Cet ouvrage fait partie de la bibliothèque YouScribe
Obtenez un accès à la bibliothèque pour le lire en ligne
En savoir plus

Determination of single base mutations related to the gene specific diseases by using electrochemical DNA biosensors in the integrated system [Elektronische Ressource] / vorgelegt von Burcu Ülker

De
103 pages
DETERMINATION OF SINGLE BASE MUTATIONS RELATED TO THE GENE SPECIFIC DISEASES BY USING ELECTROCHEMICAL DNA BIOSENSORS IN THE INTEGRATED SYSTEM Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Burcu Ülker aus Izmir Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 06.07.2005 Vorsitzender der Promotionskommission: Prof. Dr. D.-P. Häder Erstberichterstatter: Prof. Dr. Ulrich Nickel Zweitberichterstatter: Prof. Dr. Carola Kryschi Abbreviations A Adenine a Activity BSA Albumin fraction V C Cytosine CE Counter electrode σ Charge density D Diffusion coefficient DNA Deoxyribonucleic acid DPV Differential pulse voltammogram Eappl Applied potential 0Ered ox Standard redox potential EDTA Ethylene diamine tetra acetic acid F Faraday constant (96.
Voir plus Voir moins

DETERMINATION OF SINGLE BASE MUTATIONS
RELATED TO THE GENE SPECIFIC DISEASES
BY USING ELECTROCHEMICAL DNA BIOSENSORS
IN THE INTEGRATED SYSTEM







Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur Erlangung des Doktorgrades













vorgelegt von
Burcu Ülker

aus Izmir


Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der Universität
Erlangen-Nürnberg




























Tag der mündlichen Prüfung: 06.07.2005
Vorsitzender der Promotionskommission: Prof. Dr. D.-P. Häder
Erstberichterstatter: Prof. Dr. Ulrich Nickel
Zweitberichterstatter: Prof. Dr. Carola Kryschi

Abbreviations
A Adenine
a Activity
BSA Albumin fraction V
C Cytosine
CE Counter electrode
σ Charge density
D Diffusion coefficient
DNA Deoxyribonucleic acid
DPV Differential pulse voltammogram
Eappl Applied potential
0Ered ox Standard redox potential
EDTA Ethylene diamine tetra acetic acid
F Faraday constant (96.487 coulombs)
FcII Factor II
FcV Factor V
G Guanine
HET Heterozygote
I Inosine
IHP Inner Helmholz Plane
iRs Ohmic potential
2j Current density (current per unit area, A/cm )
J Flux
2j Exchange current density (A/cm ) 0
µ i Chemical potential
*µ i Electrochemical potential
MUT Mutated
n Number of electrons
NaAc Sodium acetate buffer
NAP 1-Naphthylphosphate
NC Non-complementary


NOS N-oxysuccinimide esters
OHP Outer Helmholz Plane
PCR Polymerase chain reaction
pNPP p-Nitrophenylphosphate
QMT Nexterion hybridization buffer
R Universal gas constant (8.314 JK-1mol-1)
RE Reference electrode
RNA Ribonucleic acid
RT Room temperature
SDS Sodium dodecyl sulphate
Silane 3-Glycidyloxypropyl-trimethoxysilane
SPC Screen printed chip
SPE Screen printed electrode
SSC Sodium saline citrate
T Thymine
T Temperature
TBS Tris buffered saline
WB Washing buffer
WE Working electrode
WT Wild type
η Overpotential
φ Galvanic potential

















1 Introduction 9
2 Theoretical Information 11
2.1 Electroanalytical Chemistry
2.1.1 Preface11
2.1.2 Faradaic and Non-faradaic Processes 11
2.1.3 Electrochemical Cell12
2.1.4 Electrode Reactions12
2.1.5 Nernst Equation 14
2.1.6 Electrical Double Layer15
2.1.7 The Electrode Set-up16
2.1.8 Mass Transport18
2.1.9 Overpotential 20
2.1.10 Controlled Potential Techniques21
2.1.11 Working Electrodes23
2.2 Nucleic Acids 24
2.2.1 Structure of Nucleic Acids24
2.2.2 Hybridization and Denaturation26
2.2.3 Mutations27
2.3 Nucleic Acid Diagnostics 27
2.3.1 Preface 27
2.3.2Polymerase Chain Reaction28
2.3.3Biosensors30
2.3.4Electrochemical DNA Biosensors30
3Experimental 31
3.1 Chemicals and Solutions
3.1.1 Chemicals 31
3.1.2Solutions 32
3.2 Measurement Set-ups 35
3.2.1 Electrochemical Measurement Set-ups 35
3.2.2 Colorimetric Measurement Set-up36
3.3 Preparation of Screen Printed Chips 36
3.4 Methods 39
3.4.1 Surface Preparation 39
3.4.2 Label-free Electrochemical Detection Method 42
3.4.3 Enzyme-based Electrochemical Detection Method44
3.4.4 Enzyme-based Colorimetric Detection Method46
4 Results 49
4.1 Optimisation of Detection Methods 49
4.1.1 Preface 49
4.1.2Optimisation of Surface Preparation49
4.1.2.1Preface49
4.1.2.2Screen Printing Procedure50
4.1.2.3Effect of Pre-treatment Conditions 52
4.1.2.4Silane Surface Chemistry54
4.1.2.5 Electrochemical Properties of Inosine Base57
4.1.2.6 Probe Immobilization59
4.1.3 Optimisation of Hybridization61
4.1.3.1 Preface 61
4.1.3.2Hybridization Time61
4.1.3.3Hybridization Temperature63
4.1.3.4Optimum Washing Conditions66
4.1.3.5 Sensitivity of the Detection Methods 68
4.2 Investigation of Optimum Probe Sequences 71
4.3 Determination of Single Base Mutations 74




4.4 Development of Lab-on-a-chip Technology 77
4.4.1 Preface 77
4.4.2Detection Process in the Cartridge78
4.4.3Integrated Process in the Cartridge80
5Summary 83
6References 86
7 Zusammenfassung 93















































Introduction 9
1 Introduction
Determination of specific nucleic acid sequences in biological and environmental samples can
lead to early diagnosis of inherited human diseases as well as identification and detection of
1pathogens . The determination of nucleic acids in biological samples consists of three steps:
Sample preparation, specific nucleic acid amplification and detection.
In the sample preparation step, the extraction and purification of the nucleic acids are
performed. First, the relevant cells are lysed by destroying their cellular membrane in order to
release the nucleic acids. Then the released nucleic acids are extracted and purified by using
different methods.
Usually, the amount of extracted nucleic acids is not sufficient for the determination.
Therefore, a part of the nucleic acid is amplified, for example, using polymerase chain
reaction (PCR). During the PCR, many copies of the specific DNA sequence are created. The
reaction is initiated using a pair of short primer sequences which match the ends of the
sequence to be copied. Thereafter, each cycle of the reaction copies the sequence between the
primers. Primers can bind to the copies as well as the original sequence, so in time the total
number of copies increases exponentially.
The simplest method for the detection of amplified nucleic acids is gel electrophoresis
whereby the DNA is separated according to length and stained with ethidium bromide.
However, this method is label intensive and not sequence specific. Sequence specificity can
be achieved by transferring the separated DNA to a membrane and hybridising with a
2radioactively labelled probe. This method is very sensitive but complex handling with
hazardous radioactive labels are necessary. The other methods which are achieved by
3,4 3,5 6labelling the probe with biotin , digoxigenin or fluorescent dyes in order to avoid the use
of hazardous radioactive labels are also not suitable for the routine analysis because of the
long, expensive and complicated steps of these procedures. Therefore a new, sensitive,
low-cost and sequence specific detection of nucleic acid hybridization by using
1,8-10 electrochemical DNA biosensors has recently been reported .
An electrochemical DNA biosensor is an electrode with immobilised sequence specific single
strand DNA (probe) for the identification of target DNA based on its hybridization reaction
with its complementary sequence (target) under suitable conditions. The sequence-specific
11-13hybridization events can be detected directly (label-free) or indirectly by using labels
10 Introduction
(indicator-based). The labels can be indicators which intercalate into the DNA double helix
14-16 (metal complexes, antibiotics) or which interact specifically with guanine bases of
10,17-19DNA . The other possible detection method represents the use of substrate which is
changed to an electrochemically active end product in the presence of a specific enzyme
20(enzyme-based) .
Electrochemical DNA biosensors, based on electrochemical transduction of hybridization
events, have great promise for the task of pharmaceutical, clinical, environmental and forensic
applications. Such devices couple the high specificity of DNA hybridization reactions with
21the high sensitivity, low cost and portability of electrochemical transducers . The
22electrochemical biosensors can be assembled to a miniaturised array . These miniaturised
arrays of DNA biosensors are termed DNA chips.
The development of DNA chips is motivated by their potential for application in disease
23 24diagnosis, genome sequencing , the detection of polymorphisms and single-base
25mismatches . However, such micro fabricated devices are costly and difficult to prepare and
handle. For this reason, newly developed DNA chips must offer lower cost and greater
material efficiencies to gain acceptance over traditional nucleic acid diagnostic methods.
Further reduction in the cost of performing nucleic acid diagnostics can be realized by
utulizing less expensive detection methods and electrode materials.
The aim of the current work is the development of low-cost DNA chips for the determination
of single base mutations, which lead to inherited diseases or disorders (Factor V and Factor II)
from blood samples on the fluidic platform in a disposable integrated cartridge in which the
processes of sample preparation, PCR and detection are accomplished automatically.
The use of a fully automated detection system in combination with its low cost, its simplicity
in handling and the disposability of the DNA chips make the determination methods, which
are developed for detection of gene-specific inherited diseases, suitable for routine laboratory
analysis and point-of-care diagnosis.

Un pour Un
Permettre à tous d'accéder à la lecture
Pour chaque accès à la bibliothèque, YouScribe donne un accès à une personne dans le besoin