form ed by furan - modified DNA oligonucle otides should b e used to address an
antibody previous ly conjugated wi th the short DNA (approx. 10mer) target that is required .
Then , the ICL methodology w oul d be implem ented in a m ultiplex addressable immunoarr ay
for the detection of t hree cardiac bio markers (CRP; h- FABP; TI) .
Three platform s we re going to be used to perform the experiment: Enzyme- linked
Immunosorbent Assay ( ELISA) , SPR and microarr ays. Proof - of - principle of the I CL on sur-
face will be demons trated by SPR m easurements on a Biacore syst em using complemen-
tary oligonucleotid e pairs f or hybridization and cross- li nking and an FITC/ anti - FITC probe
as a model syst em, in order to generate st rong signal . I n addition t o the SPR measure-
ments, the aim of t he ELISA experiment s wa s to know the conditio ns for ICL form ation and
to confi rm the high efficiency of the principle .
A f irst object ive wa s to know the minimum lengt h of the immobiliz ed DNA duplex that
will ensure hybridization st ability. The ne xt objective wa s to conjugate t he antibody with
these possible minimum lengths to find the optimal length of the ODN target that should
be used to address in an efficient way the antibody - DNA conj ugate . Since the future appli-
cation w ould be a mult iplex addressable immnunoar ray , the third objective is to select three
DNA sequences ver y specific with no cross - react ivity between them .
Towards the developm ent of a multiplex for cardiac markers , studies of cro ss - reac-
tivity between the diff erent components of the immunoassay w ould be perf ormed by SPR
and microar ray experiment s , as well as studies of stabil ity of the syst em using ELISA in
order to see the availabil ity to implement the ICL met hod within the mult iplex assay ).

65

3. Material s and Methods

Material and Methods
67
3.1 Phtha locy anine -f ul lerene dyads
3.1.1 Chem icals and inst rume ntation
Chemicals were purchas ed from Aldrich Ch emical Co. and Alfa Aesar Chem icals and
used as received without furt her purification. Solvents were dried in accor ding with stand-
ard procedures. Reactions were monit ored by thin - layer chromatography (TLC) on silica
gel (F 254 E. M erck). Column chromatogr aphy was carried out on silica gel Merck - 60 ( 230 -
400 mesh, 60 Å) as a solid support. UV - vis spectra were recor ded on Jasco V - 660 spec-
trophotomet er. MALDI - TOF MS spectra were obt ained by an Applied Biosyst em 4700 in-
stru ment equipped with a Nd:YA G laser oper ating at 335 nm . 1 H NMR and 13 C NMR sp ec-
tra were obtained on a Bruker AC - 300 spectrom eter. TEM and high - resolution t ransmission
electron microsc opy (HRTEM) images were r ecorded with a Philips CM 300 UT. For the
AFM inv es tigations, a Dig ital Inst rument (Veeco) Nan oscope IIIa ( Tapping Mode) with
Veeco RTESP5 Tips was used. In the case of t he UHV - STM experiments wit h dyads 18
and 20 , these exper iments were c arried out in an ult ra - high vacuu m chamber (bas e pres-
sure of 10 -10 mbar) . Dyads molecules were subj ected to thermal sublimatio n onto clean
glass surface, which was held at r oom temperature (temperat ure of deposition was raised
slowly from room temperature to 620 K, until f irst traces of deposi ted compound of gr een
color could be observed). Thin liquid chromat ography experiment s showed the depos ited
compounds to be dif ferent from initial dyad s, confirming therm al decomposition occurr ed
during sublim ation. STM measur ements were not performed.
Femtosecond tr ansient absorpti on measurements were carr ied out with a CPA - 2101
femtosecond laser (Clark MXR). The excitat ion wavelength was gener ated with an NOPA
(Clark MX R). Nanosecond transient abs orption l aser photolysis measur ements were
performed with the out put from the third harm onic (355 nm) of a Nd/YAG laser ( Brilliant B,
Quantel). The optical det ection is based on a pulsed ( pulser MSP 05 – Mülle r Elektr onik -
Optik) Xenon lamp (XBO 450, Osram), a monochroma tor (Spectra Pro 2300i, Acton Re-
search), a R928 photom ultiplier tube (Hamam atsu Photonics), and a 1 - GHz dig ital oscil lo-
scope (WavePro7100, LeCroy).

Material and Methods
68
3.1.2 Synthesis of dyad precurs ors
4- (Bromometh yl)phthalo nitr ile 24
4- Methylphthalonitr ile (2 g, 14.06 mmol), NBS (2.8 g, 15.5 mmol) and AIBN (254.5
mg, 1.55 mmol) wer e introduced t o a round bott om flask and the mixture purged w ith
argon. Degassed CCl 4 ( 100 mL) was added and the reaction mixt ure refluxed under
argon atmosphere whil e illuminating with a 200 W att tungsten lamp during 6 hours unt il
the disappearance of the starting material. The react ion mixture was then let to cool
down to room tem perature and filtered in order to remove the succinim ide. The filtr ate
was finally evaporated affording a residue consist ing of mono - , di - , and tribr omination
products at the benzyli c position (in an approximate 1/2/ 1 ratio by 1 H NMR). This mix-
ture was dissolved in dry THF (7 m L), cooled down to 0 ºC and diethyl phos phite (0.7
mL) and DIPEA (0.8 mL) added dropwise. The solution was then stirred for 30 minutes
at 0 ºC and then at room temperatur e for 4 hours while monitoring t he progress of t he
reaction by TLC and 1 H NMR until the complete disapp earance of the di - and t ribromin-
ated product s. After this time, the solvent was evapo rated, t he residue obtained dis-
solved in C HCl 3 (30 mL) and washed with an aqueous HCl 0.1M solution (30 m L), then
with a saturated solut ion of NaHCO 3 (60 mL), and finally with brine (30 m L). The organic
phase was then dried over Na 2 SO 4 , filtered and the resulting soluti on reduced to dry-
ness affording a brown solid which was purif ied by column chromatography (SiO 2 , hex-
ane/ethyl acetate 4/ 1) affording 4 - (bromomethy l) phthalonitr ile 24 as a yellow solid.
Yield: 2.3 g, 87%. 1 H NMR (300 MHz , CDCl 3 ): δ 7.84 (s, 1H, 4 J H-H = 1.7 Hz, H3), 7.80
(d, 1H, 3 J H-H = 8.1 Hz, H6), 7.75 (dd, 1H, 4 J H-H = 1.7 Hz, 3 J H-H = 8.1 Hz, H5), 4.48 ppm
(s, 2H, CH 2 ); 13 C NMR (7 5 MHz, CDCl 3 ) : d =144.0 (C5), 134.0 (C4), 133. 9 (C6), 133.7
(C3), 116.4 (C1), 115.4 (C2), 115.1 (CN), 115.0 (CN), 29.8 ppm (ArCH 2 Br).
NC
NC
Br

24

Material and Methods
69
4- (Hydroxy methyl)ph thalonitrile 25

4- (Bromomethyl)pht halonitril e 24 ( 4 g, 18.1 mmol) was added t o a suspension of
calcium carbonate (7. 24 g, 72.3 mmol) in dioxane/water (1: 1.5, 200 mL) and the mixture
stirred at reflux for 48 h. The react ion mixture was then f iltered in order to remove the
CaCO 3 , and t he filtrate washed w it h Et 2 O (3 x 250 m L). The c ombined organic layers
were then dried over Na 2 SO 4 and the filtr ate concentrated under r educed pressure ob-
taining a yellow oil that s olidified upon standing. Finally, the compound was pu rified by
column chromatogr aphy (SiO 2 , DCM /AcOEt 2/1) aff ording hydroxymethylpht halonitrile
25 as a white solid. Yield: 2.81 g, 98% yield. 1 H NMR (300 MHz, CDCl 3 ): δ 7. 85 (s, 1H,
H3), 7.81 - 7.71 (d broad, 2H, H5, H6), 4.86 (d, 2H, CH 2 ), 2.07 (t broad, 1H, OH); 13 C
NMR (75 MHz, CDCl 3 ): d = 147.4 (C4), 133.6 (C5), 131.1 (C3), 130.7 ( C6), 116.1 (CN),
115.4 (CN), 114.4 ( C1), 114.3 (C2), 63.1 ppm (CH 2 O H).

OH
NC
NC

25

Synthesi s of TBDPS -protected, hy droxy methylphthalon itrile 23

4- Hydroxymethylpht halonitrile 25 (100 mg, 0.63 mmol) was silylated by reaction with
TBDPS chloride (0. 24 mL, 0.94 mmol) in imidazole (85,8 mg, 1.26 mmol) and DMF (0. 4
mL). The reaction mixt ure was washed with a water /DCM mixture and the iso lated or-
ganic layer dried over Na 2 SO 4 and filtered. The f iltrate was then r educed in volum e and
purified by column chrom atography (SiO 2 , hexane/ AcOEt 4/0.7) affording TBDPS - pro-
tected hydroxyphthalo nitrile 23 . Yield: 170 mg, 68% yield. 1 H NMR ( 300 M Hz, DMSO -
d 6 ) δ 7.78 - 7.74 (m, 2 H, CH phthalonitrile ) , 7.68 - 7.61 (m , 5H, C H phthalonitrile + CH phen yl ), 7.50 -
7.35 (m , 6H, C H phenyl ), 4.81 (s , 2H , CH 2 ), 1.12 ppm (s, 9 H) ; 13 C NMR (75 MHz, CDCl 3 ):
 = 147.7 (C4), 135. 6 (C10), 133.6 ( C6), 132.5 (C5), 130.9 (C13), 130.4 (C12) , 130.3
(C14), 128.2 ( C11, C13, C15), 116.1 (CN), 115.6 (C2), 114.3 (C1), 64.3 ( C H 2 OH), 26. 9
( C (CH 3 ) 3 ), 19.4 (C( C H 3 ) 3 ).
NC
NC
O Si
t
Bu
23

Material and Methods
70
Synthesi s of {2- [2 -(2 - amino - ethoxy) - ethoxy] - ethyl} - carbamic acid
tert - buty l ester 28

To a solution of 2,2’ - ( ethylene - dioxy)bis(et hylamine) ( 5,18 g, 68 mmol) in anhydrous
1,4 - dioxane (80 ml), a solution of Boc 2 O (5.45 ml, 23.7 mmol) in 1,4 - dioxane (30 ml)
was added dropwise over a period of 3 hours and the reaction mi xture st irred overnight
at room temperatur e. Once finished, the reaction solv ent was removed under r educed
pressure and the residue obtain ed dissolve d in DCM (40 m l) and washed wit h water ( 3
× 100 ml). The organic layer was then dried over Na 2 SO 4 , filtered and the filtrate evap-
orated to dryness and purif ied by flash chromat ography in DCM/MeOH 8/2. Yield: 3.29
g, 56%. 1 H NMR (300 MHz, CDCl 3 ): δ 5.13 (s, 1H, NH), 3.6 2 (s , 4 H, OC H 2 C H 2 O), 3.54
(m, 4H, OC H 2 ), 3.31 (m, 2H, NH 2 C H 2 ) , 2.89 (t, 2H, 3 J H-H = 6 Hz, NHC H 2 ) , 1.89 (s, 2H,
NH 2 ), 1.44 (s, 9 H, CH 3 ).
28
N
H
O
H 2 N 2
Boc

Synthesi s of {2-[2 - (2 - benzyloxycar bonylamino - ethoxy) - ethox y] -
ethyl}- carb amic acid tert - b utyl ester 29

To a solution of 28 (1. 13 g, 4.55 mmol) in 1,4 - dioxane ( 30 ml) cooled to 0 ºC, a
solution of benzylbr omoacetat e (0.24 ml, 1.53 mm ol) in 1,4 - dioxane ( 15 ml) was added
dropwise over a per iod of 1.5 hours. The reaction mixt ure was then gently warmed up
to room temperat ure and stirred for 3 hours. After this time, the solvent was removed
under reduced pressur e and the residue obt ained dissolved in DCM (40 ml) and
washed with water (3×100 ml). The organic layer was then dried over Na 2 SO 4 , filtered
and the filtrate evaporat ed to dryness. Compound 29 was finally purified by flash chr o-
matography in CHCl 3 /M eOH 2/1.5. Yield: 0.93 g, 51%. 1 H NMR (300 MHz, CDCl 3 ): δ
7.35 (s, 5H, CH Ar ), 5.17 (s, 2H, C H 2 Ph ), 3.60 (s, 6H , OC H 2 ), 3 .52 (m, 2H , OC H 2 ), 3.50
(s, 2H , NHC H 2 CO), 3.31 (m, 2H, C H 2 NH CH 2 CO ), 2.82 (t, 2H, 3 J H-H = 6 Hz, CONHC H 2 ),
1.72 (s, 1H, N H CH 2 CO), 1.43 (s , 9H, C H 3 ).

N
H
O
N
H
O
O
29
2
Boc

Material and Methods
71
Synthesi s of {2 - [2 - (2- tert - bu toxyca rbonylami no - ethoxy) - ethoxy] -
ethylami no} - acetic acid 27

To a solution of 29 ( 0.2 g, 0.5 mmol) in methanol (10 ml), 20 mg of Pd/C (10%) were
added and H 2 bubbled, and the reac tion mixture st irred for 5 hours at room temperatur e.
The solution was then filtered through a celit e pad and the solvent was evaporated
under reduced pressur e obtaining the desired compound pur e. Yield: 0.15 g, 97%. 1 H
NMR (30 0 MHz, CDCl 3 ): δ 5.56 (s, 1H, NH), 3. 83 (m broad, 2H, OC H 2 ), 3.65 - 3.55 (m
broad, 8H, OC H 2 ), 3.48 (s, 2H , NHC H 2 CO), 3.35 (d broad, 2H, OC H 2 ), 1.43 (s, 9 H,
CH 3 ).
HOOC N
H
O N
H
27
2
Boc

Synthesi s of TBDP S-protected hy droxymethylPc 26

A solution of TBDPS - protect ed phthalonitrile 23 (3.2 g, 25 mmol) and unsubstituted
phthalonitril e (3.20 g, 25 mmol) were refluxed in DMAE (30 mL) in the presence of DBU
(0.75 mL, 5mmol), and then Zn(OAc) 2 (2.29 g, 12.5 mmol) added. The reaction was
stirred overnight at r eflux and then cooled to room temperature. To the resulting solu-
tion, 100 mL of H 2 O/MeOH (4/1) were added af fording a blue precipitate that was col-
lected by filtrat ion and dried under vacuum. The TBDPS - substit uted ZnPcs were easily
extracted from the crude m ixture in Soxhlett apparatus r efluxing in acetone over night.
The non - substitut ed ZnPc was discharged, being uns oluble in acet one. The mono - sub-
stitut ed TBDPS - ZnPc was iso lated from the mixture by column c hromatography ( SiO 2 ,
hexane/toluene/ EtOAc 2/2 /0.0 5). The product obtained was t riturated in m ethanol ob-
taining Pc 26 as blue powder. Yield: 410 m g, 10%. 1 H NMR (300 MHz, DMSO - d 6 ) δ
9.20 - 8.71 (m, 8H, P cH), 8.2 0-8.05 (m, 6H, Pc H) , 8. 01 - 7.82 (m, 5H, PcH + CH Ar ), 7.65 -
7.50 (m , 6H, A r), 5.3 5 (s, 2 H, C H 2 ), 1.30 ppm (s, 9H, CH 3 ); UV/Vis (toluene/pyridine =
2mL/10µL, [C] = 2.94 × 10 -6 M): λ max = 673, 6 47, 606, 347 nm; MALDI - TOF MS (DCTB)
(positive mode): 844,2 - 851.2 m/z (isotopic pa ttern) [M] + .

Material and Methods
72
N
N
N
N
N
N
N
Zn
O
Si
N
t
Bu
26

Synthesi s of hyd roxymethy lPc 21

A mixture of TBAF (2 mL) and glacial acetic acid (AcOH) (114. 5 µL) was added
dropwise at room t emperature to a solution of TBDPS - protect ed Pc 26 (50 mg, 0,06
mmol) in dry THF (10 mL) under stirring. The react ion was stirred for 30 min, reduced
to dryness and the resulting crude pur ified by column chr omatography (toluene/ T HF
2:0.05) obtaining Pc 21 as a blue s olid. Yield: 35. 9 mg, 98%. 1 H NMR (300 MHz, DMSO -
d 6 ), δ 9.28 - 9.03 (m, 8 H, PcH), 8.28 -8.01 (m , 7H, Pc H), 5.72 (t, 1H, 3 J H-H = 6 Hz, OH),
5.13 (d, 2H, 3 J H-H = 6 Hz, CH 2 ) ; UV/Vis (THF, [C] = 3.472 × 10 -6 M): λ max = 667, 641,
602, 342 nm; MALDI - TOF MS (dithranol) (posit ive mode): 606.2 - 614. 2 m/z (isotopic
pattern) [M] + .

N
N
N
N
N
N
N
N
Zn
HO
21

Material and Methods
73
Synthesi s of aldehy de - substituted Pc 22

DMSO (2 mL) was added at r oom temperature, under stirr ing, to a mixture of hy-
droxymethylPc 21 (45 mg, 0.074 m mol) and IBX (41.45 mg, 0.148 m mol). The react ion
was stirred for 30 min and then diluted with THF ( 40 mL), and the result ing solution
washed with a NaHCO 3 solution (40 m L) and a brine sol ution (2 × 40 mL). The organic
layer was then dried ov er MgSO 4 , filtered and the f iltrate evaporated obt aining a crude
material which was purified by column chrom atography (toluene/THF 4/ 1). Yield: 39.1
g, 87%. 1 H NMR (300 MHz, DM SO - d 6 ), δ 10.47 (s, 1H, CHO), 9.2 0-8.8 8 (m, 8 H, PcH ),
8.39 - 8.21 (m, 7H, PcH) ; UV/Vis (THF , [C] = 4.651 × 10 -6 M): λ max = 681, 628, 348 nm;
MALDI - TOF M S (dithranol) (positive m ode): 604.2 - 612.2 m/z (isotopic pattern) [M] + .

N
N
N
N
N
N
N
N
Zn
O H
22

Synthesi s of malony l- substituted Pc 31

A solution of met hyl malonyl chloride (13. 46 mg, 0.0986 mmol) in dry THF (1mL)
was added dropwise at 0 ºC to a solution of Pc 21 (30 mg, 0.0493 mmol) in pyridine/dry
THF (3 µL/5 mL). The reaction was stirred at room temperature under ar gon atmos-
phere for 15 min, and the format ion of a fine precipitate observed. Afterwards, THF (100
mL) was added to the react ion mixture, and the resulting solution washed w ith a satu-
rated solution of NH 4 Cl (2 x 50 m L) and brine (100 mL). The or ganic layer was then
dried over MgSO 4 , filter ed and the filtrate evaporated t o dryness. The resulting material
was purified by colum n chromat ography (toluene/ THF 2/ 0.2). Yield: 30 mg, 87%. 1 H
NMR (30 0 MHz, D MSO- d 6 ), δ 9 . 4 2 - 9.08 (m, 8H, Pc H), 8.3 0- 8.05 (m, 7H, P cH), 5 .78 (s,
2H, CH 2 O), 3.85 (s, 2 H, O 2 CCH 2 CO 2 ), 3.81 ppm (s , 3H , OCH 3 ) ; UV/ Vis (THF, [C] =
3.338 × 10 -6 M): λ max = 667, 637, 602, 349 nm; MALDI - TOF (DCTB) (positive m ode):
706.2 - 713.2 m/z (isotopic patt ern) [M] + .

Material and Methods
74
O
O
O
O
N
N
N
N
N
N
N
N
Zn
31

Material and Methods
75
3.1.3 Synthe sis of Zn(II) Pc -C 60 fullerene dyads
Synthesi s of Zn(II )Pc -C 60 fullerene dy a d 18

A mix ture of aldehyde - substituted Pc 22 (15 mg, 0.0025 mmol) , C 60 (45.04 mg,
0.0625 mmol), and sarcosine (7.79 m g, 0.0875 mmol) in dry o - DCB:DMF (30 /5 mL)
was refluxed under argon f or 1h. The crude reaction was diluted w ith THF (25 mL), and
washed with brine (3 × 100 mL). The organic layer was dried over MgSO 4 , filter ed and
the filtrate evaporated obtaining a crude mater ial which was purified by colum n chro-
matography (t oluene/THF 2:0.05). Finally, the product obt ained was triturated using
MeOH. Yield: 20.6 mg, 60%. 1 H NMR (300 MHz, CS 2 /THF - d 8 (1/1 )), δ 9.47 - 9. 33(m, 8H,
PcH), 8.15 - 8.02 (m, 7H, P cH), 5.7 0 (s, 1H, CH pyrrolidine ), 5.30 (d , 1H , C H H py rrolidine , 2 J H-
H = 12 Hz), 4.59 (d, 1H, C H H, 2 J H-H = 12 Hz), 1.29 (s, 3H, CH 3 ) ; UV/Vis ( toluene, [C] =
7.097 × 10 -6 M): λ max = 676, 609, 338 nm; MAL DI - TOF (DCTB) (pos itive mode): 1351. 1 -
1359.1 m/z (isotopic pat tern) [M] + , 631.0 - 637.0 m /z (isotopic pattern) [M -C 60 ] + .

N
N N
N
N
N
N
N
N
Zn
18

Material and Methods
76
Synthesi s of Zn(II )Pc -C 60 fullerene dy a d 19

A mixture of hydroxymethyl - substit uted Pc 21 (30 mg, 0.033 mmol), PCBA (20.34
mg, 0.033 mmol), 1 - etil -3-(3- dimetilaminopro pil) - carbodiim ide (EDCI) (20.49 mg, 0. 132
mmol) in o - DCB/pyridine (2/ 0.15 mL) was stirred overnight at room temperature under
argon atmosphere. Aft er this time, the solution was directly columned (t oluene/ THF
2:0.07) obtaining Pc 19 as a dark green product . Yield: 84 mg, 86%. 1 H NMR (300 MHz,
CS 2 /THF - d 8 1/1), δ 9.41 - 9.22 (m, 8H, P cH), 8.20 - 8.0 7 (m, 7H, Pc H), 7.95 (d , 2H, CH 2 O),
7.52 (t, 2H, CH ar yl ), 4.4 5 (t, 1H , CH aryl ), 7.20-7 .05 (m, 2H , CH aryl ), 5.6 8 (s , CH 2 ), 3.00
(m, CH 2 ), 2 .75 (m, CH 2 ) ; UV/Vis (toluene, [C] = 4. 626 × 10 -6 M): λ max = 673, 612, 335
nm; MALD I- TOF (DCTB) (positive mode): 1484.2 - 1492.2 m/z (isotopic patter n [M] + .

N
N
N
N
N
N
N
N
Zn
O
O
19

Synthesi s of Zn(II )Pc -C 60 fullerene dy a d 20

A solution of malonyl - subst ituted Pc 31 (20 mg, 0. 028 mmol) in dry DMF (2ml) was
added to a solution cont aining C 60 ( 40.35 mg, 0.056 m mol) and CBr 4 (33. 81 mg, 0.102
mmol) in dry toluene (15 mL) previously sonicated for 15 min. The solution was stirred
for 10 minutes and t hen DBU (25.88 mg, 0.17 mmol) added. The mixture was stirred at
room temperatur e, in the darkness, f or 48h. After this time the solvent was removed
and the resulting soli d purified by c olumn chromatogr aphy (toluene/ THF 2/0.03). The
compound obtained was finally tr iturated in M eOH obtaining a da rk green solid. Yi eld:
30 mg, 70%. 1 H NM R (300 MHz , CS 2 /THF - d 8 1/1), δ 9 . 38 - 9.12 (m, 8H, P cH), 8.19 - 8.05
(m, 7H, Pc H), 6.08 (s, 2H , CH 2 O), 4.2 0 ppm (s, 3 H, OCH 3 ) ; UV/Vis (t oluene, [C] = 1.122
× 10 -5 M): λ max = 673, 611, 332 nm; MALDI - TOF (DCTB) (positive m ode): 1424.1 -
1432.1 m/z (isotopic pat tern) [M] + .

Material and Methods
77
O
O
O
O
N
N
N
N
N
N
N
N
Zn
20

Synthesi s of Zn(II )Pc -C 60 fullerene dy a d 30

A mixture of Pc aldehy de 22 (15 mg, 0. 03 mmol), C 60 fullerene (45 m g, 0.06 mmol),
and aminoacid 27 (20.8 m g, 0.09 mmol) were added to a round bott om flask under
argon and a mixture of dry o - DCB/DMF ( 30/5 mL) added. The r eaction mixture wa s
stirred at 120 ºC overnight and t hen the solvent removed obtaining a c rude product that
was purified by column c hromatography (t oluene/THF 2/0.1) obtaining dyad 30 as a
blue solid. Yield: 20. 1 g, 58%. 1 H NMR (300 MHz, THF - d 8 ), δ 9.51 - 9.01 (m, 8H , PcH),
8.15 - 7.95 (m, 7H, P cH), 6.0 0 (s broad , 2H, C H pyrrolidine + NHC=O) , 5.61 (d, 1H, 2 J H-H =
12 Hz, CH 2 pyrrolidine ) , 4.65 (d, 1H, 2 J H-H = 12 Hz, CH 2pyrrolidi ne ), 4.05 (t, 2H, CH 2 ), 3.91 (t,
2H, CH 2 ), 3.81 (t, 2H, C H 2 ), 3.31 (t, 2H, C H 2 ), 3.2 5 (m , 4H, C H 2 ), 1.4 1 (s, 9 H, CCH 3 );
UV/Vis (toluene, [ C] = 6.061 × 10 -6 M): λ max = 675, 608, 335 nm; MALDI - TOF MS
(dithranol) (positive m ode): 1568.3 - 1576.3 m /z (isotopic patter n) [M] + , 848.3 - 853.2 m/z
(isotopic pat tern) [M -C 60 ] + , 3136.1 - 3147. 5 m/z (isotopic patt ern) [2M] + .

N
N N
N
N
N
N
N
N
Zn O
O
HN
Boc
30

Material and Methods
78
Synthesi s of Zn(I I)Pc - C 60 fullerene 17

To a suspension of 30 (9 mg, 0.0057 mmol) in CH 2 Cl 2 (2 mL), 1 mL of TFA was added
and the solution st irred overnight . The salt was precip itated f rom the reaction m ixture,
filtered, and the s olid washed with hexane and dried under vacuum obtaining fullerene
salt 17 as a blue solid. Yield: 8.8 g, 98%. UV/Vis (TH F, [C] = 5.481 × 10 -6 M): λ max =
671, 606, 346 nm; MALDI - TO F (DCTB) (positive mo de): 1468.3 - 1476. 3 m/z (isot opic
pattern) [M -H- TFA] + , 748.2 - 755.2 m/z (isotopic patter n) [M -H- TFA -C 60 ] + .

N
N N
N
N
N
N
N
N
Zn O
O
NH
3
17
TFA

Material and Methods
79
3.1.4 Exper iment al tech nique s
Atomic For ce Mic roscop y (AFM)
AFM is primar ily used to exami ne surface m orphology and to measure int eraction
forces between the t ip and sample ( i. e. force spect roscopy). 102 In a contra st to S TM, AF M
is able to measur e any solid mater ial without the condition of surface condu ctivity. Atom ic
force microscope cons ists of a sharp nanometer - s ized probe attached to a springboard or
V- shaped cantilever w hich is posit ioned over a surf ace deposited sam ple as shown in Fi g-
ure 2 8a. Piezoelectric scanners control subnanomet er movements in the x , y , and z di-
mensions, then images ar e compiled line - by - line as the sam ple is raster scanned. Addi-
tionally, a laser beam is focused on the bac k - tip of the cantilever to measure changes i n
cantilever def lection. Forc es between the sam ple and the probe caused eit her by sam ple
topography changes or the forces between t he sample and the probe result in cantilever
deflection. These cha nges in deflection are conver ted to force using Hooke’s law, which
allows for the quantification of forces on the pN scale and the employment of force spec-
troscopy.
The cantilever is excited into osc illations near the r esonant frequency of the cantilever.
The instrument t hen maintains constant oscillation am plitudes and taps along the surf ace.
Any displacement in the z dimension is a measure of the height variation on the sample.
Force spectrosc opy is used to ex amine specific inter action forces between the tip and
sample at forces as low as 10 pN. A typical measur ement is diagramed in Figure 8b, where
the tip starts far from the surface (1), comes in contact with the surface (2), applies a spe-
cific loading force to the sample (3), and then the process is repeated in reverse wh erein
the tip retracts from the surfac e. When an interaction between the tip and sample occurs ,
step (4) is obser ved. At the point the tip overcom es the force hol ding it at t he surface the
tip breaks away form the sample (5) , and retracts to its origina l position (1). Therefore, t his
process enables force s pectroscopy and allows for the direct quantification o f interact ion
strength between t he probe and the sample .

102 G. Haugstad, Atomic Force Microscopy: Understanding Basic Modes and Advance d Applica-
tions, John Wiley & Sons, Inc., 2012.

Material and Methods
80
Figure 28. Schematic representation of a) AFM components, b) AFM f orce - distance meas-
urement.

Figure 29. Different operational modes of AFM measurement.
The AFM techniques have been developed f or twenty years during which three main
operation modes have been est ablished (Figure 2 9). In a Contact (Static) mode, t he can-
tilever is in full co ntact w ith t he surface. The repulsive int eraction forces (topography) is
determined by measuring the stat ic deflection of the cantilever. Th is techniqu e is capable
of detecting the t opography of at omic scale r esolution. Due to t he fact that strong repulsi ve
inter action for ce and friction take place betw een tip and surf ace, this technique is harsh to
the sample. In a Non - contact (Dynamic) mode , the cantilever is usually driven close to it s
resonant frequency, w ith vibration amplitudes less t han 100 nm. The cantilever dr iver usu-
ally is a pie zo - electric elem ent, but many experiments have bee n perform ed with electro-
static, ma gneti c, thermo - optic or acoustical coupling driver s. The driver is m ounted to the
head of the microscope and the chip with the cant ilever is mounted on top of the driver.
The interact ion force is m odulating the vibrat ion frequen cy, amplitude and p hase. Fr om the
oscillator modulati on can be distinguished surf ace topography. Tapping mod e represents
a combination of t he stat ic and dynamic modes. It allows minimiz ing friction bet ween tip
and measured surf ace which is strongly present in the cont act mode. The oscillating ti p
only touches the m easured surfac e at maximum de t ection of t he cantilever towards meas-
ured surface. In this moment , there is direct mechanica l contact with str ong repulsive in-
teraction forces bet ween tip and surface. Due to the dynamic operation of the cantilever
and very shor t contact t ime this technique c an be considered as n on - contact. We will e m-
ploy the tapping mode t echnique in our study.

Material and Methods
81
Transmi ssion El ectr on Microsc opy (TEM)
TEM is a form of microscopy in which a be am of electr ons transmit s through an ex-
tremely thin specim en and inter acts with the specim en when p assing through it . 103 The
formation of images in a TEM can be explaine d by an optical electron beam diagram (Fig-
ure 30). Electr ons are usually generated in an electr on microsc ope by a thermionic emis-
sion from tungsten, in the same m anner as a light bul b, or alternat ively by field elect ron
emission. The electrons ar e then accelerated by an ele ctric potential (m easured in volts)
and focused by electrostat ic and electromagnetic lenses ont o the sample. The transmitted
beam contains inform ation about elect ron density, phase and per iodicity. This beam is
used to form an image.

Figu re 3 0. The optical electron beam diagram of TEM.
Transmission elect ron micr oscopes provide im ages with resolutions which are several
thousand times higher t han the highest resolution in a visible - light m icroscope because of
the smaller de Broglie wavelengt h of electr ons. Nevertheless, the magnificat ion provided
in a TEM image is in contrast to the absorption of the electrons in the material, which is
primarily due to the thickness or composit ion of the material. For a sample to be transparent
to electrons, t he sample must be thin eno ugh to transmit sufficient electr ons such that
enough intensity f alls on the screen to give an image. A detrimental effect of ionizing radi-
ation is that it can damage the specimen, particularly polymer s and most organics. So me
aspects of beam damage tur n worse at higher voltage s.

103 C. B. Carter, Transmission Electron Microscopy: A Textbook for Mat erial Science, 2nd Ed.,
Springer, New York, 2009.

Material and Methods
82
Transient ab sorptio n spectrosco py
The absorption of UV and/or visible light by an organic molecule c auses the prom otion
of an electron from an initially oc cupied, low energy orbital to a higher energy, previou sly
unoccupied or bital (Figure 3 1). 104 When such event happens, an excited st ate of the mol-
ecule is generated. Following light absorpt ion, several processes c ontribute to the relaxa-
tion of the m olecule and its ret urn to the ground state. These processes can be either
radiative (a photon is emitt ed during the transition) or no n - radiativ e (the loss of energy is
emitted as heat - infrared radiatio n - instead of UV - visib le light) .
Figure 3 1 . Yablonsk i diagram showing processes taking place in a chromophor e upon pho-
toexcitation.
After light absorption, and in the presence of other molecules in solut ion, chromo-
phores can also undergo t ransformations lik e energy transfer , PET or other chemical re-
actions. These events are among the f astest events in natur e. They occur on the t ime
scales ranging fr om femtos econds to nanoseconds. The develop ment of ult rafast laser
systems has enabled investigation of these events in real tim e. Femtosecond trans ient
absorptio n spectr oscopy , also referred t o as femtosecond pump - probe spect roscopy , is
one of the most common tools to investigate ultraf ast photochemical reactio ns. In particu-
lar, this technique all ows the observat ion of light - induced intr a - and inter molecular pro-
cesses on the time scale of the motion of atoms and electrons and, thus, provides one of

104 a) Modern Molecular Photochemistry, N. J. Turro, University Science Books, 1991 ; b) Principles
of Fluorescence Spectroscopy, 3rd Ed . , J. R. Lackowicz, Springer Science, 2006.

Material and Methods
83
the most effective methods f or studying the behavior of transient species like radicals, ions
and excited stat es. 105
The principle of transient abs orption spectros copy is shown in Figure 32a. Here, the
absorbance at a par ticular wavel ength or range of wavelengths of a sample is meas ured
as a function of time after excitation by a flash of light. In a typical experiment, both the
light for excitation – pump – and the light for measuring the absorbance – probe – are
generated by a pulsed laser . In each measurement, the absorbance of t he probe pulse by
the sample is recorded t wice, the second tim e after a certain delay wit h respect to the pump
pulse. Thus, in t he first shot, the absorbanc e of the sam ple in the ground st ate is recorded.
In contrast, the second t ime the probe pulse hits t he fraction of molecules that has been
promoted to an electronica lly excited st ate by means of t he pump pulse. A differ ence ab-
sorption spectrum is then calculated. By changing the d elay, a Δ OD pr ofile as a function
of time and wavelength is obtain ed (Figure 32b). This profile contains inform ation on the
dynamic processes occur ring in the system under study. Specifically, the analysis of t he
differential absorpt ion spectr a (ΔOD vs wavelength) allows identi fication of the excited
states or transient species form ed upon excitation of the chromophore. On the other hand,
the study of decay pr ofiles (ΔOD vs time) provides in formation on t he kinetics of phot o-
physical or photochem ical processes.
Fi gure 3 2 . (a) Schematic representation of transient absorp tion (pump - probe) spectroscopy.
(b) UV- light - induced changes in optical density (ΔOD) of pyrene s howing dy namics of the S 1 ex-
cited state absorption (ESA) and ground state bleaching (GSB ) contribution.

105 R. Berera, R. van Grondelle, J. T. M. Kennis, Photosynth. Res. , 2009 , 101 , 105 - 118.

Material and Methods
84
In general, the bands observed in a t ransient absor ption spectrum arise from thr ee
kinds of contributions:
- Ground - state bleach : as a fraction of mole cules has been prom oted to t he excited
state through the act ion of the pump pulse, population of the ground state has decreased.
As a consequence, gr ound state absorpt ion of the excited sample is lower than t he non -
excited. Hence, a negative signal in the ΔO D spectr um is observed. This signa l appears i n
the region of the gr ound state absorption (it mirr ors the UV - Vis absorption band ).
- Stimulated emiss ion : upon popu lation of the fir st excited state, stimulated emission
may take place when the second pr obe pulse passes thr ough the sam ple. This process
will occur only for optically allowed t ransitions, and its spectral profile general ly reflects the
fluorescence s pectrum of the chromophore. The photon thus generated is em itted in the
same direction as the one of the probe pulse, and bot h are detected. Hence, stimulated
emission produces a negat ive ΔOD signal. Many c hromophores present such a low Stokes
shift that both ground state bleach and st imulated emission bands overlap an d merge into
one band.
- Excited s tate absorption : optical ly allowed tr ansitions from the excited stat es (popu-
lated upon excitat ion with the pum p pulse) to higher st ates might exist in certain wave length
regions. Absor ption of the probe pulse at these r egions is then observed as a posit ive band
in the ΔOD spectru m.

Material and Methods
85
3.2 DNA int erstr and cross - l inkin g on surf ace
3.2.1 Chem icals and inst rume ntation
Chemica ls
Methylene Blue (MB) , N - hydroxysuccinimi de ( NHS), N - (3- dimethylaminopropyl) - N′ -
ethylcarbodiimid e hydrochlor ide (EDC), and molecular biol ogy grade buff er reagents wer e
purchased fr om Sigma - Aldrich and used wit hout further purif ication . The TT1 photosens i-
tizer was prepared acc ording to a method descr ibed previously. 8
Buffers used for ELISA: PBS buffer ( 1 mM sodium phosphate, 0. 015 M NaCl, pH 7.6),
substrate buffer (220 mM potass ium dihydrogen citrat e, 0.5 mM sorbic acid potassium salt,
pH 4.0), TMB solution ( 40 mM tetramethylbenzidine and 8 mM tetrabutylamm onium boro-
hydride in N,N - dimethylacetam ide), substr ate solution (540 µL TMB solution and 3 mM
hydrogen peroxi de in 21.5 mL of substrate buffer) . SPR Experiments were perfor med at
25 °C using PBST ( 0.05% (v /v) Tween™ 20) as running buff er. All buffers were filtrated
using 0. 2 µm pore filters. 3,3’,5,5’ - Tetram ethylbenzidine ( TMB) and Tween™ 20 pure were
from Serva .
The synthesis of the furan - modif ied oligonucleotides w as performed as described
previously.92 Rea gents f or the synthesis of 5’ - biotin furan - m odified ODNs were obtained
from Glen Research. The c omplementar y 5’ - FITC modified ODNs, strept avidin - coated 96 -
well microtiter plates and anti - FITC - HRP - monoclo nal antibody (Lot P319, 0.73 mg/mL)
from mouse were purc hased from BioTeZ (Berlin, Germany).
M ouse m onoclonal capt ure antibodies (FAB P 10 - 7904 : Lot 2622, 6 mg/L; Troponin I
10 - T79H : Lot 2423, 5 mg/mL; CRP 10 - C189 B: Lot 2612 , 4.5 mg/mL ), human cardiac pro-
teins ( Troponin I 30 - AT63: Lot A15032430, 0, 621 mg/ mL ; FABP 30 - 1006 , Lot A1603 14 30,
2,2 mg /mL; CR P 30 - AC05S, Lot A26031430 2,5 mg/L) and m ouse monoclonal detect ion
antibodies ( FABP 10 - 7904: Lot 2421, 1,64 mg/mL; Troponin I 10 - 7967: Lot 3526, 0,52
mg/mL; CRP 10 - C189A: Lot 2867, 1,49 mg/mL) were purchased from Fitzgerald Industr ies
International (Acton, USA) . N- Hydroxysucc inimide (NHS) activat ed glass slides were pur-
chased fr om SCHOTT NEXTE RION. Cy3 M aleimide M ono - Reactive dye (PA23031) was
obt ained from G E Healthcare Life Scienc es and their c onjugation protocol was f ollowed for
the synthesis of t he antibody - Cy 3 dye conjugate .

Material and Methods
86
I ns trumentatio n
The SPR measurement s were perform ed using a Biacore™ T200 device ( T200 Sen-
sitivity Enhanced, GE Healthc are Bio - Sciences AB, Uppsala, Swed en) and dextran - m odi-
fied chips (G - COOH- sp) from Ssens (Enschede, The Netherlands) .
The illuminator KL1500 LCD 150 W from Schott was used as a c old red ligh t source
for irradiation. The streptavidi n - coated microt iter plates were read us ing a SpectraMax-
Plus384 from Molecular Devices controlled by Soft Max® Pro software (v 5.2, M olecular
Devices). The automat ic 96 - channel plate washer, ELx405 Selec t™ from BioTek Instru-
ments was used to wash the plates and glass slides. The plate shaker was a Titr amax 101
(Heidolph, Schwabac h, Germany) . An ABI 394 DNA synthesizer was used f or ODN syn-
thesis. The glass slide was sca n ned using a n Axon GenePix 4300A r eader controlled by
GenePix Pro 7.1 sof tware. The laser power and PMT wer e set to 60% and 400 respec-
tively. M icroarray printing was per formed using a BioOd y ssey Call igrapher MiniArrayer
(Bio - RadLa boratories, I nc. USA) .

Material and Methods
87
3.2.2 Experimental pro cedures
3.2.2.1 S urface P lasm on R esonance
The following schem e shows the method used to detect the ICL of immobilized ODNs.
As a result of the ICL form ation, the short ODN should enhance its stability towards rege n-
eration conditions us ing a denatur ing Na 2 CO 3 solution. Figure 33a shows how the hybrid-
ized ODNs are eff iciently separat ed after using Na 2 CO 3 for r egeneration . O xidation of the
furan using 1 O 2 results in the formation of a r ugged ICL, rendering the resulting shor t ODN
duplex stable toward s Na 2 CO 3 (Figure 33b) . Therefore, t he binding of the antibody is still
possible. Sensorgrams were double - referenced using an em pty flow cell or a f low cell with
non - complement ary DNA for subtraction of nonspecific effects and buffer injections for cor-
rection for artifacts .

O
O
NA Au Chip

Immobilization Hybridization
ICL

1 O 2
Furan

O O O
O
a)
b)
Na
2
CO
3
Na
2
CO
3
Antibody
binding
Antibody
binding
Oxidation formation
O O

Figure 33 . Schematic comparison between hybridized and cross - linked ODN.

Material and Methods
88
General procedure for immobili zation of bi otinylate d furan - modi fied
ODNs
The gold chip was coated wit h neutravidin (NA) by EDC/ NHS coupling, follo wed by the
immobiliz ation of biot inylated OD Ns. For this, t he activation of the carboxyl ic groups was
done by injecting into the Biacore™ device channel 0. 4/0.1 M EDC/NHS for 3 min followed
by 100 μg/mL solution of NA i n 10 mM acetate buffer pH 5.5 and coupling was allowed for
13 min. Unmodified react ive sites were then blocked with 1 M ethanolamin e pH 8.5 for 7
min. The respective biotinylated and furan - modified captur e oligonucleotide (2 µM) in
PBST pH 7 .6 r unning buff er was then injected for 2 m in. Blocking of the NA reactive sit es
was perf ormed by usi ng 10 μM biotin in r unning buffer for 1 min. All injections wer e car ried
out at a fl ow rate of 1 0 μL/mi n.
Hybridiza tion proc edure
In order to obtain the association and dissociat ion curves of the DNA duplex, the com-
plementary FITC - mo dified ODNs were all owed to hybridize t o the capture O DNs by inject-
ing different concent rations (0.01 - 10 µM) in running buf fer for 2 min at a flow rat e of 30
μL/min. Dissociation of hybridize d imm obilized O DNs was achieved by injec ting r unning
buffer fo r 1 0 min at a continuous flow r ate of 30 μL/min.
Cross - linking experimen ts
Before taking the chip out of the SPR instrument for the ICL exper iments , the bindin g
of 48 nM HRP - labelled anti - FI TC antibody to hybridize d FITC - ODN was perform ed, then
50 mM Na 2 CO 3 was injected f or 1 min as a test for the instability of the DNA duplex. Hy-
bridization and cros s - linking were done in par allel by adding 100 μL of a m ixture c ontaining
1 μM FITC - ODN and the photose nsitizer (5 µM MB or 5 µM TT1) to produce the necessar y
1 O 2 . Afterwards , the chip was irradiated with r ed light (658 nm, 8 cm distance) for 45 min
at 350 rpm at room temperature. Aft er washing with running buffer and drying of the chip,
it was again mounted into the SPR instrument, where a first regeneration was carried out
by 50 mM Na 2 CO 3 for 1 min followed by th e binding of 48 nM HRP - labelled ant i - FITC
antibody in running b uffer.The immobilized ODN1 was cross - linked by incubat ion with 1
μM C.ODN1 in PBS pH 7.6 buf fer and 5 µM MB. TT1 was initially dissolv ed in (DMSO due
to its low water solubili ty; the final concentr ation of TT1 was 5 µM.

Material and Methods
89
Hybridi zation speci ficity experiments
O li gonucleotide sequ ences ODN2, 3 and 4 were im mobilized using the met hod de-
scribed before flowing 2 µM solutions in separate f low cells and tested simultaneously f or
hybridizatio n specificity. The complement ary sequences C.ODN2, 3 and 4 were tested
alone and in the mixture . For the hybridization, 1 µM solutions of the C.ODNs in running
buffer were inj ected over 2 min. Dissoc iation of hybridize d ODNs was eff ected by injecting
running buffer for 2 min. After each hybridiz ation, the surface was r egenerated flushing 1
min with 50 m M Na 2 CO 3 to prepare the c hip for the next hybridiz ation. Kinetic dat a were
obtained using f ive successive inj ections of r ecombinant G FP (Green Fluorescent Protein;
single - cycle kinet ics). Loading of the chip was calculat ed consider ing one resonance unit
(RU) corresponding to one pg/mm² of bound biologic m aterial. 106 Fur ther data analysis of
obtained sensorgr ams was perfor med using Biacore™ T200 Evalu ation Software v1. 0 .

106 Karlsson, R.; Fagerstam, L.; Nilshans, H.; Persson, B., Analysis of activ e antibody
concentration. Separation of affinity and concentration parameters. J. Imm unol. Methods, 1993,
166 , 75 - 84.

Material and Methods
90
3.2.2.2 ELISA
The streptavidin - cover ed microt iter plate was coat ed with 100 µL of ODN1 (0.1 µM) in
PBS buffer and incubat ed for 30 min on a plate shaker at 750 rpm. The plate was then
washed with wash ing buffer . The immobilize d ODN1 was allowed to hybridize f or 45 min
with 100 µL of 0.1 µM C.ODN1 at 350 rpm. The interstr and cross - linking react ion was
carried out at a final concent ration of 5 μM MB by adding 20 μL of an accordi ngly concen-
trated solution to each w ell. Afterward , the plate was irradiated with red light (658 nm; 8
cm distance) f or 45 min at 350 rpm . After discarding the mixture 50 mM Na 2 CO 3 (150
µL/well) was added and the plate was shaken at 750 rpm for regeneration. The plate was
washed after 10 min with washing buf fer. A dilution of 1:10,000 HRP - label led anti - FITC
antibody in PBS buffer ( 100 µL/well) was incubated for 45 min at 750 rpm. After washing
away unbound antibo dy, 100 µL of substrate solution was added to t he wells and incubated
while shaking at 750 r pm. The reaction was st opped after 10 m in by adding 50 µL of 1 M
sulphuric ac id to each well. The o ptical densi ty was measured at 450 nm using 650 nm as
reference. All steps were carr ied out at room t emperature. Exper iments under dark and
natura l light conditions were done in parallel.

3.2.2.3 MALDI - TOF s ample prepa ration procedures
DNA oligonuc leotide
To 20 mg of α - cyano -4- hydroxycinnamic acid ( CHCA) matrix in an Eppendorf was
added 1 ml of 90:10 acetonit rile/0,1 % trifluoracetic acid (TFA) and vortex 1minute to dis-
solve (saturat ed solution 1 , some undissolve d matrix will remain) . After, we re mixing 345 μ L
of solution 1 with 24 μ L 100Mm Amm onium dihydrogen phosph ate (NH 4 H 2 PO 4 ) and 24
μ L10% TFA solution to obtain the matrix solution. S ample solution was mix ed 50:50 with
0.025% TFA and centrifuge d usi ng a zeba spin column . The filtrate was t hen mix with
matrix solutio n at sample to matrix ratios of 50:50. For the c rystalli zation was: d eposit ed
1 μl of this final mix onto the Anchor t arget plate and allow to air dry .

Material and Methods
91
Antibod y - oligonuc leotide conjuga te
To 20 mg of Sinapinic Acid (SA) matrix in an Eppendorf was added 1 m l of 70:30
water/0,1 % TFA . and vortex 1minut e to dissolve. Sample soluti on was mixed 50:50 with
water and centrif uged using a zeba spin colum n. 0.5 - µL of matrix stock solution was pr e -
spoted on the silver anchor tar get plate. After this spot was dried , 0.5 µL of protein sample
solution was added on t op. The matrix sample cr ystals were allow ed to form through s ol-
vent evaporation.

3.2.2.4 Cross- linking in s olution
The product f rom the cros s- linking in solutio n of O DN1 - C. ODN1 using 5 µL M B as photo-
sensitizer was analysed by MALDI - TOF, RP - HPLC and PAGE experiments. Mass spectra
were acquired using a Bruker Autoflex II MALDI mass spectromet er operated with a nitro-
gen laser. RP - HPL C and PAG E exper iment s were perfor med following previously pub-
lished procedures. 95

Figure 34 . A) Reversed- phase HPLC chromatograms of the reacti on mixture of furan - modified
ODN1 and C.ODN1 (after annealing in a 1:1 ratio to obtain the duplex) with 5μM MB before reaction
and after 10 and 20 minutes of irradiation. B) Denaturing PAGE results of the cross - link reaction.
XL represents .

5 10 15 20 25 30
m AU
0
10
20
30
40

DAD1 A, Sig=260,16 Ref=off (D:\ DATA\13- 08-21\NDL000003.D)

5 10 15 20 25 30
m AU
0
10
20
30
40

DAD1 A, Sig=260,16 Ref=off (D:\ DATA\13- 08-21\NDL000004.D)

5 10 15 20 25 30
m AU
-5
0
5
10
15
20
25
30
35

DAD1 A, Sig=260,16 Ref=off (D:\ DATA\13- 08-21\NDL000005.D)

XL

C.ODN1
- ODN1

10 min

0 min

20 min

0 min

10 min

20 min

XL

XL

C.ODN1

ODN1

C.ODN1

C.ODN1

ODN1

ODN1

A)

B)

Material and Methods
92

Figure 35 . MAL DI - TOF mass spectrum of the cross - linked ol igonucleotide sequences ODN1 -
C.ODN1 in solution (calculated mass 8263.68 D a; observed mass [M+ H+]/2= 4132.332 and [M+
H+]/4= 2066.172 .

Material and Methods
93
3.2.3 Antibo dy - oligonucle otide c onjugation
The conjugation of the antibody with t he three oligonucleotides (10 - mer, 12 - mer, Poly
T 12 - mer (Sp 12mer )) was performed f ollowing the method describe Söderberg et al. 123
Compared with t he native antib ody, the MALDI - TO F m ass spectr um of all conj ugates were
shifted ~ 4 Da, which are cor responding to the m ass of the oligonucleotide, indicating suc-
cessful conjugation wit h the corr esponding oligonucleoti de.

Figure 3 6. C omparison of MALDI - T OF ma ss spectrum of the native antibody with antibody -
oligonucleotide conjugates .

Native Antibody

Antibody - 10mer

Antibody - 12mer

Antibody - Sp 12mer

Material and Methods
94
3.2.4 Cross - reactivi ty experiments o f the multi plex im-
munoassay

Surface pl asmon reson a n ce
Th e capture antibody was imm obilized o n the gold chip by EDC/NHS coup ling. For
this, the activation of the carboxyli c groups was done by injecting into the Biacore™ device
channel 0.4/0.1 M EDC/NHS for 3 min followed by 20 μg/mL solution of the cor responding
capture antibody in each flow cell , the coupling was allowed f or 13 min. Unmodified reac-
tive sites were then blocked wit h 1 M ethanolamine pH 8.5 for 7 min. The m ixture of cardiac
biomarkers (1- 500 ng/mL ) was diss olved in PBST run ning buffer and was t hen injected
for 2 min in all flow cells. Afterwar ds, 1 μg/mL of detection antibody was a dded in PBST
buffer.
Microarray
The capture antibody was dilute d to 20 μg/mL final conc entration using printin g buffer
(150 mM PBS, pH 8.5, 5 % glycerol) , spotted onto NHS glass slid es in 60% humidity and
incubated ov er night at room temper ature. The slide was then washed twice with 30 mL of
PBST buffer f or 10 min at room temperatur e and dried under a stream of Ar g on gas. Sub-
sequently, the slide was immersed in 30 mL of 70 mM et h anolamide in PBS for 1 h at room
temperature to block the surface. Blocking and washin g processes were per formed with
gentle shaking on a s haking plate. The slide was place d on a micr o array hybridizatio n
cassette and t he mixture of analyt es (100 μ L /wel l ) diluted in different concentrations r ang-
ing from 1000 to 100 n g/ mL in PB S T s olution cont aining 10% BSA. And then incubated for
1 h at room temperature. The slide was then washed manually with P BST solution. Finally ,
the slide was incubated with 1 μ g/mL of detection antibody - Cy 3 conjugat e ( 100 μ L/well) for
6 0 min at room temper ature. Fluoresce nce i ntensity ( mean value minus back ground ) of
each spot on the slid e were expr essed normalized as av erage and standard deviatio n of
three replicate spot s.

Material and Methods
95
3.2.5 Experimental tech niques

DNA hybridization can be detect ed by different techniques based on gr avimetric, elec-
trochemical , or optical detection. Two opt ical detection techniques , surface plasm on reso-
nance (SPR) and an enzym e - catalyzed colorim etric assay, are going to be described.

Surface Pl asmon Reso nance (SPR)

This technique has been used fr equently to develop biosensors, especially to detect
DNA - DNA hybridizati on. SPR is an o ptical t echnique wh ich allows the det ection and quan -
tification of molecular i nteractions - such as prot eins, nucleic acid, peptides a nd small mol-
ecules - in real tim e, without the use of labels. O ne of the interact ants (e.g. DNA probe ) is
immobilized on t he sensor surface , usually com posed of a 50 nm - layer of gold o n a glass
surface, and t he other interactant is passed o ver the gold surf ace in solution by a micr oflu-
idic flow system . The opposite, glass side of the sensor st ands has attached t o it a prism
ill uminated by polarized light fr om a near infrared LED. The focus under the sensor is set
to conditions of total internal ref lection and a det ector monit ors the intensity of the light
reflected (Figure 37 ). 107

Figure 37 . Surface Plasmon Resonance principle . Reproduced with copyrights permission
from The Nature Publising Group .
The light under conditions of total internal reflect ion leaks an electrom agnetic wave
field (evanescent ) across the gold film into the sample solution. At a certain angle of inci-
dent light, the evanes cent wave excites ele ctrons in the gold producing t he form ation of

107 M. A. Cooper, Nat Rev Drug Discov , 2002 , 1, 515 - 528.

Material and Methods
96
surface plasm ons (electron char ge density waves) within the go ld film with a drop in the
intensity of the r eflected light angl e called SP R angle. When a cha nge in mass occurs near
the sensor chip surface ( as a binding result), the angle of the light at which SPR should
occur shifts which can be described as a change in refractive index near the sensor sur-
face.
A sensorgram from an SPR exp eriment is shown in F igure 38 . It sh ows responses
measured in r esonance units (RU ) in real time. The sensorgram provides ess entially two
kinds of informat ion: the rate of interact ion (association, dissociation), which provides a)
information on kinetic r ate constants ( k ass , k dis s ) and anal yte concentrat ion, and b) the bind-
ing level, which prov ides informat ion on affinity constants. The sens orgram is a plot of
response in resonance units versus t ime in seconds, which is presented continuous ly in
real time. As the analyt e binds to the surf ace, the refractive index of the medium adjacent
to the sensor surfac e increases, which leads to an increase in the resonance signal and
the association is measured. At the end of the analyte injection, the analyte solution is
replaced by a syst em buffer , and the receptor - analyte complex is allo wed to dissociate. A
decrease in mass occurs due to dissociatio n and is measured. At equilibrium, by def inition,
the amount of analyt e that is ass ociating and dissoc iating with t he receptor molecule is
equal.
Concentration
Kinetics
Time (s)
SPR Response (RU)
Dissociation
Association Regeneration

Figure 38 . Typical phases and sensorgram of an SPR m easurement.

Material and Methods
97
One RU corresponds to 0. 0001° shift of the SPR angle, or to 1 pg/mm 2 of immobilized
receptor molecule on the sensor sur face. The response level at equilibrium is related to
the concentrat ion of an active analyte in the sample. It is possible to disturb the binding of
many complexes and regenerate the f ree receptor on the surf ace by a regeneration sol u-
tion (for example, with high salt concent ration or low pH). The aff inity of the interaction ca n
be calculated fr om the ratio of the rate constants (k d =k diss /k as s ) or by a linear or nonlinear
fitting of the response at equilibrium at varying concentrations of analyte.

Material and Methods
98
Enzyme - catalyzed colo rimetric assay
In the enzyme - catalyzed color imetric assay used the optical densi ty generated is pro-
portional to the amount of analyte. This assay was perform ed using a 96 - well microtit er
plate . For a very sensitive det ection of DNA hybridizat ion, the colour - generat ing reaction
is catalyzed by hor seradish peroxidase ( HRP) bound to a recogniti on molecule ( e.g. Strep-
tavidin) towards t he hybridized biotin - labeled DNA str and.
HRP catalyzes the reaction of the peroxidase substrat e 3,3,5,5 - tetramethylbenzidine
(TMB) in the presence of hy drogen peroxide (Figure 39a) . HRP converts the hydrogen
peroxide to water, obtaining the two hydr ogen atoms it needs for this from TMB, which acts
as a donor molecule. So, at the s ame time that HRP is being reduced, TM B is oxidized.
The TMB oxidation pathway by H 2 O 2 i s described in Figure 39 b. 108 TMB is colourless and
its oxidation produc es a blue colour wit h the maximum absor bance at 652 nm. Af ter addi-
tion of sulfuric acid, the react ion is stopped, and the blue colour changes to yellow with
maximum absorbanc e at 450 nm.

STV-HRP
Biotin-DNA
TMBred TMBox
H
2
O
H
2
O
2
a) b)

Fi gure 39 . a) Schematic illustration of DNA hybridization detection via HRP - catalyzed TMB
oxidation. b) Chemical structures resulting from the oxidation pathways of the TMB and the corre-
sponding visual colour changes on microtiter plates. Adapted from Reference 76 licens ed under a
Creative Commons Attribution 3.0 Licence.

108 Y. Lin, L. Wu, Y. Huang, J. Ren and X. Qu, Chem. Sci. , 2015 , 6, 1272 - 1 276.

Material and Methods
99
Automate d DNA s ynthesis
During the autom ated DNA synthesis of oligonucleotide s the chain is built in t he 3' - 5'
direction. The s ynthesis st arts by the attachm ent of the first nucleoside of the desir ed se-
quence to a solid support via its 3' - hydroxyl gr oup. The main step is the specific and se-
quential formation of the internucleoside 3' - 5' phosphodi ester bond. To ensure the coupling
reactions to be selective, t he 5’ - hydroxyl group of the nucleoside is prot ected with an acid -
labile dimethoxytr i t yl (DMTr) g roup . Afterwa rds, the 3’- hydroxyl group of the nucleoside is
converted into a phosphor amidite funct ion in order to << enable coupling wit h the free 5’ -
OH of th e previous nucleoside (Fi gure 40 ). 66

B
0
O
O
DMTO
B
n
O
O
HO
B
n
O
O
O
P
O O
NC
B
n+1
O
DMTO
B
n
O
O
O
P
O O
NC
B
n+1
O
DMTO
O
N
P
O O
NC
B
n+1
O
DMTO
B
n
O
O
P
HO O
B
n+1
O
HO
O
B
0
O
OH
O
P
HO O
O
n
B
n
O
O
O
O
deprotection
deprotection
cleavage
capping
COUPLING
tetrazole
OXIDATION
I
2
DEPROTECTION
TCA

Figure 40 . Scheme of automated DNA sy nthesis .

The cyclic procedur e, containing a ser ies of deprotectio n, coupling, c apping and oxi-
dation steps results in the formation of the desired oligo nucleotide sequence. The f irst step
in each chain el ongation cycle is t he deprote ction of the 5' - hydroxy l nucleoside group with
a solution of dichloroacetic acid in dichlorom ethane, after this solution is applied, t he or-
ange coloured t rityl cation is rele ased from t he reaction column i ndicating that this step
was successful. After deprotection, a new phosphoramidit e can be coupled t o the free 5’ -
hydroxyl term inal residue of the nucleic acid chain. First, the phosphoram idite is activated

Material and Methods
100
with a solution of tetrazole in acetonit rile, after which the act ivated phosphoram idite is at-
tached to the growing nucleic ac id chain with t he formation of a phosphit e triester bond
between the 5' - hydr oxyl of the terminal nucl eic acid residue and t he 3' - end of the new
monomer .
It is possible that a sm all amount of the unreacted deprotect ed terminal residue st ill
remains. This would lead to the format ion of the so - called deletion sequence, which is an
oligonucleot ide chain with a miss ing nucleotide. In order t o avoid this deletion , a capping
step is introduced into the cycle. In the capping step, the 5' - hydroxyl group of t he unreacted
ter minal nucleoside is blocked by acetylation. In the last step, t he phosphite triester is oxi-
dized to the more stable phosphodiester bond with iodi ne and water in pyridine. This cycle
is repeated until the desired oligonucleot ide chain has been obtained. The oligonucleotide
can be cleaved from the solid support by treatment with an ammonium solution and the
protective groups can be removed by heating up to 55°C. The olig onucleotide can be iso-
lated from the mixture containi ng also deletion sequenc es, cleaved p rot ecting groups and
capped oligonucl eotides by sever al purification method s, such as, for example, ethanol
precipitation, ion exchange or si ze - exclusion chrom atography, RP - HPLC and revers ed -
phase cartridge purific ation. In the last one, the DMTr - protectin g is not rem oved at the end
of the synthesis cycle. Pur ification can be obtained based on the hydrophobicity of the
DMTr group, which h as a very high affinity for t he hydrophobic column so t he full - lengt h
DMTr - Oligonucle otide is ret ained on the column, while the other components are washed
off. After cleaving of the DMTr on the cart ridge (e.g. t rifluoroacetic solution) it is possible to
elute the oligonucleot ide.

101

4. Results an d Discussi on

Results and Discu ss ion
103
4.1 Pht h al ocya nine - ful ler ene dyad s
In this chapter, I will report on synthesis and pr operties of a diff erent type of materials
– polyconj ugated nanocar bons – that possess interesting self - assembling pr operties, sig-
nificantly affect ing their physicochemical ch aracterist ics. Additionally, obj ects that will be
described in present chapter are related to the renewable energy issue. Finding sustaina-
ble energy sources is a genuine challenge of m odern society yet t o be reached . 109 I n this
connection, sc reening of art ificial photosynt hetic systems stands out as one of the hot top-
ics of contempor ary science. 110

4.1.1 Synthesis and ch aracterizati on
The tert - butyldiphenylsila ne (TBDPS) - protected hydr oxymethylphthalonit rile 10 is the
key - compound towards t he sysnthesis of all mono - substituted ZnPcs as it will be shown
further in this section. Compound 10 was obtained according to reported pr ocedure 111 in a
multistep synthes is from 4 - met hyl phthalonitr ile (Schem e 4 ). Thus, 4 - methylphthalonitr ile
was subjected to the Wohl - Ziegl er photoinduced brominat ion with N - bromosuccinimi de
(NBS) and catalyt ic amount of 2,2' - azobis(isobutyr onitrile) (AIBN) as initiator f or the selec-
tive bromination of benz ylic hydrogens (Sch eme 4 ). A mixture of mo no- , di - and tribromin-
ated compounds wer e obtained in a 1:2:1 rat io, respectively, as observed by TLC and 1 H-
NMR. Treatment of this mixture with an excess of diethylphosphit e and N , N - diisopropyl - N -
ethylamin e (DI PEA) yielded 4 - ( bromomethyl)phthalon itrile 12 . This latter compound w as
then refluxed in an aqueous solu tion of dioxane (dioxane/ water 1/1.5 ) in the presence of
calcium carbonate aff ording 4 - (hydroxymet hyl) phthalonitrile 11 with a yield of 98%. Finally,
TBDPS - protect ed hydroxymethy lpht halonit rile 10 was obtained in 92% yield upon sylilation
of phthalonitr ile 11 using TBDPS chloride in DMF in t he presence of imidazol e .

109 a) J. Twidell, T. Weir, Renewable Energy Resources , Routledge, 2015 , pp. 151 - 202; b) R.
Wengenmair, T. Bührke, Renewable energy: sustainable energy concepts for the future , W iley -
VCH, Weinheim, 2013 , pp. 1 - 170.
110 a) G.D. Scholes, G. R. Fleming, A. O laya - Cast ro, R. van Grondelle, Nat Chem. , 2011 , 3, 763 -
774; b) R. J Cogdell, T. H. P. Brotosudarmo, , A. T Gardiner , P. M Sanchez, L. Cronin, Biofuels
2010 , 1 , 861 – 876; c) Issue on “Artificial Photosynthesis and Solar Fuels”, Acc. Chem. Res ., 2009 ,
42 , 1859 - 2029.
111 R . F. Enes, J. - J. Cid, A. Hausmann, O. Trukhina, A. Gouloumis, P. Vázquez, J. A. S. Cav aleiro,
A. C. Tomé, D. M. Guldi, T. Torres . Chem. Eur. J ., 2012 , 18, 1727 - 1736.

Results an d Discu ss ion
104
NC
NC
Br
NC
NC
N O
O
AIBN/CCl
4
hv
NC
NC
Br
O
NC
NC
H
i) diethyl phosphite,
THF
CaCO
3
Dioxane/H
2
O
Si
Cl
t
Bu
DMF
92%
98%
X
X = H o Br
Br
Imidazole NC
NC
O Si
t
Bu
12
11 10
ii) DIPEA

Scheme 4 . Synthesis of TBDPS - phthalonitrile precursor 10 .
Statistical cyclotet ramerization react ion between phthalonit rile 10 (1 eq.) and unsub-
stituted phthalonitr ile (5 eq.) in refluxing N , N - dimethylam inoethanol (DMAE) in the pres-
ence of zinc acet ate and 1,8 - diazabicycl o(5.4.0) undec -7- ene (DBU) affor ded TBDPS - pr o-
tected hydroxymet hyl - ZnPc 13 in 10% yield. Worth not ing, the utilizat ion of bulky TBDPS
group as a substituent afforded a str aightforward purificat ion of the desired product by
solving the problem of solubility. Thus, the major impurity of non - sub st ituted ZnPc with
limited solubility was easily separated f rom the TBDPS - ZnPcs using a hot extraction of the
latter by acetone in a Soxhlett appar atus. The desired mono - substitut ed ZnPc was isolat ed
from the stadistic mixt ure of products by colum n chromatography. The TBD PS protect ing
group was then deprotected using tetra - n - butylammonium fluoride (TBAF) in acetic acid at
room temperatur e affording hydr oxymethyl - substituted Pc 14 in 9 8% yield. Inter estingly,
the use of present approach allowed us t o isolate 14 in it s pure form in 0. 5 g scale. This
goal would be hard to achieve considering t he stadistical condensat ion of phthalonitril e and
11 due to bad solubili ty of all the Pcs prod ucts impossible t o separate via conv entional
column chromatogr aphy or recryst allizat ion.

N
N
N
N
N
N
N
Zn
O
Si
N
N
N
N
N
N
N
N
Zn
HO
TBAF/AcOH
THF
r.t N
N
N
N
N
N
N
N
Zn
IBX
DMSO
r.t
N
NC
NC
O Si
NC
NC
Zn(OAc)
2
DBU/DMAE
140 ºC
t
Bu
t
Bu
13 14
10
98% 87%
10%
15
O H
+

Scheme 5 . Synthetic scheme leading to ZnPcs 14 and 15 .

Results and Discu ss ion
105
Finally, Pc 14 was oxidized with 2 - iodoxybenzoic acid (IBX) in DMSO obtaining, after
column chromatogr aphy, formyl - substit uted Pc 15 in 87% yield ( Scheme 4). Struct ural
characterizat ion of these compou nds was carr ied out by using mass spectrome try, 1 H NMR
and UV - vis spectroscopy analy sis. Thus, charac teristic signatur es of protons of hy-
droxymethyl gr oup in ZnPc 14 can be obse rved as the cor responding triplet at 5.72 ppm
and the doublet at 5. 13 ppm in its 1 HNMR s pectr um in DMSO - d 6 (Figure 41 ). Differen tly,
the formyl proton of 15 possesses a pronounced sing let at 10. 47 ppm. Aromatic protons of
the Pc core resonate as two broaden multiplet s between 9 and 7 ppm in both compounds.

Figure 4 1 . 1 H NMR spectra (300 MHz, DMSO - d 6 , 25 o C) of hydroxymethyl - ZnPc 14 and formyl -
ZnPc 15 . Color code is used for the assignment of pr otons and asterisks for impurities .
The synthesis of ZnPc -C 60 conj ugate 6 involved a Prato reaction between precursor
aldehyde - substitut ed ZnPc 15 , C 60 fullerene and sarcosine in o - DCB/DMF, affording dyad
6 in 60% yield as a rac emic mixture at the methine pyrr olidine carbon atom (Scheme 6 ).
The structural feat ures of compound 6 were confirmed by mass spectrometry, 1 H- NMR
and UV - vis spectroscopy. Thus , MALDI - MS studies of the ZnPc -C 60 adduct 6 revealed
peak of molecular ion at 1351 - 1381 m /z, correspondin g to the dyad of t he proposed struc-
ture (Figure 42 a) . More intense peak at 719.9 m/ z is due to the fragmentation taking place
and is attributed t o the loss of ZnPc m oiety. M ore importantly, c haracteristic s ignals of
protons for the C 60 pyrrolidine adducts have been observed in th e 1 H- NM R spectrum of 6
in CS 2 /THF - d 8 (1/1) . Thus, geminal pyrrolid ine protons r esonate as two doublet s at 5.30
and 4.59 ppm, whereas t he remaining pyrrolidine prot on appears as a singlet at 5. 70 ppm.
Singnal of the N - methy l protons can be observed as s inglet at 1.29 ppm. The UV - vis specta
14

15

ppm

Results and Discu ss ion
106
of 6 in solution and t hin film will be discussed in comparison with those of ot her dyads later
on in this chapter.

Scheme 6 . Synthesis of ZnPc -C 60 dyad 6 . Inset - 1 H NMR sp ectru m ( 300 MHz, CS 2 /THF - d 8 =
1:1 vol. , 25 o C ) with characteristic signals of the pyrrolidine unit.
The preparation of amphiphil ic analog of 6 - Pc -C 60 dyad 7 - required the synthesis of
N - functionalized glyc ine 16 that was prepared according to t he literature pr ocedure 112 as
outlined in Scheme 7. The synt hesis of 16 started with the mono prot ection of 2, 2’ - (eth-
ylene - dioxy) bis(ethylam ine) by reaction wit h di - tert - buty l - dicarbo nate (Boc 2 O) in dio xane
leading to compound 1 8 in 50% yield. The rem aining amino group in 18 was then protect ed
using benzyl - bromoacet ate affording compound 17 in 56% y ield. Compound 16 was f inally
obtained in a 97% y ield by removal of t he benzylic group in 17 in the pr esence of H 2 and
Pd/C as catalyst.
O
O
Br
N
H
O
N
H
O
O
dioxane, 12h dioxane, 12h
H 2
Pd/C
MeOH, 5h
56%
50%
97%
HOOC N
H
O N
H
18
16
Boc 2 O
17
NH 2
O
H 2 N 2 N
H
O
H 2 N 2
Boc
2
Boc 2
Boc

Scheme 7 . Synthesis of N - functionalized glycine 16 .

112 G. Pastorin, W. Wu, S. Wieckowski, J. - P. Briand, K. Kostarelos, M. Prato, A. Bianco, Chem .
Commun ., 2006 , 1182 - 1184.
ppm

15

6

Results and Discu ss ion
107
1,3 - Dipolar cycloadditi on reaction between aldehyde - functionalized Pc 15 , N - function-
alized glycine 16 and C 60 fulleren e aff orded ZnPc -C 60 dyad 19 ( Scheme 8 ).

N
N
N
N
N
N
N
N
N
Zn
O H
N N
N
N
N
N
N
N
Zn
o -DCB/DMF
120 ºC
58% N
N N
N
N
N
N
N
N
Zn O
O
NH
3
TFA/ DCM
r.t
98%
C
60
+
7
15
O
O
HN
Boc
16
19
TFA
NH
O
O
HN
Boc
COOH

Scheme 8 . Synthesis of amphiphilic ZnPc -C 60 dyad 7 .
Formation of t he dyad 19 was confirmed by MALDI TOF mass spectrometry perfor med
in a negative m ode in the presenc e of trans -2-[3-(4 - tert - Butylpheny l) -2- methyl -2- propenyl-
idene]malononitr ile (DCTB) as a m atrix. A peak of molecular ion observed at 1568.3 m/z
corresponds to the proposed struct ure of 19 ( Figure 42b). As in the case of the dyad 6 , the
more intense peak at 994.2 m /z corresponds t o the fullerenic unit after the loss of the ZnPc
moiety. Th e tert - butoxycarbonyl (Boc) - protected am ino end group in 19 was quantitatively
deprotected an excess of trifluoroacet ic acid (TFA) to afford amphiphilic Pc -C 60 conj ugate
7 in 98% yield . The pr esence of one posit ive charge co mpensated by trifluoroacet ate anion
makes this dyad unsoluble in ar omatic solvents. However, presence of the terminal am-
monium group is supp osed to assist in dispersion of this dyad in water.

Figure 42 . MAL DI - TOF MS spectra (DCTB) of a) ZnPc -C 60 dyad 6 and b) its analog 19 . Inset:
A) calculated isotopic pattern for the same peak, B) isotopic resolution of the MALDI - TOF main
peak.

Results and Discu ss ion
108
A different synt hetic strat egy was used for the pr eparation of the Pc -C 60 dyad 8 , which
involved the synthes is of a malonyl - substituted Pc 20 in 87% yield by reaction between
hydroxymethyl ZnPc 14 with c hloromalony l ester in the presen ce of pyridine. Subse -
quently, malony l - substitut ed Pc 20 disso lved in DMF was adde d to a t oluene solution of
C 60 fullerene and CBr 4 in the pres ence of an excess of DBU affording Pc -C 60 conj ugate 8
in 70% yield via a Bin gel - Hirsch react ion (Scheme 9 ).

C
60
CBr
4,
DBU
Cl
O O
O
rt, 15 min
THF/pyridine
O
O
O
O
N
N
N
N
N
N
N
N
Zn
O
O
O
O
N
N
N
N
N
N
N
N
Zn
8
14
70%
20
87%
DMF/toluene
rt

Scheme 9 . Synthesis of Pc -C 60 dyad 8 .

Figur e 43 . MALDI - TOF MS spectra (DCTB) of a) ZnPc -C 60 dyad 7 and b) its enlarged analog
8 . Inset: A) calculated isotopic pattern for the same peak, B) isotopic re solution of the MALDI - TO F
main peak.
Full spectroscopic ch aracterization of 8 has been performed, confirming its unumbig-
ous formation and is olation in a pur e form. Thus, M ALDI TOF m ass spectrum perform ed
in a positive m ode with DCTB as a matrix rev ealed the presence o f a peak at 1424.1 m /z
corresponding t o [M] + molecular ion (Figur e 43 a). 1 H NMR spectrum showed charac teristic
singlet of methylene pr otons at 6.08 ppm, and a singlet of the three m ethyl protons at 4.20

Results and Discu ss ion
109
ppm (Figure 44). In a contrast , corresponding sign als of the pr ecursor ZnPc 20 were o b-
served as the two singlets at 5. 78 and 3.81 ppm, whereas the methylene protons of the
malonyl fragment resonate as singlet at 3. 58 ppm (Figure 44). Worth noting, a 1:1 mixture
of CS 2 /THF - d 8 was found successf ul to dissolve the dyad till the concentration suitable for
NMR analy sis. Presence of the malonyl fragment in 8 was expected to enlarge the distanc e
between ZnPc and C 60 units as well as to provide a fr ee rotation within the conjugat e.

Figure 44 . 1 H NMR spectra (300 MHz, CS 2 /THF - d 8 1:1 , 25 o C) of malonatomethyl - ZnPc 20
and ZnPc -C 60 conjugate 8 . Color code is used for the assignment of protons.
Finally, an extend ed analog of 8 was design ed as ZnPc linked to C 60 fullerene in a
dyad 9 v ia a flexible oxymethylbuty ric spacer following the procedure r eported by Guldi et
all. for the synthesis of tetra - phenylpor phyrin covalently co nnected to Ce 2 @ I h -C 80
metallofulle rene. 113
The ZnPc bearing 4 - benzoylbutyr ic acid tosylhydrazone 21 has been synthesized f or
its further addition t o the fullerene m oiety. For this porpose, ZnPc 22 was reacted in Dean -
Stark apparatus wit h p - toluene - sulfonylhydraz ide (tosylhydrazone) in toluene/pyridine sol-
vents mixtur e at reflux overnight to af ford 21 in 50% yie ld (Scheme 10 ). In its turn, Zn Pc
oxymethyl ester of 4 - benzoylbutyric acid 22 was prepared v ia esterificat ion reaction of hy-

113 D. M. Guldi, L. Feng, S. G. Radhakrishnan, H. Nikawa, M. Yamada, N. Mizorogi, T. Tsuc hiya,
T. Akasaka, S. Nagase, M. A. Herranz, N. Martin, J. Am . Chem. Soc ., 2010 , 132, 9078 - 9086.
20

8

Results and Discu ss ion
110
droxymethyl ZnPc 14 and 4 - benzoyl butyric acid in a presenc e of 1 - ethyl -3-(3- dimethyla-
minopropyl)c arbodiimide (EDC) and cat alytic amounts of 4 - dimet hylaminopyridi ne (DMAP)
in THF at r.t . in 81% yield. The subseque nt addition o f sodium methoxide t o 21 in anhy -
drous reaction media was suppo sed to induce the t ransformation of the tosylhydr azone
moiety into highly rea ctive diazo group binding in s itu to C 60 v ia cyclopropanat ion reac-
tion. 114 Instead, the degradation of 21 occur red, yielding t he ZnPc 14 . However, an alter-
native route for the preparation of 9 was attempt ed as the one described belo w.

Scheme 10 . First route for the synthesis of ZnPc -C 60 dyad 9 .

Pc -C 60 conjugat e 9 was obtained in 86% yield by r eaction of ester ification between
hydroxymethyl - Pc 14 and [6, 6] - phenyl -C 61 - butyric acid (PCBA). This latter fullerene deri v-
ative was synthes ized by acid - catalyzed h ydrolysis of the methyl ester PCBM r eceived
fro m comm ercia l s upplier (Scheme 11 ). 115

114 R. K. M. Bouwer, J. C. Hummelen , Chem. Eur. J ., 2010 , 16, 11250 - 11253.
115 M. Drees, H . Hoppe, C. Winder, H. Neugebauer, N. S. Sarici ftci, W. Schwinger, F. S chffler, C.
Topf, M. C. Scharber, Z. Zhun, R. Gaudiana, J. Mater. Che m. , 2005 , 15, 5158 - 5163.
N
N
N
N
N
N
N
N
Zn
O
O
N
N
N
N
N
N
N
N
Zn
OH
9
DMAP, EDC
o -DCB/pyridine
rt
86%
PCBA
14
HO
O
AcOH,HCl
toluene
r.t, 18h
PCBM PCBA
O
O
83%

Results and Discu ss ion
111
N
N
N
N
N
N
N
N
Zn
O
O
N
N
N
N
N
N
N
N
Zn
OH
9
toluene/pyridine
15:1 vol, 75°C
C
60,
CH
3
ONa
14
DMAP,EDC
toluene
THF, r.t, 12h
81%
N
N
N
N
N
N
N
N
Zn
O
O O
N
N
N
N
N
N
N
N
Zn
O
O N
O
O
O
S NH
2
O
O
S
O
O
22
21
toluene/pyridine
10:1 vol.
reflux. 50%

Scheme 11 . Second route for the synthesis of ZnPc -C 60 dyad 9 .

MALDI TOF mass spec trum perform ed in a positive mode with DC TB as a matrix re-
vealed the presence o f a peak at 1482. 2 m/z corr esponding to [M] + molecular ion (Figure
43 b). Notably, no significant fragmentation, if any, has been observed in mass - spectromet-
ric data of 9 and 8 , in compar ison with those of the “Prat o - type” dyads 6 and 19 ( Figure
42 ), confirming a str ong influence of the retro- Prato related processes on the stability of
the ZnPc -C 60 dyads. 116 1 H NMR spectrum recorded at 300 MHz in CS 2 - THF - d 8 1:1 mixture
showed character istic multiplets of the ZnPc core protons at δ 9.41 - 9.22 and 8.20 - 8.07
ppm (Figure 45). Eight methylene protons appear ed as the four groups of signals at δ 5.68
(s, CH 2 ) , 3.00 (m, CH 2 ), 2. 75 (m, CH 2 ) and 0.98 (m, CH 2 ) ppm. Finally, the phenyl protons
were observed at δ 7.52 (t, 2H, CH ar yl ), 7.45 (t, 1H, CH aryl ) and 7. 20 -7.05 (m, 2H , CH a ryl)
ppm.

116 N. Martín , M. Altable , S. Filippone , A. Martín - Domenech , L. Ec hegoyen , C. M. Cardona , Angew.
Chem. Int. Ed ., 2006 , 45, 110 - 114.

Results and Discu ss ion
112

Figure 4 5 . 1 H NMR spectrum (300 MHz, CS 2 /THF - d 8 1:1, 25 o C) of ZnPc -C 60 dyad 9.

Results and Discu ss ion
113
4.1.2 Photo physical st udies of t he dyads

First insights into the ground state absorption featur es of the ZnPc -C 60 dyads 6 , 8 and
9 were obtained from their UV - vis spectr a in solution and in the thin films. Dyad 1 - a tri -
tert - butyl substituted analogue o f 6 - has also been prepared in this work according to the
procedure developed earlier by our gr oup. 35
I n order to utilize it as a reference c ompound. Thus, dilution spect ra in of all the dyads
in to luene are shown on the Figure 46. As it can be seen, spectr um of each dyad results
from a superposition of absorptions of fullerene and ZnPc units. Thus, they can be charac-
terized by the presence of the t wo intense bands – a sharp absorption at around 675 nm
typical for the ZnPc Q - band, and a broad peak that results fr om the sum of fullerene ab-
sorption and a ZnPc B - band at around 340 nm. More interest ingly, ZnPc and fullerene uni ts
seem to reveal minim al interaction in t he ground stat e in solution. The main indicat ion of
the latter com es from the fact that ZnPc Q - bands reve al only a slight s hift depend ing on
the nature and rigidity/f lexibility of the spacer. The ZnPc Q - bands appears at 673 nm in
UV - vis absorption spectr um of 8 and 9 , whereas those are slightly shift ed bathochromically
to 676 and 680 nm in 6 and 1 , re spectively, as a function of shorted distance between t he
two photoactive units. The latter is a direct indication of a slightly lo wer LUMO of the “prato -
type” dyads 6 and 1 .
Turning to inter molecular effect s of dyads in solution, it is ed iv enced f rom the Figure 46
that no interactions, such as π - π stacking, take place within a given range of conc entra-
tions in toluene. Thus, no shift of the ZnPc Q - band was obs erved upon dilution/addit ion of
coordinating solvent , such as pyri dine or THF, in contr ast to a study on ZnPc aggr egation
effects report ed recently by our group 117 as well as those desc ribed elsewhere. 118, 119 How-
ever, a clear depend ence of molar absor ptivity of 6 on the concentration evidences its
decreased solubility in toluene. In comparison, the other dyads remain their extinction co-
efficients unchanged and dependent on t he t ype of the spacer, revealing ε ≈ 18 × 10 4 [dm 3
mol -1 cm -1 ] for 8 and 9 , 19 × 10 4 [d m 3 m ol -1 cm -1 ] for 1 , and ε ≈ 14 × 10 4 [dm 3 mol -1 cm -1 ]
for 6 .

117 G. Bottari, O. Trukhina, A. Kahnt, M. Fr unzi, Y. Murata, A. Rodriguez - Fortea, J. M. Poblet, D .
M. Guldi, T. Torres, Angew. Chem. Int. Ed. , 2016 , 55, 11020.
118 A. W. Snow, in The Porphyrin Handbook ; K. M. Kadish, K. M . Smith, R. Guillard, Eds.; Academic
Press: San Diego, CA, 2003; Vol. 17, pp 129 - 176.
119 a) M. Katayose, S. Tai, K. Kamijima, H. Hagiwara, N. Hayashi, J. Chem. Soc., Perkin Trans. ,
1992 , 2 403 - 409; b) J. M. Kroon, B. M. Koehorst, G. M. Sanders, E. J . R. Sudholter, J. Mater.
Chem., 1997 , 7, 615 - 624.

Results and Discu ss ion
114

Figure 46 . Effect of dilution on the UV - vis absorption (toluene, 25 o C) of ZnPc -C 60 dyads a) 6 ,
b) 1 , c) 8 and d) 9 .
UV - vis ground state absorption featur es of the thin films of the dyads were also deemed
important for t he estimation of supr amolecular int eractions and s tudy of t he charge sepa-
rated events in the s olid stat e. For this purpos e, the drop cast ed films of t he dyads 1 , 6 , 8
and 9 were prepar ed on glass su bstrates , utilizing the cor responding 5 × 10 - 6 M saturated
solutions in THF. In addition, films of the dyads in a mat rix of acrylic glass, containing 5 wt.
% of poly(methyl - methacr ylate) (PMMA) were f abricated by spin coating of the correspond-
ing solutions in THF on glass substr ates at 1500 r.p.m for 10 sec. Embedding of the dyads
in PMMA matrix allowed preparation of the supercooled liquid phase at room temper ature
for revealing pos sible supram olecular eff ects in the solid stat e, if any, being mor e defined
in comparison with t hose of drop casted films. The corresponding UV - vis absorption spe c-
tra in toluene, PMM A matrix and in drop casted film are repr esented in the Figure 47 .

Results an d Discu ss ion
115
Figure 47 . UV - vis normalized absorption spectra of ZnPc -C 60 dy ads in toluene (red line), in
PMMA matrix on glass (5 wt. %, black line) and those of a thin film drop casted from THF on glass
(blue line) of ZnPc -C 60 dyads a) 6 , b) 1 , c) 8 and d) 9 .
In PMMA matrix , the c haracteri stic Z nP c Q - band experiences no shift (black lines) in
comparison with t hose in toluene (r ed lines). However, t he evident broadeni ng of the m ajor
absorption f eatures takes place, being caused by t he short er distances betwe en dyad mol-
ecules, if being comp ared with those in toluene solutio n. Interestingly, the rigid spacer be-
tween ZnPc and C 60 units in dyads 6 and 1 results in fewer chan ges of the ZnPc Q - band
in frozen solution. I n a contrast, UV - vis abs orption featur es of 8 and 9 vary significantly
when the la tter lo se their flexibility in PMMA matr ix. However, no spectral evidence, su ch
as bathochrom ic or hypsochromic shifts, of any ordered supr amolecular aggre gates driven
by intermolecular f orces or matrix itself could be obse rved. When tur ning to absorption
featu res of the dyads in drop cast ed films, the drast i c changes can be f ound (Figure 4 7 ,
blue lines).

Results an d Discu ss ion
116
Those can be charact erized by broadenin g of all the absorpt ion bands and their clear
bathochrom ic shifts in compar ison with spectr al features in tol uene and i n PM MA. A
stronger scatter ing that causes the elevation of t he baseline absorption in spectr a of 8 and
9 indicates on the precipitation of the corresponding dyads fr om their THF solution in the
form of larger aggr egated species on glass su bstrat e upon drop cast ing. Whereas, t he size
of the corresponding a gglomerate s is smaller for 6 and the least for 1 , bearing a tet ra - tert -
butyl substituted ZnPc. Obviously, the int ermolecular distances ar e the least for the drop
casted films. I n line with that, the interaction bet ween neighborin g molecules increase s
with the decrease of separat ion distances, resulting in an additional energy shif ts of the
ZnPc and C 60 correspondin g absorpt ions.
Next, ultrafast c harge generation in ZnPc - C 60 dyads has been studied by fe mtosec ond
transient absorpt ion spectr oscopy in THF solutions, PM MA matr ix and in drop casted films
with the aim t o define the possible photophy sical proce sses, such as photoe xcitation an d
charge generat ion with the corresponding charge separ ation (CS) and charge rec ombina-
tion (CR) times, in ZnPc and C 60 units. Additionally, we were interest ed in comparing the
observed phenomena in solution and in the solid st ate, with the ai m to reveal the influen ce
of the latter on charge s eparation dynam ics. The global f itting has been used f or the data
analysis a general me thod. Pump pulses at 387.5 nm (around 200 - 1200 nJ pulse energy
with about 300 μm spot size) have been used t ogether with probe pulses of 480 – 1300
nm, recorded by a double line si ngle shot capabl e spectr ometer. Due to the short pump
wavelength em ployed, the excitation of both photoactive unit s (ZnPc at its Soret band and
the C 60 ) was consider ed, accounting on (i) ener gy/charge tr ansfer from C 60 * t o ZnPc, (ii)
ultrafast charge gener ation directly fr om the ZnPc Soret band and (iii) ultrafast triplet gen-
eration from the ZnPc Soret band. Since the fact that all these ultraf ast processes leave
characterist ic spectral signatures in femtosecond tr ansient absorption spectra (TA), it
should be possible to ident ify them by global spectral analysis.
Before turning to an in depth analysis of the TA spectra of the dyads, we will def ine the
most characterist ic features of the photoinduced pr ocesses of ZnPc. In general, a global
kinetic fitting analysis results in a set of c harac teristic spectra, i.e . evolution - associat ed
differential spect ra (EADS), which possess a def inite characterist ic lifetime. From the spec-
tral shapes of the EADS, t he corresponding attribut ion to either photo - excited states or to
superpositions of them is possible.

Results and Discu ss ion
117
In Figure 47a, the two EADS of ZnPc 14 in THF are shown, having lifetimes of 13 ps
and 1.8 ns, respect ively. The similarity of their shapes indicates the existenc e of t he only
dominant photoexc ited stat e. Their definition becomes possibl e due to the char acteristic
sharp absorpt ion bands of the ZnPc molecul e.
T he preliminar y analysis of EADS incl udes the sear ch of signatur es of any excited
states, regardless their s inglet, triplet or charged nature, deplet es the number of available
molecules in the ground s tate. Therefore, the ground state is considered to be bleached t o
a certain extent, bein g called transient photobleach ( PB). Taking into accou nt the ground
state absorpt ion of ZnPc, consist ing of a sharp ( 00) transition at 1.82 eV and a clearly
resolved vibronic replic a at 2.03 eV, these two features are expected to form an integral
part of any excit ed state spectr a. In fact, looking at Figure 48, the bot h transient photo -
bleaches - PB00 and PB01 – ca n be satisfactor y found.

Figur e 48 . a) EADS from the global fitting analy sis of ZnPc 14 solution in THF, b) TA spectra
at specified time delays (thin lines) and a global fit (thick line).
From Einstein relation s, and neglecting a Stokes shif t, the oscillator strengt h for stimu-
lated absorpt ion is equal to that for s timulated emission. As a c onsequence, in the excit e d
state spectra, t he stimulated emission (SE) f eature of the (10) transition can be observe d
at 1.65 eV (Figure 48 a). Spectral analysis s hows that t he oscillator strength of SE10 is
similar to PB01, which means that the ZnPc singlet st ate is the dominating excited state,
and the only excited state occurring af ter 10 ps. Especially, the fact that the ratio SE10/
PB01 stays constant bet ween 10 and 200 ps (Figure 48b) , confirms the observat ion of
none significant triplet format ion in a given time win dow.

Results and Discu ss ion
118
The latter means that both phot oinduced absor ption (PA) bands of EADS1, namely
PAS1 at 1.95 eV, and PAS2 at 2.55 eV, can be attributed t o excited state absorptions of
the singlet state. Finally, a small band PASx at 2.3 eV in EADS0 wi th a lifetime of 13 ps
should be considered as a polaronic state, which agr ees with a pr esence of the other
sharper polaron band at 1. 4 eV. In other words, this weak band is caused by ionization
involving solvent m olecules, follo wed by geminate r ecombination with 13 ps r ec ombination
time. Singlet s tates cause only negligible PA bands in the near infr ared. M inor bands at 0.9
and at 1.1 eV are one order of magnitude weaker than PAS1 and PAS2, and thus , they
cannot be associated the ZnPc singlet state absorpt ion .

Results and Discu ss ion
119
4.1.2.1 Photo physic s i n TH F solution
When turning to photophy sical propert ies of ZnPc -C 60 dyads, we began our study from
the reference s ystem 1 , tak ing into account it s photophy sical behavior has been previous ly
reported by our gr oup. 35 Thus, three EADS of 1 i n THF are shown on the Figur e 49 a. In
overall, EA DS0 of 1 is similar to EADS1 of ZnPc 14 (Figure 49a) , showing SE10, PAS1
and PAS2. The latt er indicates t he predomin ant gener ation of singl et excitons following t he
pump at 387 nm with 150 fs pulses and 560 nJ pulse ener gy. In a contrast, EADS1 differs
strongly from EADS0 indicating t he occurrence of photophysical processes. I n EADS1,
singlet excited st ate related bands, namely SE10, PAS1 and PAS2, disappear, whereas ,
the new absorption feat ures evolve at 1.25, 1.4, and 2.35 eV, associat ed with photoind uced
absorption by the char ged acceptor (PAA) and that of the c harged donor - PAD1 and
PAD2, respectively. Since EADS0 and EADS1 are w ell distinct, the r ate constant k1 can
be directly associated with charge separat ion. EADS2, on t he other hand, is similar to
EADS1, which leads to the conclusion that EADS2 is still the same state, namely the
charge separat ed state, but that charge rec ombination is non - exponentia l with two life-
times, 1/k2 and 1/k3. Being struct urally close to 1 , dyad 6 in a similar manner reveals
EADS0 dominated by singlet excited states, whereas EADS1 repr esents only polarons and
no visible contribution of sing le t states ( Figure 49b). EADS1 is only polarons, so we con-
sider the presence of PCBM excitons as m inor. Charge recom bination divide s into a fast
and a slow part, like in 1 .
When turning to the dyads 8 and 9 , possessing flexible spacers and a certain degree
of rotational fr eedom, the photophysical even ts become more com plex. Thus, in TA spectra
of 8 , EADS0 contains character istic featur es of the ZnPc singlet excited stat e, whereas the
EADS2 evolves as fi ngerprints of the charge separat ed state (Figure 49 c). However ,
EADS1 appears as an intermediate st ate between those two: PAD1 is not yet fully devel-
oped, whereas the band at 2.4 eV seems to be a compromise between PAS2 and PAD2.
EADS1 clearly shows some SE10 which EADS2 lack s. Importantly, t he total integral of
PB01 seems to increase from EADS0 to EADS1. All these observations can be explain ed
by the following sce nario. M ost dyades are excited on t he ZnPc side, and perf orm charge
separation as sec ond step. Some dyades ar e however excited on t he C 60 side. Since the
neutral C 60 * excited s tate causes ver y little PA or PB, these dyades r emain invisible i n
EADS0 but then perform energy transfer to ZnPc, creat ing ZnPc singlet excite d states.

Results and Discu ss ion
120
In other words, while the initia l singlet states of ZnPc site ar e charge separated, new
ones get evolved fr om the C 60 site. O nce all, the C 60 singlet states have bee n transferred
to ZnPc, the recombination of the charge separat ed state occurs in 320 ps, much slower
than in 1 , where it occurs in 40 ps.

Figure 49 . a) EADS from global fits for TA spectra of ZnPc -C 60 dyads a) 1 , b) 6 , c) 8 and d) 9
in THF, obtained after pumping at 387 nm with 150 fs, pulses and 560 nJ pulse energy In panel
(a), the nomenclature of the photoinduced bands is introduced as PB, SE, PAS, PA A, PAD c orre-
sponding to photobleaching, stimulated emission, photoinduced absorption of singlet, photoin-
duced absorption of acceptor and photoinduced absorption of donor, respectively.
Regarding the dyad 9 , the situat ion is similar to 8 , in that charge sep aration occurs with
two lifetimes. EADS1 shows evid ence of both - singlet and polaronic stat es, showing that
the 387 nm pump pulse creates som e C 60 excitons which are tr ansferred to Zn Pc from
EADS0 to EADS1 (Figur e 49 d).

Results and Discu ss ion
121
An important diff erence between 8 and 9 is that in the latter, the integral of PB01 is
reduced going fr om EADS1 to EADS2.A lso the integr als of the PA D1 and PAD2 bands a re
clearly reduced, whic h is explained by a certain amount of geminate recombination on a
10 ps time scale, ty pical for such a process. On the other hand, the integr al of PAA at 1.25
eV is not reduced, but even increased, which agrees with our model of del ayed charge
separation after exciton transfer from C 60 t o ZnPc. In summary, in THF solution, we f ind
charge separation times from 1 - 2 ps for all samples, and r ecombination times ar ound 50
ps for 1 and 6 , and around 500 ps for 8 and 9 .

4.1.2.2 Photo physic s i n P MMA films on glass
TA spectra of the dy ads in PMMA matrix are shown o n the Figure 5 0 . In the cas e of 1 ,
subpicosecond charge separ ation and slo w recombination of about 900 ps tak e place (Fi g-
ure 50 a). No sign of non - geminate r ecombination could be observed. In Figure 50 b, 6 is
reproduced without residuals even using only two stat es. In overall, subpicosecond char ge
separat ion and r ecombination on a time scale of roughly 1 ns evolve, being indepen dent
of pump intensity. In Figure 5 0 c, the EA DS of a global fit of 8 in PMAA mat rix are given.
Notably, in thin f ilms, the PB 00 / SE 00 band at 1.87 eV is fully rendered because t h e
material was not fully absorbing at the Q band m aximum like the THF solut ions. This makes
it easy to trace ZnPc singlet excit ons in PMMA films. As ment ioned previously, a PB band
represents always t he sum of all possibl e photoexcitatio ns of ZnPc, while a n S E is specif ic
for the singlet exciton. As it can be clearly obser ved from the Figure 5 0 c, PB01 is incr easing
from EADS0 EADS1, showing th at the tot al number of excited Zn Pc species incr eases.
Thus, the PB00 accordingly incr eases. On the other hand, decreasing SE00/ PB00 band
indicates the cor responding decreas e of the singlet sta tes. The decr ease of singlets con-
tinues from EAD S1 to EADS2, sh owing on - going ch arge separatio n. In summary , the be-
havior of 8 is similar in s olution and PMMA films. Table 1 however shows that charge r e-
combination proc eeds more slowly in PMMA films. The recom bination coefficie nt does not
depend strongly on int ensity, showing that non - geminat e recombination is a m inor effect
in PMMA dissolve d 8 . Thus, the slower recombinat ion of 8 i n PMMA might point t o the
necessity of flexible link ers between the ZnPc and C 60 moieties. In PMMA, the moieties
can no longer move so fr eely .

Results and Discu ss ion
122

Figure 5 0 . a) EADS from global fits for TA spectra of ZnPc -C 60 fi lms a) 1 , b) 6 , c) 8 and d) 9 in
PMMA matrix on glass, obtained after pumping at 387 nm with 150 fs, pulses and 560 nJ pulse
energy. In panel (c), the nomenclature of the photoinduced bands is introduced as P B, SE, PAS,
PAA, PAD corresponding to photobleaching, stimulated emission, photoinduced absorption of sin-
glet, photoinduced absorption of acceptor and photoinduced absorption of donor, respectively .
Finally, EADS of 9 are shown on the Figure 5 0 d. Comparison of the EADS of 8 and 9
shows that the photophy sics might be m ore complex than assumed pr eviously. The rat io
(SE00+PB00)/PB01 i s ascribed to t he presence of singlet st ates. In Figure. 50d, we can
see the SE10 band in EADS0 (at 1.65 eV) , so we can test this hypothesis. The SE10 b and
is completely gone in EADS1, and at t he same time, the strength of the PB01 band i n-
creases relative t o the 00 transitions of SE and PB. Both observat ions are consist ent with
singlet disappear ance and an incr ease of the overall populati on of ZnPc excit ed states.
However, by com paring EADS1 and EADS2: from the r atio of PB01 and the 00 t ransitions
of PB and SE, we would conc lude a furt her dramatic decrease of singlet pop ulation. This
is however not confirmed by t he SE10 band, which is already compl etely gone in EADS1.

Results an d Discu ss ion
123
The picture becom es clearer upon look ing at 2. 15 eV. There, a shoulder is present
which agrees with the posit ion of the second v ibronic of the gr ound state absorpt ion, and
therefore can be associated with PB02. The strength of this PB02 transition incr eases
strongly from EADS0 via EADS 1 to EADS2, and espec ially fr om EADS1 to E ADS2, where
the relative increase is m uch more than of the respective PB01 transition. We conject ure
therefore that apart from the singlet/polaro n ratio, also the Huang - Rh ys f actor is changing
over time. This gives import ant insight into morphology. A larger Huang - Rhys factor m eans
that the potentials of gr ound and excited state becom e more dissimilar over time. A bleach
of one molecule cannot change over time, since irr espective of the geometr y that the ex-
cited state att ains over t ime, it is always the relaxed ground st ate which is the ref erence
for the bleach in the t ransient absorption exp eriment. Changing bleach is therefor e always
a sign of excited state mobility towards specific s ites. An alternative explanati on could be
the built - up of a PA band over time, superposing the 00 transitions m ore than the 01 one.
In Figure 5 0 d, we see that the PAA band of C 60 (- ) becomes s tronger from EADS1 to
EADS2, while the correpondin g PAD2 band at 2.35 eV does not get stronger. This might
point to a contribution of a two step s charge transfer , where first Pc+;Pc - charged pairs are
formed (this is not possible in solution but possible in films although we would expect it
more in dense films and not so much in PMM A films), and later on the ZnPc( - ) is tran sferred
to PCBM, where it is energeticall y stabilized. As in the case of 8 , we find no clear sign of
non - geminate rec ombination in 9 by looking at the c harge recombination coef ficient as
f unction of pump intensit y.
In summary, in PMMA film s, the spectr al signatures remain sim ilar to those of t he so-
lutions, which mean t hat intermolecular interaction, which would lead to excitonic feat ures,
is weak. Subpicoseco nd charge separation and recombinat ion on a 1 ns time scale was
observed for all samp les. In term s of their photophys ical behavior, dyads can be easily
divided in the two groups, a “prato - type” compounds 1 and 6 , and those with a f lexible
linker 8 and 9 . The slower r ecombination in PMM A fo r 8 and 9 was ascribed t o hindered
mobility of the link ing groups and l arger distance bet ween the two si tes. Int erestingly, pho-
toexcitation dyanamics follow a similar tr end in 1 and 6 despite tert - buty l peripheral substi -
tution of ZnPc in 1 . It se ems, int ramolecular interactions play a bigger role in affecting t he
photoexcitat ion dynamics than int ermolecular ones.

Results and Discu ss ion
124
4.1.2.3 Photo phys i cs i n drop ca sted fil ms on glas s
The EADS spectra of 1 in drop casted films (Figure 51a) and in PMMA (Figure 5 0 a)
are similar which means that even in dense f ilms, 1 cannot find an intermolecular arrange-
ment that leads to st rong excitonic interact ions. In EADS of 1 , PAD1 at 1.46 eV is clear ly
visible, which is absen t in 8 and 9 . Comparing EADS 1 and EAD S 2 of 1 , we f ind that PAD1
and PAD2 do not sho w the same decay, unex pectedly . This m eans, the band at 1. 46 eV
was equivocally assigned t o PAD1. Possibly, a triplet state gives the same PA band at 2.3
eV as a charged state. Additionally, t he doublet at 1.15 and 1.25 eV is appears being bett er
separated than in the ot her dyads. No intensity dependence is obser ved for 1 , meaning
that the charge recombination f lows fully geminate. Surprisingly, 6 behaves diff erently from
1 in drop casted films. Those features find more resemblance with those of 8 which will be
discussed below.

Fi gure 5 1 . a) EADS from global fits for TA spectra of ZnPc -C 60 films a) 1 , b) 6 , c) 8 and d) 9
in drop casted films on glass, obtained after pumping at 387 nm with 150 fs, pulses and 560 nJ
pulse energy.

Results and Discu ss ion
125
Figures 51 c refers to drop - cast fi lms of 8 . I t is evident that the bleach contr ibutions are
vastly different from those in PMM A films, pointing to inter molecular interact ions taking
place. The main bleac h is shifted from 1.85 eV to 1.65 eV, explaine d by J aggregation. 118, 119
There is a strong PA band at 2.2 eV, which is very similar f or all EADS in Figur e 5 1 c, from
which we conclude that even in EADS0, there is no singlet state. There ar e two possible
explanations f or this observation: (i) c harge separation in dense films occur s on a time
scale shorter t han 200 fs, our instrumental resolution, or (ii) t he charged state is the primary
excitation, direct ly generated f rom the pump pulse. These obser vations are similar t o the
ones in P3HT - PCBM bulk heterojunc tions, and so this study might add clues to the ongoing
discussion. Another observation does not have an obv ious explan ation: at the position of
the C 60 (- ) band, we seem to have a doublet, and this doublet has a strongly diff erent decay
kinetics than t he PAD2 band at 2.2 eV. PAD2 decays on a 400 ps time scale while t he
doublet at 1.2 eV decays on a 10 ps time scale. However, for donor - acceptor charge sep-
aration, both donor and acceptor charged ba nds should decay with the same kinetics. As
a possible solution t o explain suc h an event, we assum e that one of the bands const ituting
the doublet is in fact a triplet state. The band at 1. 2 eV decays faster t han the one at 1.15
eV. Intensity depende nt measurem ents do not show a str ong change of the charge c arrier
lifetime. This means t hat even in drop cast films, most of the charge r ecombination is gem-
inate, which is undesirable f or photovoltaics. EADS of 9 in drop casted fil m i s represent ed
on the Figure 5 1 d. I n overall, 8 and 9 behave very s imilarly, wit h respect t o the spectral
shapes of the EADS as well as the resulting time constant s. As in the case of 8 , in 9 the
charge recombination is mostly geminate.
In summary, in dense films fabr icated by drop casting of 5 × 10 -6 M solutions of dyads
in THF, we find strong excitonic eff ects for all systems, with the exception for 1 , from en-
hanced intermolecular interaction. Charge carrier formation in all cases is so fast that we
cannot resolve it (< 200 fs) . Interestingly, it might even be the case of a direct excitation of
charges. Charge rec ombination happens fa ster than in PMMA matr ix. However, the gemi-
nate recombinatio n remains str ong which means that the charge pairs do not become re-
ally separated but stay spatially corr elated. Finally, in drop casted films we find evidence
for the occurrence of a third state, which might be a triplet s tate. More prof ound studies are
deemed necessary to understand such kinetics. However, they were not undertaken within
a time framework of this t hesis.

Results and Discu ss ion
126
Table 4. Transient charge separation (CS) and recombination (CR) time c onstants in picosec-
onds (ps) of the dyads in THF solutions, PMMA matrix and drop casted films (films).
Compound CS (THF) CR (THF) C S ( PMMA) CR (PMMA ) CS (film) CR (film )
1 1.15 234 0. 40 849 < 200 f s 568
6 1.28 403 0. 19 982 < 200 f s 666
8 1.92 320 0. 31 757 < 200 f s 471
9 1.61 669 0. 43 1152 < 200 f s 529

4.1.3 Microscopic ch aracterizati on
Next, the a mphiphilic dyad 7 , bearing charg ed ammonium functionality, was synthe-
sized with the aim to study its ability towards self - assembling in aqueous solutions in a
similar manner to how it has been report ed before by our group. However, according to
TEM studies, extensive sonicatio n in water did not afford formation of any nanorod - like/fi-
ber struct ure on the TEM grid, except t hose round - shape r epr esented in t he Figure 52 .
Lower dispersiveness of the dyad 7 in aqueous media might be due to the lack of the tert -
butyl substitutent s, making stron ger an inter molecular interact ion between th e nanocar-
bonic units.

Figure 52 . TEM image of the round - shape agglomerates form ed by dispersion of conjugate
17 in H 2 O at a) 2.5 µm, b) 500 nm, c) 300 nm scale.
T he drop casted films t hat were studied by fem tosecond TA were exami ned by AFM
in order to draw the c orrelation be tween the s urface morphology an d photophys ical behav-
iour of the systems. I n the case of the dyad 1 , a uniform coverage of the glass surface by

Results and Discu ss ion
127
tens of nanometer - size agglomerates has b een found. The corresponding t opographic im-
age and phase pict ure are r epresented on the Figure 53a and 53 b. The profile inf ormation
(Figure 53c) suggest s the formation of nearly 200 nm layer pack ed with approximat ely 100
nm size ZnPc -C 60 spherical agglomerates. In a contrast, being depos ited in the same c on-
ditions, dyad 6 revealed stronger tendency to segr egation (Figure 53d and 53e). The latter
afforded the format ion of interconnected few micr ometres - long, 300 nm - height islands,
comprising appr oximately 100 nm size spherical agglom erates of ZnPc -C 60 dyad 6 . Th e
observed morpholog y stands in agreement with the charge dynamics revealed by femto-
second TA. In ot her words, absence of an ordered st ructure, that would allow an efficie nt
charge delocalizat ion and extraction, brings t hese systems to a geminal recombination as
a governing mechani sm after charge separat ion. More tight intr amolecular distances in 6,
when compared with 1, are m ostly dictated by less affinity of the molecules towards t he
glass surfac e. The latter br ings 6 to a non - structur ed formation of am orphous aggregates
that prefer to form stacks giving bigger amorphous agglom erates rather than to remain on
the surf a ce.
Figure 53 . AFM topographic image of a spin casted solutio n of a) 1 and d) 6 in TH F on glass.
The corresponding profiles along the dashed white line of c) 1 and f) 6 . Phase image for b) 1 of the
area shown in a), and for e) 6 of the area shown in d).

Results and Discu ss ion
128
Examination of the morphology of the films of the remaining two dyads with f lexible
linkers 8 and 9 afforde d informativ e ima ges represented in the Figure 54 . Inte restingly, th e
fairly similar structures have been found in both cases – unifor m spherical dots of approx-
imately 400 nm av erage size. Obviously, a certain degree of geometrical freedom allowed
their self - assembly, avoidi ng an extensive contact with hydrophilic surface of a glass sub-
strate. At this point, it would be extremely diff icult to predict t he packing geom etry of the
dyads 8 and 9 within these spher es. One can suggest those to have a micelle structure
with fullerenes poi nting inwar d the sp h ere whereas phthalocya nines – out ward, as a con-
sequence of being imm ersed in a coordinating THF solution. How ever, such suggestion s
have only a speculative char acter and are not supported by any strong experimental evi-
dences. It’s worthy to ment ion that presence of such nano - sized par ticles might be the
reason of an extremely pronounces sc attering in UV -vis spect ra of drop casted films of 8
and 9, earlier shown on the Figure 54c and 54d. Not a surprise, a geminate recom bination
was observed as a major mechanism of relaxation in TA experiment s.

Figure 54 . AFM topographic image of a spin casted solutio n of a) 8 and d) 9 in THF on glas s.
The corresponding profiles along the dashed white line of c) 8 and f) 9 . Phase image for b) 8 of the
area shown in a), and for e) 9 of the area shown in d).

Results and Discu ss ion
129
4.2 DNA i nt erstr and cross - linking on surfa ce
Several methods for cross - linking of DNA strands have been described . 120 Exploiting
a technique for covalent c ross - linking of furan - modifie d ODNs in solution this was the first
study to demonstrat e this principle on flat surfac es by integration of t hree components:
furan - modified short oli gos (12mers) , oxidative coupling using a “ residue - free” oxidant , sin-
glet oxygen, and an efficient solubl e phthalocyan ine photosensit izer functio nal with red
light. In the following results is dem onstrated that covalent ICL format ion can be applied to
surface - imm obilized oligonucleo tides using 1 O 2 on furan - carry ing ODNs allowing for the
use of considerably short er oligonucleotides than in class ical hybridization - on ly based DDI.
Proof - of - principle is d emonstrated by surf ace plasmon r esonance (SPR) meas urements
on a Biacore system using com plementary oligonuc leotide pairs for cross - linking and an
FITC/anti - FITC probe as a m odel system in order to generat e strong SPR signals . 121 In
addition , the high efficiency of t he principle is c onfirmed by an Enz yme - linked Im muno-
sorbent Assay (ELI SA).
Addressable s urfaces are pr omising tools for bioana lytical and diagnost ic applica-
tions and for positioni ng of biomolecules on chips, in microf luidic channels or ot her struc-
tures. Si te - specificity allo ws e. g. f or multiplexed assays. DDI has been explored in this
context as an easi ly accessible a ddress gener ator. For follow - up assay steps or m anipu-
lations a stable bond i s desirable . The second part of the res ults show the developm ent
towards the impleme ntation of t his ICL methodology into a multipl ex immunoassay for car-
diac markers, studies of the effect of the singlet oxygen on the antibody as well as cross -
reactivity tests on SPR and microarrays are shown. Table 4 identifies the ODNs used in
the experiments.

120
a) K. Ichikawa, N. Kojima, Y. Hirano, T. Takebayashi, K. Kowata and Y. Komatsu, Chem.
Commun. ,
2012
, 48, 2143 - 2145; b) M. Shelbourne, T. Brown, A. H. El - Sagheer and T. Brown,
Chem. Commun. ,
2012
, 48, 11184 - 11186; c) N. E. Price, K. M. Johnso n, J. Wang, M. I. Fekry,
Y. Wang and K. S. Gates, J. Am. Chem. Soc. ,
2014
; d) S . Ghosh and M. M. Greenberg, J Org.
Chem. ,
2014
, 79, 5948 - 5957 .
121 a) J. A. Sch enk, F. Sellrie, V. Böttge r, A. Menning, W. F. Stöcklein and B . Micheel, Biochimie,
2007 , 89, 1304 - 1311 ; b) M. Hovestädt, H. Memczak, D. Pleiner, X. Zhang, J. Rappich, F. F. B ier
and W. F. Stöcklein, J Mol Recognit, 2014 , 27, 707 - 713 .

Results and Discu ss ion
130
Table 4. Oligonucleotide sequences and nomenclature .
DNA
name

DNA
Sequen ce

Structure of furan-
contai ning nucleo side X

ODN1

5' bioti n CTG ACG GX G TGC 3'

HO OH
O
O

ODN2

5' bioti n CAG TCG GX G AGC 3'

ODN3

5' biotin GAC TG C CXC ACG 3'

ODN4

5' biotin CAC AG C CXC TCG 3'

ODN10

5' bioti n G ACG GXG TGC 3'

ODN8

5' bioti n ACG GXG TG 3 '

C.ODN1

3' GAC TGC CCC ACG - F ITC 5'

C.ODN2

3' GTC AGC CCC T CG - FIT C 5'

C.ODN3

3' CTG ACG GXG TGC - FITC 5'

C.ODN4

3' GTG TCG GX G AGC - FIT C 5'

C.ODN10

3' C TGC CCC ACG - F ITC 5'

C.ODN8

3' GC CCC ACG - F ITC 5'

4.2.1 DNA - directed immobiliz ation on SP R biosensor
chips
The limit of det ection of hybridization of the C.ODN1 at different concent rations with the
immobilized O DN1 on the neutravidin - coated gold chip are shown in Figure 55. Conce n-
tration- dependent binding signals were obtained as a funct ion of the injection time.

Figure 55 . Exemplary sensorgrams of injections of different concentrations (10 µM - 0.01 µM
in a 1:3 dilution series) on C.ODN1 functionalized flow cell.
The formation of a covalent bond between the immobilized ODN on a surface and
the C.ODN target should enhance the st ability of the duplex. This duple x should be
stable even in the presence of Na 2 CO 3 which has a strong anion effect and stabilizes

Results and Discu ss ion
131
the single str anded conformation . 122 Us ing ODN1, DNA c ross - linking on t he surfac e
was investigated usin g 5 µM MB at the conditions descr ibed above. Detect ion of hy-
bridized ODN1 by binding of the HRP - labell ed anti - FITC antibody showed si milar SPR
response to the cr oss - linked ODNs aft er regeneration of the surface with Na 2 CO 3 ( Fig-
ure 56). This is due to t he increased stab ility of the ODN duple x by the cross - link
formed. The somewh at lower signal (B1 vs . A1) may be due to som e singlet oxygen -
induced DNA damage. When ODN1 was only hybridiz ed, thus allowing dehybridizat ion ,
the duplex was not stable to t he regeneration step.

O
O O
Na
2
CO
3

1
O
2

Na
2
CO
3
A1 B1
O
A2 B2

Figure 56 . Comparison of the SPR signal obtained from non - cross - linked DNA and cross -
linked DNA duplex (ODN1 - C.ODN1 ). The signal obtained from the hybridized duplex (A1) de-
creases after surface regeneration (A 2). The binding of the cross - linked DN A duplex with the
recognition antibody is stable aft er regeneration following I CL formation (B1 - B2). Detection of
DNA ICL formation using ODN1 by 48 nM of H RP - labelled anti - FITC antibody, its binding is
stable after regeneration using 50 mM Na 2 CO 3 .

122 E. N. Galyuk, D. Y. Lando, V. P. Egorova, H. Dai and Y. M. D osin , J. Biomol. Struct. Dyn. ,
2003 , 20, 801 - 809 .

Results and Discu ss ion
132
Covalent cross - linki ng in solution was proven us ing polyacry lamide gel el ectr opho-
resis and MALDI - TOF - MS, resulting in the observ ation of [M+H + ]/2 at m/z 4,13 2.332 fo r
ODN1 - C.ODN1 .
Control experim ents confirm ed that the anti - FITC HRP ant ibody does not bind to
the immobilized bi otinylated and f uran - modified capt ure oligonucleo tide ODN1, the an-
tibody only recognizes t he FITC from the respectively labeled sequence C.ODN1 onc e
hybridizatio n has occurr ed The sensorgram obser ved during the B iacore exper iments
in shown in Figure 57.

Figure 5 7 . Control experiment. The anti - FITC HRP antibody does not bind to the immobi-
lized biotinylated and furan - modified capture oligonucleotide ODN1, the antibody only recog-
nizes the FITC from the r espectively labelled sequence C.O DN1 once hybridization has oc-
curred.

The ICL of the novel furan - modif ied sequences, ODN2, 3 and 4 to the complemen-
tary C. ODNs showed similar SPR respons e, the increase in t he SPR response cor re-
sponding to the bindi ng of FITC - C.ODN sequences w ith the recognition an tibody is
preserved aft er regeneration condit ions due to the covalent bond formed, which in-
creased the stability of the ODN duplexes.

Results and Discu ss ion
133
Fi gure 58 shows the overlay of t he sensorgrams obtained and a schem atic represen-
tation of the steps during the exper iment .

O O O
Na
2
CO
3
Na
2
CO
3

1
O
2
O
O

ICL
a b c d e

Figure 58 . SPR sensorgram from non - cross - linked DNA and cross - l inked DNA (ODN2, 3 and
4). The injection of the anti - FITC - HRP detection antibody (48 nM) on the immobiliz ed hybridized
ODN duplexes (a) produced an incre ase of the signal (b). Thi s signal decreased after surface re-
generation using 50 mM Na 2 CO 3 (c). Then, the chip was taken out of the SPR instrument for hy-
bridization and irradiation for ICL formation (5 µM MB). The chip was introduced into the SP R
instrument again and the surface was regenerated again (d). Even after surface regeneration the
detection antibody still recognised th e FITC la bel C. ODN of the stable duplex leading to an increase
in the SPR response (e). The binding of the OD Ns and the detection antibody was disturbed again
using Na 2 CO 3 (f) .
SPR experiment s indicates t hat the yield of DNA Cross - linking form ation by using MB
was 30 - 40 % and for TT1 ca. 5 - 10 %. As expected, the MB was more ef f icient in promoting
ICL formation than the phthalocy anine TT1, whose lower photodynamic activity can result
from the format ion, to some extent, of molecular aggregat es in water . 7

Results and Discu ss ion
134
In order to determ ine suitable sequences which can be used in platfor ms for multi-
plexed diagnostic assays, hybridization sp ecificity meas urements were per formed . The
binding of the novel fur an - modified sequences, ODN2, 3 and 4 to the complementar y
C.ODNs are presented in Table 5. All three sequence s are very specific and show no
cross- reactivity. M oreover, the specific det ection of ODNs in solution was possible even
for mixtures, laying the basis f or a selective platform for multiplex bioanalytical applicatio ns.
Table 5 . Binding signals in RU obtained by injection of 1 µM C.ODNs 2, 3, & 4. The three
sequences yield highly specific binding, unspecific effects ar e < 2 % .
C.ODNs

2

3

4

2+3

2+4

3+4

2+3+4

ODN2

351 1 - 10 331 319 -3 327
ODN3

5 324 -7 318 1 292 308
ODN4

1

2

287

2

282

284

288

T he studies focuss ing on findin g out the minimum oligonucleoti de length that can
achieve an ef ficient crosslink on a surfac e are presented in F igure 59 . The res ults show
that a 12 or 10mer ODNs can be used because t he affinity decreas es drastically in th e
case of an 8mer, as was expected because its length is under the 10 bases that a turn of
the DNA helix require s.

F igure 59 . SPR response of the oligonucleotide affinity on the surface. The 10 - and 12mer
ODNs are suitable for addressing an ODN - antibody conjugate.

Results and Discu ss ion
135
The 10 - and 12 mers were selected for the synthesis o f the a ntibody - oligon ucleotide
conjugate required. The conjugat es were synthes ized by a method alr eady described, 123
and the best result was obtained for the conjugate that has a spacer (Sp (poly (T) tail of 10
bases)) between the antibody an d the recognition sequ ence ( Figure 60 ).

Ab -Sp- 12mer >> Ab - 12mer > Ab - 10mer
Figu r e 6 0 . A ntibody - oligonucleotide conjugate affinity over the time.

123 O. Söderberg, K. - J. Leuchowius, M. Gullberg, M. Jarvius, I. Weibrecht, L. - G . Larsson and U.
Landegren, Methods , 2008 , 45 , 227 - 232.

Results and Discu ss ion
136
4.2.2 Light - induc ed immobiliz ation on microplat e sur-
faces
The results in Fig ure 6 1 clear ly show that bot h MB and irradiati on are necessar y for
the ICL formation, being both limit ing factors for t he formation of singlet oxyge n necessary
to oxidize the f uran nucleoside which can t hen form the covalent link with the com plemen-
tary strand. The wells under dar k control conditions pr oduce insi gnificant opt ical density
(OD) values aft er regeneration of the s urface with sodium car bonate. The OD after dehy-
bridization att ack almost reaches the values observed befor e surface regeneration .

1
O
2
Na
2
CO
3
B1
O
B2

Figure 6 1 . Signals (OD) obtained for a microtiter plate - based E LISA based on duplex systems
(ODN1 - C.ODN1) B1 and B2 and a HRP - labelled detection antibody. Com parison of the values
obtained before and after regeneration with 50 mM Na 2 CO 3 and irradiated vs. protected parts with
photosensitized (5 µM MB ) cross - linking. High signals indicate cross - linking requires a photo sen-
sitizer (PS) and light .

Results and Discu ss ion
137
4.2.3 Multiplex immu noassay cross - re activity

The results in Figure 62 shows the cr oss - reactivity experim ents performed in microar -
ray and surface plasmon res onance. The concentrat ion choosed of the analized pr oteins
were in function of their differ ents levels of clinical cut - o ff, which are summar ized in the
chapter 1.1.1 Cardiac Biomar kers. SPR experiment s were performed first to determine that
all reactans of the immnuassay reaction ar e suitable for the use in the futur e address -
multiplex diagnostic as say. The biding signal s are represented in Figure 62 .a) . All compo-
nents showed a high s pecificity a nd no cross - r eactivity with the ot hers reactans and only
showed biding with their corresponding matc h. However, when the multiplex im munoassay
is performed in microarray its presents a high cross - reactivity of the T1 protein wit h a l l
captures antibodies (Figure 62.b) . Therefore, TI shouldn´t be combine with FABP and CRP
proteins on microar ray detection platform. Although have been used bef ore in multianalyte
detection using differ ents transduction platf orms. 124 The higher florescence intens ity in mi-
croarray of CRP corresponds wit h the high resonance units of SRP. As well as for FABP,
it shows few resonanc e units in comparison with values obtained for T1 and CRP .

Multiplex Sandwich Immunoassay
Capture
antibody
Analyte
Detection
antibody
conjugate

Figure 62 . Cross - reactivity of the multiplex san dwich immunoassay. a) Binding signals in RU
obtained by injection of 1 µ g/mL detection antibody - FABP; TI & CRP. Thr ee a ntibodies yield highly
specific binding, unspecific effe cts are < 1 %. b) N ormalized intensity fluorescence signals obtained
from a microarray .

124 a) A. Qureshi, Y. Gurbuz and J. H. Niazi, Sens. Actuators B , 2012 , 171 – 172, 62 - 76; b) D. P .
Matta, S. Tripathy, S. R. Krishna Vanjari, C. S. Sharma and S. G. Singh, Biomed. Microdevices ,
2016 , 18, 111; c) N. Radha Shanmugam, S. Muthukumar, S. Chaudhry, J. Anguiano and S. Prasad,
Biosens. Bioelectron. , 2017 , 89, 764 - 772.

Detecti on Ab

Captur e Ab +
A naly te

F ABP

T1 CRP
TI

1,7 325 1,8
F ABP

46

0,6

1,1

CRP 1,3 1,6 872
b) Microarr ay

a) Surface Pl asmon Resonan ce

Results and Discu ss ion
138
4.2.4 Imple mentation of ICL in immunoa ssay s
The OD values of the imm unoassay decrease s ignificant over time after irradiat ion of
the capture antibody i n the presence of photosensitizer s (Figure 63 ) . The singlet oxy gen
oxidate the disulfide b onds, which ar e critical to maintain t he protein struc ture and are
particularly susceptible to oxidative modificat ion and loss of function ver y fast. 125

Figure 6 3 . OD values obtained by variation of the irradiation time with red light and methylene
blue ( 5, 1 and 0.1 µM). The OD values are reduced after irradiation of the capture antibody.
The results of study the ICL on surface at lower concentration of photosensit iser and
irradiation time conf irmed that the ICL formation is fast er than in solut ion . On the surface
the irradiation time required for ICL formation is only 10 min . In Figure 64 it is shown that
a concentration of 5 µM MB is more eff icient.
Figure 64 . OD values obtained by variation of the concentration of methylene blue (5, 1 and
0.5 µM) and the irradiation time in minutes.

125 M. Karimi, M. T. Ignasiak, B. Chan, A. K. Croft, L. Radom, C. H. Schiesser, D. I. Pattison and
M. J. Davies, Sci. Rep ., 2016 , 6, 38572.

139

5. C onclusion s

C onclusions
141
5.1 Pht h al ocyan ine -C 60 fullerene d yads
In conclusion, f our novel ZnPc -C 60 dyads 6 - 9 have been designed and sy nthesized,
bearing peripherally un substitut ed ZnPc units. To ac hieve this goal, two m ain synthetic
strategies for t he covalent modification of C 60 fullerene have been successfully employed ;
namely Prato - type 1,3 - dipolar c yacloaddition and Bingel - Hirsch m ethod of cyclopropana-
tion. Preparation of novel TBDPS - substituted ZnPc was deem ed important for obtaining all
these conjugates . T he synt hesis of monos ubstituted Pcs in suff icient amount and p urity for
their furt her covalent modificatio n has been a bottleneck in chem istry and materials science
employing non - substit uted Pcs. It was obtai ned in enough quantit ies for t he studies even
the difficulties of t he synthesis and purificat ion .
Next, th e st udy of photoexcitation dynamics of donor - acceptor conj ugates 6 , 8 and 9
in comparison with t ert - butyl substit uted analog 1 , perf ormed in THF solution, PMM A matrix
and drop casted film, allowed us to estimate the influence of the condensed phase of the
life time of charge separ ation (CS) and charge r ecombination (CR ). This is a valuable in-
formation for OPV commun ity. B riefly, a longer spacer between ZnPc and C 60 and the use
of PMMA matrix was found to extend t imes of CS and CR, especially for t he dyads 8 and
9 – by “ freezing” them in one of the conformations. In contra st, the CS and CR times were
significantl y dropped down for the dyads in the drop casted films as a consequence of
closer inter molecular cont acts and the lack of any supram olecular order in the solid sta te.
In fact, all the systems suff ered a geminal recombination, extr emely undesirable in O PV
applications, since it means excitons are not successfully split into f ree charges.
In addition, all the f ilms studied b y TA were i nvestigated by AFM , revealing a globular
morphology of the amorphous agglomerat es having a size of hundreds of nanometer s.
Additiona lly, the am phiphilic dyad 7 was studi ed by TEM, however, no sign of a pr esence
of ordered struct ures similar to fibers or rods was found. Despite an apparen t strong ten-
dency of the dyads to int ermolecular inter action and a dense packi ng in the solid state, no
evidence could be fo und in solution in the range of concent rations f rom 10 -7 to 10 -5 M,
employing UV - vis spect roscopy. The results of our study suggest the nec essary use of
nanocomposite polym ers a s p ossible templates for enhancing the supr amolecular or dering
and consequent OPV per formance of the ZnPc -C 60 covalent dyads .

Conclusions
142
5.2 DNA Int erst rand cr oss - linking on s urf ace
The steps to develop a new stable platf orm based on IC L methodology wer e described
and all objectives wer e successfully accom plished. It was shown that I CL formation of fu-
ran - modified DNA is a very useful method to incr ease the st ability of short immobilized
DNA duplexes on a s urface. SPR and ELISA experim ents confirmed the ICL for mation and
the stability of the DNA after regeneration compar ed to the non -cross- linked O DN.
Two photosensitizer s were compared and it was determ ined that methylene blue (MB)
is more efficient in promoting ICL for mation than the phthalocyanine TT 1. Its lower
photodynamic act ivity can result from the formation, to some extent, of molecular
aggregates in water . The minimum optimum length for addressing t he antibody -
oligonucleot ide conjugate was found to be 12mer with a poly( T)10 tail. Fur thermore, thi s
work demonstrates that it is possible to simultaneously use thr ee different specific
sequences for selective immobilizat ion on a microarray surf ace.
Concluding it can be said that this techniq ue represents a biomolecu le - compat ible
immobilization met hod that could improve the robust ness of array - based detection
metho ds, but t he effect of the s inglet oxygen on the change of t he bi n ding site of t he
antibody needs to be taken into account. Nevert heless, it i s a very prom ising tool to
increase the stability o f other surfaces based on DNA hybridiziat ion which usu ally ha ve the
drawback of being r ather unstable .

Phthaloyanines pr oved to be versatile compounds in achieving nano chemical
structur es that can be used in novel, innovativ e devices.

143

6. Conclusi on es

C onclusiones
145
6.1 Diadas ft alocia nina -ful ler eno

En conclusión, c uatro nuevas diadas ZnPc - C 60 6 - 9 han sido diseñ adas y sintetizadas,
las cuales contienen unidades ZnPc sin sustituyentes per iféricos. Para esto, se emplearon
dos principales es trategias d e sint é tica para la modificación cov alente de C 60 fu l lereno: la
cicloadició n 1,3 - dipolar de tipo - Prato, y el método de ciclopropanación B ingel - Hirsch. La
preparación de est a nueva ZnPc TBDP S - sustituida fue c onsiderada import ante para la
obtención de los conjugados. Un obstáculo de la química y de la ciencia de los mate riale s
ha sido la sínt esis d e Pc s mono sustituidas en s uficiente cant idad y pureza a partir de Pc s
no sustituidas para poder realizar una m odificación covalente adici onal. A pesar de estas
dificultades , esta Pc fue obtenida en cant idades suficient es para los estudios .
Además, el est udio de la dinám ica de foto exc itación de conj ugados dador - acep tor 6,8
y 9 en com paración con el aná logo tert - butil sustituido 1 , realizad a en solución THF, matriz
PMMA y película de gota fundida, nos permitió estim ar la influencia de la fase condens ada
en el tiempo de v ida de la separación de car ga (CS) y la r ecombinación de carga ( CR).
Esta es información e s de gran valor para la comunidad O PV. En resumen, se encontró
que un mayor espac io entre la ZnPc y C 60 y e l uso de la m atriz PMMA extendia los tiempos
de CS y CR, especial mente para las dia das 8 y 9 , congelándolas en una de sus confor -
maciones. De modo c ontrario, los t iempos de separación y re combinación de carga dismi-
nuyeron significat ivamente para las diadas en las películas como consecu encia de con-
tact os intermoleculare s más cercanos y la falt a de cualquier or den supramolecular en e l
estado sólido. De hecho t odos los sistemas sufren una r ecombinación gemin al, algo ex-
tremadamente indese able en las aplicacione s OPV, puesto que significa que los excitone s
no se separaron exitosam ente en cargas libr es.
Adicionalmente, todas las películas estudia das por TA fueron invest igadas por AFM,
revelando una mor fología globular de los aglomerados am orfos que tienen un tamaño de
cientos de nanómet ros. La diada anf ifílic a 7 fue estudiada por TEM. Sin em bargo, no se
encontraron signos de la existencia de est ructuras or denadas similar es a las fibras o bas-
tones. A pesar de una aparente tendenci a fuerte de interacción molecular de las diadas y
un empaquetado denso en el estado sólido, al emplear espectr oscopía UV - vis no se pudo
encontrar evidencia en solució n en el rango de concentraciones de 10 -7 a 10 -5 M . Los
resultados de este estudio sugi eren el uso necesario de polímer os nanocompuestos com o
plantilla para m ejorar el ordenamiento s upramolecular y el consecuent e desempeño OPV
de las diadas covalent es ZnPc -C 60 .

C onclusion es
146
6.2 E nlaces q uímicos interca tenar ios de DN A
en super ficie
Este estudio describe l os pasos para desarrollar una nueva platafor ma estable basado
en la metodología ICL (enlaces quí micos intercat enarios), cum pliéndose exitosamente sus
objetivos. Se d emostró que la form ación ICL de ADN modif icado con un furano es un
método muy útil para incrementar la est abilidad de los dúplex cortos de ADN inmovilizados
en una superficie. Experimentos de SPR y ELI SA confirmar on la formación ICL y la est a-
bilidad del ADN después de regener ación compar ado con el ADN no enlazado quí mica-
mente.
Así mismo, dos fot osensibilizadores f ueron comparados , determ inándose que el azu l
de metileno (M B) es más eficiente que la T T1 ftalocianina par a promover la formación de
ICL. Su baja actividad fotodinám ica puede resultar , hasta cierto punto, como consecuencia
de la formación de agregados m oleculares en agua. Se encont ró que la longitud óptim a
mínima necesaria par a dirigirse a los conjugados ol igonucleotido - anticuerpo es de 12mer
con una cola de poli - T 10. Por otra parte, se demostr ó que es posible utilizar tres secuen-
cias específicas difer entes para una inmovil ización sele ctiva en una superficie de chip.
En conclusión, es tá técnica repre senta un mét odo de inmovilizació n compatible con
biomolécula s que pu ede mejorar la impor tancia de mét odos de detección basados en
chips de ADN. Sin embargo, el efect o del oxígeno singlet e en el cambio del sitio de unión
del anticuerpo debe ser tomado en cuenta. No obstant e, es una herramient a prometedora
que incrementa la estabilidad de otr as superficies basada en la hibridación de ADN, la cual
usualmente t iene la desventaja de ser algo inestable.

Ftalocianinas han pr obado ser compuestos versátiles en conseguir est ructuras nano
químicas que pueden ser usadas en dispositivos innov ativos.

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