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

0 Ered 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 j Current density (current per unit area, A/cm2) J Flux 2 j0 Exchange current density (A/cm ) 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 11

2.1.1 Preface 11 2.1.2 Faradaic and Non-faradaic Processes 11 2.1.3 Electrochemical Cell 12 2.1.4 Electrode Reactions 12 2.1.5 Nernst Equation 14 2.1.6 Electrical Double Layer 15 2.1.7 The Electrode Set-up 16 2.1.8 Mass Transport 18 2.1.9 Overpotential 20 2.1.10 Controlled Potential Techniques 21 2.1.11 Working Electrodes 23

2.2 Nucleic Acids 24

2.2.1 Structure of Nucleic Acids 24 2.2.2 Hybridization and Denaturation 26 2.2.3 Mutations 27

2.3 Nucleic Acid Diagnostics 27

2.3.1 Preface 27 2.3.2 Polymerase Chain Reaction 28 2.3.3 Biosensors 30 2.3.4 Electrochemical DNA Biosensors 30

3 Experimental 31 3.1 Chemicals and Solutions 31

3.1.1 Chemicals 31 3.1.2 Solutions 32

3.2 Measurement Set-ups 35

3.2.1 Electrochemical Measurement Set-ups 35 3.2.2 Colorimetric Measurement Set-up 36

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 Method 44 3.4.4 Enzyme-based Colorimetric Detection Method 46

4 Results 49 4.1 Optimisation of Detection Methods 49

4.1.1 Preface 49 4.1.2 Optimisation of Surface Preparation 49 4.1.2.1 Preface 49 4.1.2.2 Screen Printing Procedure 50 4.1.2.3 Effect of Pre-treatment Conditions 52 4.1.2.4 Silane Surface Chemistry 54 4.1.2.5 Electrochemical Properties of Inosine Base 57 4.1.2.6 Probe Immobilization 59 4.1.3 Optimisation of Hybridization 61 4.1.3.1 Preface 61 4.1.3.2 Hybridization Time 61 4.1.3.3 Hybridization Temperature 63 4.1.3.4 Optimum Washing Conditions 66 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 4.4.2 Detection Process in the Cartridge 78 4.4.3 Integrated Process in the Cartridge 80

5 Summary 83 6 References 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 pathogens1. 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 radioactively2 labelled probe. This method is very sensitive but complex handling with hazardous radioactive labels are necessary. The other methods which are achieved by labelling the probe with biotin3,4, digoxigenin3,5 or fluorescent dyes6 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 electrochemical DNA biosensors has recently been reported1,8-10 . 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 hybridization events can be detected directly (label-free)11-13 or indirectly by using labels

10 Introduction

(indicator-based). The labels can be indicators which intercalate into the DNA double helix (metal complexes, antibiotics)14-16 or which interact specifically with guanine bases of DNA10,17-19. 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 (enzyme-based)20.

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 the high sensitivity, low cost and portability of electrochemical transducers21. The electrochemical biosensors can be assembled to a miniaturised array22. These miniaturised arrays of DNA biosensors are termed DNA chips.

The development of DNA chips is motivated by their potential for application in disease diagnosis, genome sequencing23, the detection of polymorphisms24 and single-base mismatches25. 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.

Theoretical Information 11

2 Theoretical Information

2.1 Electroanalytical Chemistry35,36

2.1.1 Preface

Electroanalytical chemistry is concerned with the interplay between electricity and chemistry, namely the measurements of electrical quantities, such as current, potential and charge and their relationship to chemical parameters. The factors effecting the transport of charges across the interfaces between two phases are the important issue in electroanalytical chemistry. One of these two phases contributing to the interface is an electrolyte, through which charge is carried by the movements of ions. Electrolytes can be liquid solutions or fused salts, or they can be ionically conducting solids such as sodium β-alumina, which has mobile sodium ions. The second phase at the interface might be another electrolyte, or it might be a conductor, through which charge is carried by electronic movement. The conductors can be solid or liquid. The transition of the charges, the crossing from one conducting phase into the other at the interface leads to a difference in potential. These two-phase systems can be formed by using two solid (metal-metal) or one solid and one liquid phase (metal-electrolyte). The metal-electrolyte systems describe the basis of electrochemistry whereas the metal-metal systems are used commonly for the measurement of temperature.

2.1.2 Faradaic and Non-faradaic Processes35

Two types of processes occur at the electrodes. One of these is the transferration of charges over the metal-solution interface. This electron transfer causes oxidation or reduction to occur. Since these reactions are governed by Faraday’s law, they are called faradaic processes. Faraday's law relates the amount of charge involved in an electrochemical reaction with the number of moles of reactant and the number of electrons required for the reaction. The faradaic processes are usually important in investigation of electrode reactions in the electrochemical cells. Non-faradaic processes involve the accumulation of charges at the metal-solution interface. In case of non-faradaic processes such as adsorption or desorption, the structure of the electrode and solution interface can change, influenced by changing potential or solution composition.

12 Theoretical Information

Non-faradaic processes occurring at electrodes cause a flow of non-faradaic currents (charging currents). 2.1.3 Electrochemical Cell36-38

The system which is formed by electrode-electrolyte phases is called electrochemical half- cell. The connection of two half-cells through a salt bridge forms an electrochemical cell (Scheme 1). The electrodes of both half-cells must be connected to a voltmeter in order to measure the cell potential.

Voltmeter

Salt bridge Electrode Electrode

Electrolyte

Half cell 1 Half cell 2

Scheme 1. Electrochemical Cell

2.1.4 Electrode Reactions35-39

Electrode reactions are heterogeneous chemical processes. These reactions occur due to the charge transfer through the interface of both phases, electrode and electrolyte. The heterogeneous charge transfer is caused by the varying chemical potentials (µ) of metal ions (Mez+) in the solution (L) and on the metal surface (M).

Due to the contact between two phases, with time, a chemical equilibrium develops at the interface.

Theoretical Information 13

µ ()L = µ (M ) with µ = µ o + RT ⋅ln a (1) i i i i i

In the systems featuring a difference of the chemical potentials as depicted in equation 2, the ions are transferred from the metal to the solution.

µ ()M > µ (L) (2) Mez+ Mez+

The ion transition leads to a negative charge on the metal surface, while the positively charged metal ions are assembled at the solution boundary. Thus, an electrical double layer is formed to compensate for the excess of charge on the electrode (qe), since the interface must be neutral. Due to the formation of an electrical double layer, the difference in the galvanic potential in both phases will be increased and the transfer of the metal ions will be more difficult.

The work necessary for the transition of the ions against the double layer is equal to the difference of the chemical potential in both phases and depends on the chemical environment.

∆µ = µ ()M − µ (L) (3) Mez+ Mez+

Another additional work is required to move the metal ions against the electrical field. This work is proportional to the galvanic potential (φ) and hence depends on the electrical properties of an environment that is very much larger than an ion itself. These two amounts of work for a single species can not be separated experimentally, but the differences in the scales of the environments responsible for them, make it possible to separate them mathematically. Butler and Guggenheim developed the conceptual separation and introduced the * electrochemical potential (µ ), for species i with charge zi in phase α:

*α α α µi = µi + zi Fϕ (4)

α Where µ is the familiar chemical potential, zi Fϕ is the amount of electrochemical work.

In the case of equal electrochemical potentials in both phases, an electrochemical equilibrium balances at the interface.

14 Theoretical Information

* * µi ()L = µi (M) (5)

The difference of the galvanic potential in both phases in a half cell is given by equation 6.

∆φ = φ M − φ L (6)

This difference of the potential between metal (M) and solution (L) without a passing current can be calculated by means of the concentration dependency of the chemical potential as given in equation 7.

µ 0 ( M ) − µ 0 ( L ) a ( M ) z+ z+ RT z+ ∆φ = φ − φ = Me Me + ln Me (7) M L zF zF a ( L ) Mez+

The galvanic potential consists of two different terms. One is dependent on the concentration whereas the other one is not. The term which is not dependent on the concentration is called standard potential, where the value of the activity coefficients of the components in both phases are one (a=1) in the reference conditions. The resulted equation which explains the concentration dependency of galvanic potential for metal/metal ions half cell is Nernst Equation (equation 8).

o RT a ( L ) ∆φ = ∆φ + ln i (8) zF ai ( M ) R is the universal gas constant (8.314 JK-1mol-1), T is the temperature in Kelvin, n is the number of electrons transferred in the reaction, and F is the Faraday constant (96.487 coulombs).

2.1.5 Nernst Equation35-37,40

Redox processes proceed in the half cells, which are formed by dipping an electrode into the electrolyte containing redox active components. Redox processes are reversible processes fast enough to be considered stable in thermodynamic equilibrium. The basic electrode reaction occurring at the redox electrode is,

ne − + Ox ⇔ Re d

Theoretical Information 15

where Ox and Red are the oxidised and reduced forms, respectively, of the redox couple. Such reactions occur in a potential region which makes the electron transfer thermodynamically or kinetically favourable. The relationship between the potential of the electrode and the concentration of the electroactive species at the surface is described with Nernst Equation (equation 9).

0 RT [Ox ] E = E Red Ox + ln (9) Red Ox nF [Re d ]

o where E red/ox is the standard potential for the redox reaction.

The Nernst Equation is only valid if the system is in an electrochemical equilibrium. The redox potential will adjust slowly if the transition of the electrons at the corresponding electrode is slow. In this case, the Nernst Equation is not valid.

2.1.6 Electrical Double Layer36, 39, 40

The electrical double layer is the array of the charged particles and/or oriented dipoles which exists at every material interface. In electrochemistry, such layers reflect the ionic zones formed in the solution to compensate for the excess of charge on the electrode. Accordingly, such a counter-layer is made of ions of opposite sign to that of the electrode. The electrical double layer consists of three main parts: the metallic phase, an inner layer and an outer or diffuse layer.

The layer closest to the electrode, inner layer (Helmholtz Layer), contains solvent molecules and specifically adsorbed ions. The locus of the electrical centers of the specifically adsorbed ions is called the Inner Helmholz Plane (IHP). The total charge density (µC/cm2) from specifically adsorbed ions in this layer is σi. Whenever the interaction between ion and the metal is not strong enough for the desolving process, the ion can not come as close to the metal as the specifically adsorbed ions. The imaginary plane passing through the electrical centers of the closest approaching solvated ions is known as the Outer Helmholtz Plane (OHP). The interaction of the solvated ions with the charged metal involves only long-range electrostatic forces, thus, this interaction is essentially independent from the chemical properties of the ions. These ions are said to be non-specifically adsorbed. Unlike specifically adsorbed ions at the IHP which form a two-dimensional monolayer, the non-specifically adsorbed ions are not all located at the OHP but are distributed in a three-dimensional region, called the diffuse layer, which extends from the OHP all the way into the bulk of the solution.

16 Theoretical Information

The excess charge density in the diffuse layer is σd, so that the total excess charge density on the solution side of the double layer, σs, is given by:

σ s = σ i + σ d = −σ M (10) where σM is the charge density on the metal.

In controlled-potential techniques, the charging of the double layer is responsible for the background current known as the charging current, which limits the detectability of redox active substances. Such a charging process is non-faradaic because electrons are not transferred across the electrode-solution interface. It occurs when a potential is applied across the double layer, or when the electrode area or capacitance is changing. Note that the current is the time derivative of the charge. Hence, when such processes occur, a residual current is flowing based on the differential equation:

dq dE dA dC i = = C A + C ( E − E ) + A(E − E ) dl (11) dt dl dt dl pzc dt pzc dt where dE/dt is the potential scan rate, dA/dt is the rate of the change of the area, Cdl is the capacitance per unit area, Epzc is the potential at zero charge and dCdl/dt is the rate of capacitance change. The term dCdl/dt yields importance if adsorption processes change the double layer capacitance.

2.1.7 The Electrode Set-up37

The overall chemical reaction taking place in an electrochemical cell is made up of two independent half reactions, which constitute the real chemical changes at the two electrodes. Each half reaction responds to the interfacial potential difference at the corresponding electrode. In electrochemistry, one of these specific electrochemical reactions is important, and the electrode at which it occurs is called the working electrode, whereas the other is called the reference electrode.

Since the reference electrode exhibits constant conditions, the potential difference between the reference electrode and the measurement buffer is constant. Therefore, the working electrode is responsible for any changes within the cell. Thus, the observed potential of working electrode is evalued with respect to the reference electrode. If the passage of current does not effect the potential of the reference electrode, the potential (E) of working electrode is given by equation (12).

Theoretical Information 17

E appl = E + iR s = E eq + η + iR s (12)

The term iRs is the ohmic potential drop in the solution, the Eeq is the potential of the working electrode in an open circuit, η is the overpotential, Eappl is the applied potential and E is the new potential value of the working electrode. Under conditions where iRs is small (less than 1-2 mV), the two electrode set-up can be used to determine the i-E curve. On the other hand, when currents or solution resistance are high (e.g., in large scale, electrolytic cells or galvanic cells or in experiments involving non-aqueous solutions with low conductivities), the iRs term may be much larger. In experiments where iRs may be high, a three electrode set-up is preferable in order to minimise the iRs (Scheme 2).

Power supply

I

Auxiliary electrode Working electrode Reference electrode V In cell notation

Working electrode

Reference electrode

Auxiliary electrode

Scheme 2 . Three-electrode cell and notation for the different electrodes.

In this setup, the current is passed between the working electrode and auxiliary (so-called counter) electrode. The auxiliary electrode can be any electrode desired, because its electrochemical properties do not influence the behaviour of the electrode of interest. It is usually chosen to be an electrode that does not produce substances by electrolysis which can reach the working electrode surface and cause interfering reactions there. The potential of the working electrode is measured relatively to a separate reference electrode. The device used to

18 Theoretical Information

measure or monitor the difference in potential between the working and reference electrodes has a high input impedance so that a negligible current is passed through the reference electrode. In the three electrode set up, the current which is caused from the high input impedance of the device is passed not through the reference electrode but through the auxiliary electrode, so that the potential of the reference electrode remains constant and, the iRs contribution to the measurement will be small.

2.1.8 Mass Transport35, 36, 38

The Nernst Equation is limited to the surface region of the electrode. For a more realistic description of real redox experiments, the mass transport of the redox pair species between the bulk solution and the surface area has to be considered. Mass transfer is the movement of material from one location within a solution to another. It arises either from differences in electrical or chemical potential between the two locations, or from movement of a volume element of the solution. The modes of mass transfer are diffusion, convection and migration.

The flux (J) is a common measure for the mass transport at a fixed point. It is defined as the number of molecules penetrating a unit area of an imaginary plane in a unit of time, and has -2 -1 the units of mol cm s . The flux of species i (Ji) at a distance x from the surface is governed by the Nernst-Planck equation, written for one dimensional mass transfer along the x-axis as:

∂C ( x ) z F ∂φ ( x ) J ( x ) = − D i − i D C + C v( x ) (13) i i ∂x RT i i ∂x i

2 where Di is the diffusion coefficient (cm /sec), ∂Ci (x)/ ∂x is the concentration gradient at distance x, ∂φ()x / ∂x is the potential gradient, zi and Ci are the charge and concentration of species I, respectively and v(x) is the velocity (m/sec) with which a volume element in solution moves along the axis.

Diffusion

Diffusion is the movement of a species under the influence of a concentration gradient from regions of high concentrations to regions of lower ones. The aim is to compensate the concentration differences. It occurs in all solutions and arises from local uneven concentrations of reactants. Entropic forces act to smooth out these uneven concentration distributions and are therefore the main driving force for this process.

Theoretical Information 19

The rate of diffusion can be predicted within a solution of constant viscosity using Fick's First Law (for linear diffusion):

∂[ c ] ⎛ ∂ 2 [ c ] ⎞ = D ⎜ ⎟ (14) c ⎜ 2 ⎟ ∂t ⎝ ∂x ⎠

The rate of change in the concentration as a function of time is related to the change in the concentration gradient. So the steeper the change in concentration the greater the rate of diffusion. In practice diffusion is the most significant transport process for the majority of electrolysis reactions.

Convection (stirring or hydrodynamic transport)

Convection is the transport to the electrode by a gross physical movement. The fluid flow occurs by stirring or flowing the solution and by rotating or vibrating the electrode (forced convection) or because of density gradients (natural convection).

The natural convection is generated by small thermal or density differences and mixes the solution in a random and therefore unpredictable manner. In the case of electrochemical measurements, these effects tend to lead to problems for longer measurement periods. It is possible to drown out the natural convection effects from an electrochemical experiment by deliberately introducing convection into the cell. This form of convection is termed forced convection. It is typically several orders of magnitude greater than any natural convection effects and therefore effectively removes the random aspect from the experimental measurements. This, of course, is only true if the convection is introduced in a well defined and quantitative manner.

Migration Migration is the movement of charged particles along an electrical field. This is essentially an electrostatic effect which arises due the application of a voltage on the electrodes. This effectively creates a charged interface (the electrodes). Any charged species near that interface will either be attracted or repelled from it by electrostatic forces. Due to ion solvation effects and diffuse layer interactions in solution, migration is notoriously difficult to calculate accurately for real solutions. Consequently, most voltammetric measurements are performed in solutions which contain a background electrolyte (e.g. KCl) that does not undergo electrolysis itself but helps to shield the reactants from migratory effects. By adding a large quantity of the electrolyte (relative to the reactants) it is possible to ensure that the

20 Theoretical Information

electrolysis reaction is not significantly effected by migration. The purpose of introducing a background electrolyte into a solution is not, however, solely to remove migration effects as it also acts as a conductor.

2.1.9 Overpotential36, 40, 41

The overpotential (η) is the difference between the electrode potential and the equilibrium potential at open circuit when a current (i) passes between the electrodes. An overpotential is generally caused by a kinetic inhibition of one reaction step of the electrochemical process. There are different overpotential contributions associated with different reaction steps:

1) Charge transfer overpotential ()ηct

The charge transfer through the Helmholtz layer is a rate determining step.

2) Mass transport overpotential ()ηmt

Mass transport is a rate determining step. The mass transport overpotential can be considered as a sum of different overpotentials which are observed in case of the diffusion (D), convection (C) and migration (M) modes of mass transport.

η mt = η D + η M + η C (15)

3) Reaction overpotential ()ηR

Reactions proceeding or following the electrode reaction are rate determining.

The total overpotential can be considered as a sum of different contributions.

η = η CT + η MT + η R (16)

For a given overpotential, the magnitude of current flow can depend on a number of factors, including the mass transfer by diffusion, convection or migration, as well as the charge transfer rate between solution species and the electrode (called electrode kinetics). Positive overpotentials (i.e. potentials more positive than the equilibrium potential) cause oxidation reactions at the electrode, whereas negative overpotentials cause reduction reactions to occur.

If the solution is well stirred or currents are kept so low that the surface concentrations do not differ appreciably from the bulk values (kinetics-limited regime), the current-potential relationship is given by the Butler-Volmer Equation (15):

Theoretical Information 21

αnF −()1−α nF − ⎛ η η ⎞ for a reaction ne + Ox ⇔ Red (17) j = j ⎜e RT − e RT ⎟ 0 ⎜ ⎟ ⎝ ⎠

2 where j is the current density (current per unit area, A/cm ), j0 is the exchange current density (A/cm2), α is the transfer coefficient and n is the number of electrons transferred.

Since mass transfer effects are not included here, the overpotential associated with any given current serves solely as activation energy. It is required to drive the heterogeneous process at the rate reflected by the current. The lower the exchange current, the more sluggish the kinetics are and hence the larger is the activation overpotential for any particular net current. If the exchange current is very large, then the system can supply large currents, perhaps even the mass transfer limited current, with insignificant activation overpotential. In that case, any observed overpotential is associated with changing surface concentrations of species Ox and Red. It is called a concentration overpotential and can be viewed as activation energy required to drive mass transfer at the rate needed to support the current.

2.1.10 Controlled Potential Techniques

The basis of all controlled potential techniques is the measurement of the current response to an applied potential. A multitude of potential excitations (including a ramp, potential steps, pulse trains, a sine wave, and various combinations thereof) exist.

Pulse voltammetry35 Pulse voltammetric techniques are aimed at lowering the detection limits of voltammetric measurements. By substantially increasing the ratio between the faradaic and non-faradaic currents, such techniques permit convenient quantitation down to the 10-8 M concentration level. The various pulse techniques are all based on a sample current potential-step experiment. A sequence of such potential steps, each with duration of about 50 ms, is applied onto the working electrode. Following the stepping of the potential, the charging current decays rapidly (exponentially) to a negligible value, while the faradaic current decays more slowly. Thus, by sampling the current late in the pulse life, an effective discrimination against the charging current is achieved. Two different pulse voltammetric techniques exist: Normal pulse and differential pulse voltammetry.

22 Theoretical Information

Differential pulse voltammetry35 In differential pulse voltammetry, fixed magnitude pulses are applied to the working electrode (Scheme 3).The current is sampled twice, just before the pulse application (at 1) and again late in the pulse life (at 2, when the charging current is decayed).

∆t m ∆Um L IA T N ∆U TE 1 s O P

∆t i 2 measurements

TIME

Scheme 3. Schematic potential waveform of a differential pulse voltammetric (DPV) measurement.

The potential steps ∆Us with an interval time ∆ti are modulated by a modulation potential ∆Um for

the modulation time ∆tm.

The first current is instrumentally subtracted from the second, and this current difference (∆i = i − i ) is plotted vs. the applied potential. The major component of the current ()t2 (t1 ) difference is the faradaic current which characterises oxidations or reductions at the electrode. The capacitive component of the current, originating from the electrical charging of the double layer, is largely removed. The resulting differential pulse voltammogram consists of current peaks with heights directly proportional to the concentration of the corresponding analytes:

Theoretical Information 23

1 / 2 nFAD C ⎛ 1 −σ ⎞ = ⎜ ⎟ (18) i p 1 + σ π (t2 − t1 ) ⎝ ⎠ where σ = exp (nF/RT ∆E/2) (∆E is the pulse amplitude) and (t2-t1) is time measured from the pulse rise. The maximum value of the quotient (1-σ)/(1+σ), obtained for large pulse amplitudes, is unity.

The selection of the pulse amplitude and potential scan rate usually requires a trade-off among sensitivity, resolution and speed. Larger pulse amplitudes, for example, result in stronger response, but also broader peaks and lowered potential resolution.

2.1.11 Working Electrodes

The performance of the voltammetric procedure is strongly influenced by the working electrode material. The working electrode should provide high signal-to-noise characteristics, as well as a reproducible response. Thus, the selection of working electrodes depends on the redox behaviour of the target analyte and the background current over the potential region required for the measurement. Other considerations include the potential window, electrical conductivity, surface reproducibility, mechanical properties, cost, availability and toxicity. A wide range of materials are used as working electrodes for electroanalytic applications. The most popular ones are those involving mercury, carbon, or noble metals (platinum and gold).

Carbon Electrodes Solid electrodes based on carbon are currently in widespread use in electroanalysis, primarily because of their broad potential window, low background current, low cost, chemical inertness, and suitability for various sensing and detection applications. In contrast, electron transfer rates observed at carbon surfaces are slower than those observed at metal surfaces. The electron transfer activity is effected by the carbon surface structure. A variety of electrode pre-treatment procedures have been proposed to increase the electron transfer rates. The type of carbon, as well as the pre-treatment method, has a profound effect upon the analytical performance. The most popular carbon-electrode materials are glassy carbon, carbon paste, carbon fibre, carbon films, or carbon composites.

24 Theoretical Information

2.2 Nucleic Acids

2.2.1 Structure of Nucleic Acids42, 43

A nucleic acid consists of a chemically linked sequence of subunits (Scheme 4). Each subunit contains a nitrogenous base (a heterocyclic ring of carbon and nitrogen atoms), a pentose

sugar and a phosphate group.

Scheme 4. Subunits of nucleic acids.

Two types of pentose are found in nucleic acids. They distinguish deoxribonucleic acid (DNA) and ribonucleic acid (RNA) and give rise to the general names for the two types of nucleic acids. In DNA the pentose is 2-deoxyribose, whereas in RNA it is ribose. The difference lies in the absence/presence of the hydroxyl group at position 2 of the sugar ring.

The nitrogenous bases fall into two types. Pyrimidines have a six-member ring and purines have fused five- and six-member rings as shown in Scheme 5. DNA containes two purines, adenine and guanine and two pyrimidines, cytosine and thymine. The bases are usually referred to by their initial letters; so DNA contains A,G,C,T.

Theoretical Information 25

Scheme 5. Bases of DNA.

A base linked to a sugar is called a nucleoside, when a phosphate group is added, the base sugar-phosphate is called a nucleotide. Nucleotides provide the building blocks out of which nucleic acids are constructed. The nucleotides are linked into a polynucleotide chain by a backbone consisting of an alternating series of sugar and phosphate residues. The 5’ position of one pentose ring is connected to the 3’ position of next pentose ring via a phosphate group, as shown in Scheme 6.

Scheme 6. A polynucleotide chain consists of a series of 5´-3´ sugar-phosphate links that form a backbone.

26 Theoretical Information

Watson and Crick44, 45 proposed that the two polynucleotide chains in the double helix associate by hydrogen bonding between the nitrogenous bases. In their usual forms, G can only specifically bond with C, while A can only bond specifically with T (Scheme 7). These reactions are described as complementary base pairing46.

Scheme 7. Base pairing of DNA.

2.2.2 Hybridization and Denaturation

The formation of double stranded nucleic acid hybrids by complementary base pairing is called hybridization, which is reversible and depends on various conditions, such as temperature and buffer. The non-covalent forces that stabilize the double helix are disrupted by applying heat or by exposure to low salt concentration. The two strands of a double helix separate entirely when all hydrogen bonds between them are broken. The process of strand separation is called denaturation or melting. The temperature at which half of the oligonucleotides form a duplex with complementary target is called melting temperature, denoted Tm. Tm depends on the proportion of G-C base pairs. Because each G-C base pair has three hydrogen bonds, it is more stable than an A-T base pair, which has only two hydrogen bonds. The more G-C base pairs are contained in a DNA, the greater is the energy necessary 42 to separate the two strands. The Tm increases ~0.4 °C for every 1% increase in G-C content .

Theoretical Information 27

2.2.3 Mutations42, 43, 47

Mutations are changes in the sequence of nucleotides which occur naturally or through the effect of mutagens. Any base pair of DNA can be mutated. The existing of mutations can be differentiated by comparing the properties of normal gene (wild-type) with a defective gene (mutated).

A point mutation changes only a single base pair, and can be caused by either of two types of events:

- Chemical modification of DNA directly changes one base into a different base.

- A malfunction during the replication of DNA causes the wrong base to be inserted into a polynucleotide chain during DNA synthesis.

Point mutations can be divided into two types, depending on the nature of the change when one base is substituted by another:

- Transition: Comprising the substitution of one pyrimidine by the other, or of one purine by the other: thus a G-C pair is exchanged with an A-T pair or vice versa. For example: Factor V Leiden and Factor II point mutations.

- Transversion: A purine is replaced by a pyrimidine or vice versa, so that an A-T pair becomes a T-A or C-G pair.

2.3 Nucleic Acid Diagnostics

2.3.1 Preface

One fundamental method for the nucleic acid diagnostics is the determination of medically relevant nucleic acid sequences by detecting the sequence specific hybridization. The detection of specific nucleic acid sequences in human, viral and bacterial samples provide the basis for the diagnosis of infectious and inherited diseases48,49. Detection of infectious disease agents and genetic mutations at the molecular level opens up the possibility of performing reliable diagnosis even before any symptoms of a disease appear50.

Nucleic acid diagnostics consist of several steps. These are necessary for the diagnosis of specific DNA sequences from the biological samples: nucleic acid extraction and purification (sample preparation), amplification and detection.

28 Theoretical Information

2.3.2 Polymerase Chain Reaction

Polymerase chain reaction (PCR) is an in vitro method of nucleic acid synthesis by which a particular segment of DNA is specifically replicated. It needs two oligonucleotide primers that flank the DNA fragment to be amplified and repeated cycles of heat denaturation of the DNA, annealing of the primers to their complementary sequences, and extension of the annealed primers with DNA polymerase. These primers hybridize to the opposite strands of the target sequence and are oriented so that DNA synthesis by the polymerase proceeds across the region between the primers (Scheme 8). Since the extension products themselves are also complementary to and capable of binding primers, successive cycle of amplification essentially double the amount of the target DNA synthesized in the previous cycle. The result is an exponential accumulation of the specific target fragment, theoretically 2n, where n is the number of cycles of amplification performed51.

Theoretical Information 29

Polymerase Chain Reaction: PCR

Scheme 8. Each PCR cycle consists of three reaction steps. Firstly, the template DNA is denaturated. Secondly, the primers anneal (hybridise) to specific regions of the appropriate single stranded template chain and thirdly, the primers are extended by the polymerase.

30 Theoretical Information

2.3.3 Biosensors

A chemical sensor is a device that transforms the chemical information into an analytically useful signal. Chemical sensors usually contain two basic components connected in series: a chemical recognition system and a physical transducer. Biosensors are chemical sensors in which the recognition system utilizes a biochemical mechanism52,53. Biosensors combine the specificity of a biological recognition mechanism with a physical transduction technique, which can be based either on electrochemical, mechanical, thermal or optical sensing principles. The most usual aim of the biosensor is to produce continuous signals that are proportional to the amount of molecules bound to or reacting at the sensor surface54. Biosensors can be classified according to their recognition system or the transduction process. There are a variety of recognition mechanisms and transducers used in biosensors. The recent developments focus on electrochemical DNA biosensors, whereas the more traditional enzyme sensors, immunosensors and microbial sensors have undergone several decades of development.

2.3.4 Electrochemical DNA Biosensors55 In an electrochemical DNA biosensor, a short (20-40mer) single-strand synthetic oligonucleotide (probe) is immobilized onto an electrode surface to create the recognition layer. The probe immobilised electrode is then immersed into a solution of PCR-amplified sample which contains the target oligonucleotide to be tested. When the sequence of target matches exactly that of the probe, the hybrid is formed at the electrode surface. The specific hybridization event between DNA probe and complementary target is detected by using an electrochemical transducer. The immobilization of probe onto the surface is performed by using non-covalent as well as covalent binding. Adsorption forces56-58, hydrophobic interactions of bases with the mercury surface59, electrostatic binding of DNA to the positively charged carbon electrode60,61 are some of the methods which are used for the non-covalent binding of probe. Various kinds of covalent binding of probe to carbon, gold and mercury surfaces are also performed62,63. The hybridization event can be detected by using redox indicators which interact preferentially with double stranded DNA, such as simple intercalators and minor-groove binders64 or which bind covalently to DNA65, or directly measure the oxidation signal of guanine which is present in the DNA structure (label-free electrochemical detection)66,67.

Experimental 31

3 Experimental

3.1 Chemicals and Solutions

3.1.1 Chemicals

Table. 1 List of used chemicals

Substance Source Purity M (g/mol)

Carbon paste ink Gwent - - Ag/AgCl paste Gwent - - Ethylenglycolmonobuthylether Aldrich 99+% 118.18 Sodium dodecyl sulphate (SDS) Sigma ≥99% 288.4 Ethanol Merck p. a. 46.07 3-Glycidyloxypropyl-trimethoxysilane Fluka ≥97% 236.34

Sodium carbonate monohydrate (Na2CO3·H2O) Fluka ≥99% 124.01 Hydrochloric acid (HCl) Fluka ≥32% 36.46 Nexterion hybridization buffer (QMT) Peqlab p. a. - Sodium chloride (NaCl) Merck p. a. 58.44 Sodium citrate dihydrate Riedel-de Häen p. a. 294.10 Acetic acid Fluka ≥99.5 60.05

Sodium acetate-trihydrate (NaAc·3H2O) Fluka ≥99% 136.08 Potassium chloride (KCl) Fluka ≥99.5% 74.56 Tris (hydroxymethyl) aminomethan Merk p. a. 121.14 Tween 20 Fluka p. a. 522.669 Triton-X 100 Fluka p. a. - Albumin fraction V (BSA) Roth ≥98% ≈69 1-Naphthylphosphate (NAP) Aldrich ≥99% 224.15 Streptavidine Alkaline Phosphatase Pharmingen p. a. - Phosphatase substrate Sigma p. a. 371.14 Diethanolamine Sigma-Aldrich 98% 105.14

32 Experimental

Ethylenediaminetetraaceticacid (EDTA) Fluka ≥99% 292.25 di-sodium hydrogen phosphate (Na2HPO4) Fluka ≥99% 177.99 10xReactions buffer Y (Buffer Y) Peqlab p. a. -

Magnesium chloride-hexahydrate (Mg2Cl·6H2O) Merck p. a. 203.30

3.1.2 Solutions

10% Sodium Dodecyl Sulphate Solution:

100 g SDS crystals are dissolved in the 900 ml deionised water (Mili-Q). It is stored at room temperature (RT).

5% Silane Solution:

For 10 ml 5% silane solution, 9 ml ethanol, 0.5 ml 3-Glycidyloxypropyl-trimethoxysilane and 0.5 ml Mili-Q are mixed in a plastic tube. It is prepared daily before use.

1 M Sodium Carbonate Solution :

24.8 g Na2CO3·H2O is dissolved in 200 ml Mili-Q. It is stored at RT. This solution is the stock solution for the 0.1 M Na2CO3 binding buffer.

0.1 M Sodium Carbonate Binding Buffer (pH 9.5):

0.1 M Na2CO3 binding buffer is prepared by diluting the 1 M Na2CO3 solution. Then, the pH is adjusted to 9.5 with HCl. It is prepared daily before use.

500 mM Phosphate Binding Buffer (pH 8.5):

44.98 g. Na2HPO4 and 0.15 g EDTA are dissolved in 300 ml Mili Q and adjusted to 500 ml. Then, the pH is adjusted to 8.5 with HCl. It is stored at RT.

0.2 M Acetic Acid Solution:

11.55 ml glacial acetic acid is mixed in 500 ml Mili-Q and adjusted to 1 litre with Mili-Q. This solution is one component of the stock solution for the 0.1 M sodium acetate (NaAc) measurement buffer. It is stored at RT.

Experimental 33

0.2 M Sodium Acetate Solution:

27.21 g sodium acetate·3H2O is dissolved in 800 ml Mili-Q and adjusted to 1 litre with Mili-Q. This solution is another component of the stock solution for the 0.1 M NaAc measurement buffer. It is stored at RT.

0.1 M NaAc Measurement Buffer (pH 4.6):

51 ml 0.2 M sodium acetate, 49 ml 0.2 M acetic acid and 100 ml Mili-Q are mixed. Then, the pH is adjusted to 4.6 with HCl. It is stored at RT.

PCR Dilution Buffer:

40 mg albumin fraction V (BSA) is dissolved in 6 ml 1xBuffer Y and 5 µl Tween 20 is added to this solution. Then, the volume is adjusted to 10 ml. It is stored at –20 °C.

2xSodium Saline Citrate Buffer:

17.53 g NaCl and 8.82 g sodium citrate·2H2O are dissolved in 800 ml Mili-Q and the pH is adjusted to 7 with HCl. Then, the volume is adjusted to 1 liter with Mili-Q. It is stored at RT.

20xSodium Saline Citrate Buffer:

175.3 g NaCl and 88.2 g sodium citrate·2H2O are dissolved in 800 ml Mili-Q and the pH is adjusted to 7 with HCl. Then, the volume is adjusted to 1 liter with Mili-Q. It is stored at RT and used as a stock solution for the preparation of washing buffers.

Washing Buffer 1 (WB 1):

5 ml 20xSSC is mixed in 80 ml Mili Q, then 1 ml 10% SDS is added. The volume is adjusted to 200 ml with Mili-Q. It is stored at RT.

Washing Buffer 2 (WB 2):

0.5 ml 20xSSC is mixed in 80 ml Mili Q, then 1 ml Tween 20 is added. The volume is adjusted to 200 ml with Mili-Q. It is stored at RT.

Enzyme Dilution Buffer:

20 ml 20xSSC is mixed in 80 ml Mili Q, then 1 ml Tween 20 is added. The volume is adjusted to 200 ml with Mili-Q. It is stored at RT.

34 Experimental

1% Bovin Serum Albumin Solution:

1 g albumin fraction V (BSA) is dissolved in 80 ml 2xSSC or TBS measurement buffer (pH 8.0) and adjusted to 100 ml with the same buffers. 1% BSA in 2xSSC is used for the enzyme- based electrochemical detection method and in TBS measurement buffer (pH 8.0) is used for the optical detection method. It is prepared daily before use.

TBS Measurement Buffer (pH 9.6 and pH 8.0):

8 g. NaCl, 0.2 g KCl and 3 g tris-(hydroxymethyl)-aminomethan are dissolved in 800 ml Mili- Q and the pH is adjusted to 9.6 or 8.0 with HCl. Then, the volume is adjusted to 1 liter with Mili-Q. It is stored at 4 °C.

Diethanolamine Buffer (pH 9.8):

The buffer consist of 1 M diethanolamine and 0.5 mM MgCl2. For 1 L diethanolamine buffer,

97 ml diethanolamine is mixed with 600 ml Mili-Q containing 100 mg MgCl2-6H2O. Then, the volume is adjusted to 1 L with Mili-Q and the pH is adjusted to 9.8 with HCl. The solution is filtrated and stored at 4 °C.

2 mM 1-Naphthylphosphate (NAP) Substrate Solution:

For 10 ml 2 mM NAP solution, 2.24 mg NAP is dissolved in TBS buffer (pH 9.6). It is prepared daily before use. The tube in which NAP is prepared is covered with aluminium foil in order to protect the solution from light.

Colorimetric Substrate Solution:

One tablet (5 mg) of phosphatase substrate (p-Nitrophenylphosphate, pNPP) is dissolved in 5 ml diethanolamine buffer (pH 9.8). It is prepared daily before use. The tube in which the solution is prepared is covered with aluminium foil in order to protect the solution from light.

Experimental 35

3.2 Measurement Set-ups

3.2.1 Electrochemical Measurement Set-ups

For the label-free electrochemical detection method

The measurements are performed using Ag/AgCl reference and platinum (Pt) counter electrodes. The utilised potentiostat is an Ecochemie AUTOLAB PGSTAT 12. The parameters for the differential pulse voltammetry are: 5s equilibration time, 0.05s modulation time, 0.5s interval time, 8mV step potential and 50mV modulation amplitude. The measurements are performed in 0.1 M NaAc buffer (pH 4.6). The resultant measurements are background-corrected by a Savitzky-Golay filter algorithm with a moving average of 0.01.

For the enzyme-based electrochemical detection method

The measurements are performed using screen printed Ag/AgCl reference and carbon counter electrodes on the chip itself. A multi-potentiostat allows the measurement of eight working electrodes simultaneously. The same differential pulse voltammetry parameters as in label- free electrochemical detection method are used for the detection of hybridization event. The measurements are accomplished directly in TBS buffer with 2 mM NAP after an accumulation period of 1 min. The measurement chamber is shown in Scheme 9.

A B

Scheme 9. Measurement chamber (A) and contact array (B) for the measurements with multi- potentiostat.

36 Experimental

For the measurements in the cartridge

The label–free as well as the enzyme-based electrochemical detections in the cartridge are carried out using the same measurement chamber (Scheme 9-A) and conditions as explained for the enzyme-based electrochemical detection method.

3.2.2 Colorimetric Measurement Set-up

The measurements are processed by reading the optical density of the DNA-BIND (Costar/USA) wells at 405 nm on a Elx 808 IU 96-well plate reader following the addition of substrate.

3.3 Preparation of Screen Printed Chips

The preparation of chips is performed by using pencil leads in order to provide the electrical contact between the both sites of the chip. The electrode material, carbon paste, is applied onto the chip surface by using screen printing technology. The screen printed electrodes on the chip are contacted from the opposite site of the chip by using conductive needles. The preparation of chips using pencil leads is illustrated in Scheme 10.

Scheme 10. Preparation of chips by using pencil leads.

Experimental 37

The chips are produced by arranging 18 pencil leads with a diameter of 0.5 mm in an octagonal formation by sticking the leads into drilled holes of two oppositely positioned teflon plates(1). The locations of the holes define the position of the electrodes. Then the side faces are encased by teflon plates (4). One of the teflon plates exhibits a hole at the top (3) in order to fill the case with a stycast epoxy resin. After the curing of the stycast, the side plates are removed and the epoxy block with the embedded pencil leads is cut into 5 mm thick slices. The chips are finished by grinding them with different grain sized wheels and burnishing them on a polishing cloth with a polishing machine Saphir 320. The appearance of the chips after preparation is shown in Scheme 11.

Scheme 11. Appearance of a chip after the preparation.

After these prearrangements the carbon paste ink is printed onto the chip surface by using an ISIMAT SD 05 screen printing machine in order to prepare the screen printed electrodes on the chips. The screen is separated from the substrate by a small gap of around 0.5 mm. The substrate then is clamped by the substrate holder, which is specifically designed for the epoxy chips. The carbon paste ink is applied to the upper surface of the screen and the flexible rubber squeegee is traversed across the stencil. As the squeegee is passed across the screen, the mesh fabric is pressed onto the substrate surface. Thus, the ink is forced through the permeable areas of the screen mesh. Directly behind the squeegee, the screen peels away from the substrate and leaves the ink on the substrate surface. The principle of the screen printing process is also shown in Scheme 12.

38 Experimental

Direction of squeege

Substrate holder Squeegee

Ink

Substrate Screen fabric Screen frame

Scheme 12. Principle of screen printing process.

After the printing process, the screen printed chips are cured in an oven at 80 °C for 1 h. The appearance of the screen printed chips with eight working electrodes (WEs) in octagonal arrangement, six counter electrodes (CEs) and four reference electrodes (REs) is depicted in Scheme 13. The silver ink for the REs is applied onto the surface manually and the chips are cured again at 60 °C for 30 min.

Counter Electrodes

Working Electrodes Reference Electrodes

Scheme 13. Chip after screen printing.

Experimental 39

3.4 Methods

3.4.1 Surface Preparation

Two different electrochemical methods are used68 and each step of these methods is optimized. The procedure for both label-free and enzyme-based electrochemical methods consists of four general steps: Surface preparation, hybridization, washing and transduction. Surface preparation is identical for both cases, but the other steps are adapted to the detection system. The surface preparation part comprises four steps: Screen printing, pre-treatment, silane chemistry and probe immobilization.

Screen Printing

The SPCs are printed as described in section 3.3.

Pre-treatment

Pre-treatment is performed by immersing the SPCs into 10% SDS solution for 15 min at RT. After pre-treatment, the surface is washed with Mili-Q.

Silane chemistry

Silane chemistry is performed by immersing the SPCs into a 5% silane solution for 15 min. The SPC is then incubated at 80 °C for 15 min.

(3-Glycidyloxypropyl)-trimethoxysilane is used for the silane surface chemistry. During the formation of the silane layer, the methoxy groups of silane couple to free hydroxyl groups of the electrode surface and the silane forms a crosslinking structure by coupling two methoxy groups (Scheme 14). The crosslinking process increases the stability of the formed silane layer.

40 Experimental

O

O OH CH3 H3C O O Si O CH3 OH O

O CH3 H3C O O Si O O O Si O O CH3

OH

Scheme 14. The methoxy groups of the (3-Glycidyloxypropyl)-trimethoxy-silane react with hydroxyl groups of the electrode surface and with each other. Silane forms a stable layer on the electrode surface.

(3-Glycidyloxypropyl)-trimethoxysilane contains an epoxy group, which enables an easy covalent coupling of the aminolinked-oligonucleotides (Scheme 15).

Experimental 41

R

O O O Si O O CH3 oligonucleotide H2N OH

R OH O O Si O NH O oligonucleotide CH3 OH

Scheme 15. The oligonucleotides are attached to the surface via the silane layer.

Probe immobilization

The aminolinked-probe is covalently immobilized on a SPC surface covalently via the silane layer, by pipetting the 10 nmol/ml probe solution prepared in binding buffer onto the surface and waiting for 5 min at RT.

42 Experimental

3.4.2 Label-free Electrochemical Detection Method

The schematic characterization of label-free electrochemical detection is given in Scheme 16.

Scheme 16. In the label-free electrochemical detection method, the guanine oxidation signal is measured for the detection of the hybridization event.

The label-free electrochemical detection basically consists of four steps: Surface preparation, hybridization, washing and voltammetric transduction of guanine oxidation.

Surface Preparation

The surface preparation is basically as introduced in section 3.4.1, but the guanine bases of the aminolinked probe are substituted by inosine in order to avoid the background signal which is caused by the probe. Inosine has similar base–pairing properties to guanine. It also forms base pairs with cytosine, but with two hydrogen bonds instead of three.

Experimental 43

The electrochemical properties of inosine are totally different from guanine and its oxidation signal is well separated from the guanine response. The chemical structure and binding properties of inosine are displayed in Scheme 17.

NH2 O N 7 8 5 4 6 5

6 4 N N HN 9 3 1 Sugar N 2 2 N Sugar 1 3 O

cytosine inosine

Scheme 17. The chemical structure and binding properties of inosine.

Hybridization

The hybridization is achieved by pipetting the 1 nmol/ml synthetic target or 1:1 diluted PCR- amplified sample solution prepared in QMT hybridization buffer or PCR dilution buffer onto the SPC surface and waiting for 30 min at 45 °C.

Washing

The washing step is accomplished by immersing the SPC into the WB 1 (1xSSC+0.1%SDS) (WB 1) for 15 min at RT in order to avoid non-specific binding of the target to the electrode surface.

Voltammetric Transduction

The oxidation of guanine bases on the surface only takes place after the hybridization event and is measured in 0.1 M NaAc measurement buffer (pH 4.6) by using DPV under the conditions introduced in section 3.2.1. The guanine leads to an oxidation peak at 1.0 V (vs. Ag/AgCl).

44 Experimental

3.4.3 Enzyme-based Electrochemical Detection Method

The schematic characterization of the enzyme-based detection method is demonstrated in Scheme 18.

Scheme 18. In the enzyme-based electrochemical detection method, the oxidation peak of the end product (1-naphthol) which is derived from the substrate (1-naphtylphosphate) in the presence of a specific enzyme (alkaline phosphatase) is used for the determination of the hybridization event.

The enzyme-based electrochemical detection includes seven steps: Surface preparation, hybridization, washing step 1, enzymatic labelling, washing step 2, substrate reaction and voltammetric transduction. The used enzyme is streptavidine conjugated alkaline phosphatase and the utilised substrate is 1-naphthylphosphate.

Surface Preparation

Surface preparation is carried out as described in section 3.4.1.

Experimental 45

Hybridization The hybridization is performed as described in section 3.4.2, but for this method synthetic target or PCR products are modified with biotin, because the streptavidine conjugated enzyme binds strongly and specifically to biotin. Thus, the probe hybridised with the biotin-modified target can easily be labelled with enzyme. The synthetic target, as well as the dilution of the PCR-amplified sample is prepared in dilution buffer.

Washing step 1

The washing step 1 is performed by immersing the SPC into the WB 2 (0.1xSSC+0.1% Tween 20) solution for 5 min at RT in order to avoid the non-specific binding of target to the electrode surface. This step is repeated twice.

Enzyme labelling

This process is performed by immersing the SPC into the 1:1000 diluted streptavidine alkaline phosphatase solution prepared in enzyme dilution buffer and waiting for 30 min at RT. Due to the specific interaction between streptavidine and biotin, the probe which is hybridised with the biotin-modified target is labelled with enzyme.

Washing step 2

Washing step 2 is performed as in Washing step 1 and removes non-specifically adsorbed enzyme from the electrode surface. After washing step 2, the electrodes are dipped into the TBS measurement buffer (pH 9.6) until the substrate reaction is carried out.

Substrate reaction

The substrate reaction is performed by immersing the SPC into the 2 mM 1-Naphthylphosphate solution prepared in TBS measurement buffer (pH 9.6) and waiting for 1 min at RT. During the enzymatic reaction, the end product 1-Naphtol is produced.

Voltammetric transduction

The voltammetric transduction of the end product 1-Naphtol, which is obtained from the substrate in the presence of enzyme alkaline phosphatase, takes place directly in the same solution after the substrate reaction. DPV is utilised and the conditions given in section 3.2.1 are applied. The 1-Naphtol leads to an oxidation peak at 0.2 V.

46 Experimental

3.4.4 Enzyme-based Colorimetric Detection Method

The enzyme-based colorimetric detection method is performed using the DNA-BIND 96-well plates which are specifically designed for the immobilization of aminolinked DNA for use in immuno-PCR and other nucleic acid hybridization assays. DNA-BIND surface chemistry enables covalent attachment of aminolinked-probe to the surface of the wells. The surface of the DNA-BIND plate is coated with a layer of reactive N-oxysuccinimide esters (NOS) which react with nucleophiles such as primary amines. These NOS groups are covalently linked to the polystyrene surface and thus cannot be washed off the plate. The aminolinked-probe can be directly coupled to the NOS surface This coupling is specific and not affected by the amines attached to the adenine, guanine and cytosine rings. The reaction of coupling is shown in Scheme 19.

O O O H PH 8.5 R C O N + R NH R C N R 1 2 2 1 2 O

DNA-BIND Surface Biomolecule Immobilisation

R = Spacer arm R = DNA, Protein, etc. 1 2

Scheme 19. The immobilization of DNA onto the DNA-BIND plates.

The enzyme-based colorimetric detection method consists of seven steps: Probe immobilization, washing step 1, blocking, washing step 2, hybridization, enzymatic labelling, washing step 3, substrate reaction and colorimetric measurement. With one plate, 96 different tests can be carried out and measured at the same time.

Probe immobilization

The probe immobilization is performed by pipetting 1 nmol/ml aminolinked-probe solution prepared in binding buffer (pH 8.5) into the wells for 20 min at RT.

Experimental 47

Washing step 1

The wells are washed with TBS buffer (pH 8.0) for three times at RT in order to avoid non- specific binding of probe to the surface.

Blocking

The blocking is performed by incubation of 1% BSA solution prepared in TBS buffer (pH 8.0) in the wells for 30 min at RT in order to avoid non-specific adsorption of target.

Washing step 2

The washing step 2 is performed the same way as washing step1.

Hybridization

The hybridization is performed by adding 1:10 diluted biotin-modified PCR product prepared in dilution buffer into the wells for 30 min at 45 °C .

Enzyme labelling

This step is performed by incubation of 1:1000 diluted streptavidine alkaline phosphatase solution prepared in 2xSSC+0.1% Tween 20 in the wells for 30 min at RT.

Washing step 3

The wells are washed with 0.1xSSC+0.1% Tween 20 solution for 5 min at RT. This step is performed two times. After washing step 3, diethanolamine buffer is added into the wells and incubated for 5 min at RT. This incubation is also performed twice.

Substrate reaction

The substrate reaction is performed by incubating the wells with 2.6 mM p-nitrophenylphosphate solution (pNPP) prepared in diethanolamine buffer (pH 9.6) for 20 min at RT.

Colorimetric measurement

The colorimetric measurement is performed as described in section 3.2.2. A miscoloured scan of DNA-BIND microtiter plate after performance of an enzymatic reaction is shown in Scheme 20. The reaction is visualised by the darkness of the wells.

48 Experimental

Scheme 20. DNA-BIND microtiter plate after the enzymatic reaction. The reaction in the wells can be seen as dark spots.

Results 49

4 Results

4.1 Optimisation of Detection Methods

4.1.1 Preface

As described in details in other sections, the electrochemical detection methods consist basically of three main steps: Surface preparation, hybridization and voltammetric transduction. During this work, it was necessary to optimize the experimental parameters which effect the surface activity of the biosensor and the sensitivity of the system in order to develop reliable and sensitive detection methods. Therefore, different experimental parameters were investigated in order to find the optimum surface preparation and hybridization conditions.

4.1.2 Optimisation of Surface Preparation

4.1.2.1 Preface

The investigation of parameters which effect the electrochemical surface activity is an important issue for the optimization of surface preparation. The efficiency of the silane surface chemistry as well as probe immobilization depend on the efficiency of the electrochemical surface activity. Thus, to control the electrochemical activity of the surface, it is necessary to measure the guanine signal of the probe which should be immobilized on the surface via the silane layer. Basically, the protocol of the label-free electrochemical detection method is used to perform optimization studies for the surface preparation, but with a small difference in the hybridization step. The hybridization step is performed in the hybridization buffer without target oligonucleotide in order to prevent the hybridization event allowing adjustment of the experimental conditions. Therefore, the voltammetric transduction for the optimization studies of the surface is performed by measuring the guanine signal of 1 nmol/ml TNF2k aminolinked-probe (N-TNF2k) instead of hybrid. The surface preparation for the optimization studies is performed as described in section 3.4.1 by using an aminolinked-probe without inosine substitution.

50 Results

4.1.2.2 Screen Printing Procedure

Since the screen-printing technique is not instrumentally complex, the production parameters which can be adjusted and optimized in the electrode production are limited. The pressure and speed of squeegee which effect the viscosity of the carbon paste ink can be optimized by applying high pressure and low speed in order to achieve effective coverage of the electrode material69. Therefore, the important optimization study that effects the surface activity is the curing temperature of the inks after printing.

The Effect of Curing Temperature After Printing Process

The exact formulation of the carbon paste ink used is not known. However, it is known that the ink is comprised of three basic constituents: Synthetic grade graphite, a vinyl or epoxy based polymeric binder and a solvent to improve the viscosity for the printing process. Since the solvent component of the ink evaporates in the early stages of heating, any increases of the curing temperature effect the binder and graphite particles, thus, the electroanalytical properties of the surface. According to the reported results70 by using electron microscopy, at lower curing temperatures the surfaces of the electrodes are smoother, with no obvious definition of graphite particles. When the temperature of curing is increased, the microparticulate nature of the carbon paste increases accordingly. The appearance of cracks and grooves in the paste is consistent with the composition of the polymeric binder, resulting in greater definition of the graphite particle surface area.

With respect to these results, the electrochemical performance of the SPE surface which is produced by using different curing temperatures is investigated by using label-free electrochemical detection method with a small difference in the hybridization step as described in section 3.4.2. Surface preparation is performed as described in section 3.4.1.

Results 51

. 300 n1 250 n2 n3 200 n4 150

100

CURRENT (nA) 50

0 60 80 100 120 CURING TEMPERATURE (°C)

Figure 1. Effect of curing temperature on the guanine signal of immobilised probe. Number of measurements is given with n. Pre-treatment: 15 min in 10% SDS, silane chemistry: 15 min in 5% silane, curing: 30 min at 80C°, probe immobilization: 1 h in 1 nmol/ml N-TNF2k probe solution prepared in 0.1 M Na2CO3 binding buffer (pH 9.5), hybridization: 30 min in QMT hybridization buffer without target, washing: 5 min in 1xSSC+0.1%SDS, measurement: in 0.1 M NaAc measurement buffer (pH 4.6) by using DPV with an equilibration time of 5 s, a modulation time of 0.05 s, an interval time of 0.5 s, a step potential of 8 mV and a modulation amplitude of 50 mV in a potential range of 0.6-1.2 V.

Within this study, a high electrochemical activity of the surface area is observed with the curing temperature of 80 °C as shown in Figure 1. 80 °C seems to be the optimum curing temperature for the carbon paste which is used for screen printing of electrodes. The greater definition of the graphite particles which play important role for the binding of oligonucleotides onto the electrode surface is probably obtained at this temperature.

52 Results

4.1.2.3 Effect of Pre-treatment Conditions

The active electrode surface can be reduced and electrochemically active pollutions can be adsorbed to the surface according to the contamination of the surface. This reduces the sensitivity and effects the reproducibility. For this reason it is necessary to activate the electrode surface before use. The most common activation is electrical pre-treatment and it is performed by applying a positive voltage to the working electrodes versus the reference electrode. But the classical electrical pre-treatment has a major disadvantage. The process can hardly be integrated in an automated system. Therefore, in this work, chemical pre-treatment, which has a similar effect to an electrical pre-treatment of carbon electrodes, is used instead of electrical pre-treatment68. Because the major effect of the electrical pre-treatment is the cleaning of the surface, the cleaning can also be achieved by a chemical procedure and strong ionic detergents turned out to be ideal for the chemical cleaning of the carbon surfaces71.

Comparison of Different Ionic Detergents for the Chemical Pre-treatment

The effect of different ionic detergents on the electrochemical surface activity is investigated in order to find an optimum and efficient compound for the chemical pre-treatment. For the pre-treatment step 10% Tween 20 and 10% Triton-X 100 as well as 10% SDS are used. The pre-treatment is performed for 15 min at RT. The investigation is performed by using the label-free electrochemical detection method with a small difference in the hybridization step as described in section 3.4.2. Surface preparation is performed as described in section 3.4.1. 1 nmol/ml aminolinked-probe (N-TNF2k) is immobilised on the surface. The effect of different detergents on guanine oxidation signal of the immobilised probe is given in Figure 2. All measurements are performed triplet.

Results 53

350

300

A) 250 (n T

200 RREN CU 150

100 ABCD

Figure 2. Comparison of different detergents applied for the chemical pre-treatment. (A) without pre-treatment,(B) 10% SDS, (C) 10% Tween 20 and (D) 10% Triton X 100. Other conditions are as in Figure 1.

After a chemical pre-treatment with 10% SDS, the greater resolution of guanine oxidation peak of the immobilised probe, N-TNF2k is obtained. SDS pre-treatment seems to improve the electrochemical activity of the surface and the efficiency of the probe immobilization. 10% SDS is chosen for further experiments.

Effect of Pre-treatment Time

The effect of pre-treatment time on the efficiency of the surface activity is also investigated. The surface preparation is performed as in section 3.5. The chemical pre-treatment is done for 7.5 , 15 , 30 and 60 min with 10% SDS. The investigation is performed using the label-free electrochemical detection method with a small difference in the hybridization step as described in section 3.4.2. Surface preparation is performed as described in section 3.4.1. 1 nmol/ml aminolinked-probe (N-TNF2k) is immobilised onto the surface. The effect of pre- treatment time on the guanine signal of the immobilised probe is given in Figure 3. All measurements are performed in triplet.

54 Results

400

. n1 n2 300 n3

200

100 CURRENT (nA) 0 7,5153060 PRE-TREATMET TIME (min)

Figure 3. Effect of pre-treatment time on the guanine signal of the immobilised probe. Number of measurements is given with n. Pre-treatment: 7.5, 15, 30, 60 min in 10% SDS. Other conditions are as in Figure 1.

After 15 min pre-treatment of surface, the highest oxidation signal of guanine is obtained.

4.1.2.4 Silane Surface Chemistry

A stable covalent binding of the probe onto the electrode surface is important for the development of a reliable, specific and sensitive biosensor. Therefore, in this work, the immobilization of probe is achieved covalently by using silane surface chemistry. Because dense layers of silane are insulating, it is expected that the silane layer passivates the electrode surface and inhibits the activity of the surface. But the reported results68 demonstrate that the silane layer only inhibits the activity of the surface very slightly. The inhibition effect is actually dependent on the percentage of silane solution. For carbon electrodes (3- Glycidyloxypropyl)-trimethoxysilane is an ideal silane. Its surface binding and crosslinking reactivity is high because of the maximum number of three active methoxy groups.

The Percentage of Silane To obtain the effective silane layer on the surface for the probe immobilization, different silane percentages are compared by measuring the guanine signal of 10 nmol/ml immobilized probe (N-TNF2I) after hybridization with 1 nmol/ml complementary target (TNF2k). 0.5, 1, 5, 7.5, 10 % silane solutions are used for the silane surface chemistry. The surface

Results 55

preparation is performed as described in section 3.4.1. The investigation is performed by using the label-free electrochemical method as described in section 3.4.2. The comparison of different percentages of silane is given in Figure 4.

140 n1 120 n2

. n3 100 n4 n5 80

60

40 CURRENT (nA) 20

0 0.5 1 5 7.5 10 SILANE PERCENTAGE (%)

Figure 4. Comparison of different silane percentages. Number of measurements is given with n. Silane chemistry: 15 min, in 0.5, 1, 5, 7.5, 10% silane solution, curing: 15 min at 80 °C, probe

immobilization: 5 min in 10 nmol/ml N-TNF2I probe solution prepared in 0.1 M Na2CO3 binding buffer (pH 9.5), hybridization: 30 min. in 1 nmol/ml TNF2k target solution prepared in QMT hybridization buffer at RT. Other conditions are as in Figure 1.

The optimum conditions for the surface chemistry are obtained by using 5% silane solution as seen in Figure 4. The affectivity of the silane layer is probably increased with the increasing of silane percentage up to 5%, but a further increase results in the passivation of the electrode surface and inhibition of the surface activity.

Effect of Curing Time After Surface Chemistry The curing of the electrodes at 80 °C after the silane modification is necessary for the covalent coupling of the silane to the surface and the crosslinking of the methoxy groups of silane. The surface preparation is performed as described in section 3.4.1 but with different curing times of 15, 30, 45 and 60 min. 10 nmol/ml aminolinked-probe (N-TNF2I) is immobilised on the surface. The investigation is performed by using the label-free

56 Results

electrochemical detection method as described in section 3.4.2. The comparison of different curing times is given in Table 2. One chip is used for each measurement. One chip consists of eight electrodes.

Table 2. Effect of different curing times after silane chemistry on the hybridization signal. Silane chemistry:15 min in 5% silane solution, curing: 15, 30, 45, 60 min at 80 °C, probe immobilization:

5 min in 10 nmol/ml N-TNF2I probe solution prepared in 0.1 M Na2CO3 binding buffer (pH 9.5), hybridization: 30 min in 1 nmol/ml TNF2k target solution prepared in QMT hybridization buffer at RT. Other conditions are as in Figure 1.

Curing Times Chip 1 Chip 2 Chip 3 Chip 4 Chip 5 5min 15min 30min 45min 60min Guanine Signal

Electrode 1 98.22 154.20 108.20 89.45 81.13 Electrode 2 102.12 172.20 95.23 90.76 82.18 Electrode 3 87.95 149.60 131.30 101.00 78.72 Electrode 4 111.2 140.20 107.00 80.88 72.19 Electrode 5 85.27 165.70 128.40 70.53 90.64 Electrode 6 72.39 117.60 80.86 98.28 67.05 Electrode 7 92.13 169.20 102.60 105.10 97.57 Electrode 8 82.34 118.40 93.97 102.7 95.28 Average: 91.45 148.39 105.95 88.48 83.10 Standard Deviation : 12.23 21.55 17.12 11.30 10.79

The curing time after silane chemistry is the critical step. It is necessary for the formation of the silane layer. But a curing time longer than the one which is necessary for the formation of the layer can result in the decreased affectivity of electrode surface for probe immobilization, because the epoxy groups of silane layer can react with the impurities in the room atmosphere. As seen in Table 2, the greater oxidation signal of immobilised probe after hybridization with the complementary target is obtained at a curing time of 15 min. When applying shorter curing time, the formation of silane layer is probably not completed and thus, the smaller signal is obtained.

When applying longer curing times, the silane layer may be deformed because of interaction with impurities in the room atmosphere and the immobilization can not be performed successfully. For this reason, a smaller signal is obtained.

Results 57

4.1.2.5 Electrochemical Properties of Inosine Base

To avoid the background guanine signal which occurs due to the immobilised probe, the guanines of the probe are replaced by an alternative base which has a similar base-pair recognition but different electrochemical behaviour. To ensure that there is no interference between the redox activity of inosine and guanine, the electrochemical properties of inosine are investigated. Within this experiment, the poly inosine (Poly[I]) containing only inosines and TNF2k oligonucleotide containing guanines and adenines are adsorbed to the SPE surface by immersing the electrodes into the 10 nmol/ml Poly[I] or TNF2k solutions prepared in 0.1 M NaAc buffer (pH 4.6) in an open current circuit for 5 min. The electrochemical measurement is performed directly after accumulation in 0.1 M NaAc measurement buffer (pH 4.6) by using DPV under the given conditions as described in section 3.2.1. The inosine oxidation signal appears at a potential of 1.36 V which is totally different than the potential of guanine, as shown in Figure 5 .

The immobilised inosine-substituted probe is also measured directly after the immobilization step in order to control the background signal which may be caused by the inosine base in the probe sequence. No signal is observed at the position of guanine as shown in Figure 6.

58 Results

Figure 5. The oxidation peaks of (A) adenine (a) and guanine (b), (B) inosine. Adsorption: 5 min 10 nmol/ml poly[I] and 10 nmol/ml N-TNF2k in 0.1 M NaAc buffer (pH 4.6), measurement: in 0.1 M NaAc measurement buffer (pH 4.6) by using DPV with an equilibration time of 5 s, a modulation time of 0.05 s, an interval time of 0.5 s, a step potential of 8 mV and a modulation amplitude of 50 mV in a potential range of 0.6-1.5 V.

0.6

0.5

A) b

µ 0.4 NT ( 0.3

URRE

C 0.2 a

0.1

0.0 0.6 0.7 0.8 0.9 1.0 1.1 1.2 POTENTIAL (V)

Figure 6. The differential pulse voltammogram for 10 nmol/ml FcV inosine-substituted probe, (a) before hybridization and (b) after hybridization with complementary target in the PCR-amplified samples. Hybridization: 30 min, 1:1 diluted PCR amplified amplicons in dilution buffer, at 45 °C. Other conditions are as in Figure 1.

Results 59

It is verified that there is no interference between the redox activity of guanine and inosine bases and that the replacement of guanine with inosine can be used safely for the label-free electrochemical detection method.

4.1.2.6 Probe Immobilization

Probe Concentration

To optimise the probe concentration during the coupling step, the dependency of the concentration of aminolinked-probe N-TNF2k is investigated. Therefore, the probe solutions with different concentrations between 0.4-6.4 nmol/ml are pipetted onto the silane modified surfaces. The surface preparation is performed as described in section 3.4.1. The probe immobilization is performed for 1 h. In the development of an integrated system, it is important to make the process time as short as possible. Thus, different immobilization times are also investigated in order to obtain effective immobilization of probe within a shorter time period. The change in the guanine signal of the probe according to the different probe concentrations is given in Figure 7.

600

525 . 450

375

300

225

150 CURRENT (nA) 75

0 0 0,8 1,6 2,4 3,2 4 4,8 5,6 6,4 7,2 PROBE CONCENTRATION (nmol/ml)

Figure 7. Electrochemical characterisation of the coupling efficiency of the silane mediated probe immobilization in dependency of its concentration. The conditions are as in Figure 1.

60 Results

The guanine signal of probe increases quickly with increase in the probe concentration and starts to saturate at a concentration of 1.6 nmol/ml. Because of the occupation of all available binding sites of the electrode surface, the guanine signal of probe starts to saturate at 1.6 nmol/ml. Immobilization Time

The immobilization time of probe to the silane modified surface as well as probe concentration has an influence on the sensitivity of the electrochemical detection systems. The effect of immobilization time on the sensitivity is investigated by performing the probe immobilization step for 5, 15, 30 and 60 min. The surface preparation is performed as described in section 3.4.1. The investigation is performed by applying the label-free electrochemical detection method as described in section 3.4.2. The effect of different immobilization times on the guanine signal of 1 and 10 nmol/ml aminolinked-probe (N-TNF2I) after hybridization with 1 nmol/ml complementary target (TNF2k) are given in Figure 8. Three WEs of the chip are used for each of the probe concentrations and two WEs are used for the immobilization of noncomplementary probe (NC) which is completely different from complementary probe.

70 . 60

50

40

30 a b CURRENT (nA)

20 0 5 10 15 20 25 30 35 40 45 50 55 60 65 IMMOBILISATION TIME (min)

Figure 8. Electrochemical characterisation of the coupling efficiency of probe immobilization in dependency of immobilization time at probe concentrations of (a) 1 nmol/ml and (b) 10 nmol/ml. Hybridization: 30 min in 1 nmol/ml target (TNF2k) solution prepared in QMT hybridization buffer, at RT. Other conditions are as in Figure 1.

Results 61

According to the obtained results in Figure 8, it is clear that the surface is covered with 10 nmol/ml probe even in 5 min and there is no necessity to perform the immobilization for a longer time period with a probe concentration of 10 nmol/ml. The specificity of the system is also proved by measuring the hybridization signal of immobilised NC probe and no non- specific hybridization signal is obtained (not shown).

4.1.3 Optimisation of Hybridization 4.1.3.1 Preface

The determination of specific gene sequences is performed by detecting the hybridization event between the immobilised probes and their complementary targets which are present in the PCR-amplified amplicons and discriminating the wild type (WT) or mutated (MUT) probe signals after hybridization with WT or MUT PCR-amplified biological samples. For this reason, the hybridization event is the main part of the electrochemical detection systems. To perform the effective hybridization, the effect of experimental parameters on the detection and discrimination of hybridization signals such as hybridization time, hybridization temperature and washing steps are investigated. The optimisation of washing is also investigated as an experimental parameter for hybridization, because it is performed directly after hybridization in order to avoid non-specific adsorption of target to the surface. The stringency of the washing steps also plays an important role in the differentiation of single base mutations. Control experiments are also performed to assess whether the hybridization is formed selectively. A noncomplementary (NC) probe which is completely different from WT or MUT probe is used in order to control the non-specific adsorption or binding of target.

4.1.3.2 Hybridization Time

The time required for adequate probe-target hybridization depends on the type of the probe and the sensitivity of the system. The influence of the hybridization time is investigated by using the enzyme-based colorimetric detection method as described in section 3.4.4. The FcV WT, MUT and NC probes at a concentration of 1 nmol/ml are immobilised on the surface of the DNA-BIND wells. The hybridization is accomplished with FCV WT PCR-amplified amplicons by waiting for 5, 15, 30, 60, 120 and 240 min at 45 °C. The substrate is incubated for 10 min. The influence of the hybridization time is shown in Figure 9. Five wells of the plate are used for each probe immobilization.

62 Results

The OD signals of the hybridization with WT and MUT probe immobilised electrodes increase according to the increase of the hybridization time and then saturate slightly at a hybridization time of 120 min. This effect reflects that the probes are saturated by hybridised PCR-amplified amplicons. Figure 9 also shows that the hybridization and washing conditions are optimum to avoid the non-specific adsorption and binding of target. The NC probe immobilised electrode features almost no hybridization signal.

1.4

1.2 aa

1

0.8

OD 0.6

0.4 b 0.2 c 0 0 20 40 60 80 100 120 140 160 180 200 220 240 260 HYBRIDISATION TIME (min)

Figure 9. The effect of hybridization time on the hybridization signals of FcV (a) WT, (b) MUT and (c) NC probe immobilised electrodes with FcV WT PCR-amplified amplicons. Probe immobilization: 20 min 1 nmol/ml probe solutions prepared in binding buffer ( pH 8.5), hybridization: 30 min with 1:10 diluted PCR-amplified samples in dilution buffer, enzyme binding: 30 min 1:1000 diluted streptavidine alkaline phosphatase in 2xSSC+0.1% Tween 20, substrate reaction: 10 min 2.6 mM pNPP solution, measurement: at 405 nm on a 96- well plate reader.

As is mentioned before, a shorter process period is preferred for integrated systems. Therefore, the hybridization time of 30 min. is chosen for further experiments in order to shorten the process period even though the highest signal is obtained after the hybridization for 120 min.

Results 63

4.1.3.3 Hybridization Temperature

The important factors concerning the improvement of the hybridization process are the hybridization conditions and their respective stringency. Increasing temperature generally leads to increased stringency. At high stringency, duplexes are formed only between strands with perfect one-to-one complementarity. This allows easy differentiation of WT and MUT PCR-amplified amplicons and the determination of FcV and FcII. The optimal hybridization temperature is influenced by the homology of the probe to the target and the GC content of the probe.

The optimal hybridization temperature (Topt) is 20-25 °C below the melting temperature of the probe-target hybrid72. The melting temperature under the salt-containing hybridization conditions can be calculated approximately by using equation 19.

+ Tm = 81.5 + 16.6(log M [ Na ]) + 0.41(%G + C ) − 0.72(% formamide ) (19) With respect to this calculation, the optimal hybridization temperatures are calculated approximately for the hybridization of FCV and FCII probes with their complementary sequences in the PCR-amplified samples and different temperatures, within a range that include these calculated temperatures, are investigated by using enzyme-based colorimetric detection method as described in section 3.4.4. The hybridization is performed at 35, 45 and 50 °C for FcV and FcII. The optical density results for FcV and FcII WT and MUT probes after hybridization with WT or MUT PCR-amplified samples at the hybridization temperatures of 35, 45 and 50 °C is given in Table 3 and 5, respectively. The effect of hybridization temperature on the hybridization signal of FcV and FcII is also given in Table 4 and 6.

64 Results

Table 3. Optical density results for FcV WT and MUT probes after hybridization with FcV WT or MUT PCR-amplified samples at the hybridization temperatures of 35, 45 and 50 °C. Probe immobilization: 20 min 1 nmol/ml probe (WT VB, MUT VB) solutions prepared in binding buffer (pH 8.5). Other conditions are as in Figure 9.

Number of FcV WT FcV MUT Temperature measurements PCR-amplified sample PCR-amplified sample (°C) WT probe MUT probe WT probe MUT probe

35 n1 0.612 0.234 0.242 0.867

n2 0.621 0.224 0.249 0.72

45 n1 1.427 0.158 0.101 0.592

n2 1.377 0.165 0.09 0.605

50 n1 0.204 0.024 0.031 0.065

n2 0.186 0.022 0.034 0.066

Table 4. Effect of hybridization temperature on the differentiation of FcV WT and MUT probes after hybridization with FcV WT and MUT PCR-amplified samples. Signal averages for WT and MUT probes are given as SWT and SMUT, respectively.

Difference (%) Temperature FcV WT PCR-amplified sample FcV MUT PCR-amplified sample (°C)

100*(SWT-SMUT)/SWT 100*(SMUT-SWT)/SMUT

35 62.88 69.00

45 88.45 83.97

50 88.21 50.00

Results 65

Table 5. Optical density results for FcII WT and MUT probes after hybridization with FcII WT or MUT PCR-amplified samples at the hybridization temperatures of 35, 45 and 50 °C. Probe immobilization: 20 min 1 nmol/ml probe (WT IIC, MUT IIC) solutions prepared in binding buffer (pH 8.5). Other conditions are as in Figure 9.

Number of FcII WT FcII MUT Temperature measurements PCR-amplified sample PCR-amplified sample (°C) WT probe MUT probe WT probe MUT probe

35 n1 0.387 0.573 1.107 2.112

n2 0.424 0.635 0.999 1.917

45 n1 0.609 0.71 1.312 2.07

n2 0.637 0.731 1.184 2.194

50 n1 1.715 0.157 0.686 1.778

n2 1.728 0.185 0.693 1.857

Table 6. Effect of hybridization temperature on the differentiation of FcII WT and MUT probes after hybridization with FcII WT and MUT PCR-amplified samples.

Difference (%)

Temperature (°C) FcII WT PCR-amplified sample FcII MUT PCR-amplified sample

100*(SWT-SMUT)/SWT 100*(SMUT-SWT)/SMUT

35 32.78 47.74

45 13.59 41.46

50 90.06 62.07

Since the best differentiation of WT and MUT probes, after hybridization with WT and MUT PCR-amplified samples is obtained at hybridization temperatures of 45 °C (Table 5) for FcV and 50 °C (Table 7) for FcII, 45 and 50 °C are determined as optimum hybridization temperatures for FcV and FcII , respectively. The specificity of the system is also controlled by using an NC probe. The optical density obtained for the NC probes is under 0.01 (not shown).

66 Results

4.1.3.4 Optimum Washing Conditions

The stringency of washing buffer has a profound effect on the hybridization results. Stringency level of the washing buffer need to be high enough to permit only complementary sequences to remain bound to the immobilised probe and the washing buffer needs to contain enough ionic detergent to prevent non-specific binding. Stringency can be altered by changing the salt concentration, adding formamid or changing the temperature. In this work, the stringency is altered by changing the salt concentration. The investigation of optimum washing buffer is performed for both the label-free as well as the enzyme-based electrochemical detection methods.

The effect of wash stringency on the differentiation of the WT, MUT and NC probes after hybridization with FcV WT PCR-amplified amplicons by using label-free (A) and enzyme- based (B) electrochemical detection methods is shown in Figure 10. The surface preparation is performed as described in section 3.4.1. With respect to the obtained results, 1xSSC+0.1%SDS and 0.1xSSC+0.1%Tween 20 are chosen as washing buffers for the label- free and enzyme-based electrochemical detection methods, respectively.

Results 67

A a 100 a a 80 ) A

n b 60

NT ( b 40 b CURRE 20 c c c 0 3xSSC 2xSSC 1xSSC W ASHING BU FFERS (+0.1% SDS)

B 4.0 a

a a 3.2 b

b b b

(µA) 2.4 T

a

REN 1.6 c CUR 0.8 c c c 0.0 2xSSC 1xSSC 0.1xSSC 0.01xSSC WASHING BU FFERS (+0.1% TWEEN)

Figure 10. Effect of wash stringency on the differentiation of (a) WT, (b) MUT and (c) NC probes after hybridization with FcV WT PCR-amplified amplicons by using (A) label-free and (B) enzyme-based electrochemical detection methods. The conditions for label-free method are as in Figure 6. The conditions for enzyme-based method are as following: Hybridization: 30 min in 1:1 diluted FcV WT PCR amplified amplicons prepared in dilution buffer, enzyme labelling: 30 min 1:1000 diluted streptavidine alkaline phosphatase in 2xSSC+0.1% Tween 20, substrate reaction: 1 min 2 mM NAP solution prepared in TBS buffer (pH 9.6), measurement: directly after the substrate reaction, by using DPV with an equilibration time of 5 s, a modulation time of 0.05 s, an interval time of 0.5 s, a step potential of 8 mV and a modulation amplitude of 50 mV in a potential range of 0-0.5 V

68 Results

4.1.3.5 Sensitivity of the Detection Methods

The sensitivity of the label-free, as well as of the enzyme-based electrochemical detection methods are determined by investigation of the respective hybridization signals in dependency of the dilution of the sample. Therefore, various dilutions of FcV PCR-amplified samples are prepared with dilution buffer. The surface preparation is performed as described in section 3.4.1. Both electrochemical detection methods adhere to the conditions introduced in section 3.4.2 and 3.4.3, respectively. The effect of sample dilution on the hybridization signal by using label-free and enzyme-based electrochemical methods is given in Figure 11. One chip with eight electrodes is used for each sample dilution. Four electrodes of the chip are modified with the FcV WT probe, four electrodes are modified with the NC probe.

As seen in Figure 11A, the hybridization between FcV WT probe and FcV WT PCR- amplified samples can be detected down to a sample dilution of 1:8 as far as the label-free electrochemical detection method is concerned. In contrast, the hybridization detection is feasible even at a sample dilution of 1:100 when applying the enzyme-based electrochemical method (Figure 11B). The sensitivity limit of the label-free system is clearly lower than the enzyme-based system. Since the guanine signal of the hybrid is measured for the detection of the hybridization event in the case of the label-free system, the sensitivity limitation is proportional to the number of guanine bases existing on the electrode surface after the hybridization. The sensitivity of the label-free system can be increased by changing the length of the amplicons reproduced by PCR. But along with increasing length, the signal could decrease because of a slower hybridization kinetics and steric hindrance due to the self- coupling of the bases to the amplicons. A larger number of guanine bases, achieved by increasing the quantity of cytosine bases of the probe also lead to an enhanced signal. But in the case of determination of mutations, the probe must be selected from the region where the mutation occurs. For this reason, it is not always possible to have a mutation region with a large number of cytosine bases. Recently, label-free electrochemical detection systems are preferred due to their abilities for rapid and low-cost hybridization detection but the use of these systems is limited because of their low sensitivity. Regarding the enzyme-based electrochemical method, the sensitivity is proportional to the amount of end product, which is derived from the substrate in the presence of a specific enzyme and less evidently to the activity of the enzyme. The higher the enzyme activity is, the more end product is yielded and thus, the oxidation signal is higher. Therefore, it is important

Results 69

to adjust the most appropriate pH to achieve optimum enzyme activity. In enzyme reactions, the end product concentration increases linearly with time for an extended period of time. Later on the substrate is depleted, so the signal strength starts to level off. Eventually, the concentration of end product reaches a plateau and doesn’t change any more with time. The concentration of the end product can be increased over the period of time under optimum conditions for the enzyme. Thus, the sensitivity limit of the enzyme-based electrochemical detection method exceeds the one associated to the label-free method due to the high concentration of the end product available in the measurement buffer.

70 Results

A 60 a 50 b

40 ) A n

( 30

NT

RE 20 CUR 10

0

1:1 1:1.5 1:2 1:4 1:8 SAMPLE DILUTION

B 3.5

a 3.0 b

2.5 ) A

µ 2.0

ENT ( 1.5 RR

CU 1.0

0.5

0.0 1:6.25 1:12.5 1:25 1:50 1:100 SAMPLE DILUTION

Figure 11. Sensitivity of the (A) label-free and (B) enzyme-based electrochemical detection methods. Hybridization: 30 min at 45 °C with FcV PCR-amplified amplicons diluted in dilution buffer. Other conditions for the label-free method is as in Figure 1 and for the enzyme-based method is as in Figure 10 .

Results 71

4.2 Investigation of Optimum Probe Sequences

Since the specific inherited disorders related to the FcV as well as FcII are caused by single base mutations in their specific gene regions, it is important to differentiate the hybridization signals of probes with (MUT) and without (WT) single base mutation after hybridization with the accordant WT or MUT amplicons in order to identify these two blood disorders. Therefore the investigation of optimum probe sequences for the label-free as well as the enzyme-based electrochemical detection method is also an important issue. An optimum WT probe sequence must lead to higher signal with WT PCR-amplified amplicons and smaller signal with MUT PCR-amplified amplicons, whereas an optimum MUT probe sequence must react vice versa. Therefore, different probe sequences are designed and different parameters effecting the differentiation between WT and MUT probe signals (such as hybridization temperature, wash stringency) are investigated. While this investigation is performed using SPCs and the electrochemical detection systems, the task of discovering the optimum probe sequences demands a long time. Therefore the investigation is carried out using enzyme- based colorimetric method as described in section 3.4.4. This method allows the rapid testing of many different probe sequences and many different parameters ascribable to the simultaneous testing and measurement abilities.

The specific WT and MUT probes for FcV and FcII are chosen by comparing the optical signals of these probes after the hybridization with WT and MUT PCR-amplified amplicons at different hybridization temperatures. The optical signal differences of FcV and FcII probes after hybridization with PCR-amplified samples are given in Figure 12 and 13, respectively. Two wells of the DNA-BIND plate are used for every probe.

72 Results

. 100

80

60

40

20

SIGNAL DIFFERENCE (%) 0 MUT VA MUT VB MUT VC WT VA WT VB WT VC

IMMOBILISED PROBES

Figure 12. Optical signal differences of FcV WT and MUT probes after hybridization with WT and MUT PCR amplified samples. Other conditions are as in Figure 9.

2.5 . WT PCR 2.0 MUT PCR

1.5

1.0

0.5 CURRENT (µA) 0.0 MUT IIA MUT IIB MUT IIC MUT IID MUT IIE WT IIA WT IIB WT IIC WT PCR 0.035 0.027 0.315 0.051 0.024 1.02 1.555 0.702

0.028 0.024 0.362 0.068 0.027 1.216 1.685 0.726

MUT PCR 0.287 0.157 2.163 1.287 0.151 0.169 0.943 0.088

0.267 0.147 2.077 1.302 0.159 0.127 0.972 0.102

IMMOBILISED PROBES Figure 13. Enzyme-based colorimetric analysis of optimum FcII WT and MUT probe sequences after hybridization with WT (a) and MUT (b) PCR amplified amplicons. Hybridization: 30 min in 1:10 diluted PCR-amplified sample at 50 C°. Other conditions are as in Figure 9.

Results 73

Since the determination of single base mutations and the discrimination of genotypes are the two important aims of this study, the selection of optimum probe sequences are performed in order to find a probe-pair (WT-MUT) which enables the achievement of these two goals successfully. For the determination of mutations, the signal obtained for the immobilised WT probe after hybridization with WT target should be higher than the one obtained after hybridization with MUT PCR-amplified samples and vice versa for the immobilised MUT probe. Concerning the discrimination of genotypes, the ratio between the hybridization signals of WT probe-WT PCR and WT probe-MUT PCR should resemble the ratio between MUT probe-WT PCR and MUT probe-MUT PCR. WT VB/MUT VB and WT IIB/MUT IIC probe-pairs showing the expected features at 45 °C and 50 °C are chosen for FcV and FcII, respectively.

74 Results

4.3 Determination of Single Base Mutations

The determination of single base mutations related to the FcV and FcII blood disorders and the discrimination of genotypes by using electrochemical DNA biosensors rely on the differential pulse voltammetric transduction of the hybridization event between the FcV WT or MUT probes and their complementary target sequences present in the PCR-amplified samples. The surface pre-treatment is performed as described in section 3.4.1. The detection of hybridization is carried out by label-free and enzyme-based electrochemical detection methods as described in section 3.4.2 and 3.4.3, respectively.

In the label-free electrochemical detection method, the detection of hybridization is accomplished by the appearance of the guanine oxidation signal, whereas no guanine signal is obtained from inosine-substituted probes. In the enzyme-based electrochemical detection method, the hybridization is detected by the oxidation peak of the end product which occurs only in the presence of enzyme alkaline phosphates. The specific enzyme alkaline phosphatase is conjugated with streptavidine which allows the specific binding of the enzyme to the biotinylated target. The end product can occur in the presence of specific enzyme. Thus, the oxidation signal of the end product can only be measured when the hybridization occurs.

The detection of the hybridization event between FcV WT and MUT probes and their complementary targets which are present in the FcV WT, heterozygous (HET) and MUT PCR-amplified amplicons by using label-free electrochemical detection methods is shown in Figure 14.

The detection of single base mutation related to FcII is achieved with the enzyme-based electrochemical detection method. The detection of hybridization between FcII WT and MUT probes and the corresponding complementary targets existent in the FcII WT, HET and MUT amplified amplicons by using the enzyme-based electrochemical method is given in Figure 15.

Results 75

A

30 29%

20 MUT

10 WT CURRENT (nA) 71% 0 0.75 0.8 0.85 0.9 0.95 POTENTIAL (V)

B

30 33% 20 MUT WT

10

CURRENT (nA) 67% 0 0.75 0.8 0.85 0.9 0.95 POTENTIAL (V)

C

30

46% 20 MUT WT 54% 10 CURRENT (nA)

0 0.75 0.8 0.85 0.9 POTENTIAL (V)

Figure 14. Differential pulse voltammograms for FcV WT (—), MUT (----) and NC (—) probe immobilised electrodes after hybridization with (A) WT (B) MUT and (C) HET PCR-amplified amplicons. The ratio between WT and MUT probes is also given in percentage. The calculations are made with the formal of 100*(SWT / SWT+ SMUT). Signal averages for WT and MUT probes are given as

SWT and SMUT, respectively.

76 Results

A

2 25% 1.6 1.2 MUT

0.8 WT 0.4 CURRENT (µA)

0 75% 0.05 0.15 0.25 0.35 POTENTIAL (V)

B

2 25% 1.6

1.2 WT 0.8 MUT

CURRENT (µA) 0.4

0 75% 0.05 0.15 0.25 0.35 POTENTIAL (V)

C

2

1.6 A) µ 1.2 WT 47% 0.8 53% MUT 0.4 CURRENT ( 0 0.05 0.15 0.25 0.35 POTENTIAL (V)

Figure 15. Differential pulse voltammograms for the FcII WT (—), MUT (----) and NC (—) probe immobilised electrodes after hybridization with (A) WT (B) MUT and (C) HET PCR-amplified amplicons. The ratio between WT and MUT probes is also given in percentage. The calculations are made with the formal of 100*(SWT / SWT+ SMUT). Signal average for WT and MUT probes are given as

SWT and SMUT, respectively.

Results 77

4.4 Development of Lab-on-a-chip Technology

4.4.1 Preface

The goal of lab-on-a-chip technology is to integrate multiple processes, including sample preparation (DNA extraction and purification), amplification, hybridization and detection on a microfluidic platform. The ability to perform all the steps of the biological assay in a disposable cartridge promises significant advantages in terms of speed, cost, efficiency and automation. That makes the electrochemical molecular methods more suitable for routine laboratory diagnosis or even centralized models such as point-of-care systems. The preparation of these microlaboratories commonly relies on advanced microfabrication and micromachining technologies.

Within this work, an integrated diagnostic device which consists of sample preparation, amplification and electrochemical detection modules is introduced for the first time. The total process is performed automatically on a microfluidic platform in an integrated system (cartridge). The cartridge consists of the microchannels through which the sample and reagents are transferred, the small containers in which the reagents are stored and a chip on which the detection process is performed. The schematic presentation of the cartridge is shown in Scheme 21.

sample injection

DNA & RNA isolation

reagents waste

amplification

detection

Scheme 21. Schematic presentation of the integrated cartridge.

78 Results

The sample preparation takes place as a first step after the biological sample is injected into the cartridge. The extracted sample is then transferred to the amplification step. The amplified sample is hybridised with the immobilised probe on the surface of the chip. The detection is accomplished by detecting the hybridization event with label-free or enzyme-based electrochemical detection methods by using a novel simplified multi-potentiostat.

This integrated diagnostic system, in connection with the DNA chip on which different probes are immobilised, enables the simultaneous and rapid detection of several single base mutations which lead to inherited diseases or disorders within one measurement chamber.

4.4.2 Detection Process in the Cartridge

The integrated device implements three separated processes, including sample preparation, amplification and detection as described above. Before the integrated process is performed, every partial stage is investigated separately in the cartridge in order to optimize the conditions for the processes on the microfluidic system. Therefore, the detection process is performed in the cartridge by using PCR-amplified samples. The amplification of the samples is not accomplished within the cartridge but injected into it. The chip preparation is performed as explained in section 3.4.1. The FcV or FcII WT (WT VB, WT IIC), MUT (MUT VB, MUT IIC ) and NC (I-cap-FcVmut2-N for FcII and I-cap-FcIImut-N for FcV) probes are immobilised onto the chip surface. The detection for FcV and FcII is performed by using label-free, as well as enzyme-based electrochemical detection methods as described in section 3.4.2 and 3.4.3. In the cartridge, the position of chip is anchored. Thus, instead of immersing the chip into the solution, the solutions are rather inserted into the chip through microchannels. The eight WEs of the chip are measured with the multi-potentiostat simultaneously as described in section 3.2.1. Both, firstly, the determination of single base mutations and, secondly, the discrimination of genotypes are successfully accomplished by the detection process within the cartridge. The ratio of the hybridization signals between WT and MUT probe immobilised electrodes is shown in Figure 16 for FcV and Figure 17 for FcII, respectively.

Results 79

4.0 3.5 3.0

T 2.5 U M

2.0 / 1.5 WT 1.0 0.5 0.0 WT HET MUT

FcV PCR-AMPLIFIED AMPLICONS

Figure 16. Ratio of hybridization signals between FcV WT and MUT probe immobilised electrodes after the detection process in the cartridge.

4.0 3.5 3.0

T 2.5 MU

2.0 /

WT 1.5 1.0 0.5 0.0 WT HET MUT

FcII PCR-AM PLIFIED AM PLICONS

Figure 17. Ratio of hybridization signals between FcII WT and MUT probe immobilised electrodes after the detection process in the cartridge.

80 Results

4.4.3 Integrated Process in the Cartridge

After optimization of each partial steps within the cartridge, the complete integrated process is performed. The biological sample (blood sample) for FcV and FcII is injected into the cartridge, after which the sample preparation, amplification and detection are implemented automatically. The amplicons of FcV and FcII which are amplified in the cartridge, are transferred to the chip and hybridised with the immobilised probes. The detection of the hybridization for FcV and FcII is performed by using label-free and enzyme-based electrochemical detection methods, by supplying the chip with the adequate reagents. The chip preparation is performed as defined in section 3.4. The detection methods are described in section 3.5.1 and 3.5.2. The FcV and FcII WT (WT VB, WT IIC), MUT (MUT VB, MUT IIC ) and NC (I-cap-FcVmut2-N for FcII and I-cap-FcIImut-N for FcV) probes are immobilised onto the chip surface. The placement of the probes on the chip for FcV and FcII is shown in Scheme 22. The fully integrated process within the cartridge by using SPCs was developed recently and hence the sensitivity of the label-free detection method is low. Thus, regarding the integrated process in the case of the FcV system, the electrochemical activity of every WE surface of the chip has to be controlled. A well-known synthetic probe-target system is used to conform to this demand and in order to prove the functionality of the immobilization and hybridization steps. Therefore, the synthetic target (TNF2k) which is complementary to the immobilised probe (N-TNF2I), is spiked into the hybridization buffer during the integrated process of FcV. Two WEs of the chips are used for the immobilization of N-TNF2I probe. All eight available WEs of the chip are measured simultaneously with the multi-potentiostat as described in section 3.2.1.

A B

Scheme 22 . Assignment of the probes on the chip for (A) FcV and (B) FcII in the case of fully integrated processes.

Results 81

The ratio of acquired hybridization signals between WT and MUT probe immobilised electrodes for FcV and FcII is shown in Figure 18 and 19, respectively.

4.0 3.5 3.0 T

U 2.5 M 2.0

WT / 1.5 1.0 0.5 0.0 WT HET MUT

FcV BLOOD SAMPLES

Figure 18. The ratio of hybridization signals between FcV WT and MUT probe immobilised electrodes after the integrated process in the cartridge.

2.5

2.0

1.5 T

MU / 1.0 WT

0.5

0.0 WT HET MUT FcII BLOOD SAMPLES

Figure 19. The ratio of hybridization signals between FcII WT and MUT probe immobilised electrodes after the integrated process in the cartridge.

Summary 83

5 Summary

DNA biosensors based on nucleic acid hybridization processes are rapidly being developed to achieve the goal of fast and inexpensive diagnosis of inherited and infectious diseases. Electrochemical biosensors, coupling the inherent specificity of DNA recognition reactions with the electrochemical transducers show great promise for sequence specific detection. Electrochemical transducers are often used for detecting the DNA hybridization event due to their high sensitivity, small dimensions, low cost and compatibility with micro fabrication technology.

In this work, an electrochemical DNA biosensor for the detection of single base mutations was developed by using low cost screen-printed electrodes. It was shown that the DNA biosensors were able to detect single base mutations in the genes of FcV and FcII proteins which are involved in coagulation. The optimisation studies are performed in order to increase the electrochemical activity of the surface and enhanced the sensitivity of the detection methods.

The screen printed carbon paste electrodes were selected for the development of DNA biosensors. The carbon paste material was applied to the surface of the chip by using screen printing technology, which is particularly suitable for the development of DNA chips. The DNA chip designed within the scope of this study consists of eight working electrodes (WEs), four reference electrodes (REs) and six counter electrodes (CEs).

Label-free and enzyme -based electrochemical methods were used to detect single base mutations. It was even possible to discriminate genotypes. The procedures for label-free and enzyme-based electrochemical detection methods in each case consist of surface preparation, hybridization, washing and transduction steps. Concerning the label-free electrochemical detection, the guanine bases of the probe oligonuclotides were substituted by inosine. Inosine has similar base-pairing properties to guanine but its oxidation signal distinctly differs from that of guanine. Thus, the detection of the hybridization was accomplished with the appearance of the guanine oxidation signal, whereas no guanine signal was obtained from inosine substituted probes. In the enzyme-based electrochemical detection method, the hybridization event was detected indirectly by measuring the signal of the electrochemically active end product which occurred from the substrate in the presence of a specific enzyme, alkaline phosphatase. The label-free electrochemical detection system was preferred due to its

Summary 84

characteristics of rapid and low-cost hybridization detection without the use of external redox indicators, but the use of this system is limited because of its low sensitivity. The enzyme- based electrochemical method provides increased sensitivity due to the huge amount of end product obtained from the substrate in the present of the specific enzyme.

The electrode surface was activated chemically by using the ionic detergent SDS. Specific probe oligonucleotides were covalently immobilised on the working electrode surface using Silane mediated modification chemistry. The electrochemical detection methods were optimised by investigating different experimental parameters and the optimum conditions were chosen.

The efficiency of the electrochemical surface activity is effected by the conditions of screen printing, pre-treatment, silane surface chemistry and probe immobilization. Different curing temperatures and curing times after screen printing were compared by measuring the guanine signal of the aminolinked probe in order to increase the electrochemical activity of the surface. The unavoidable adsorption of electrochemically active contaminations to the surface reduces the sensitivity and effects the reproducibility. For this reason, the activation of the electrode surface by chemical pre-treatment was necessary. Different detergents were analysed and the pre-treatment time was optimised. The optimum conditions for the silane chemistry were constituted according to the obtained results.

The hybridization and wash stringency profoundly influence the determination of single base mutations. The stringency of the hybridization was mainly attained by optimising the hybridization temperature, also taking the respective theoretical melting temperature into consideration . In this regard, the hybridization was performed at different temperatures to determine the most suitable one for resolving one base mutations.

It is well-known that DNA strongly adsorbs to most carbon surfaces. This imposes the necessity to reduce the adsorption of target on the transducer surface during the hybridization detection assay. Concerning this, the utilized ionic detergent content of the washing buffer was exposed to be inevitable to prevent adsorption and non-specific binding of the target. A high stringency of the washing buffer is essential for enhancing the selectivity to enable a reliable detection of single base mutations. The ionic detergent content and the stringency of washing buffer for label-free as well as enzyme-based detection methods were investigated. The detection of single base mutations with both of the detection systems were successfully accomplished by using optimum washing buffers.

Summary 85

The label-free and enzyme-based determination of single base mutations related to FcV and FcII were also implemented successfully in a disposable integrated cartridge consisting of the fluidic platform. The blood sample was injected into this cartridge and the processes sample preparation, PCR and electrochemical detection were performed fully automatically for the first time. The eight WEs were measured simultaneously by a multi-potentiostat.

86 References

6 References

1. Milan, K. M., Mikkelsen, S. R. Sequence-selective biosensor for DNA based electroactive hybridization indicators. Anal. Chem., 65, pp 2317-2323, 1993.

2. Su, H., Kallury, K. M. R., Roach, A. and Thompson, M. Interfacial nucleic acid hybridization studied by random primer 32P labelling and liquid-phase acoustic network analysis. Anal. Chem., 66, pp 769-777, 1994.

3. Herrington, C. S., Burns, J., Graham, A. K., Evans, M. and McGee, J.O. Interphase cytogenetics using biotin and digoxigenin labelled probes I: relative sensitivity of both reporter molecules for detection of HPV16 in CaSki cells. Journal of Clinical Pathology, 42, pp 592-600, 1989.

4. Sha, M., Griffith, B. P., Raveh, D., Isom, H. C., Ward, D. C., Hsiung, G. D. Detection of guinea pig cytomegalovirus nucleic acids in cultured cells with biotin-labelled hybridization probes. Virus Res., 6(4), pp 317-329, 1987.

5. Gentilomi, G., Ferri, E., Girotti, S. Direct quantitative chemiluminescent assays for the detection of viral DNA.Anal. Chim. Acta, 255, pp 387-394, 1991.

6. Piunno, P. A. E., Krull, U. J., Hudson, R. H. E., Damha, M. J. and Cohen, H. Fiber optic DNA sensor for fluorometric nucleic acid determination. Anal. Chem., 67, pp 2635- 2643, 1996.

7. Sevall, J. S., Prince, H., Garratty, G., O’Brien, W. A., Zack, J. A. Rapid enzymatic analysis for human immunodeficiency virus type 1 DNA in clinical specimens. Clin. Chem., 39, pp 433-439, 1993.

8. Okahata, Y., Matsunobu, Y., Ijiro, K., Mukae, M., Murakami, A., Makino, K. Hybridization of nucleic acids immobilized on a quartz crystal microbalance. J.Am. Chem. Soc., 114, pp 8299-8300, 1992.

9. Hashimoto, K., Keiko, I. and Ishimori, Y. Sequence-specific gene detection with a gold electrode modified with DNA probes and an electrochemically active dye. Anal. Chem., 66, pp 3830-3833, 1994.

10. Erdem A., Kerman, K., Meric, B., Akarca, U. S., Ozsoz, M. Novel hybridization indicator methylene blue for the electrochemical detection of short DNA sequences related to the hepatitis B virus. Anal. Chim. Acta, 422, pp 139-149, 2000.

87 References

11. Wang, J., Rivas, G., Cai, X., Dontha, N., Shiraishi, H., Luo, D., Valera, F. Sequence- specific electrochemical biosensing of M. tuberculosis DNA. Anal. Chim. Acta, 337, pp 41-48, 1997.

12. Kara, P., Kerman, K., Ozkan, D., Meric, B., Erdem, A., Nielsen, P. E., Ozsoz, M. Label- free and label-based electrochemical detection of hybridization by using methylene blue and peptide nucleic acid probes at chitosan modified carbon paste electrodes. Electroanalysis, 14, pp 1685-1690, 2002.

13. Meric, B., Kerman, K., Ozkan, D., Kara, P., Ozsoz, M. Indicator-free electrochemical DNA biosensor based on adenine and guanine signals. Electroanalysis, 14, pp 1245- 1250, 2002.

14. Erdem, A., Meric, B., Kerman, K., Dalbasti, T., Ozsoz, M. Detection of interaction between metal complex indicator and DNA by using electrochemical biosensors. Electroanalysis, 11, pp 1372-1376, 1999.

15. Meric, B., Kerman, K., Ozkan, D., Kara, P., Erensoy, S., Akarca, U. S., Mascini, M., Ozsoz, M. Electrochemical DNA biosensor for the detection of TT and hepatit B virus from PCR amplified real samples by using methylene blue. Talanta, 56, pp 837- 846, 2002.

16. Carter, M. T., Bard, A. J. Voltammetric studies of the interaction of tris-(1,10- phenanthroline)-cobalt (III) with DNA. J. Am. Chem., 109, pp 7528-7530, 1987.

17. Wang, J., Rivas, G., Cai, X. Screen-printed electrochemical hybridization biosensor for the detection of DNA sequences from the Escherichia coli pathogen. Electroanalysis, 9(5), pp 395-398, 1997.

18. Liu, S., Ye, J., He, P., Fang, Y. Voltammetric determination of sequence-specific DNA by electroactive intercalator on graphite electrode. Anal. Chim. Acta, 335, pp 239-243, 1996.

19. Palecek, E., Tomschik, M., Stankova, V., Havran, L. Chronopotentiometric stripping of DNA at mercury electrodes. Electroanalysis, 9, pp 990-997, 1997.

20. Campbell, C. N., Gal, D., Cristler, N., Banditrat, C., Heller, A. Enzyme-amplified amperometric sandwich Test for RNA and DNA. Anal. Chem., 74(1), pp 158-162, 2002.

21. Wang, J., et al. DNA electrochemical biosensors for environmental monitoring-A review. Anal. Chim. Acta, 347, pp 1-8, 1997.

88 References

22. Edman, C. F., et al. Electric field directed nucleic acid hybridization on microchips. Nucl. Acid. Res., 25, pp 4907-4914, 1997.

23. Steel, A. B., Herne, T. M., Tarlov, M. J. Electrochemical quantitation of DNA immobilized on gold. Anal. Chem., 70, pp 4670-4677, 1998.

24. Wang, D. G., et al. Large-scale identification, mapping and genotyping of single- nucleotide polymorphisms in the human genome. Science, 280, pp 1077-1082, 1998.

25. Sosnowski, R.G., Eugene, T. U., Butler, W. F., O’Connell, J. P., Heller, M. J. Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control. Proc. Natl. Acad. Sci. USA, 94, pp 1119-1123, 1997.

26. Hoheisel, J., Vingron, M. DNA chip technology. Biospektrum, 4, pp 17-20, 1998.

27. Castaman, G., Lunghi, B., Missiaglia, E., Bernardi, F., Rodeghiero, F. Phenotypic homozygous activated protein C resistance associated with compound heterozygosity for Arg506Gln (Factor V Leiden) and His1299Arg substitutions in Factor V. J. Haematol., 99, pp 257-261, 1997.

28. Castoldi, E., Kalafatis, M., Lunghi, B., Simioni, P., Ioannou, P. A., Petio, M., Girolami, A., Mann, K. G., Bernardi, F. Molecular bases of pseudo-homozygous APC resistance: the compound heterozygosity for FV R506Q and a FV null mutation results in the exclusive presence of FV Leiden molecules in plasma. Thromb. Haemost., 80, pp 403- 406, 1998.

29. Van Cott, E. M., Soderberg, B. L., Laposata, M. Activated protein C resistance, the factor V Leiden mutation, and a laboratory testing algorithm-A review. Arch. Pathol. Lab. Med., 126(5), pp 577-582, 2002.

30. Royle, N. J., Irwin, D. M., Koschinsky, M. L., et al. Human genes encoding prothrombin and ceruloplasmin map to 11p11-q12 and 3q21-24, respectively. Somat. Cell Mol. Genet., 13, pp 285-292, 1987.

31. Pollak, E. S., Lam, H. S., Russell, J. E. The G20210A mutation does not affect the stability of prothrombin mRNA in vivo. Blood, 100(1), pp 359-362, 2002.

32. Poort, S. R., Rosendaal, F. R., Reitsma, P. H. A common genetic variation in the 3'- untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood, 88(10), pp 3698-3703, 1996.

89 References

33. Margaglione, M., Brancaccio, V., Giuliani, N. Increased risk for venous thrombosis in carriers of the prothrombin G-->A20210 gene variant. Ann. Intern. Med., 129(2), pp 89- 93, 1998.

34. Varga, E. A., Moll, S. Prothrombin 20210 Mutation (Factor II Mutation). Circulation, 110, pp e15-e18, 2004.

35. Wang, J. Analytical Electrochemistry. Wiley-VCH, New York 1994.

36. Bard, A. J., Faulkner L. R. Electrochemical Methods, Fundamentals and Applications. John Wiley & Sons, Canada 1980.

37. Nickel, U. Lehrbuch der Thermodynamik. Carl Hanser Verlag, München 1995.

38. Skoog D. A., West D. M., Holler F. J. Fundamentals of Analytical Chemistry.Saunders College Publishing, New York 1992.

39. Atkins P. W. Physical chemistry. Oxford University Press, Oxford 1990.

40. Hamann, C. H., Vielstich, W. Electrohemie. 3. Auflage. Wiley- VCH Verlag GmbH, weinheim 1998.

41. Fokker, W. Elektrochemische Kinetik. Akademie Verlag, Berlin 1996.

42. Lewin, B. Genes. Oxford University Press, Oxford 1997.

43. Neidle, S. Oxford Handbook of Nucleic Acid Structure. Oxford University Press, Oxford, 1999.

44. Watson, J. D., Crick, F. H. C. A structure of desoxyribonucleic acid. Nature, 171, pp 737- 738, 1953.

45. Watson, J. D., Crick, F. H. C. Genetical implications of the structure of desoxyribonucleic acid. Nature, 171, pp 964-967, 1953.

46. Marmur, J., Rownd, R. and Schildkraut, L. Denaturation and renaturation of DNA. Prog. Nuc. Acid Res., 1, pp 232-300, 1963.

47. Ralph, C., Williams J. R. Molecular Biology In Clinical Medicine. Elsevier Science Publishing Co., New York 1991.

48. Keller, G. H., Manak, M. M. DNA Probes. Stocton Press, New York 1989.

49. Loewy, Z. G., Pottathilin, R., Singh, P., Sharma, B. P., Tyle, P. Antibody, Biosensor and Nucleic Acid Technologies. Van Nostrand Reinhold, New York 1993.

90 References

50. Lucarelli, F., Marazza, G., Turner, A. P. F., Mascini, M. Carbon and gold electrodes as electrochemical transducers for DNA hybridization sensors. Biosens. Bioelectron. 19, pp 515-530, 2004.

51. Wink, M., Wehrle, H. PCR (Polymerase Kettenreaktion) in medizinischen und biologischen Labor. GIT Verlag GmbH, Germany 1994.

52. Turner, A.P.F., Karube, I. and Wilson, G.S. Biosensors: Fundamentals and Applications. Oxford University Press, Oxford 1987.

53. Cammann, K. Fresenius Z. Anal. Chem. 287(1), 1977.

54. Nilsson, P. Biosensor Technology Applied to Hybridization Analysis and Mutation Detection. Doctoral Dissertation, Stockholm, 1998.

55. Thevenot, D. R., Toth, K., Durst, R. A., Wilson, G. S. Electrochemical biosensors: Recommended definitions and classification. Pure Appl. Chem., 71(12), pp 2333-2348, 1999.

56. Wang, J., Rivas, G., Cai, X. H. Screen-printed electrochemical hybridization biosensor for the detection of DNA sequences from the Escherichia coli pathogen. Electroanalysis, 9, pp 395-398, 1997.

57. Wang, J., Fernandes, J. R., Kubato, L. T. Polishable and Renewable DNA Hybridization Biosensors. Anal. Chem., 70, pp 3699-3702, 1998.

58. Pang, D.W., Abruna, H. D. Micromethod for the Investigation of the Interactions between DNA and Redox-Active Molecules. Anal. Chem., 70, pp 3162-3169, 1998.

59. Cai. X., Rivas, G., Shiraischi, H., et al. Electrochemical analysis of formation of polynucleotide complexes in solution and at electrode surfaces. Anal. Chim. Acta, 344, pp 64-76, 1997.

60. Wang, J., Rivas, G., Cai, X. H., et al. Adsorption and detection of peptide nucleic acids at carbon paste eletrodes. Electroanalysis, 9, pp 120-124, 1997.

61. Wang, J., Palecek, E., Nielsen, P. E., et al. Peptide Nucleic Acid Probes for Sequence- Specific DNA Biosensors. J. Am. Chem. Soc., 118, pp 7667-7670, 1996.

62. Kelly, S. O., Barton, J. K., Jackson, N. M., et al. Orienting DNA Helices on Gold Using Applied Electric Fields. Langmuir, 14, pp 6781-6784, 1998.

91 References

63. Kelley, S. O., Jackson, N. M., Hill, M. G., et al. Long-Range Electron Transfer through DNA Films. Angew. Chem. Int. Ed., 38, pp 941-945, 1999.

64. Hashimoto, K., Ito, K., Ishimori, Y. Novel DNA sensor for electrochemical gene detection. Anal. Chim. Acta, 286, pp 219-224, 1994.

65. Ihara, T., Nakayama, M., Murata, M., et al. Gene sensor using ferrocenyl oligonucleotide. Chem. Commun., pp 1609-1610, 1997.

66. Kara, P., Kerman, K., Ozkan, O., Meric, B., Erdem, A., Nielsen, P. E., Ozsoz, M. Label- Free and Label Based Electrochemical Detection of Hybridization by Using Methylene Blue and Peptide Nucleic Acid Probes at Chitosan Modified Carbon Paste Electrodes. Electroanalysis, 14, pp 1685-1690, 2002.

67. Napier, M. E., Thorp, H. H. Modification of Electrodes with Dicarboxylate Self- Assembled Monolayers for Attachment and Detection of Nucleic Acids. Langmuir, 13, pp 6342-6344, 1997.

68. Schülein, J. DNA sensor based on graphite electrodes. Doctoral Dissertation, Erlangen, 2002.

69. Prudenziati, M. Handbook of sensors and actuators 1. Elsevier Science, Oxford, 1994.

70. Grennan, K., Killard, A. J., Smyth, M. R. Physical characterizations of a screen-printed electrode for use in an amperometric biosensor system. Electroanalysis, 13, pp 745-750, 2001.

71. Schülein, J., Graßl, B., Krause, J., Schulze, C., Kugler, C., Müller, P., Bertling, W. M., Hassmann, J. Solid composite electrodes for DNA enrichment and detection. Talanta, 56, pp 875-885, 2002.

72. Anderson, N. L. M., Young, B. D. Quantitative filter hybridization, in: Nucleic acid hybridization: a practical approach. IRL Press, Oxford, 1985.

92

93

7 Zusammenfassung

In dieser Arbeit wurde ein elektrochemischer DNA-Biosensor zur Detektion von Einzelbasenmutationen unter Verwendung von kostengünstigen Siebdruckelektroden entwickelt. Es wurde die Detektion von Einzelbasenmutationen in den Genen für das Faktor II- und Faktor V-Protein gezeigt. FcII und FcV sind Proteine aus der Blutgerinnungskaskade. Optimierungsarbeiten wurden durchgeführt, um die elektrochemische Aktivität der Elektrodenoberfläche zu steigern. Mit der Steigerung der Oberflächenaktivität konnte auch die Sensitivität der Detektion erhöht werden.

Für die Entwicklung der DNA-Biosensoren wurden Siebdruckelektroden eingesetzt. Die Aufbringung der Kohlenstoffpaste auf die Oberfläche der Chips erfolgte mit der Siebdruck- Technologie, welche besonders geeignet ist für die Entwicklung von DNA-Chips. Die in dieser Arbeit entwickelten DNA-Chips wiesen acht Arbeitselektroden, vier Referenzelektroden und sechs Gegenelektroden auf.

Zur Detektion der Einzelbasenmutationen wurden markierungsfreie und enzymbasierte elektrochemische Methoden eingesetzt. Auch eine Genotypisierung konnte durchgeführt werden. Die Schritte für die markierungsfreie und die enzymbasierte Methode bestanden jeweils aus Oberflächenvorbehandlung, Hybridisierung, Waschen und Auslesung. Bei der markierungsfreien Methode war bei dem Fänger-Oligonukleotid das Guanin ersetzt durch das Inosin. Inosin hat die gleichen Basenpaarungs-Eigenschaften wie Guanin. Jedoch unterscheidet sich das Oxidationssignal von Inosin von dem von Guanin. Damit war eine Korrelation zwischen dem Guanin-Oxidationssignal und der Hybridisierung geschaffen. Die Messung von einem Guanin-Oxidationssignal war damit ein Hinweis auf eine Hybridisierung, da das Fänger-Oligonukleotid kein Guanin-Signal generieren konnte. Bei der enzymbasierten Methode erfolgte die Detektion des Hybridisierungsereignisses über die Messung des Signals von einem elektrochemisch aktiven Endprodukt, welches von einem spezifischen Enzym, der alkalischen Phosphatase, bei Vorhandensein des Substrats generiert wurde. Das markierungsfreie elektrochemische Detektions-System wurde bevorzugt, weil es schneller und kostengünstiger ist, da es ohne Redox-Indikatoren auskommt. Jedoch ist es wegen der geringen Sensitivität begrenzt einsetzbar. Die enzymbasierte Methode zeigt eine höhere Sensitivität in Abhängigkeit von der Höhe des Endproduktes, welches bei Vorhandensein des spezifischen Enzyms generiert wird.

94

Die Elektrodenoberfläche wurde chemisch aktiviert durch das ionische Detergenz SDS. Unter Benutzung von Silan-Oberflächenchemie wurden spezifische Fänger-Oligonukleotide auf die Oberfläche der Arbeitselektrode immobilisiert. Über die Untersuchung der verschiedenen experimentellen Parameter wurden die elektrochemischen Detektionsmethoden optimiert und die optimalen Parameter ausgewählt.

Die Effektivität der elektrochemischen Oberflächenaktivität ist abhängig von den Bedingungen beim Siebdrucken, bei der Vorbehandlung, der Silan-Oberflächenchemie und der Immobilisierung. Um die elektrochemische Aktivität der Oberfläche zu erhöhen wurden verschiedene Aushärtetemperaturen und Aushärtezeiten über das Auslesen des Guanin- Signals von aminomarkierten Fänger-Oligonukleotiden untersucht. Die unvermeidbare Adsorption von elektrochemisch aktiven Verunreinigungen an die Oberfläche reduzieren die Sensitivität und beeinflussen die Reproduzierbarkeit. Aus diesem Grunde war die Aktivierung der Elektrodenoberfläche über eine chemische Vorbehandlung erforderlich. Dazu wurden verschiedene Detergenzien getestet und die Dauer der Vorbehandlung optimiert. Von den erhaltenen Resultaten wurden die optimalen Bedingungen für die Silanchemie abgeleitet.

Bei der Bestimmung von Einzelbasenmutationen ist die Stringenz bei der Hybridisierung und beim Waschen von großer Bedeutung. Die Stringenz bei der Hybridisierung wurde vor allem erreicht über die Optimierung der Hybridisierungstemperatur. Die theoretische Schmelztemperatur diente dabei als Anhaltswert. Dazu wurde die Hybridisierung bei verschiedenen Temperaturen durchgeführt und die mit den besten Resultaten im Hinblick auf die Einzelbasendiskriminierung ausgewählt.

Es ist bekannt, dass DNA an die meisten Kohlenstoffoberflächen stark adsorbiert. Aus diesem Grunde war es wichtig, die Adsorption der Ziel-DNA an die Sensoroberfläche während der Hybridisierung zu reduzieren. Es stellte sich heraus, dass der verwendete Anteil an einem ionischem Detergenz in dem Waschpuffer für eine Verhinderung der Adsorption und der unspezifischen Bindung der Ziel-DNA unerlässlich ist. Eine hohe Stringenz des Waschpuffers ist essentiell für eine Steigerung der Selektivität, die wiederum notwendig ist für eine zuverlässige Detektion von Einzelbasenmutationen. Der Anteil an ionischem Detergenz und die Stringenz des Waschpuffers wurden sowohl für den markierungsfreie als auch für die enzymbasierte Detektionsmethode untersucht. Bei Verwendung von optimalen Waschpuffern konnte mit beiden Detektionssystemen die Bestimmung von Einzelbasenmutationen erfolgreich durchgeführt werden.

95

Die markierungsfreie und die enzymbasierte Bestimmung von Einzelbasenmutationen beim FcII- und FcV-Gen konnte erfolgreich in eine integrierte Cartridge zur Einmalverwendung, welche die fluidische Plattform enthielt, implementiert werden. Zum ersten Mal war es möglich, die Prozesse der Probenaufbereitung, PCR und elektrochemische Detektion vollständig automatisch in einer integrierten Cartridge durchzuführen. Dabei wurden die acht Arbeitselektroden simultan über ein Multipotentiostat ausgelesen.

DANKSAGUNG

Mein besonderer Dank geht an Herrn Dr. Jürgen Schülein, der einen großen Anteil an dem Zustandekommen dieser Promotionsarbeit hat. Auch fachlich war er mir eine große Hilfe. Großer Dank gilt Herrn Dr. Thomas Pöhmerer, der ebenfalls viel für das Zustandekommen dieser Arbeit beigetragen hat. Danken möchte ich auch für seine fachliche Unterstützung und stetige Diskussionsbereitschaft. Herrn Prof. Dr. Ulrich Nickel danke ich, dass er mir als Doktorvater die Möglichkeit gegeben hat, über dieses sehr interessante Thema zu promovieren. Für die Anfertigung der für diese Arbeit erforderlichen Messgeräte möchte ich Herrn Dr. Björn Graßl meinen Dank aussprechen. Neben seiner fachlichen Hilfe habe ich seine lustige und angenehme Art sehr genossen. Herrn Dr. Dirk Kuhlmeier danke ich für viele wertvolle Diskussionen und Anregungen. Auch für seine fröhliche und herzliche Art danke ich ihm sehr, mit der er immer für eine angenehme Atmosphäre sorgte. Frau Christine Schülein danke ich für die fröhlichen Gespräche und für ihre Unterstützung, sowohl privat als auch fachlich. Herrn Dr. Andre Josten und Herrn Dr. Georg Bauer danke ich für ihre Hilfestellungen in chemischen Fragen. Herrn Dr. Roland Barten danke ich für die kritische Durchsicht dieses Manuskripts. Herrn Dr. Olaf Weiner und Herrn PD-Dr. Wolfgang Bertling danke ich, dass sie es mir ermöglicht haben, bei der directif GmbH zu promovieren. Diese Doktorarbeit ist in einer kooperativen und offenen Atmosphäre entstanden, in der die Arbeit Freude gemacht hat. Dafür bedanke ich mich bei: Andrea Fenzl, Anja Weiland, Hans Kosak, Ingrid Weigel, Jürgen Krause, Marcela Pöhmerer, Maria Kölber, Marion Kramer, Markus Neugebauer, Martin Behrens, Mathias Belzner, Ralph Dominik, Sandra Strich und Sonja Simon. Ein großer und lieber Dank geht an meine Freundin Eva Josten für ihre moralische Unterstützung und herzliche Art. Meinen Eltern und meinen Schwiegereltern danke ich für ihr Vertrauen und ihre ständige Ermutigung. Meinem Mann, Uğur Ülker, danke ich herzlich für die liebevolle Unterstützung, seine Geduld und ständige Aufmunterung, auch frustrierende Tage zu überstehen.

CURRICULUM VITAE

Personal Information

Name and Surname: Burcu Ülker (Meric) Date of Birth: 17.06.1977 Place of Birth: Izmir, TURKEY Marital Status : Married

Education

1983-1988 Elementary school (Karsilkokulu –Izmir) 1988-1994 Secondary school (Suphi Koyuncuoglu Lisesi-Izmir) 1994-1998 B.Sc. in Faculty of Pharmacy, Ege University-Izmir 1998-2001 M.Sc. in Faculty of Pharmacy, Department of Analytical Chemistry, Ege University-Izmir 2001-2004 PhD in Faculty of Pharmacy, Department of Analytical Chemistry, Ege University-Izmir 2004-2005 PhD in Faculty of Physical Chemistry, University of Friedrich- Alexander Erlangen- Nürnberg and directif GmbH Career 1999- 2004 Teaching and Research Assistant in Faculty of Pharmacy, Ege University, Izmir 2000 Research collaboration for one month (19.09–19.10.2000) in “University of Florence,Department of Analytical Chemistry” under supervision of Prof. Marco Mascini. 2001-2003 Research collaboration for two years (15.09.2001-15.09.2003) in “University of Friedrich Alexander Erlangen-Nürnberg, Institute of Physik III ” under supervision of Prof. Paul Müller.

Projects Directed And Involved

Burcu Meric, “Detection of DNA sequences by using electrochemical hybridization indicators”, Ege University, Faculty of Pharmacy Project, (Project number: 2000 / ECZ / 024), 2000. Mehmet Ozsoz, Arzum Erdem, Kagan Kerman, Burcu Meric, Emrah Kilinc, Electrochemical DNA biosensors, (Project number: TBAG - 1871), Turkish Research Council Project, 2000.

Awards Arzum Erdem, Kagan Kerman, Burcu Meric, Ulus Salih Akarca, Mehmet Ozsoz. A Novel Hybridization Indicator Methylene Blue for the electochemical Detection of Short DNA Sequences Related to the Hepatitis B Virus. Project Exhibition, Ege University, EBILTEM, 25-27 October 1999, Izmir.

This project won the 3rd Best Project Award.

Arzum Erdem, Burcu Meric, Kagan Kerman, Dilsat Ozkan, Mehmet Ozsoz. Electrochemical DNA biosensor for the environmental pollutant Microcystis spp. Project Exhibition, Ege University, EBILTEM, 23-27 October 2000, Izmir.

This project won the 2nd Best Project Award.

Burcu Meric, Kagan Kerman, Dilsat Ozkan, Pinar Kara, Arzum Erdem, Ulus S. Akarca, Selda Erensoy and Mehmet Ozsoz. Electrochemical DNA biosensor for the determination of genetically and inherited diseases from PCR products. Project Exhibition, Ege University, EBILTEM, Izmir, 15-26 October 2001.

This project won the 1st Best Project Award.

PUBLICATIONS

1. Emrah Kilinc, Tayfun Dalbasti, Gunay Yetik, Kagan Kerman, Burcu Meric, Mehmet Ozsoz. Amperometric Microelectrode Design by Rhodium Deposition For Improved Nitric Oxide Measurement, Acta Pharmaceutica Turcica, 41, pp. 141, 1999.

2. Arzum Erdem, Kagan Kerman, Burcu Meric, Ulus Salih Akarca, Mehmet Ozsoz. DNA Electrochemical Biosensor For The Detection of Short DNA Sequences Related To The Hepatitis B Virus, Electroanalysis, 8, pp. 586, 1999.

3. Arzum Erdem, Burcu Meric, Kagan Kerman, Tayfun Dalbasti, Mehmet Ozsoz. Detection of Interaction Between Metal Complex Indicator and DNA by Using Electrochemical Sensor, Electroanalysis, 11, pp. 1372, 1999.

4. Arzum Erdem, Kagan Kerman, Burcu Meric, Ulus Salih Akarca, Mehmet Ozsoz. A Novel Hybridization Indicator Methylene Blue for the electrochemical Detection of Short DNA Sequences Related to the Hepatitis B Virus, Analytica Chimica Acta, 422, pp. 139, 2000.

5. Arzum Erdem, Aysun Pabuccuoglu, Burcu Meric, Kagan Kerman, Mehmet Ozsoz. Electrochemical Biosensor Based on Horseradish Peroxidase for the Determination of Oxidizable Drugs, Turkish Journal of Medical Sciences, 30, pp. 349, 2000.

6. Arzum Erdem, Kagan Kerman, Burcu Meric, Dilsat Ozkan, Tayfun Dalbasti, Mehmet Ozsoz. Ion-selective Membrane Electrode for the Determination of A Novel Phenylpiperazine Anti-Depressant, Nefazodone, Turkish Journal of Chemistry, 24, pp. 353, 2000.

7. Arzum Erdem, Burcu Meric, Kagan Kerman, Dilsat Ozkan, Mehmet Ozsoz. Amperometric Biosensor based on Mushroom Tissue Polyphenol Oxidase for the Elimination of Metal Inhibitory Effects, Turkish Journal of Chemistry, 25, pp. 231, 2001.

8. Arzum Erdem, Burcu Meric, Kagan Kerman, Dilsat Ozkan, Pinar Kara and Mehmet Ozsoz. Electrochemical DNA Biosensor for the Determination of Benzo[A]pyrene - DNA Adducts, Analytica Chimica Acta, 450, pp. 45, 2001.

9. Arzum Erdem, Kagan Kerman, Burcu Meric, Mehmet Ozsoz. Methylene Blue as a Novel Electrochemical Hybridization Indicator, Electroanalysis, 13, pp. 219, 2001.

10. Arzum Erdem, Dilsat Ozkan, Burcu Meric, Kagan Kerman, Mehmet Ozsoz. Incorporation of EDTA for the Elimination of Metal Inhibitory Effects in an Amperometric Biosensor Based on Mushroom Tissue Polyphenol Oxidase, Turkish Journal of Chemistry., 25, pp. 231, 2001.

11. Fei Yan, Arzum Erdem, Burcu Meric, Kagan Kerman, Mehmet Ozsoz, Omowunmi A Sadik. Electrochemical DNA Biosensor for the Detection of Specific Gene Related to microcystis species, Electrochem. Commun., 3, pp. 224, 2001.

12. Burcu Meric, Kagan Kerman, Dilsat Ozkan, Pinar Kara, Mehmet Ozsoz. Indicator-free Electrochemical DNA Hybridization Biosensor Based on Guanine and Adenine Signals. Electroanalysis, 14, pp. 1245, 2002.

13. Pinar Kara, Kagan Kerman, Dilsat Ozkan, Burcu Meric, Arzum Erdem, Peter E. Nielsen, Mehmet Ozsoz. Label-Free and Label Based Electrochemical Detection of Hybridization by Using Methylene Blue and Peptide Nucleic Acid Probes at Chitosan Modified Carbon Paste Electrodes, Electroanalysis,14, pp. 1685, 2002.

14. Burcu Meric, Kagan Kerman, Dilsat Ozkan, Pinar Kara, Selda Erensoy, Ulus Salih Akarca, Marco Mascini, Mehmet Ozsoz. Electrochemical DNA Biosensor for the Detection of TT and Hepatitis B Viruses from PCR Amplified Real Samples by using Methylene Blue. Talanta, 56(5), pp. 837, 2002.

15. Arzum Erdem, Kagan Kerman, Burcu Meric, Dilsat Ozkan, Pinar Kara, Mehmet Ozsoz. DNA Biosensor for Microcystis spp. Sequence Detection by Using Methylene Blue and Ruthenium Complex as Electrochemical Hybridization Labels, Turkish Journal of Chemistry, 26, pp. 851, 2002.

16. Kagan Kerman, Dilsat Ozkan, Pinar Kara, Arzum Erdem, Burcu Meric, Peter E. Nielsen, Mehmet Ozsoz. Label-Free Bioelectronic Detection of Point Mutation by Using Peptide Nucleic Acid Probes, Electroanalysis, 15, pp. 667, 2002.

17. Burcu Meric, Nazife Kilicarslan, Kagan Kerman, Dilsat Ozkan, Umran Kurun, Nejat Aksu, Mehmet Ozsoz. Performance of Precision G Blood Glucose Analyser with a new test strip G2B on Neonatal Samples, Clinical Chemistry, 48, pp. 176, 2002.

18. Burcu Meric, Kagan Kerman, Dilsat Ozkan, Pinar Kara, Ozlem Kucukoglu, Ercin Erciyas, Mehmet Ozsoz. Electrochemical biosensor for the interaction of DNA with an alkylating agent, 4,4'-dihydroxy chalcone based on guanine and adenine signals, Journal of Pharmaceutical and Biomedical Analysis, 30, pp. 1339, 2002.

19. Pinar Kara, Dilsat Ozkan, Kagan Kerman, Burcu Meric, Erdem Arzum, Mehmet Ozsoz. DNA sensing on glassy carbon electrodes by using hemin as the electrochemical indicator, Analytical And Bioanalytical Chemistry, 373, pp. 710, 2002.

20. Kerman Kagan, Ozkan Dilsat, Kara Pinar, Meric Burcu, Gooding Justin, Ozsoz Mehmet. Voltammetric determination of DNA hybridization using methylene blue and self- assembled alkanethiol monolayer on gold electrodes, Anal. Chim. Acta, 462, pp. 39, 2002.

21. Dilsat Ozkan, Arzum Erdem, Pinar Kara, Kagan Kerman, Burcu Meric, Jorg Hassmann, Mehmet Ozsoz. Allele Spesific Genotype Detection of Mutations from Polymerase Chain Reaction Amplicons Based on Label-Free Electrochemical Genosensor, Analytical Chemistry, 74 (23), pp. 5931, 2002.

22. Dilsat Ozkan, Pinar Kara, Kagan Kerman, Burcu Meric, Arzum Erdem, Frantisek Jelen, Peter E. Nielsen, Mehmet Ozsoz. DNA and PNA sensing on mercury and carbon electrodes by usi

23.

24. ng methylene blue as an electrochemical label, Bioelectrochemistry, 58, pp. 119, 2002.

25. Dilsat Ozkan, Kagan Kerman, Burcu Meric, Pinar Kara, Hasan Demirkan, Mihai Polverejan, Thomas J. Pinnavaia, Mehmet Ozsoz. Heterostructured Fluorohectorite clay as an Electrochemical Sensor for the Detection of 2,4-Dichlorophenol and the Herbicide 2,4-D, Chemistry of Materials, 14, pp. 1555, 2002.

26. Pinar Kara, Burcu Meric, Aysin Zeytinoglu, Mehmet Ozsoz. Electrochemical DNA Biosensor for the detection and discrimination of Herpes symplex type1 and type2 viruses from PCR amplified real samples, Anal. Chim. Acta, 518 , pp. 69, 2004.

27. Burcu Meric, Kagan Kerman, Giovanna Marrazza, Ilaria Palchetti, Marco Mascini, Mehmet Ozsoz. Disposible genosensor, a new tool for the detection of NOS-terminator, a genetic element present in GMOs, Food Control, 15, pp. 621, 2004.