amperometric cholesterol and alcohol biosensors based on conducting polymers a thesis ...

133 Pages·2010·1.4 MB·English

AMPEROMETRIC CHOLESTEROL AND ALCOHOL BIOSENSORS
BASED ON CONDUCTING POLYMERS

A THESIS SUBMITTED TO
THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES
OF
MIDDLE EAST TECHNICAL UNIVERSITY

BY
ÖZLEM TÜRKARSLAN

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY
IN
CHEMISTRY

APRIL 2010
Approval of the thesis:

AMPEROMETRIC CHOLESTEROL AND ALCOHOL
BIOSENSORS BASED ON CONDUCTING POLYMERS

submitted by ÖZLEM TÜRKARSLAN in partial fulfillment of the
requirements for the degree of Doctor of Philosophy in Chemistry
Department, Middle East Technical University by,

Prof. Dr. Canan Özgen
Dean, Graduate School of Natural and Applied Sciences

Prof. Dr. İlker Özkan
Head of the Department, Chemistry

Prof. Dr. Levent Toppare
Supervisor, Chemistry Dept., METU

Examining Committee Members:

Prof. Dr. Leyla Aras
Chemistry Dept., METU

Prof. Dr. Levent Toppare
Chemistry Dept., METU

Prof. Dr. Kadir Pekmez
Chemistry Dept., Hacettepe Univ.

Prof. Dr. Jale Hacaloğlu
Chemistry Dept., METU

Prof. Dr. Ahmet Muhtar Önal
Chemistry Dept., METU

Date: April 6, 2010
PLAGIARISM

I hereby declare that all information in this document has been
obtained and presented in accordance with academic rules and ethical
conduct. I also declare that, as required by these rules and conduct, I
have fully cited and referenced all material and results that are not
original to this work.

Name, Last name :

Signature :

iii
ABSTRACT

AMPEROMETRIC CHOLESTEROL AND ALCOHOL BIOSENSORS
BASED ON CONDUCTING POLYMERS

Türkarslan, Özlem
Ph.D., Department of Chemistry
Supervisor: Prof. Dr. Levent Toppare

April 2010, 115 pages

Cholesterol and ethanol biosensors based on conducting polypyrrole
(PPy), poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(3,4-
ethylenedioxypyrrole) (PEDOP) were constructed. Cholesterol oxidase
(ChOx, from Pseudomonas fluorescens) and alcohol oxidase (AlcOx, from
Pichia pastoris) were physically entrapped during electropolymerization of
the monomers (Py, EDOT, EDOP) in phosphate buffer containing sodium
dodecylsulfate (SDS) as the supporting electrolyte. The amperometric
responses of the enzyme electrodes were measured monitoring oxidation
current of H O at +0.7 V in the absence of a mediator. Kinetic parameters,
2 2
such as K and I , operational and storage stabilities, effects of pH and
m max
temperature were determined for all entrapment supports. Based on
Michaelis-Menten (K ) constants, it can be interpreted that both enzymes
m
immobilized in PEDOT showed the highest affinities towards their
substrates. Before testing the alcohol biosensors on alcoholic beverages,
iv
effects of interferents (glucose, acetic acid, citric acid, L-ascorbic acid)
which might be present in beverages were determined. The alcohol content
of the distilled beverages (vodka, dry cin, whisky, rakı) was measured with
these biosensors. A good match with the chromatography results (done by
the companies) was observed.

Keywords: Amperometric Biosensors, Conducting Polymers, Enzyme
Electrodes, Cholesterol Biosensors, Alcohol Biosensors

v
ÖZ

İLETKEN POLİMER TABANLI
AMPEROMETRİK KOLESTEROL VE ALKOL BİOSENSÖRLERİ

Türkarslan, Özlem
Doktora, Kimya Bölümü
Tez Yöneticisi: Prof. Dr. Levent Toppare

Nisan 2010, 115 sayfa

Polipirol (PPy), poli(3,4-etilendioksitiyofen) (PEDOT) ve poli(3,4-
etilendioksipirol) iletken polimer matrislerinde Pseudomonas
fluorescens’tan elde edilen kolesterol oksidaz (ChOx) ve Pichia pastoris’ten
elde edilen alkol oksidaz (AlcOx) enzimleri fiziksel yolla tutuklanmıştır.
Destek elektrolit olarak sodium dodesilsulfatın, monomerin ve enzimin
bulunduğu fosfat tampon çözeltisine gerilim uygulanmasıyla monomerler
elektrot yüzeyinde polimerleşirken enzimler de hapsolmuştur. Enzim
elektrotlarının amperometrik cevapları medyatörsüz ortamda H O ’nin
2 2
yükseltgenme akımının izlenmesiyle elde edilmiştir. K , I gibi kinetik
m max
parametreler, kullanım kararlılığı ve raf ömrü tayinleri, pH ve sıcaklıktaki
değişimlerin etkileri tüm tutuklama matrisleri için incelenmiştir. K
m
değerlerine dayanılarak PEDOT matrisinde her iki enzimin de sübstratlarına
karşı daha ilgili olduğu kanısına varılmıştır. Alkol biosensörleri piyasada
bulunan çeşitli alkollü içkilerde sınamadan önce içkilerde bulunabilen ve
vi
sonuçları etkileyebileceği düşünülen glükoz, asetik asit, sitrik asit ve L-
askorbik asit içeren ortamlarda test edilmiştir. Daha sonra bu biosensörlerle
votka, cin, viski, rakı gibi distillenmiş içkilerde bulunan alkol miktarı tayin
edilmeye çalışılmıştır. Bulunan sonuçlar şişelerin üzerinde yazılı çeşitli
kromatografik yöntemlerle belirlenmiş sonuçlarla uyum içerisindedir.

Anahtar sözcükler: Amperometrik biosensörler, İletken polimerler, Enzim
Elektrotları, Kolesterol biosensörleri, Alkol Biosensörleri

vii
ACKNOWLEDGMENTS

I would like to thank my “doctorate father” Prof. Dr. Levent Toppare
for all his guidance, support and encouragement over the last six years.
I would like to express my thanks to Dr. Senem Kayahan Kıralp ,
Dr. Ayşe Elif Böyükbayram and MSc. Sevinç Tunçağıl for providing an
orientation to biosensor preparation and evaluation as well as for sharing
their knowledge on enzymes.
I wish to thank all the past and present members of Toppare
Research Group for the nice working environment.
I would like to acknowledge METU Chemistry Department for the
scientific and technical support.
I would like to thank my chemistry teacher at high school, José
Romero for making me love chemistry.
I am also grateful to Simge Tarkuç, Funda Oğuzkaya, Fadile
Kapaklı, Yusuf Nur, Gülben Ardahan, Balam Balık, Abidin Balan, Dr. Pınar
Çamurlu and Dr. Yasemin Arslan Udum for their real friendship.
I owe great thanks to my family for moral and financial support
during my entire career. I would also like to thank İlker-Jale Tuksal for
listening and advising me at every time.

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TABLE OF CONTENTS

ABSTRACT ................................................................................................. iv
ÖZ ................................................................................................................. vi
ACKNOWLEDGMENTS .......................................................................... viii
TABLE OF CONTENTS ............................................................................. ix
LIST OF FIGURES .................................................................................... xiv
LIST OF ABBREVIATIONS .................................................................. xviii
1. INTRODUCTION ..................................................................................... 1
1.1 Conducting Polymers .......................................................................... 1
1.1.1 Historical Review of Conducting Polymers ................................. 2
1.1.2 Conduction Mechanism in Conducting Polymers ........................ 5
1.1.2.1 Conductivity .......................................................................... 5
1.1.2.2 Band Theory .......................................................................... 6
1.1.2.3 Doping ................................................................................... 8
1.1.2.4 Hopping ............................................................................... 13
1.1.3 Synthesis of Conducting Polymers ............................................. 14
1.1.3.1 Chemical Polymerization..................................................... 15
1.1.3.2 Electrochemical Polymerization .......................................... 17
1.1.4 Applications of Conducting Polymers ........................................ 20
1.1.5 Characterization of Conducting Polymers .................................. 22
1.1.6 Polypyrroles and Polythiophenes ............................................... 23
ix
1.2 Enzymes ............................................................................................. 24
1.2.1 Enzymes as Proteins ................................................................... 24
1.2.2 Zwitterionic Character ................................................................ 27
1.2. 3 General Properties of Enzymes ................................................. 28
1.2.4 Enzyme Classification ................................................................ 31
1.2.5 Enzyme Activity ......................................................................... 33
1.2.6 Factors Affecting Enzymatic Speed ........................................... 33
1.2.7 Basic Enzyme Kinetics ............................................................... 36
1.2.8 Enzyme Immobilization.............................................................. 41
1.2.9 Immobilization Methods ............................................................. 42
1.2.9.1 Enzyme Immobilization via Adsorption .............................. 43
1.2.9.2 Enzyme Immobilization via Gel Entrapment ...................... 44
1.2.9.3 Enzyme Immobilization via Intermolecular Crosslinking ... 44
1.2.9.4 Enzyme Immobilization via Covalent Attachment.............. 45
1.2.10 Enzyme Immobilization via Electrochemical Polymerization
......................................................................................................... 46
1.3 Biosensors .......................................................................................... 48
1.3.1 Biosensors Based on Conducting Polymers ............................... 51
1.3.2 Amperometric Biosensors .......................................................... 53
1.3.3 Amperometric Biosensors Based on Conducting Polymers ....... 55
1.3.4 Applications of Biosensors ......................................................... 57
1.4 Cholesterol Biosensors ...................................................................... 57
1.5 Alcohol Biosensors ............................................................................ 60
1.6 Aim of the Study ................................................................................ 62
x