BOSTON UNIVERSITY

SCHOOL OF MEDICINE

Thesis

CHARACTERIZATION OF CARBON ELECTRODE SURFACES:

DEVELOPMENT OF BIOSENSORS FOR FORENSIC DNA APPLICATIONS

by

CANDACE RENEE CHURINSKY

B.S. Colorado School of Mines, 2010

Submitted in partial fulfillment of the

requirements for the degree of

Master of Sciences

2013

© Copyright by

CANDACE RENEE CHURINSKY

2013

Approved by

First Reader

Catherine Grgicak, Ph.D. Assistant Professor of Biomedical Forensic Sciences

Second Reader

Javier Giorgi, Ph.D. Associate Professor of Chemistry University of Ottawa

iv

v

CHARACTERIZATION OF CARBON ELECTRODE SURFACES:

DEVELOPMENT OF BIOSENSORS FOR FORENSIC DNA APPLICATIONS

CANDACE RENEE CHURINSKY

Boston University School of Medicine, 2013

Major Professor: Catherine Grgicak, Ph.D., Assistant Professor of Biomedical

Forensic Sciences

ABSTRACT

Quantitative polymerase chain reaction (qPCR) techniques are currently

used to quantify samples containing deoxyribonucleic acid (DNA) in forensic

analyses. This technology can provide valuable information to an analyst

regarding the amount of DNA present but lacks the ability to determine the

quality of the sample. Electrochemistry-based biosensors that utilize screen-

printed electrodes may provide a method to determine the number of DNA

molecules and the length of those molecules in a single assay. This work aimed

to create a biosensor by electrostatically loading TPOX oligonucleotides onto a

carbon screen-printed electrode for the purpose of quantifying genomic DNA.

Electrochemical signal was obtained via the indicating molecule bis-benzimide

H33258, which preferentially interacts with double-stranded DNA and would

indicate a hybridization event. Cyclic voltammetry was chosen to measure the

vi

current signal; peaks obtained using this technique can be analyzed with the

Randles-Sevčik equation, which relates current signal with concentration of the

target species.

A large amount of signal variation and background charging current was

observed when H33258 was used as the redox probe. This led to a study of the

surface characteristics of the carbon electrodes themselves (i.e. effective surface

area) by utilizing the reversible and well-characterized redox couple

hexaammine-ruthenium. The effect of electrode activation at high anodic

potentials was also studied. Though highly recommended in the literature,

activation of the carbon surface caused effective surface area and charging

current to increase. While a larger electro-active surface is often desirable, the

high background current generated when activation is used within the protocol

can mask the signal of interest. Due to the low signal-to-noise ratio and inability

to reuse the carbon electrode, it was concluded that carbon screen printed

electrodes are not optimal forensic DNA biosensors.

vii

TABLE OF CONTENTS

Title Page .............................................................................................................. i

Copyright ............................................................................................................... ii

Reader’s Approval Page ....................................................................................... iii

Acknowledgements .............................................................................................. iv

Abstract ................................................................................................................ v

Table of Contents ................................................................................................ vii

List of Tables ........................................................................................................ x

List of Figures ....................................................................................................... xi

List of Abbreviations ............................................................................................ xv

1.0 Introduction .................................................................................................... 1

1.1 Forensic DNA Analysis ........................................................................... 1

1.2 Quantitative PCR (qPCR) ....................................................................... 2

1.2.1 The Chemistry of qPCR ................................................................ 2

1.2.2 Challenges Associated with qPCR ................................................ 7

1.2.2.1 Accuracy of the Standard Curve .......................................... 7

1.2.2.2 Degraded DNA .................................................................... 8

1.3 Biosensor ................................................................................................ 9

1.3.1 Creating a Biosensor ................................................................... 10

1.3.2 Electrodes ................................................................................... 13

1.3.3 Considerations for Loading the Oligonucleotide .......................... 16

viii

1.3.4 Producing a Signal ...................................................................... 17

1.4 Electrochemistry Theory: Measuring and Interpreting a Signal ............. 18

1.4.1 Proposed Mechanisms for Detection .......................................... 20

1.4.2 Electrochemical Technique: Cyclic Voltammetry ......................... 22

1.5 Purpose ................................................................................................ 28

2.0 Materials and Methods ................................................................................ 29

2.1 Materials and Reagents ........................................................................ 29

2.2 Instrumentation and Software ............................................................... 30

2.3 Electrodes and Voltammetry Glassware ............................................... 31

2.4 Analytical Procedures for Carbon SPE ................................................. 33

2.4.1 Evaluating SPCE Performance Using [Ru(NH3)6]3+/2+ ................. 33

2.4.2 Direct Quantification of DNA in Solution ...................................... 35

2.4.3 Electrostatic Adsorption of DNA onto SPCE for the Manufacture

of a Carbon-DNA Biosensor ....................................................... 35

2.4.3.1 Protocol ............................................................................. 36

2.4.3.2 SEM/EDX Analysis ............................................................ 38

2.4.4 Monitoring Surface Kinetic Changes Using Hexaammine

ruthenium ................................................................................... 39

2.4.4.1 Monitoring Changes when Applying the Adsorption

Protocol ............................................................................. 39

2.4.4.2 Oxidizing Bis-benzimide H33258 ....................................... 41

2.4.4.3 Estimating Real Surface Area and the Effect of Activation 42

ix

2.4.4.3.1 Protocol .................................................................... 42

2.4.4.3.2 Double-Layer Capacitance Method.......................... 46

2.4.4.3.3 Tafel/Randles-Sevčik Method .................................. 47

3.0 Results and Discussion ............................................................................... 50

3.1 SPCE Performance............................................................................... 50

3.1.1 Stability of the Electrode Cards ................................................... 50

3.1.2 Reproducibility of the Electrode Cards ........................................ 53

3.1.3 Reusability of the Electrode Cards .............................................. 55

3.1.4 Effect of the Electrode Cards on Reversibility of a Known

Reversible Redox Couple ........................................................... 58

3.2 Oxidation of Guanine and Adenine ....................................................... 61

3.3 DNA-Modified SPCE ............................................................................. 63

3.4 De-convoluting the Adsorption Protocol ................................................ 70

3.5 Analysis of Real Surface Area .............................................................. 76

3.5.1 Double-Layer Capacitance .......................................................... 76

3.5.2 Exchange Current Density and Activation Coefficient ................. 78

3.5.3 Real Surface Area ....................................................................... 85

3.6 Recommendations of Carbon Electrodes for Forensic Biosensors ....... 92

4.0 Conclusion ................................................................................................... 92

5.0 Future Research .......................................................................................... 94

6.0 References .................................................................................................. 96

7.0 Curriculum Vitae ........................................................................................ 104

x

LIST OF TABLES

TABLE 1. Data from nine CV cycles of one measurement in 0.0018 M

[Ru(NH3)6]3+/2+ in 0.02 M Tris-HCl. The baselines used to calculate values

of ip were defined by the analyst. Data is presented in terms of current

densities (µA/cm2).

53

TABLE 2. Data from cycle ten of CVs from four electrode cards: peak

potential (Vf), maximum current (Im) and peak height (ip). Peak height was

calculated using baselines defined by the software and the analyst. Data

is presented in terms of current densities (µA/cm2).

54

TABLE 3. Data from cycle ten of six repetitive CV measurements from

one electrode card, taken over a three-day period: peak potential (Vf),

maximum current (Im) and peak height (ip). Peak height was calculated

using baselines defined by the software and the analyst. Data is

presented in terms of current densities (µA/cm2).

56

TABLE 4. Peak current data for three different electrode cards used in the

electrostatic adsorption protocol; each had been previously used in one

experiment with [Ru(NH3)6]3+/2+. ‘DNA1,’ ‘DNA2,’ and ‘DNA3’ data

correspond to the panels shown in Figure 17. IND = indeterminable.

65

TABLE 5. Cathodic peak current (ip) of each measurement normalized to

the initial measurement in hexaammine ruthenium. Ratio =

ip,measurement/ip,initial.

74

TABLE 6. Capacitance calculated from the current envelope in blank

buffer.

77

xi

LIST OF FIGURES

FIGURE 1. Schematic of qPCR chemistry in one cycle for TaqMan®-

based qPCR. The DNA template is melted, primers and probe anneal

(A). DNA polymerase creates the complement of the target sequence by

adding dNTPs to the primers (B, C). When DNA polymerase reaches the

probe it degrades the probe and releases the reporting functional group,

which fluoresces because it is no longer inhibited by the quenching

functional group (D, E).

5

FIGURE 2. Recognition layer of a biosensor where the gray strand

represents the short DNA oligonucleotide and the black strand represents

the complementary target of the oligonucleotide.

12

FIGURE 3. Schematic of a screen-printed patterned electrode card. The

cards are usually tens of millimeters wide and long, and less than 1 mm

thick. This example shows a carbon working and counter electrode, and

a silver reference electrode.

15

FIGURE 4. Schematic representation of a biosensor with an intercalating

indicator.

21

FIGURE 5. Schematic representation of a biosensor with an electrostatic

indicator.

22

FIGURE 6. Triangular waveform illustrating a potential sweep rate of 0.05

V/s between -0.5 V and +0.3 V.

23

FIGURE 7. An example of a CV of a reversible redox couple, 0.0018 M

[Ru(NH3)6]3+/2+ in 0.02 M Tris-HCl. Potentials are with respect to a

Ag/AgCl reference electrode. The arrows indicate the portions of the

curve that display the background current (black dashed arrow), the

region of activation control (black solid arrow), and the region where

current tails off (gray dashed arrow).

25

xii

FIGURE 8. An example illustrating the extraction of Ep, Im and ip from a CV

curve. This system is 2 mM Fe(CN)63-/4- in 10 mM H2SO4 and 1 M NaCl,

measured at 100 mV/s with a platinum electrode[48] (vs. SCE).[49]

26

FIGURE 9. Pine Instrument screen-printed carbon electrode cards. One

version has a round working electrode surface (top) and the other has a

rectangular working electrode surface (bottom).

31

FIGURE 10. Flow chart depicting experimental design for electrode cards

A – D. CV measurements in [Ru(NH3)6]3+/2+ were cycled from -0.5 V to +

0.3 V (vs. Ag/AgCl) at 0.05 V/s for 10 cycles. Adsorption occurred at +0.5

V (vs. Ag/AgCl) for 3 hrs. Hybridization occurred overnight at room

temperature. Indicator (incubation) occurred for 1 hr at room temperature.

A blank box indicates that the step was intentionally skipped.

41

FIGURE 11. Flow chart depicting experimental design for electrode cards

I – M, not activated. Open circuit potential was recorded for 1 min.

Uncompensated resistance through solution was measured prior to each

CV. CV measurements in Tris-HCl or [Ru(NH3)6]3+/2+ were cycled from -

0.5 V to + 0.3 V (vs. Ag/AgCl) at 0.05 V/s for 10 cycles.

44

FIGURE 12. Flow chart depicting experimental design for electrode cards

N – Q, activated. Cards were activated at +1.7 V (vs. Ag/AgCl) for 1 min.

Open circuit potential was recorded for 1 min. Uncompensated resistance

through solution was measured prior to each CV. CV measurements in

Tris-HCl were cycled from -0.5 V to + 0.3 V (vs. Ag/AgCl) at 0.05 V/s for

10 cycles.

45

FIGURE 13. Nine CV cycles from one measurement on one electrode

card in 0.0018 M [Ru(NH3)6]3+/2+ in 0.02 M Tris-HCl.

52

FIGURE 14. Cycle ten of CVs from four electrode cards in 0.0018 M

[Ru(NH3)6]3+/2+ in 0.02 M Tris-HCl.

54

FIGURE 15. Cycle ten of six repetitive CV measurements from one

electrode card in 0.0018 M [Ru(NH3)6]3+/2+ in 0.02 M Tris-HCl.

56

xiii

FIGURE 16. Example of the automatic baselines created by the software

program Echem AnalystTM. The solid arrow is indicating a baseline that is

inaccurate, and the dashed arrow is indicating a baseline that is properly

aligned to the charging current preceding the peak.

58

FIGURE 17. CVs from electrode cards used in the electrostatic DNA

adsorption protocol. The small dashed lines (- - -) represent the signal

from bare electrodes, the long dashed (– – –) lines represent the signal

from the single-strand modified electrodes, and the solid lines ( )

represent the signal from the double-strand modified electrodes. ‘DNA1’

data were collected using a card previously used in a [Ru(NH3)6]3+/2+

experiment that scanned from -0.5 V to +0.3 V (vs. Ag/AgCl). ‘DNA2’ data

were collected using a card that scanned down to -0.6 V and ‘DNA3’ data

were collected using a card that scanned down to -0.7 V in previous

[Ru(NH3)6]3+/2+ experiments.

64

FIGURE 18. CVs of double-strand signal from three repetitive

measurements on one electrode card (in chronological order). The arrow

shows where the peak maximum of curve 3 is masked.

66

FIGURE 19. (A) SEM image with a scale of ~ 50 μm (white bar) and (B)

EDX elemental analysis of the working electrode of Pine SPCE. The EDX

graph shows the presence of carbon (C) and chlorine (Cl).

70

FIGURE 20. Overlay of the curves obtained from each measurement on

Card A and Card B.

72

FIGURE 21. Initial measurements in hexaammine ruthenium (pre-

adsorption) of Cards A – D.

73

FIGURE 22. (A) CVs (cycle 1) of background signal and post-H33258-

incubation in blank buffer and (B) CVs (cycle 10) of hexaammine

ruthenium of each card after the experiment. Background CVs are

dashed (- - -).

75

xiv

FIGURE 23. (A) Measurements of eight different cards, four of which were

activated, run in 0.0018 M [Ru(NH3)6]3+/2+/0.02 M Tris-HCl from -0.5 V to

+0.3 V (vs. Ag/AgCl). (B) Four consecutive measurements of two cards,

one card was activated prior to each measurement. Gray curves are from

cards that were activated, black curves are from cards that were not

activated.

79

FIGURE 24. Exchange current density data obtained from the cathodic

curve of 0.0018 M [Ru(NH3)6]3+/2+. Runs 1 – 4 represent between-

electrodes data, Runs 4 – 7 represent within-electrode data. HFC = High

Field Cathodic, LFC = Low Field Cathodic, Linear Reg = Linear

Regression, Χ2 = Chi-Squared Minimization.

82

FIGURE 25. (A) Activation coefficient data obtained from the cathodic

curve of 0.0018 M [Ru(NH3)6]3+/2+. Runs 1 – 4 represent between-

electrodes data, Runs 4 – 7 represent within-electrode data. Linear Reg =

Linear Regression, Χ2 = Chi-Squared Minimization. The gray dashed line

represents the theoretical value of α. (B) Tafel plots; gray lines represent

non-activated cards, black lines represent activated cards, solid lines

( ) represent runs on new electrodes, and dashed lines (- - -)

represent repeated runs on one electrode.

84

FIGURE 26. Active surface area (SA) calculated using Equation 12, and

the measurements taken in blank buffer.

86

FIGURE 27. Active surface area (SA) calculated using Equations 10 and

13 (Tafel analysis and Randles-Sevčik equation), and the measurements

taken in 0.0018 M [Ru(NH3)6]3+/2+ in 0.02 M Tris-HCl. (A) displays the

data from cards that were used once, (B) displays the data from the

cards that were reused.

87

xv

LIST OF ABBREVIATIONS

A amperes

bp base pairs

© copyright

CCD charge coupled device

CT cycle threshold

c-TPOX complementary TPOX

CV cyclic voltammetry/voltammogram

DC direct current

DNA deoxyribonucleic acid

dNTP deoxynucleotide triphosphate

dsDNA double-stranded DNA

DTT dithiothreitol

e- electron

EDX energy dispersive X-ray spectroscopy

F farad

IPC internal PCR control

MB Methylene Blue

MΩ mega-ohm

OCV open circuit potential

PBS phosphate buffered saline

xvi

PCR polymerase chain reaction

PET polyethylene terephthalate

PF positive feedback

qPCR quantitative polymerase chain reaction

® registered trademark

R2 coefficient of determination

RFLP restriction fragment length polymorphism

RSD relative standard deviation

SCE saturated calomel electrode

SDS sodium dodecyl sulfate

SE standard error

SEM scanning electron microscopy

SPCE screen-printed carbon electrode

SPE screen-printed electrodes

ssDNA single-stranded DNA

STR short tandem repeat

TCEP tris(2-carboxyethyl)phosphine

TE Tris-EDTA (ethylene-diamine-tetra-acetic acid)

TM trade mark

V volt

VNTR variable number of tandem repeats

1

1.0 INTRODUCTION

Current forensic methods to detect DNA have low limits of detection, are

robust and allow for accurate discrimination between individuals. This forensic

DNA process also needs to be successful with small and occasionally degraded

samples. Although robust and powerful, human identification with DNA is

successful only within a specified mass range, i.e. typically 0.25 – 1 ng. As a

result, quantification of an unknown/evidence DNA extract is an important step

that provides the information necessary for successful downstream human

identification analysis.

1.1 Forensic DNA Analysis

Forensic DNA analysis became standard practice in the 1980s with a

technique developed by Jeffreys et al.[1] This technique analyzed segments of

DNA that are highly variable between individuals. Identification between humans

ultimately was achieved by digesting regions of DNA that contained segments of

variable numbers of tandem repeats (VNTRs) with restriction enzymes. The

VNTRs, also known as minisatellites, contain approximately 10 to 1000 repeating

units comprised of 10 to 100 base pairs each. When followed with a Southern

blot analysis, distinction between individuals was possible since individuals have

different numbers of VNTRs and could therefore be distinguished by the

fragment length of the DNA between the two restriction sites. Although highly

discriminating, this technology is limited by requirements of high DNA mass input

2

and quality. Further, this process is laborious and mixture interpretation is

difficult.[2][3]

Some of these issues were solved with the application of the polymerase

chain reaction (PCR) to forensic DNA analysis, a technology first described by

Mullis and Faloona.[4] This technology replicates segments of template strands of

DNA at an exponential rate when the reaction proceeds efficiently. The process

of amplifying DNA is used with variable microsatellite regions, or short tandem

repeats (STRs), which are typically comprised of approximately 5 to 40[5] repeats

of 2 to 6 base pair units.[2] Since this technique requires less time and has a

lower limit of detection than RFLP-based methods, it has become the mainstay of

forensic ‘fingerprinting’ or ‘typing.’[3] A set of loci (regions of the genome) have

been chosen as a standard for analysis of forensic samples and include:

CSF1PO, FGA, TH01, TPOX, VWA, D3S1358, D5S818, D7S820, D8S1179,

D13S317, D16S539, D18S51, and D21S11.[6]

1.2 Quantitative PCR (qPCR)

1.2.1 The Chemistry of qPCR

The PCR process involves copying template strands to amplify the

amount present to levels that can be detected via fluorescence. The reaction is

cycled through a temperature regimen that causes the template to melt into its

single strands. The temperature is then decreased to allow primer annealing and

the polymerase to polymerize the complementary strand of the target. The end

result is a doubling of the DNA strand at the targeted locations. The reaction

3

undergoes successive cycles and the process is repeated until the feed-stocks

are consumed and/or the cycling is stopped.

Quantitative PCR, also known as real-time PCR or qPCR, uses a thermal

cycler to control the temperatures and can measure fluorescence changes during

cycling.[7] The components necessary for this process include deoxynucleotide

triphosphates (dNTPs), a DNA polymerase, fluorescently-labeled probe

molecules, primers, and other chemicals such as a magnesium source, salts and

buffers for stability. During one cycle, the template DNA molecule is melted and a

primer (short DNA sequence) anneals to its complement on the strand preceding

the segment of DNA to be amplified. This occurs for both of the original template

strands so each locus has a primer for either direction. The sequences that the

primers recognize must be highly conserved within the population so that the

reaction is reproducible each time the assay is performed.

Some assays use an intercalating molecule (i.e. SYBR green) as the

fluorescent signal,[8] while others use a labeled probe molecule that anneals to its

complement on one of the strands, in between the two flanking primers (Figure 1,

A).[9] This is known as TaqMan®-based qPCR. In TaqMan® chemistry the probe

contains a reporting fluorophore and a quenching molecule. The fluorescence

from the reporting moiety is quenched by the moiety on the opposite end of the

probe and is prevented from reaching the transduction element of the system.

The element is typically a charge-coupled device (CCD) that reads the

fluorescence, which results in an amplification curve that describes the

4

accumulation of product over time. After the primers and probes have annealed,

the DNA polymerase adds free dNTPs to the primers such that a new

complementary DNA strand is polymerized (Figure 1, B and C). When the

polymerase reaches the probe, its exonuclease activity allows it to cleave the

reporting moiety as it degrades the probe. Since the reporter is no longer near

the quenching molecule the fluorescence is now detectable, where detection of

one fluorophore represents the polymerization of one new double stranded DNA

target (Figure 1, D and E).[8][9] The accumulating fluorescence is recorded after

each PCR cycle and is used to determine the starting quantity of DNA[7][8] as per

the following equation:

Cn C0 (1 + EPCR)n (Eqn. 1)

where n is the cycle number, Cn is the concentration (measured via fluorescence)

of the sample at cycle n, C0 is the initial concentration of the sample, and EPCR is

the PCR efficiency.[9]

5

FIGURE 1. Schematic of qPCR chemistry in one cycle for TaqMan®-based qPCR. The DNA

template is melted, primers and probe anneal (A). DNA polymerase creates the complement of

the target sequence by adding dNTPs to the primers (B, C). When DNA polymerase reaches the

probe it degrades the probe and releases the reporting functional group, which fluoresces

because it is no longer inhibited by the quenching functional group (D, E).

6

When the reaction first starts there is an excess of reactants and very few

copies of the DNA sequence to be amplified, so the reaction proceeds with high

efficiency. The growth of the number of amplicons (the specific sequences that

are amplified) occurs at an exponential rate and the fluorescence level starts to

increase.[7][8] The efficiency of the reaction declines as the reaction proceeds and

the feed-stocks are consumed, and amplification enters a linear rate of growth.

Eventually the rate plateaus and the reaction no longer produces additional

amplicons.[8]

A threshold level for the fluorescence is set for the assay and is the point

where the quantity of initial DNA is determined. This level must be high enough

so as not to be confused with background noise but low enough to occur during

the exponential phase of the amplification. The fluorescence from each sample

crosses the threshold value in the exponential phase at a particular cycle (n,

commonly symbolized as CT) which, when compared back to a standard curve

developed from samples of known quantity, can be used to determine the initial

amount of DNA. A lower value of n for a particular sample would indicate a larger

starting template amount.[7]

To create the standard curve, which is a necessary tool for this analysis, a

serial dilution of a genomic DNA sample that spans several orders of magnitude

is run on the instrument.[9] A relationship between cycle number and initial DNA

concentration is established. As per Equation 1, if the amplification efficiency of

the target amplicon is 100% (EPCR = 1), Equation 2 is obtained.

7

Cn C0 (2)n (Eqn. 2)

By taking the logarithm of both sides and defining n as the cycle threshold,

a log-linear relationship between n and C0 is obtained:

n logCn

log 2 -

logC0

log 2 (Eqn. 3)

where the y-intercept is logCn

log 2 and the slope is -

1

log 2 or -3.32. According to

Equation 3, if optimal conditions are met and the standards contain accurate

DNA concentrations, the y-intercept and slope derived from the standards should

exhibit insignificant differences between runs. This standard equation is then

applied to unknown samples and the starting concentration is determined.[7-10]

The quantification step is important and necessary for processing forensic

case samples. The downstream STR amplification assay is sensitive to input

DNA quantity and failure to adhere to an optimum range can cause problems

when analyzing profiles. Typically, the optimal targets are in the vicinity of 0.25 –

1 ng. If less DNA is amplified, stochastic effects in the profile due to imbalanced

allele amplification may result. If too much DNA is amplified, artifacts such as

bleed through and stutter may be exacerbated due to higher levels of

fluorescence.[11]

1.2.2 Challenges Associated with qPCR

1.2.2.1 Accuracy of the Standard Curve

Accurately quantifying unknown samples rests heavily on the accurate

creation of the standard curve. For forensic purposes the serial dilution often

8

extends down to very low concentrations. For example, the manufacturer’s

instructions for the Quantifiler® Duo DNA Quantification Kit suggest creating a

serial dilution from 50 ng/µL to 0.023 ng/µL. The lower end represents only a few

cell’s worth of DNA.[9] When working with this level of DNA, accuracy in the

amount that is pipetted into the reaction well becomes important and can affect

the parameters obtained from the standard curve.

Challenges associated with accurately producing the standard series are

well-documented. For example, the manufacturer of the Quantifiler® Duo

quantification kit explicitly states that discrepancies in the dilution series and the

quality of pipettes can greatly affect the accuracy of the assay.[9] Grgicak et al.

found that significant variability was introduced due to errors in pipetting and

suggested that standard curves be generated based on data from one set of

serial dilutions.[10] This was supported by Smith and Osborn who showed that

significant errors in initial quantification of the standard caused large sample-to-

sample variation.[8] Both groups point out that the error is propagated through the

analysis due to the logarithmic relationship; a small error at the beginning of the

process can compound to large errors in final quantification.

1.2.2.2 Degraded DNA

In order for amplification to occur, the sequence to which primers and

probes anneal and the sequence to be amplified must be present in one

continuous strand. Therefore, degraded DNA poses a problem, as the strands

may be broken in these critical areas and the reaction will not be able to proceed.

9

Quantitative PCR will typically target one sequence of a specified length, and if

the sample is severely degraded and very few intact strands remain, the

calculated concentration may not be representative of the true amount present.

In extreme cases, amplification may fail altogether and the sample will appear to

contain no DNA.[7]

Typical qPCR cannot determine quality of input DNA, which is important

information for the analyst to determine appropriate downstream processing (i.e.

miniSTR versus traditional STR processing). Variation of the qPCR techniques

which amplify multiple DNA lengths have been proposed, but developing this

multiplex is cost-prohibitive for most forensic DNA laboratories and only gives

levels of relative degradation.[12] Since genotyping success is highly dependent

on characterizing the amount and quality (i.e. length) of the DNA, developing a

quantitative technique that can do both efficiently and accurately can have

significant implications to criminal justice policy and practice.

1.3 Biosensor

Recent designs for quantifying DNA using modified electrodes have been

proposed in the literature and have recently garnered much attention. Solid

electrodes become biosensors when they are modified with a biochemical

molecule that targets an analyte in a sample. The interaction between the

biological components at the electrode surface produces a signal that is

correlated to properties of the sample. The biosensor design provides a sensitive

analytical technique that can be applied to many fields of study involving clinical

10

and research-based molecular diagnostics. The application of the technology to

forensic DNA analysis as an alternative to the current technique of qPCR is

described here. Additionally, biosensors may be used to determine the extent of

DNA degradation by analyzing strand length. As previously stated, typical

forensic qPCR techniques lack the ability to detect DNA degradation, making

biosensors a desirable alternative.

1.3.1 Creating a Biosensor

A biosensor is typically created on a solid electrode surface by chemically

or electrostatically attaching biochemical molecules such as proteins and nucleic

acids. If the surface is chemically modified, the molecules bind in a monolayer

such that the biochemical molecules cover the electrode surface in a layer that is

one molecule thick. This layer is termed the ‘recognition layer’ because the

molecules are specific to a target in the liquid sample undergoing analysis. When

the electrode is placed into a sample the analyte and recognition layer interact,

and an electrical signal is obtained that characterizes the analyte.[13]

When creating a forensic DNA biosensor the recognition layer is formed

using human sequence-specific oligonucleotides that target a particular region of

genomic DNA. Oligonucleotides are short DNA molecules, usually 18 to 40

bases long,[14] and are complementary to a sequence on the target molecule. In

qPCR the oligonucleotides, also known as primers, provide a scaffold on which

the DNA polymerase can synthesize the complement of the template, and anneal

directly preceding the sequence to be copied.[9] The primers on the biosensor

11

serve the same purpose; to specifically target particular DNA sequences. The

notable difference is that amplification does not occur. The oligonucleotide is

engineered to recognize a sequence that is highly conserved so that the assay

can be reproduced with high fidelity between individuals. The target of the

oligonucleotide sequence must also occur only once in a single copy of genomic

DNA so that absolute quantity of the molecules present can be determined per

sample. A series of oligonucleotides that fit these qualifications are already in use

by PCR applications in forensic DNA analysis and are ideal candidates for the

recognition layer of a biosensor. This work uses primers that target the

conserved region of the TPOX locus,[15] which is one of the 13 core loci used in

forensic DNA analysis.

Figure 2 illustrates the recognition layer of a theoretical forensic biosensor.

The small DNA oligonucleotide is directly attached to the electrode surface.

When a targeted DNA molecule is present in the sample being analyzed the

natural hybridization affinity of complementary single-stranded DNA molecules

causes the target to hybridize to the short DNA strand attached to the electrode

surface. Once hybridization has occurred the sample can be measured to

determine how much DNA is present. Notice that one DNA molecule binds per

one recognition layer molecule.[13]

12

FIGURE 2. Recognition layer of a biosensor where the gray strand represents the short DNA

oligonucleotide and the black strand represents the complementary target of the oligonucleotide.

The efficiency of this assay will be affected by two factors. First, the DNA

strands must be denatured so that single strands can interact with the probe

molecules on the biosensor. The quantification of the sample will not be accurate

if the DNA strands re-anneal to their original complement before measurement.

Selective melting and annealing can be controlled by altering the temperature of

the sample.[16][17] Formamide and urea will also denature DNA strands, however

these chemicals may then interfere with the annealing step to the probe.[18][19]

Second, the dynamic range of concentration that the biosensor can detect will be

determined by the maximum loading capacity of probe molecules onto the

electrode surface. The maximum loading capacity will be limited by the maximum

surface area of the electrode,[20] the fractional surface coverage of electro-active

13

sites with which the probe specifically interacts,[21-23] and steric and electrostatic

hindrance between closely loaded probe and DNA molecules.[24]

1.3.2 Electrodes

Early work involved detection of nucleic acids on mercury electrodes, but

the toxicity of mercury has motivated researchers to seek alternative electrode

types.[25] Carbon and gold are common materials used for solid electrodes in

more recent DNA applications, however classic forms like disk electrodes are

bulky and expensive. Carbon is often used because it is cheaper than gold,

relatively inert, and has a wide potential window in which it can operate. It comes

in various forms including glassy carbon, carbon paste, carbon fiber

microelectrodes, and thick-film screen-printed patterned electrodes.[14][26]

Patterned electrodes have also garnered attention because they offer

advantages such as lower cost, disposability, and miniaturization.[14] They are

commercially produced in carbon, gold and platinum forms,[20][27] whereby the

metal is deposited as a viscous ink onto a plastic or ceramic card. The electrical

contacts that output to the instrument, the leads that connect the contacts with

the electrodes, and the electrodes are all printed in this fashion. This printed

layer is then covered by a non-conducting layer that defines the exposed

electrode areas. If the electrode is carbon, the ink is generally composed of

graphite particles suspended in a matrix containing a vinyl or epoxy binder and a

solvent. The binder aids in adhesion of the ink to the card and the solvent

maintains the desired viscosity of the ink. When printed, spaces between the

14

graphite particles will partially fill in with binder, but the surface will still appear

rough.[28-30] Some companies that produce SPE include Kanichi Research

Services Lt. (UK), DropSens Ltd. (Spain), Zensor Ltd. (Taiwan),[28] Alderon

Biosciences (NC, USA),[30] and Pine Instrument Company (PA, USA).[31]

Typically, commercially available patterned electrodes are a three-

electrode system laid out in a planar array. The three-electrode system consists

of a working electrode, a counter electrode, and a reference electrode. The

counter electrode is larger than the working electrode, and the reference

electrode is situated between the working and counter electrodes (Figure 3). In a

potentiostatic experiment, where potential is controlled and current is measured,

the potential is applied to the working electrode with respect to the reference

electrode. Any current generated as a result of the applied potential will pass

between the working and counter electrodes. Since no current passes through

the reference electrode, its potential remains constant at the value specified by

the analyst. This is a theoretical model and in reality there is a drop in potential

between the working and reference electrodes, since they cannot be placed

infinitely close to each other.[32] Commercial instruments offer utilities that can

measure and largely correct for this (called iRu/IR compensation).[33]

15

FIGURE 3. Schematic of a screen-printed patterned electrode card. The cards are usually tens of

millimeters wide and long, and less than 1 mm thick. This example shows a carbon working and

counter electrode, and a silver reference electrode.

The reference electrode on patterned cells is typically silver/silver chloride

and is made of printed silver ink.[29] Known as an indicator electrode, it is

stabilized by the presence of chloride ions in solution and samples must contain

a chloride ion source for the applied potential of the system to be reliable.[32]

While the size, cost, and design are attractive, it should be noted that

screen-printed electrodes cannot be mechanically cleaned to produce highly

uniform and flat surfaces like their more expensive solid counterparts.[30]

Cleaning procedures instead involve electrochemical pretreatment by cycling the

electrode from large positive to negative potentials multiple times,[34] in buffer for

carbon SPE[21] and in sulfuric acid for gold or platinum SPE.[27] As a result, the

recognition layer created upon these surfaces may be less ordered and can

affect accessibility of the probe to the target.[14]

16

1.3.3 Considerations for Loading the Oligonucleotide

The oligonucleotide can be loaded onto the electrode surface via several

immobilization techniques. The technique will determine the orientation of the

oligonucleotides, the accessibility to target DNA, and whether the recognition

layer is a monolayer.

To electrostatically adsorb DNA, a positive potential is applied to the

electrode in a solution containing the oligonucleotide. This technique is typically

used with mercury and carbon electrodes. The probe can be loaded in two

orientations, either with the phosphate backbone or with the bases directly

adjacent to the electrode surface. Hydrophobic interactions with the solvent may

cause the bases to load adjacent to the surface, which would prevent

hybridization and decrease efficiency of the assay. The purpose of applying a

potential is to promote loading the negative phosphate backbone of the DNA

onto the positive electrode surface, leaving the bases free to hybridize with the

target sequence.

Chemisorption is a technique most often used with gold electrodes but can

also be used with other electrodes like silver, platinum and mercury.

Immobilization occurs through the formation of a self-assembled monolayer in

which thiolated probes containing a sulfur atom at one end covalently attach to

the gold atoms of the electrode surface upon contact. This process orients the

probe perpendicular to the electrode surface, which when used in conjunction

with spacing alkanethiol molecules, will allow for a high level of accessibility

17

between the probe and target. However, the procedure for creating a biosensor

using this technique is more complicated than that used for electrostatic

adsorption.

Other methods can include affinity binding between a biotinylated probe

and an avidin-modified electrode surface, trapping in polymer films or gels, and

covalent binding with carbodiimide bonds.[35]

1.3.4 Producing a Signal

Many sources of signal to produce the final quantification signal for

biosensors have been proposed. For example, guanine residues can be

irreversibly oxidized to produce an electrochemical signal that is proportional to

their concentration; this is the most direct method to measure the target because

it does not require the addition of an indicating molecule or redox probe.[14][26][35-

38] Other sources include indicating molecules that are either added to the

sample or are attached to the probe. Such indicating molecules must have an

affinity for either double-stranded (dsDNA) or single-stranded DNA (ssDNA) to

allow for quantitation of the target. These molecules can be separated into

different groups based on the way they interact with nucleic acids.

Intercalating compounds are a popular choice because they insert in

between the bases of a dsDNA molecule. Since ssDNA molecules do not provide

the appropriate structural environment for an intercalating molecule, the affinity is

specific to duplexed nucleic acids and the intercalator indicates a hybridization

reaction.[35] Examples include acridine orange[38] and daunomycin.[14][35][39][40]

18

Groove binders also exhibit a greater affinity for duplex DNA molecules because

the groove binder interacts with the double-helical structure. Examples include

cationic metal complexes ([Co(phen)3]3+, [Co(bpy)3]

3+)[14][38] and bis-benzimide

H33258.[14][35][41] Bis-benzimide H33258 has also been reported as an intercalator

in the literature.[13] For these types of indicators the signal observed will be

greater in the presence of hybridized DNA because the molecules will

concentrate near the electrode surface with the DNA. In contrast, Methylene Blue

(MB) dye preferentially interacts with guanine bases of ssDNA[14][35] and the

signal decreases in the presence of hybridized DNA.

Another option is electrostatic indicators like anionic hexacyanoferrate

([Fe(CN)6]3-/4-) and cationic hexaammine ruthenium ([Ru(NH3)6]

3+/2+), which will

interact differently with dsDNA and ssDNA due to charge density differences

from the negative sugar-phosphate backbone. Finally, the probes can be labeled

directly with molecules like ferrocene[35] or MB during oligonucleotide synthesis.

When hybridized, the probe-target duplex would exhibit restricted movement,

thereby preventing the label from interacting with the electrode surface and

causing a decreased signal.[42]

1.4 Electrochemistry Theory: Measuring and Interpreting a Signal

The field of electrochemistry provides a battery of sensitive analytical

techniques with which to process a sample. It combines an oxidation-reduction

chemical reaction that occurs in a liquid phase with an electrical circuit that is

completed by solid conductive materials. Depending on the type of analysis,

19

either potential or current may be controlled while changes in the other are

measured. In this way the kinetics of the reaction occurring in solution are

described by the signal produced from electron transfer through the system.[32]

The appeal of potentiostatic techniques is that the thermodynamics of the

system can be controlled by adjusting the potential. For a general

electrochemical reaction (Eqn. 4), this relationship is described by the Nernst

equation (Eqn. 5):

xidant + ne- Reductant (Eqn. 4)

E E - RT

nFln (Eqn. 5)

where E is the potential difference between the electrodes (V), E° is the standard

reduction potential (V), R is the universal gas constant (R 8.314 J/mol∙K), T is

the temperature (K), n is the number of electrons transferred in the half reaction,

F is Faraday’s constant (F 96,484.6 C/mol), and is the reaction quotient,

which is the ratio of activities of the species.[43]

An advantage to electrochemical assays is that the techniques are highly

sensitive to very low concentrations. Wang et al. estimated detection limits as low

as 25 pg/μL ssDNA and 30 pg/μL dsDNA on thick-film carbon sensors,[37] and

120 pg/μL dsDNA on carbon paste electrodes using potentiometric stripping

analysis.[38] Pedano and Rivas estimated a detection limit of 126 pg/μL ssDNA

and 219 pg/μL dsDNA on a glassy carbon electrode using potentiometric

stripping analysis.[26] To offer some perspective, the lowest concentration point

used to generate the standard curve in typical forensic qPCR assays is 23

20

pg/μL.[9] The sensitivities obtained with electrochemical techniques as reported in

the literature are comparable to those obtained with the fluorescence-based

qPCR assay, and may result in less error propagation, as amplification efficiency

variation is a known source of signal variability in PCR-based methods.[44]

1.4.1 Proposed Mechanisms for Detection

The primary goal for the design of a forensically-relevant biosensor is that

quantity and quality of the sample DNA can be determined in one assay, in a

matter of minutes, and with a high level of accuracy. The following

electrochemical mechanisms are proposed to accomplish this goal.

First, absolute DNA quantity can be assessed by using an

intercalating/groove-binding compound as the redox indicator (i.e. H33258).

Since these compounds have a higher affinity for dsDNA over ssDNA, the

resulting current signal should be proportional to the number of target DNA

molecules that hybridize with the oligonucleotides on the surface of the

biosensor. Figure 4 displays the schematic for an intercalating indicator (not to

scale). The amount of intercalating molecules that collect near the electrode

surface will be independent of the length of target DNA strands and dependent

on the number of hybridized strands.

21

FIGURE 4. Schematic representation of a biosensor with an intercalating indicator.

In contrast, quality of the sample can be determined with the use of

electrostatic redox-active molecules (for example, [Ru(NH3)6]3+/2+). Degraded

DNA will contain strands that are shorter in length compared to pristine genomic

DNA samples. Electrostatic molecules are sensitive to changes in charge and

can thus be used to differentiate strand lengths. A relationship between the

current developed from electrostatic indicators and strand length can provide

valuable information on the degree of degradation of a sample. Figure 5

illustrates this concept. If the two indicating molecules are chosen carefully, this

assay could provide separate current signals for each property (multiplex).

22

FIGURE 5. Schematic representation of a biosensor with an electrostatic indicator.

1.4.2 Electrochemical Technique: Cyclic Voltammetry

Cyclic voltammetry (CV) is a direct current (DC) potentiostatic technique

where potential is swept between two limits at a constant rate and current is

monitored. The limits are chosen such that they flank the potential at which the

species is expected to oxidize/reduce. The relationship between potential and

time is described by a triangular waveform, as shown in Figure 6.[45]

23

FIGURE 6. Triangular waveform illustrating a potential sweep rate of 0.05 V/s between -0.5 V and

+0.3 V.

Figure 7 displays an example of a cyclic voltammogram, and was

measured at the rate described by the triangular waveform in Figure 6 (ν = 0.05

V/s). The analyte providing the current response is the reversible redox couple

hexaammine ruthenium, [Ru(NH3)6]3+/2+, at a concentration of 0.0018 M in 0.02 M

Tris-HCl buffer. The voltammogram contains nine cycles that overlay to a large

degree. A ‘steady-state’ voltammogram, which traces nearly the same path with

each cycle, is often obtained when using a reversible redox couple. The curve

observed as potential increases toward more positive potentials represents the

oxidation of the analyte and is described by the following chemical equation.

[Ru(N 3)6]2+ [Ru(N 3)6]

3+ + e- (Eqn. 6)

24

This is the anodic sweep of the reaction. The curve observed as potential

decreases toward more negative potentials (cathodic sweep) represents the

reduction of the analyte and is the reverse reaction of Eqn. 6.[46] These data are

often presented in terms of current density, in which absolute current values are

normalized to the surface area of the electrode.

The voltammogram in Figure 7 displays a few aspects of the cell that are

important for analysis. The solvent of a sample will usually create a background

current that is comparable to what would be considered noise in other assays.

This current, known as charging current,[47] is apparent in Figure 7 and is

indicated by the black dashed arrow. When the peaks are quantitatively

analyzed, the background current contribution is typically subtracted out. The

scan range limits are sufficiently negative and positive so that the oxidation and

reduction peaks are fully developed. The portion of the curve indicated by the

black solid arrow displays the region of ‘activation control’ for that sweep.

Activation potentials are the range of potentials at which the reactants are

oxidizing or reducing without influences associated with diffusion or transport.

Within this range, there is a steep increase in current due to the conversion of the

reactants at the electrode surface. This current is called faradaic current and is

the result of direct electron transfer to or from the analyte. Eventually the reaction

is limited by the diffusion of reactants toward the surface or products away from

the surface, so the current reaches a maximum and then tails off (dashed gray

25

arrow).[32][45] Though these points are indicated on the cathodic curve in Figure 7,

they occur for each sweep direction in reversible systems.

FIGURE 7. An example of a CV of a reversible redox couple, 0.0018 M [Ru(NH3)6]3+/2+

in 0.02 M

Tris-HCl. Potentials are with respect to a Ag/AgCl reference electrode. The arrows indicate the

portions of the curve that display the background current (black dashed arrow), the region of

activation control (black solid arrow), and the region where current tails off (gray dashed arrow).

The axes shown in Figure 7 represent one of the conventions used for

displaying CVs. Another convention involves reversal of the axes such that

cathodic current is in the positive direction, anodic current is in the negative

direction, and the x-axis displays more negative potentials to the right. As a result

it is important to specify whether the current is from oxidation or reduction of the

analyte, as opposed to relying on sign conventions alone. Figure 8 illustrates how

the parameters of interest are extracted from a cyclic voltammogram. A baseline

is extrapolated from the region preceding the peak for each curve. The peak

26

potential (Ep) and maximum current (Im) are found at the maximum of each curve.

The peak height (ip) is measured vertically from Im down to the baseline. Potential

is measured in volts (V). Maximum and peak current values are measured in

amperes (A) and may be displayed in terms of current density (A/cm2).[46]

FIGURE 8. An example illustrating the extraction of Ep, Im and ip from a CV curve. This system is 2

mM Fe(CN)63-/4-

in 10 mM H2SO4 and 1 M NaCl, measured at 100 mV/s with a platinum

electrode[48]

(vs. SCE).[49]

Cyclic voltammetry is useful for quantitative experiments because the

current signal is directly proportional to the concentration of analyte being

measured, following the Randles-Sevčik equation for a reversible reaction:

ip 0.4463 nFAC (nFνD

RT)1 2⁄

(Eqn. 7)

where n is the number of electrons transferred in the half reaction, A is the

electrode surface area (cm2), C is the bulk concentration of the analyte

(mol/cm3), ν is the scan rate (V/s), and D is the diffusion coefficient of the analyte

27

in solution (cm2/s).[46] The peak current (ip, in amperes) is measured relative to a

baseline extrapolated from the background signal so as not to include charging

current in the analysis.[32][45] Real systems with reversible redox couples will

rarely exhibit true reversibility, especially if electrodes with rough surfaces are

used. In these cases the quasi-reversible Randles-Sevčik equation offers a better

approximation of the system:

ip (2.99x105) n(αn)1 2⁄ AC(D)1 2⁄ (ν)1 2⁄ (Eqn. 8)

and incorporates the activation coefficient (α) into the calculation.[50] The

activation coefficient, also known as the transfer coefficient, is an experimental

parameter that is related to the activation energy barrier of a system and is a

function of the relationship between potential and current. For many systems a

value of α 0.5 is a reasonable and commonly-used approximation, but if a

kinetic analysis is performed, an experimental value for α can be incorporated

into the quantification scheme.[32]

A kinetic relationship between overpotential and current density is

described by the Butler-Volmer equation and includes both the oxidation and

reduction half reactions:

i i0 [eαaF

RT - e- αcF

RT ] (Eqn. 9)

where i is current density (A/cm2), i0 is exchange current density (A/cm2), α is the

activation coefficient, and is overpotential (V). Overpotential is given as:

Ecell - E (Eqn. 10)

28

where E° is the equilibrium potential (V) for the system under study. Therefore,

is a measure of the extent of deviation from equilibrium.[32][51] It should be noted

that Equation 9 applies only to the region of the curve where the system is

activation controlled.[51] At large values of overpotential within this region, one of

the terms will become negligible and the equation will simplify to the Tafel

equation. Experimental data from one sweep direction can then be used to solve

for the exchange current density (i0) and the activation coefficient (α). This

analysis is appropriately named high-field analysis and is generally used at

overpotentials greater than 52 mV for reactions proceeding at room

temperature.[32][51]

If Equations 7 or 8 are used to determine concentration, then the

biosensor method would require the development of a standard curve, much like

that in qPCR. A standard curve would define the relationship between

concentration of analyte and current signal for application to samples of unknown

DNA quantity and quality. However, stochastic variation is expected to have less

impact than in thermal cycling-based methods, and the propagation of signal

error is not expected to occur to the same extent.

1.5 Purpose

Given the aforementioned issues associated with PCR-based methods, it

would be of interest to create a biosensor that will offer a robust technique for

quantifying DNA in forensic samples and to introduce a duplexed assay that can

simultaneously qualify DNA to determine the extent of degradation. Therefore,

29

the purpose of the research described herein is to examine the ability to load a

screen-printed carbon patterned electrode with single-stranded oligonucleotides

that will recognize human genomic DNA and transduce a signal that is directly

correlated with the number of DNA copies present in the sample. Carbon SPE

were chosen over gold SPE for the present work because they are cost-effective

and the loading protocol is simpler. Specifically, carbon SPEs were tested for

their ability to be reused over multiple runs. Reproducibility and repeatability were

assessed by measuring changes in current densities and evaluating any changes

in effective electrode surface area. Additionally, a detailed examination into the

sources of error via the processing scheme was performed in an effort to isolate

the sources of variability.

Electrochemical techniques are appealing alternatives to fluorescence-

based detection assays because they are comparable in sensitivity,[52] are as

specific as qPCR and do not require costly fluorescence detection systems.[13]

Recent research has shown promising applications for biosensors in many fields

including clinical diagnostics, DNA analysis and sequencing, food and

environmental testing.[37][53] This research seeks to extend the application to

forensic DNA analysis.

2.0 MATERIALS AND METHODS

2.1 Materials and Reagents

Buffers were prepared with materials purchased from Sigma Aldrich (St.

Louis, M ) and deionized water of 18.2 MΩ∙cm resistivity from a Millipore

30

Synergy® Synergy UV Water Purification System (Billerica, MA). Hexaammine

ruthenium (III) chloride and Bis-benzimide H33258 were also purchased from

Sigma Aldrich. Hexaammine ruthenium (III) chloride was prepared in 0.02 M Tris-

HCl buffer to a final concentration of 0.0018 M. Bis-benzimide H33258 was

prepared in 0.045 M phosphate buffered saline (PBS) and 0.009 M sodium

chloride to a final concentration of 100 µM.

Two synthetic TPOX locus oligonucleotides were purchased from

Invitrogen (Grand Island, NY), and the sequences are shown below. Both

samples were received in the lyophilized form and were reconstituted in TE

buffer.

TPOX: 5’-CGGGAAGGGAACAGGAGTAAG-3’

c-TPOX: 5’-CTTACTCCTGTTCCCTTCCCG-3’

2.2 Instrumentation and Software

Voltammetric measurements were performed with a Series GTM 750

Potentiostat/Galvanostat/ZRA instrument in conjunction with the FrameworkTM,

version 5.60, and Echem AnalystTM, version 5.60, software (Gamry Instruments,

Warminster, PA). The Framework software provides the interface with the

potentiostat for data acquisition and the Echem Analyst software analyzes data

with techniques that are specific to electrochemical measurements. Numerical

data were exported from Echem AnalystTM into Microsoft Excel® 2010 for

additional analysis.

31

2.3 Electrodes and Voltammetry Glassware

Screen-printed carbon electrode (SPCE) cards were purchased from Pine

Instrument Company (Grove City, PA). Each card consists of a carbon working

electrode, carbon counter electrode, and silver reference electrode printed in a

planar array on a polyethylene terephthalate (PET) polymer card (Figure 8).[54]

The cards measure approximately 61 mm long, 15 mm wide and 0.36 mm

thick.[20] A layer of blue insulating material covers the electrical leads that connect

the exposed electrode surfaces to the grip mount. The composition of the blue

insulating material is proprietary to the manufacturer, however it is hydrophobic in

nature and is designed for use in aqueous solvents.[54] The grip mount is a device

that has an edge card connector to hold the electrode card, and mini-B USB

connectors that communicate via cables with the potentiostat.

FIGURE 9. Pine Instrument screen-printed carbon electrode cards. One version has a round

working electrode surface (top) and the other has a rectangular working electrode surface

(bottom).

The surface area of the working electrode differs between the two

electrode cards shown in Figure 9, however this does not affect data analysis

32

and comparison because current (measured in amperes, A) is normalized to the

surface area and reported as current density in amperes per square centimeter

(A/cm2).

Two types of voltammetry glassware were purchased from Pine

Instrument Company. The first container is a glass vial that holds approximately

20 mL of liquid, and was used for cleaning procedures. The second container is a

glass vial that has a Teflon insert at the bottom which contains a slit-like void in

the center that holds approximately 1 mL of liquid and is designed such that

when the screen-printed card is inserted into the slit the electrolyte/sample

covers the electrodes. This setup was used during sample analysis.

Before the card was used in an experiment, the silver reference electrode

was prepared into a Ag/AgCl electrode. This was accomplished by incubating the

reference electrode in 1 µL of 6.15% sodium hypochlorite (Clorox® Ultra bleach,

The Clorox Company, Oakland, CA) for approximately five minutes. The

electrode card was then rinsed with deionized water and dried in air. This

process formed a layer of silver chloride on the surface of the silver electrode

that appeared black in color. In Figure 9, the top card illustrates the appearance

of the silver reference electrode and the bottom card illustrates the appearance

of the Ag/AgCl reference electrode.

33

2.4 Analytical Procedures for Carbon SPE

2.4.1 Evaluating SPCE Performance Using [Ru(NH3)6]3+/2+

The stability of the screen-printed carbon electrodes and silver reference

electrode was examined using CV and a reversible redox-active ion,

hexaammine ruthenium ([Ru(NH3)6]3+/2+), at a concentration of 0.0018 M in 0.02

M Tris-HCl buffer. Four different electrode cards were used; each once to

determine stability and reproducibility, and then one of the cards was used in an

additional five measurements to determine repeatability. The results were used

to indicate the level of variation 1) between ten CV cycles in one measurement

(stability), 2) between identical CV measurements performed on four different

electrode cards (reproducibility), and 3) between six identical CV measurements

performed consecutively on one electrode card (reusability).

The electrodes were pretreated by cycling the potential from -0.1 V to +1.6

V (vs. Ag/AgCl) in 0.02 M Tris-HCl buffer at a rate of 0.1 V/s for 40 cycles. This

process was applied to remove any organic compounds or other contaminants

that may be present in the carbon ink from the printing process.[21] Sample

measurements were also preceded by a ‘blank’ measurement to monitor the

buffer and cell for potential contamination, and to record the background signal of

the buffer. The ranges, rates and number of cycles were consistent between

blank and sample measurements for each experiment so that the only difference

between the two was the presence/absence of analyte. Every electrode card

34

used in this research was pretreated and tested in this way, though the buffer

differed depending on the experiment.

For the stability study, the card was first equilibrated at open circuit

potential for 60 s and then cycled in 1 mL of 0.02 M Tris-HCl buffer from -0.5 V to

+0.3 V (vs. Ag/AgCl) at a rate of 0.05 V/s for 10 cycles. Oxidation and reduction

signals of the redox couple were obtained when the electrodes were measured in

1 mL of 0.0018 M [Ru(NH3)6]3+/2+ in 0.02 M Tris-HCl using the same

specifications as the blank measurement.

Echem AnalystTM software was then used to view the CVs and determine

parameters that represent the system being studied. These parameters include

the peak potential (Vf, in mV) and maximum current (Im, in µA) of a peak, and the

height of the peak (ip, in µA). Peak height is calculated relative to a baseline that

is extrapolated from the portion of the graph that directly precedes the peak. The

three parameters listed above were compared between 1) the 10 cycles within a

CV measurement for each electrode card (stability), 2) between cycle ten of each

CV measurement of four electrode cards (reproducibility), and 3) between cycle

ten of six different CV measurements taken using one electrode card

(reusability). The average, standard deviation and percent relative standard

deviation (RSD) were calculated for the peak potentials, maximum currents and

peak currents of each experimental set.

35

2.4.2 Direct Quantification of DNA in Solution

Theoretically, the simplest method to analyze DNA using electrochemistry

is in solution and by direct electrooxidation of the bases, specifically guanine and

adenine. This protocol was tested first because it would be preferable over the

more time-consuming and difficult protocols mentioned previously.

To obtain a signal from DNA directly, the card was first pretreated in 0.1 M

PBS, rinsed with deionized water and allowed to dry. A blank measurement was

recorded in 1 mL of 0.05 M PBS and the sample measurement was recorded in 1

mL of 25 µM (~165 ng/µL) c-TPOX oligonucleotides in 0.05 M PBS. For each

measurement the card was equilibrated at open circuit for 60 s, cycled from +0.4

V to +1.4 V (vs. Ag/AgCl) at a rate of 0.05 V/s for 15 cycles, rinsed with deionized

water and allowed to dry. The oxidation peaks of DNA bases are expected at

approximately +0.75 V and +1.10 V for guanine and adenine, respectively (+0.8

V and +1.15 V vs. SCE[36]). Additionally, samples containing 37.2 ng/µL of

QuantifilerTM Human DNA standard and 59.5 ng/µL of extracted genomic DNA

were tested using the procedure described herein.

2.4.3 Electrostatic Adsorption of DNA onto SPCE for the Manufacture of a

Carbon-DNA Biosensor

The method described in this section is based on protocols published in

the literature with slight modifications.[13][55][56] These protocols typically start with

a cleaning procedure followed by activation of the working electrode surface and

immobilization of the probe molecule. Next, the target sequence hybridizes to the

36

probe, the hybrid is incubated with a molecule that serves as an indicator for the

hybridization event, and finally the signal produced by the indicating molecule is

measured. The method described herein has additional steps in between those

listed above in which the working electrode was incubated with the indicating

molecule, bis-benzimide H33258, and then measured. The purpose of these

additional measurements is to determine how H33258 interacts with the bare and

probe-modified electrode surfaces. Bis-benzimide H33258 is an

electrochemically active, DNA groove-binding or intercalating molecule that is

oxidized at approximately +0.6 V (vs. Ag/AgCl). According to theory, minimal

signal should be observed in these measurements because H33258 has a

greater affinity for dsDNA than ssDNA or a bare electrode surface.[13]

2.4.3.1 Protocol

A bare electrode was assessed first. It was pretreated in 0.1 M PBS,

rinsed with deionized water and allowed to dry. The card was placed into a vial

containing 1 mL of 0.475 M PBS and 0.5 M sodium chloride, equilibrated at open

circuit for 60 s, and cycled from +0.25 V to +0.8 V (vs. Ag/AgCl) at a rate of 0.05

V/s for five cycles to obtain a blank measurement of the bare electrodes. All

measurements were taken using these input parameters and buffer. Next, an

aliquot of 3 µL of 100 µM H33258 was placed on the working electrode surface

and incubated for 1 hr in a humidity chamber. The electrode card was rinsed with

buffer for 5 s, placed into the vial and measured. The resulting voltammograms

display the interaction of the indicating molecule with a bare SPCE.

37

The next portion of the protocol created the biosensor recognition layer.

Before the probe oligonucleotide could be immobilized, the working electrode

was activated by applying a potential of +1.7 V (vs. Ag/AgCl) for 1 min in blank

buffer. This step increases the hydrophilicity of the surface by creating oxygen

and hydroxyl functional groups,[23] removing the organic binder from the printing

process,[57] and has been shown to facilitate nucleic acid loading.[37] The

electrode card was then rinsed with deionized water, allowed to dry, and

immersed into a cell that contained 25 µM c-TPOX oligonucleotides in 0.475 M

PBS and 0.5 M sodium chloride. A potential of +0.5 V was applied to the working

electrode for 3 hr to electrostatically immobilize the probe molecules onto the

surface. The card was rinsed with blank buffer for 10 s to remove excess

oligonucleotides that had not adsorbed. The working electrode was incubated in

H33258 for 1 hr in a humidity chamber, rinsed with buffer and measured. The

resulting voltammograms display the interaction of the indicating molecule with a

probe-modified SPCE (single-stranded).

The final portion of the protocol involved the formation of double-stranded

DNA on the electrode surface. The working electrode was incubated in a 4 µL

aliquot of 20 µM TPOX oligonucleotides in 0.475 M PBS and 0.5 M sodium

chloride in a humidity chamber overnight (˃ 12 hr) to form a hybrid on the

working electrode surface. The card was rinsed with blank buffer for 10 s to

remove any oligonucleotides that had not hybridized, was incubated with

H33258, rinsed with buffer and measured. The resulting voltammograms display

38

the interaction of the indicating molecule with a duplex-modified SPCE (double-

stranded). Theoretically this signal should be significantly larger than that

measured on the bare and probe-modified SPCE.[13]

Three carbon SPE cards were used once in this protocol, and then the

first card was reused an additional two times in the same protocol. Prior to this,

each card was used in a measurement with hexaammine ruthenium following the

protocol described in the performance study of the Pine SPCE. The first card was

cycled in the same scan range as in the performance study, -0.5 V to +0.3 V (vs.

Ag/AgCl). The scan ranges of the second and third cards extended farther to -0.6

V and -0.7 V respectively, for the purpose of investigating the signal observed at

more negative potentials. These previous experiments were not suspected to

affect the creation of the biosensor because hexaammine ruthenium does not

foul electrode surfaces upon oxidation/reduction. The absolute values of Im and ip

were compared, as were the ratios of double-strand signal to single-strand

signal. The average, standard deviation and RSD were calculated.

2.4.3.2 SEM/EDX Analysis

Two additional electrode cards were analyzed with scanning electron

microscopy/energy dispersive X-ray spectroscopy (SEM/EDX) for the presence

of DNA. Specifically, the working electrodes were evaluated for the presence of

phosphorus from the sugar-phosphate backbone of the DNA using EDX. Both

cards were run through the protocol until the end of the adsorption step. One

card underwent adsorption in blank buffer and the other card in the 25 µM DNA

39

sample. Phosphate buffers were replaced with Tris-HCl buffers to eliminate the

presence of phosphorus from sources other than the oligonucleotides. A third

card straight from the manufacturer was also examined using SEM/EDX.

The cards were placed on the stage of a JEOL JSM-6100 SEM/EDX

instrument and imaged with a 30 mm aperture, 10 kV acceleration voltage and

15 mm working distance. For the EDX analysis the aperture was decreased to 20

mm and the acceleration voltage was increased to 20 kV.

2.4.4 Monitoring Surface Kinetic Changes Using Hexaammine Ruthenium

This portion of the study sought to determine whether the working

electrode surface changed after it was subjected to each step from the

electrostatic adsorption protocol described in Section 2.4.3. Hexaammine

ruthenium is a stable, reversible redox-active molecule that can be measured

reproducibly with low variation between experiments. It also provides an ideal CV

for analyses using the Butler-Volmer and Randles-Sevčik equations. The

[Ru(NH3)6]3+/2+ system provided a useful metric of surface changes when the

electrostatic adsorption protocol was dissected into its individual steps for

analysis, and the SPCE surface was characterized.

2.4.4.1 Monitoring Changes when Applying the Adsorption Protocol

Four electrode cards (A – D) were pretreated in 0.02 M Tris-HCl buffer,

rinsed with deionized water and dried in air. A blank measurement was recorded

in a vial containing 0.02 M Tris-HCl buffer by equilibrating at open circuit for 5

40

min and cycling from -0.5 V to +0.3 V (vs. Ag/AgCl) at a rate of 0.05 V/s for 10

cycles. Next, a baseline measurement was recorded in a sample of 0.0018 M

[Ru(NH3)6]3+/2+ in 0.02 M Tris-HCl with the same input parameters as the blank

measurement. The uncompensated resistance (Ru, in ohms) was measured prior

to each run and positive feedback (PF) iRu compensation was applied at 90% of

the measured value, as recommended by the manufacturer.[33]

Each electrode card was then subjected to a different sequence as shown

in the flow chart in Figure 10. Cards A and B underwent the adsorption,

hybridization and indicator-incubation steps with intermediate measurements in

hexaammine ruthenium. The process for Card A involved only blank buffer

whereas Card B involved samples containing DNA and H33258. Cards C and D

were restricted to only two measurements in hexaammine ruthenium in order to

minimize the number of cycles of applied potential. Card C underwent the

adsorption step in blank buffer but the hybridization step in DNA, and Card D

underwent the adsorption and hybridization steps in blank buffer but the

incubation step in H33258. Activation of the electrode surface at +1.7 V (vs.

Ag/AgCl) for 1 min preceded the adsorption steps.

The CVs of the [Ru(NH3)6]3+/2+ couple were compared to those obtained

during the initial performance studies. Larger variation than that expected from

random error would indicate that the electron transfer kinetics of the working

electrode surface had changed as a result of processing.

41

FIGURE 10. Flow chart depicting experimental design for electrode cards A – D. CV

measurements in [Ru(NH3)6]3+/2+

were cycled from -0.5 V to + 0.3 V (vs. Ag/AgCl) at 0.05 V/s for

10 cycles. Adsorption occurred at +0.5 V (vs. Ag/AgCl) for 3 hrs. Hybridization occurred overnight

at room temperature. Indicator (incubation) occurred for 1 hr at room temperature. A blank box

indicates that the step was intentionally skipped.

2.4.4.2 Oxidizing Bis-benzimide H33258

The final step of the electrostatic adsorption protocol involves incubating

the working electrode in an aliquot of the indicating molecule H33258. The

previous experiment considered only the effect of the incubation but not the

effect of oxidation of H33258 on the electrode surface. To determine if oxidation

of the indicating molecule fouled the electrode surface and affected the electron

A

CV [Ru(NH3)6]

3+/2+

Adsorb Blank

CV [Ru(NH3)6]

3+/2+

Hybridize Blank

CV [Ru(NH3)6]

3+/2+

Indicator Blank

CV [Ru(NH3)6]

3+/2+

B

CV [Ru(NH3)6]

3+/2+

Adsorb DNA

CV [Ru(NH3)6]

3+/2+

Hybridize DNA

CV [Ru(NH3)6]

3+/2+

Indicator H33258

CV [Ru(NH3)6]

3+/2+

C

CV [Ru(NH3)6]

3+/2+

Adsorb Blank

Hybridize DNA

CV [Ru(NH3)6]

3+/2+

D

CV [Ru(NH3)6]

3+/2+

Adsorb Blank

Hybridize Blank

Indicator H33258

CV [Ru(NH3)6]

3+/2+

42

transfer kinetics of the working electrode, the last experiment was repeated in the

same format on Cards E – H with a few changes. The measurements in

hexaammine ruthenium were replaced with measurements in 0.475 M PBS and

0.5 M sodium chloride and the blank measurement at the beginning was

removed. The cards were equilibrated at open circuit for 5 min and cycled from

+0.25 V to +0.8 V (vs. Ag/AgCl) at a rate of 0.05 V/s for 10 cycles. At the end,

each card was measured in hexaammine ruthenium from -0.5 V to +0.3 V (vs.

Ag/AgCl). The cyclic voltammograms of the [Ru(NH3)6]3+/2+ couple were

compared to those obtained during the initial stability studies.

2.4.4.3 Estimating Real Surface Area and the Effect of Activation

Nominal surface area of the working electrode is known when purchased

from the manufacturer. On a nanoscale level, however, the surface is not pristine

or atomically flat. Little is known about how much of the surface area is

electrochemically active, and this can be confounded by the presence of other

chemicals such as the binder that is used in the printing process for SPE.[21]

Pretreatment and activation can make the surface more amenable to electron

transfer by increasing the amount of active sites.[21][55] This next study estimated

the electroactive surface area of the carbon working electrode and the effect of

activation on the surface.

2.4.4.3.1 Protocol. This study was designed to collect data sets consisting of at

least four data points for six specific categories: new electrodes (as from the

43

manufacturer), reused electrodes, activated electrodes, non-activated electrodes,

electrodes measured in blank buffer, and electrodes measured in hexaammine

ruthenium. A total of thirteen electrode cards were used for this experiment. Each

card was first pretreated in 0.02 M Tris-HCl buffer. The card may then have been

activated in 0.05 M PBS at +1.7 V (vs. Ag/AgCl) for 1 min depending on which

category it was in. Next, it was placed in a vial containing the sample of interest,

either 0.02 M Tris-HCl blank buffer or 0.0018 M [Ru(NH3)6]3+/2+. A measurement

of the open circuit potential (OCV) was recorded for 1 min. The uncompensated

resistance through solution was measured and 90% of this value was applied as

PF iRu compensation. The electrodes were equilibrated at open circuit for 60 s

and a measurement was taken from -0.5 V to +0.3 V (vs. Ag/AgCl) at a rate of

0.05 V/s for 10 cycles.

The flow chart in Figure 11 describes the procedure for the non-activated

group of electrode cards, I – M. The flow chart in Figure 12 depicts the procedure

for the activated group of electrode cards measured in blank buffer (N-Q). The

procedure outlined in Figure 12 was also performed on cards R – U, except CV

measurements were taken in [Ru(NH3)6]3+/2+ as opposed to Tris-HCl. Repeat

measurements on the same card were collected once per day over consecutive

days.

44

FIGURE 11. Flow chart depicting experimental design for electrode cards I – M, not activated.

Open circuit potential was recorded for 1 min. Uncompensated resistance through solution was

measured prior to each CV. CV measurements in Tris-HCl or [Ru(NH3)6]3+/2+

were cycled from -

0.5 V to + 0.3 V (vs. Ag/AgCl) at 0.05 V/s for 10 cycles.

I

Clean

CV Tris-HCl

CV

[Ru(NH3)6]3+/2+

CV

[Ru(NH3)6]3+/2+

J

Clean

CV Tris-HCl

CV

[Ru(NH3)6]3+/2+

K

Clean

CV Tris-HCl

CV

[Ru(NH3)6]3+/2+

L

Clean

CV Tris-HCl

CV

[Ru(NH3)6]3+/2+

M

Clean

CV Tris-HCl

CV Tris-HCl

x3

x3

45

FIGURE 12. Flow chart depicting experimental design for electrode cards N – Q, activated. Cards

were activated at +1.7 V (vs. Ag/AgCl) for 1 min. Open circuit potential was recorded for 1 min.

Uncompensated resistance through solution was measured prior to each CV. CV measurements

in Tris-HCl were cycled from -0.5 V to + 0.3 V (vs. Ag/AgCl) at 0.05 V/s for 10 cycles.

After these measurements, quantitative analysis was performed on the

data to approximate the active surface area of the carbon working electrodes and

how it changed when an electrode was reused and/or activated. Two methods

were used to calculate functional surface area. One method involved obtaining

the double-layer capacitance from measurements in blank buffer and the other

involved a Tafel analysis used in conjunction with the quasi-reversible Randles-

Sevčik equation, both of which are described below.

N

Clean

Activate

CV

Tris-HCl

Activate

CV

Tris-HCl

O

Clean

Activate

CV

Tris-HCl

P

Clean

Activate

CV

Tris-HCl

Q

Clean

Activate

CV

Tris-HCl

x3

46

2.4.4.3.2 Double-Layer Capacitance Method. When a ramping potential was

applied to the working electrode in blank buffer without the presence of a redox

couple, a charging current was observed because the interface between the

electrode and the solution acted as a capacitor that was being charged.[47]

Therefore, a value for the double-layer capacitance was calculated from the CV

using:

Cdl a - c

2 (Eqn. 11)

where Cdl is the double-layer capacitance (in farads, F), Ia is the anodic current

(A), Ic is the cathodic current (A), and ν is the scan rate (ν = 0.05 V/s).[58] The

scan rate was known and the current values correspond to the steady-state

potential measured at open circuit. The OCV was not reproducible between

electrode cards and fell outside the scan range for half of the measurements.

Since the purpose of this study was for comparison and the equations used are

approximations of the active surface area, one value of potential was chosen to

determine Ia and Ic for all measurements. Use of this equation is preceded by the

assumption that the system is modeled as a real capacitor and resistor in parallel

(i.e. the resistance is from the solution).[58]

Once Cdl was determined, the active surface area of the electrode was

calculated using the following equation which utilizes the Helmholtz model[47]:

A Cdl l

0 (Eqn. 12)

47

where A is the active surface area (m2), is the dielectric constant of the solution,

0 is the permittivity of free space ( 0 = 8.854x10-12 F/m), and l is the separation

between the ‘plates’ of the double-layer capacitor (m). The dielectric constant for

ionic liquids is between 10 and 12,[59] so a value of 11 was used. The plates

of the double-layer capacitor are the electrode surface and the first layer of ions

in solution, which are approximately l = 3x10-10 m (3 Å) apart.[47]

2.4.4.3.3 Tafel/Randles-Sevčik Method. The Tafel slope and Randles-Sevčik

equation were used to calculate active surface area for the experiments that

involved faradaic current, which is current from direct electron transfer like that

which occurs with the hexaammine ruthenium redox couple.

Before the Tafel slope analysis was performed on the data, the CV plots

were transformed from potential (V) versus current (A) to overpotential ( , in V)

versus the natural logarithm of current density (ln(i), where i was in A/cm2).

Overpotential was calculated as the potential of the cell less the potential at

which current equals zero, = E – E0 (applying Eqn. 10). When overpotential is

plotted against ln(i) the resulting Tafel plot exhibits two curves that are

asymptotic to = 0 V, one of which occurs over negative overpotentials and the

other over positive overpotentials. A CV has an anodic sweep and a cathodic

sweep which run in opposite directions; each sweep creates its own Tafel plot.

The purpose of calculating the Tafel slope was to determine two constants

of the system, the activation coefficient (α) and the exchange current density (i0).

The activation coefficient was then used in the quasi-reversible Randles-Sevčik

48

equation (Equation 8), along with the peak height, to calculate the functional

surface area of the working electrode. The cathodic curve was used for analysis.

When using the cathodic data of a CV, the appropriate section of the

curve used for a Tafel analysis is at negative overpotentials. The linear region of

this curve, which typically begins around - 52 mV and continues in the negative

direction, is fitted via linear regression analysis and corresponds to the equation:

ln(i) ln(i0) + αF

RT (Eqn. 13)

where i is current density (A/cm2), i0 is exchange current density (A/cm2), α is the

activation coefficient, F is Faraday’s constant (F 96,484.6 C/mol), R is the

universal gas constant (R 8.314 J/mol∙K), T is the temperature (T 298.15 K),

and is overpotential (V). This is a simplified form of the Butler-Volmer Equation

for high-field analysis at large overpotential. The coefficient multiplying

overpotential is the inverse Tafel slope (or the Lefat slope) and is determined

with linear regression. t can then be used to solve for α.

The error for i0 and α was propagated[60][61] though the equations using the

standard error determined from the linear regression analysis. The error of i0 from

the high-field analysis was calculated using Equation 14 and the error of α was

calculated using Equation 15.

i0 F i0 ln (i0) (Eqn. 14)

α RT

F slope F (Eqn. 15)

nce α was calculated the quasi-reversible Randles-Sevčik equation[49]

was used to calculate electroactive surface area:

49

A ip

(2.99 x 105) n(αn)1 2⁄ C(D)1 2⁄ (ν)1 2⁄ (Eqn. 16)

where A is the electroactive surface area (cm2), ip is the peak current relative to

the baseline (amperes, A), n is the number of electrons transferred in the half-

reaction (1), α is the activation coefficient, C is the concentration of the analyte (C

= 1.8x10-6 mol/cm3), D is the diffusion coefficient of [Ru(NH3)6]3+/2+ ions in solution

(D = 8.8x10-6 cm2/s),[62] and ν is the scan rate (ν = 0.05 V/s).

In addition to the Tafel analysis via linear regression in the high-field

potential range, a Tafel analysis was also performed using the Echem AnalystTM

software. The cathodic sweep of the curve from the tenth cycle of each

voltammogram was imported into a sample potentiodynamic data file and was

analyzed in the software program using the ‘Tafel Fit’ tool and the provided

default seed values. The open circuit potential was input prior to analysis. This fit

routine uses a non-linear chi square minimization using four adjustable

parameters: Icorr, Ecorr, αA and αB. Both methods were used to determine i0 and α,

and the values were compared.

A low-field approximation was also used to calculate the exchange current

density (i0) via:

i i0 nF

RT (Eqn. 17)

and linear regression analysis. In this case the CV data were transformed into a

plot of overpotential (V) versus current density (A/cm2). The low-field

50

approximation applies only to small overpotentials, typically ± 5 mV about the

origin.[32]

The error of i0 from the low-field analysis was propagated[60][61] using the

standard error determined from the linear regression analysis via Equation 18.

i0LF RT

F slopeLF (Eqn. 18)

3.0 RESULTS AND DISCUSSION

3.1 SPCE Performance

The CVs of the [Ru(NH3)6]3+/2+ redox couple from this study were analyzed

in three parts. First, the cycles within a measurement were compared to

determine if the electrodes were stable. Second, the tenth cycle was compared

between measurements of four electrode cards to determine reproducibility.

Third, the tenth cycle was compared between six consecutive measurements on

one electrode card to determine reusability.

3.1.1 Stability of the Electrode Cards

When performing a CV experiment, the system must pass through a few

cycles before it reaches a steady-state. At this point the data should be

consistent and the curve should trace the same path, as is evident in Figure 13.

The voltage that is applied to the working electrode is controlled with respect to

the reference electrode[32] so the stability of the reference electrode was

measured based on the consistency between cycles of the potential (Vf) at which

the peaks occurred. The average, standard deviation and RSD were calculated

51

for the peak potentials within nine successive CV cycles of one measurement.

The first cycle was not used. The values of Vf for the nine CVs are tabulated in

Table 1 and show little variation between cycles, suggesting the reference

electrode was stable over the time period in which the measurement was

collected. Vanysek and Gauthier obtained similar results when using Pine SPCE

and reported that potentials applied to the working electrode did not deviate in

excess of a 20 mV range, including when chloride ions were not present in the

sample measured.[31]

The current in the system passes between the carbon working and

counter electrodes so the stability of these electrodes was determined by

examining the variation in maximum current of each peak (Im) and the peak

height (ip). The average, standard deviation and RSD were calculated for these

parameters using nine successive CV cycles within one measurement (Table 1).

Like Vf, Im and ip exhibited low levels of variation during the measurement

suggesting that these electrodes are also stable. It should be noted, however,

that more variation was observed in the peak heights (ip) as opposed to the

maximum current (Im) values. Peak height is solely a factor of the faradaic current

generated by the reaction of the analyte, since the contribution from charging

current is subtracted out. The larger variability observed in values for peak height

is indicative of variability in charging current, as the maximum current values

remained relatively constant between cycles. Further, the current data from the

anodic sweep varied more than that from the cathodic sweep. This stability

52

analysis was repeated for three measurements on three new cards and five

repeated measurements on one card, which all showed similar results (variation

never exceeded 5.4%).

FIGURE 13. Nine CV cycles from one measurement on one electrode card in 0.0018 M

[Ru(NH3)6]3+/2+

in 0.02 M Tris-HCl.

53

TABLE 1. Data from nine CV cycles of one measurement in 0.0018 M [Ru(NH3)6]3+/2+

in 0.02 M

Tris-HCl. The baselines used to calculate values of ip were defined by the analyst. Data is

presented in terms of current densities (µA/cm2).

Cycle Anodic Peak (Oxidation) Cathodic Peak (Reduction)

Vf (mV) Im (µA/cm2) ip (µA/cm2) Vf (mV) Im (µA/cm2) ip (µA/cm2)

2 -197.9 168.98 202.64 -311.8 -258.09 -261.05

3 -197.9 168.54 205.76 -311.8 -258.41 -261.21

4 -197.9 168.89 206.85 -313.7 -258.57 -261.56

5 -197.8 169.17 207.26 -311.8 -258.63 -261.21

6 -197.8 169.62 208.41 -313.7 -258.57 -261.75

7 -197.9 169.81 209.39 -313.8 -258.41 -261.43

8 -197.9 170.13 209.65 -311.7 -258.15 -261.62

9 -197.9 170.38 209.90 -313.8 -257.93 -261.34

10 -197.9 170.57 207.55 -311.7 -257.68 -260.67

Avg -197.9 169.56 207.49 -312.6 -258.27 -261.32

StDev 0.0 0.71 2.29 1.1 0.33 0.33

RSD (%) 0.02 0.42 1.10 0.34 0.13 0.13

3.1.2 Reproducibility of the Electrode Cards

Four new cards, as from the manufacturer, were used to measure the

current response of the [Ru(NH3)6]3+/2+ redox system. Variation between

electrode cards was determined by comparing the tenth CV curve of each

measurement (Figure 14) and calculating the average, standard deviation and

RSD (Table 2). A range in which 95% of values are expected to fall was

calculated as the average plus/minus two standard deviations. This range was

later used as a standard to determine if observed signal varied significantly from

expected signal. Qualitatively the CVs are nearly identical, which is confirmed by

the low values of RSD for the peak parameters. The SPCE appear to exhibit little

variation between cards when measuring this system. However, when compared

54

to the RSD values from within a run (Table 1), different electrode cards do exhibit

a greater variability, where the RSD increased by at least a factor of 3.

FIGURE 14. Cycle ten of CVs from four electrode cards in 0.0018 M [Ru(NH3)6]3+/2+

in 0.02 M Tris-

HCl.

TABLE 2. Data from cycle ten of CVs from four electrode cards: peak potential (Vf), maximum

current (Im) and peak height (ip). Peak height was calculated using baselines defined by the

software and the analyst. Data is presented in terms of current densities (µA/cm2).

Run

Anodic Peak (Oxidation) Cathodic Peak (Reduction)

Vf (mV)

Im (µA/cm

2)

ip (µA/cm2) Vf

(mV) Im

(µA/cm2)

ip (µA/cm2)

software analyst software analyst

1 -197.9 170.57 196.18 207.55 -311.7 -257.68 -262.07 -260.67

2 -195.9 178.95 207.64 217.01 -311.7 -263.25 -267.07 -267.48

3 -197.7 165.73 191.05 200.41 -311.6 -250.45 -253.47 -253.41

4 -193.8 175.76 201.21 213.76 -313.7 -259.04 -263.12 -263.44

Avg -196.3 172.75 199.02 209.68 -312.2 -257.60 -261.43 -261.25

StDev 1.9 5.82 7.09 7.32 1.0 5.33 5.73 5.93

RSD(%) 0.97 3.37 3.56 3.49 0.33 2.07 2.19 2.27

2·StDev ± 3.8 ± 11.64 ± 14.18 ± 14.64 ± 2.0 ± 10.66 ± 11.46 ± 11.86

55

3.1.3 Reusability of the Electrode Cards

Next, a single card was used repetitively for six measurements to

determine the effect of reuse. The card was rinsed with deionized water and

allowed to dry in between each measurement. Variation between measurements

was determined as in the reproducibility portion of the study. The CVs vary to a

larger extent than in the previous section because the baseline and the maximum

current increase with successive measurements (Figure 15). The peak potential

(Vf) and the cathodic current remain stable throughout cycling. The cathodic

curves produce similar peak heights (ip) because the baseline slope increases

consistently with the maximum current (Im) value (Table 3). Therefore, peak

height variation is less than 2% RSD. Conversely, the slope of the anodic curve

baseline is changing to a larger extent with each measurement due to some type

of interference and therefore cannot be reliably measured (i.e. the ip measured

by the software).

56

FIGURE 15. Cycle ten of six repetitive CV measurements from one electrode card in 0.0018 M

[Ru(NH3)6]3+/2+

in 0.02 M Tris-HCl.

TABLE 3. Data from cycle ten of six repetitive CV measurements from one electrode card, taken

over a three-day period: peak potential (Vf), maximum current (Im) and peak height (ip). Peak

height was calculated using baselines defined by the software and the analyst. Data is presented

in terms of current densities (µA/cm2).

Repeat

Anodic Peak (Oxidation) Cathodic Peak (Reduction)

Vf (mV)

Im (µA/cm

2)

ip (µA/cm2) Vf

(mV) Im

(µA/cm2)

ip (µA/cm2)

software analyst software analyst

1 -197.9 170.57 196.18 207.55 -311.7 -257.68 -262.07 -260.67

2 -195.8 177.55 132.07 168.25 -315.9 -260.16 -256.27 -257.77

3 -195.8 195.35 57.04 158.18 -317.7 -282.13 -274.27 -265.86

4 -191.8 191.21 0.28 142.61 -321.8 -278.47 -256.11 -256.82

5 -191.8 204.04 -23.73 149.75 -319.7 -290.89 -266.53 -265.03

6 -185.8 211.08 -173.44 126.21 -329.7 -302.52 -264.36 -261.62

Avg -193.2 191.63 31.40 158.76 -319.4 -278.64 -263.27 -261.30

StDev 4.3 15.42 129.55 27.83 6.1 17.40 6.85 3.68

RSD(%) 2.25 8.04 412.57 17.53 1.91 6.24 2.60 1.41

2·StDev ± 8.7 ± 30.84 ± 259.1 ± 55.66 ± 12.2 ± 34.80 ± 13.70 ± 7.36

57

Echem AnalystTM was initially used to extract Im and ip from the CVs, but it

could not anchor the baseline to calculate ip correctly if the linear region

preceding the peak was too short. This is a result of the conditions under which

the measurement was taken, such as the electrodes, electrolytes, and scan

range used. Figure 16 illustrates an example. The height of the bottom peak (the

cathodic sweep) was measured correctly because the baseline (indicated by the

dashed arrow) is parallel to and overlays the linear region prior to the peak. The

height of the top peak (the anodic sweep) was not measured correctly because a

large enough linear region in which to anchor the baseline (indicated by the solid

arrow) could not be found. For some of the anodic peaks the baseline passes

above Im and the resulting ip value is negative when it should be positive (see

Table 3, portion shaded in gray). Due to this information the reduction peak was

used for analysis. The inability to establish appropriate CV baselines for the

anodic curves in these samples, both by the software and the analyst, is a direct

result of the changes in CV shape (Figure 14) and suggests an increase in the

effect of the interference as the card is re-used.

58

FIGURE 16. Example of the automatic baselines created by the software program Echem

AnalystTM

. The solid arrow is indicating a baseline that is inaccurate, and the dashed arrow is

indicating a baseline that is properly aligned to the charging current preceding the peak.

3.1.4 Effect of the Electrode Cards on Reversibility of a Known Reversible

Redox Couple

The hexaammine ruthenium redox couple is a reversible system and

provides a fair indication of the basic kinetic performance of the SPCE. For the

reversible reaction:

[Ru(N 3)6]3+ + e- [Ru(N 3)6]

2+ (Eqn. 19)

that involves the transfer of one electron, the difference between the potentials of

the cathodic and anodic peaks should equal ΔEpeak = 59 mV. Also, the ratio of the

peak heights, ipa/ipc, should equal unity.[46] For measurements between electrode

cards the average values are ΔEpeak = 115.9 mV ± 5.7 mV (2.5% RSD) and ipa/ipc

= 0.80 ± 0.02 (1.3% RSD). The peak potential difference is nearly twice of that

expected for the reversible system and the peak height ratio is 80% of the

expected value, indicating that the redox couple exhibits quasi-reversible

59

behavior on the SPCE. For repetitive measurements on a single electrode card,

ΔEpeak increases and ipa/ipc decreases with each consecutive measurement. This

suggests that some ions may be accumulating over time.

These results have been corroborated by others. For example, for other

brands of SPCE Cui et al. observed values of ΔEpeak ranging from 73 mV to 134

mV and values of ipa/ipc ranging from 0.79 to 1.01.[63] Gornall et al. observed

ΔEpeak = 87 mV and 80 mV.[29] The deviation from 59 mV for the ΔEpeak is

expected on printed carbon electrodes as they do not have the pristine surfaces

that allow for the truly reversible nature of hexaammine ruthenium to be

observed.[46] In contrast, other carbon electrodes have approached theoretical

values. Fanjul-Bolado et al. observed values of ΔEpeak = 63, 64, 59, 59 mV and

ipa/ipc = 0.85, 0.94, 0.91, 0.94 with a conventional carbon paste electrode and

three other brands of SPCE.[30] The reproducibility of the SPCE tested in this

study is similar to that of Kadara et al. who report a RSD in signal of 3% - 8%

when electrodes were studied from three different brands of SPCE.[28] It should

be noted that the electrolyte concentration of these four studies is at least five

times more concentrated than that used in this study, which can affect ΔEpeak.

The half peak-potential of an analyte is given as the average between the

oxidation and reduction peak potentials, or E1/2 = (Epa + Epc)/2. Unlike ΔEpeak, the

half-peak potential should be independent of the type of electrode used. The half-

peak potential of 1.8 mM [Ru(NH3)6]3+/2+ in 20 mM Tris-HCl buffer on Pine SPCE

is E1/2 = -254.25 ± 1.10 mV (vs. Ag/AgCl), and is consistent with some of the

60

literature. Cheng observed a half-peak potential of -249 ± 8 mV (vs. Ag/AgCl) for

0.005 mM [Ru(NH3)6]3+ in 10 mM Tris buffer (pH 7.4) on a gold substrate chip.[64]

Tadayyoni et al. observed a half-peak potential of -290 ± 10 mV (vs. SCE) for

0.05 mM [Ru(NH3)6]3+ in 0.1 M KCl on silver electrodes; this converts to

approximately -244 mV vs. Ag/AgCl.[65][66] Conversely, Grzybowski et al. noted a

half-peak potential of -160 mV (vs. Ag/AgCl) in 1 mM [Ru(NH3)6]3+/0.1 M LiClO4

on an array of gold minielectrodes and Fathi et al. obtained a value of -170 mV

(vs. Ag/AgCl) in 1 mM [Ru(NH3)6]3+/1 M KNO3 on a silver nanostructure

electrode.[67][68]

Work by Muzikar and Fawcett examined the effect of varying electrolyte

concentrations on the half-peak potential, and the authors found that as

electrolyte concentration increased the half-peak potential became less negative.

Their results were congruent with those on Pine SPCE. The potential was -310

mV (vs. SCE) in 5 mM [Ru(NH3)6]3+/0.02 M HCLO4 on a gold disk electrode,

which converts to approximately -264 mV vs. Ag/AgCl.[66][69] They suggested that

the activity of the reactants changed depending on the electrolyte concentration.

The variation observed in E1/2 between studies using differing electrolytes

indicates that the choice of electrolyte may also affect the intrinsic behavior of the

redox couple.

Many reasons other than the electrolyte concentration have been

proposed to explain the quasi-reversible behavior of what is expected to be a

reversible redox couple, and they all center on the composition and performance

61

of the carbon ink. While the composition of the ink is proprietary, it is reported

that the ink contains graphite particles, vinyl or epoxy polymer binder, and a

solvent.[29][30][55] The relative proportions of these components likely affect

electron transfer kinetics of the surface. For example, the binder may fill spaces

or cover carbon particles and thus insulate the carbon from electron transfer.[28-30]

Smaller amounts of binder and higher graphite loading with more edge-plane

sites create a rougher surface which will increase the electron transfer kinetics

but also decrease the reproducibility between electrodes.[28][29] Finally, curing

temperature used to harden the ink on the cards can affect the presence and

amount of solvent.[28]

These preliminary studies provided a range of values that can be

expected when only random variation plays a role. Repeatability studies suggest

that a 6.2% RSD is expected between reused electrodes while the variation is

smaller between new electrodes. This information was used in following studies

to determine if the procedures used to develop the biosensor caused changes to

the electrode surfaces and affected the kinetics of the system under study.

3.2 Oxidation of Guanine and Adenine

The first experiment to measure DNA with a carbon SPE involved placing

the electrode into a solution containing oligonucleotides and using CV to

measure oxidation signals from guanine and adenine bases. This design is

termed ‘DNA stick’[13] and is desirable over the biosensor design in the sense that

62

it would eliminate the need to modify the electrode. The sample could be

measured directly and the time required to run the assay would decrease.

Gonçalves et al. observed well-defined oxidation peaks from guanine and

adenine residues on basal plane pyrolytic graphite electrodes. They studied low-

and high-molecular weight dsDNA, with an average length of 300 bp and 2000

bp respectively. They calculated the amount of guanine bases oxidized to

produce the peak signal in the measurement involving low-molecular weight DNA

(116 ± 4 out of approximately 150 bases, using a modified Randles-Sevčik

equation). It should be noted that this estimation was predicated on the

assumption that the four bases were equally distributed in the DNA molecules

that were analyzed. Gonçalves et al. were less successful when measuring high-

molecular weight DNA and suggested that larger DNA molecules will not diffuse

to the electrode surface as quickly as smaller molecules.[36]

The Gonçalves study used samples containing 10 mM of DNA, which is

much higher than what would typically be seen in forensic casework. For the

experiment using carbon SPE a concentration of 25 µM DNA was used as it is

more congruent with the levels observed in forensic DNA analysis. This equates

to approximately 165 ng/µL, which is over three times more concentrated than

the most concentrated sample used to develop the standard curve for qPCR

assays (50 ng/µL). No peaks were observed on the resulting CVs (data not

shown).

63

Two genomic DNA samples were also analyzed using the DNA stick

design at concentrations less than the first sample. These samples were diluted

to reach a total volume of 1 mL, as required by the cell design. The reasoning

behind investigating a genomic DNA sample was that the oligonucleotide used

was relatively cytosine- and thymine-rich, and the genomic DNA was expected to

contain higher levels of guanine and adenine residues. The disadvantage of

using genomic DNA is that it is comprised of long strands, longer than the high-

molecular weight DNA studied by Gonçalves et al., and may suffer from a low

diffusion coefficient. No peaks were observed for either of these samples as well

(data not shown).

As a result of the need to utilize high quantities of DNA and the inability to

detect signal at even 165 ng/μL, the DNA stick design was discarded as a

possible method for quantifying forensic DNA samples and the focus of this study

migrated to developing a biosensor.

3.3 DNA-Modified SPCE

Three electrode cards were used to create biosensors with TPOX

oligonucleotides. Although previously used in experiments with [Ru(NH3)6]3+/2+,

the cards were assumed to be equivalent for comparison after creating the

biosensor on the working electrodes.

Figure 17 displays the CVs obtained for each electrode card used to

create a biosensor in the first three panels. Three measurements were collected

on each card representing the signal observed for bare, single-strand and

64

double-strand modified electrodes after incubation with the indicating molecule.

The fourth panel overlays the CVs measured using double-strand modified

electrodes.

FIGURE 17. CVs from electrode cards used in the electrostatic DNA adsorption protocol. The

small dashed lines (- - -) represent the signal from bare electrodes, the long dashed (– – –) lines

represent the signal from the single-strand modified electrodes, and the solid lines ( )

represent the signal from the double-strand modified electrodes. ‘DNA1’ data were collected

using a card previously used in a [Ru(NH3)6]3+/2+

experiment that scanned from -0.5 V to +0.3 V

(vs. Ag/AgCl). ‘DNA2’ data were collected using a card that scanned down to -0.6 V and ‘DNA3’

data were collected using a card that scanned down to -0.7 V in previous [Ru(NH3)6]3+/2+

experiments.

The peak representing the oxidation of the H33258 molecules using the

biosensor occurs at approximately +0.5 V (vs. Ag/AgCl). The literature has

reported the oxidation peak at approximately +0.6 V using a SPCE and a glassy

carbon electrode.[13][70] Differences in buffer concentration, scan rate and

65

electrode type suggest that the difference of 0.1 V between these results and

those previously reported on glassy carbon are expected.[70]

As indicated in Figure 17, the double-strand modified curves appear

significantly different from each other and do not show the same consistency in

maximum current (Im) as the hexaammine ruthenium experiments. Additionally,

the current continued to increase after the peak ‘maximum’ was reached for

several curves, thereby preventing quantitative analysis due to peak masking

(Table 4).

TABLE 4. Peak current data for three different electrode cards used in the electrostatic adsorption

protocol; each had been previously used in one experiment with [Ru(NH3)6]3+/2+

. ‘DNA1,’ ‘DNA2,’

and ‘DNA3’ data correspond to the panels shown in Figure 17. IND = indeterminable.

Measurement DNA1 DNA2 DNA3

Vf (mV) ip (μA) Vf (mV) ip (μA) Vf (mV) ip (μA)

Bare IND IND IND IND IND IND

Single-Strand IND IND 491.9 1.49 IND IND

Double-Strand 494.8 2.38 495.9 1.97 IND IND

A peak was expected for measurements of the double-strand modified

electrodes but peak-like features were also observed for the single-strand

modified and bare electrode measurements. Though the H33258 is rinsed off of

the electrode surface prior to measurement, the presence of signal on the bare

electrode would suggest that some of the indicator remained. In theory, this

would not be a problem so long as the signal obtained from a DNA-modified

electrode was larger and discriminable from the bare electrode-H33258 signal.

However, this could not be determined from the data.

66

The procedure was repeated for the first electrode card (data signified by

DNA1 in Figure 17) an additional two times to determine if the signal could be

reproduced on a single card, despite apparent irreproducibility between cards.

The single-strand and double-strand modified current signals increased in each

repetition, and the charging current increased enough to mask the maximum of

the peak, which was previously identifiable. The double-strand signals are

overlaid in Figure 18 to illustrate the consecutive increases in current density,

and the arrow indicates the point where the peak maximum is masked by the

background current. This result was confounding since the experiment followed

the prescribed cleaning procedures for SPCE. There should not have been

residual sample from the previous run, considering that electrostatic adsorption is

fragile compared to other attachment methods.[14]

FIGURE 18. CVs of double-strand signal from three repetitive measurements on one electrode

card (in chronological order). The arrow shows where the peak maximum of curve 3 is masked.

67

Variation like that observed in the electrostatic adsorption experiments

would prevent an accurate and reliable standard curve for DNA quantitation. It

was not possible to decouple the sources of variation for this data, however, and

would impact the weight of the comparison. The variation may be random, from

simply running the same protocol on different electrode cards, and/or the

variation may stem from preceding hexaammine ruthenium experiments the

cards were used in. The increase in scan range to more negative potentials may

have affected the working electrode surface. When compared to the expected

values of current density from the [Ru(NH3)6]3+/2+ couple determined in the

performance study, one of the electrode cards used with an extended scan range

fell outside of the calculated limits for random variation. The peak height from the

reduction of [Ru(NH3)6]3+ observed with the first card (DNA1), with the regular

scan range, was the same value as the average that was calculated in the

reproducibility study, at -261 µA/cm2. The peak heights from reduction of

[Ru(NH3)6]3+ using the second (DNA2) and third (DNA3) cards were -249 µA/cm2

and -252 µA/cm2 respectively, and the value from the second card fell just

outside of the expected -261 ± 12 µA/cm2 range. The SPCE are more sensitive

to small changes in potential than previously predicted or reported.

Additionally, each run involved an activation step directly prior to

oligonucleotide adsorption. Activation, by holding the working electrode at a high

anodic potential for a short period of time, has been shown to increase the

electron transfer kinetics and improve the performance of the electrode. This step

68

purportedly makes the electrode surface more amenable to DNA adsorption by

increasing hydrophilicity,[37][63] increasing the amount of carbon-oxygen functional

groups on the surface,[28-30] increasing surface roughness, and removing possible

contaminants.[29][30][55] Signal magnitude and reversibility are improved because

there is an increase in the amount of active sites on the electrode. This

necessary step may have introduced additional variation, and is discussed later

(Section 3.5.3).

The SPCE are designed to be disposable and only a few studies have

examined the effects of reuse. Vanysek and Gauthier performed a longevity

study of the Pine SPCE and found that the electrodes could reproduce the CVs

of the ferricyanide couple after 6000 cycles. After pretreating the electrodes by

cycling at 4 V/s in 1 M sulfuric acid they report that the peak separation

decreased from 216 mV to 69.5 mV (at cycle 4). After 2000 cycles the peak

separation had increased to 74 mV and remained steady for the remaining 4000

cycles. They concluded that the electrodes can be used for approximately one

week if used in a single type of assay.[31] Wang et al. used homemade SPCE in

conjunction with potentiometric stripping analysis and reported a 6.9% RSD in

signal after using a sensor in 14 repetitive measurements. It should be noted that

potentiometric stripping analysis is more sensitive, with lower limits of detection,

than other voltammetric methods.[37] Both of these articles indicate that the

electrodes are used with a single type of assay. The electrostatic adsorption

protocol described in this work is comprised of several steps that apply different

69

potentials or scan over differing ranges. This type of multi-step processing may

affect the electrode surface and lead to compounding variation with each

consecutive step.

Further, it is unclear whether the H33258 is interacting with DNA, or

simply the carbon surface and the signal is being amplified with each

measurement. Imaging via SEM and EDX analysis were performed in an attempt

to determine if DNA was in fact adsorbed on the working electrode. Two

electrodes were assessed for phosphorus, which would confirm the presence of

DNA because the other materials used in the experiment did not contain this

element. One electrode was used as a negative sample and was not expected to

contain phosphorus because it underwent adsorption in blank buffer. The other

electrode underwent adsorption in DNA and might exhibit a phosphorus peak.

Neither electrode contained phosphorus but both exhibited an unexpected, large

chlorine peak.

A new electrode, which was not used in the adsorption protocol, was

examined using SEM/EDX to determine if the chlorine observed was an original

component of the electrode or a residue from the NaCl and Tris-HCl buffers used

in the adsorption protocol. The chlorine peak was observed for this electrode as

well, suggesting that chlorine may be a component of the binder used in the

printing process. Since the ink formulation is proprietary the exact composition is

unknown, however Kadara et al. performed elemental analysis on several

commercial carbon SPE using X-ray photoelectron spectroscopy. The authors

70

observed that some of the sensors have chlorine levels as high as 23% – 27%

and postulated that the binder may be poly(vinyl chloride).[28] The presence of the

chlorine peak on the electrodes that were used in the adsorption protocol

indicates the initial cleaning procedure did not remove all of the binder. The

binder may block potential electro-active sites on the surface and inhibit electron

transfer kinetics. Figure 19 shows the SEM image and elemental composition of

the working electrode of the SPCE, as from the manufacturer. EDX analysis of

the reference electrode resulted in the expected silver (Ag) and chloride (Cl)

peaks (data not shown).

FIGURE 19. (A) SEM image with a scale of ~ 50 μm (white bar) and (B) EDX elemental analysis of

the working electrode of Pine SPCE. The EDX graph shows the presence of carbon (C) and

chlorine (Cl).

3.4 De-convoluting the Adsorption Protocol

The aforementioned data suggests that the working electrode surface is

modified during the DNA adsorption process. The evidence to support this is

apparent in Figure 18, where a repeat of the same process results in a curve with

a different shape and size. To study this, four electrode cards (A – D, Figure 10)

71

were subjected to a step in the process and then probed with the hexaammine

ruthenium redox couple. The resulting current responses were compared to the

variability data described in Section 3.1 and analyzed separately for variation

within a card and between cards. In addition, differences in baseline noise and

peak shape were compared as they might also indicate changes in surface

kinetics.

The cathodic peak current throughout the process on Card A (all-blank

run) and Card B (DNA, H33258 present) did not significantly differ between

measurements, suggesting the electrodes were not being modified during the

course of one experiment. The percent RSD of peak height between the four

measurements on each card was 1.0% and 0.9% respectively, less than that

obtained during the stability study for within-electrode variation (1.4%). These

results also suggest that DNA is not being adsorbed and hybridized on the

surface or that the amount of DNA present on the surface is too low to affect the

kinetics of the hexaammine ruthenium redox couple. Figure 20 shows the

overlaid curves for the four measurements on Card A and Card B.

72

FIGURE 20. Overlay of the curves obtained from each measurement on Card A and Card B.

A difference between the measurements before and after the adsorption

step on Cards A and B would signify that application of a prolonged potential

changed the electrode surface. A difference between the measurements on Card

C would signify that hybridizing with the DNA complement changed the surface,

and a difference between the measurements on Card D would signify that

incubating the working electrode in the indicator changed the surface. The first

measurement on Card D was atypical when compared to the first measurements

on Cards A – C (Figure 21). The peak separation (ΔEpeak) is larger, suggesting

slower electron transfer kinetics that might be seen if, for example, residual

organic binder was blocking potential electroactive sites. The comparisons as

described above are for the purpose of finding a trend and comparing absolute

values is not necessary. Instead, and because the first measurement of Card D

was atypical, the cathodic peak current (ip) values of subsequent measurements

were normalized to the peak current value of the first measurement in the

73

sample. A value close to unity would indicate that the peak current values did not

differ significantly from each other. All ratios were greater than 95%, suggesting

that significant differences were not observed. Table 5 summarizes the results. In

addition, peak shape and the baseline preceding each peak were consistent

between and within all measurements, except in the initial measurement of Card

D. All of the data are not shown here but the similar baseline and peak shape is

evident in Figure 21.

FIGURE 21. Initial measurements in hexaammine ruthenium (pre-adsorption) of Cards A – D.

74

TABLE 5. Cathodic peak current (ip) of each measurement normalized to the initial measurement

in hexaammine ruthenium. Ratio = ip,measurement/ip,initial.

Card Post-Adsorp Post-Hybrid Post-H33258

A 1.017 1.007 1.023

B 0.996 1.003 0.982

C N/A 0.962 N/A

D N/A N/A 1.035

The next variable of interest is the oxidation of H33258 and whether it

fouled the electrode surfaces. Sufen et al. expressed the concern of build-up of

H33258 oxidation products and the effect on electrode performance.[70] Since this

occurs at a higher scan range than the hexaammine ruthenium studies it was

also of interest to determine if repeated cycling at the more positive scan range

affected the surface. The reasoning behind the experimental setup was to

simulate the intermediate measurements used to detect the bare, single-strand

and double-strand responses of the indicating molecule. Card E was exposed

only to blank buffer and Card F was exposed to DNA and H33258 samples. Card

G underwent the adsorption step in a blank sample and the hybridization step in

a DNA sample. Card H underwent the adsorption and hybridization steps in blank

samples but was exposed to the indicating molecules. Figure 22 shows the

curves from the measurements in blank buffer of Cards E, F and H and the final

measurement of each card in hexaammine ruthenium (Cards E – H).

75

FIGURE 22. (A) CVs (cycle 1) of background signal and post-H33258-incubation in blank buffer

and (B) CVs (cycle 10) of hexaammine ruthenium of each card after the experiment. Background

CVs are dashed (- - -).

As expected, an oxidation peak is observed for cards F and H, which were

both exposed to the indicator. Figure 22 B suggests that the electrode surface is

not being fouled by the oxidation of H33258, at least not enough to inhibit the

electron transfer kinetics of the hexaammine ruthenium redox couple, because

the voltammogram for Card E, which incubated in blank buffer, nearly completely

overlays the voltammogram for Card F, which incubated in H33258. The percent

RSD of the cathodic peak current from the [Ru(NH3)6]3+/2+ couple is 5.7% and is

larger than that expected for variation between electrode cards (reproducibility

study, 2.3%). The curves for the electrode cards that were measured twice (G, H)

have smaller peak current values than the electrode cards that were measured

four times (E, F). A possible explanation for this could be that repetitive cycling at

the higher scan range changes surface properties/kinetics such that the peak

current is larger when measured in the same system. This phenomenon was not

76

observed during the previous study where the electrode cards were cycled at

more negative scan ranges.

These experiments were useful in separating the variables of the complex

process of creating a biosensor. The results indicate that the steps of the process

do not cause electrode surface kinetics to change but the repetitive cycling,

particularly at the more positive scan range, may confound the results and make

the production of a standard curve for quantitation difficult. The following section

discusses functional surface area of the working electrodes and how it changes

upon reuse. It also delves more into the effect of activation.

3.5 Analysis of Real Surface Area

The final study on Pine’s SPCE approximated surface area by measuring

charging current in blank buffer and by measuring peak current in a hexaammine

ruthenium sample. Variability due to reuse and activation were evaluated.

3.5.1 Double-Layer Capacitance

Measurements in blank buffer consisted of an activated and non-activated

set of carbon electrode cards. Each set contained four measurements on

different cards, and then one card was used for three additional measurements,

culminating in four measurements for each category of interest. The curves were

analyzed to determine the double-layer capacitance, Cdl in µF, between the

electrode surface and solvent. The results of this study are shown in Table 6.

77

Standard error (SE) was calculated as the standard deviation divided by the

square root of the number of measurements.

TABLE 6. Capacitance calculated from the current envelope in blank buffer.

Non-Activated Activated

Between

Electrodes Within

Electrode Between

Electrodes Within

Electrode

Cdl (µF)

0.725 0.975 1.500 1.500

0.725 1.025 1.175 1.525

0.825 1.025 1.200 2.250

0.775 1.000 1.300 3.325

0.975

Avg 0.805 1.006 1.294 2.150

StDev 0.104 0.024 0.148 0.857

RSD(%) 12.9 2.4 11.4 39.9

SE 0.046 0.012 0.074 0.428

The calculation of capacitance depends on the range of the current

envelope, which did not exceed 600 nA in any measurement. Capacitance is

reported in µF/cm2 and generally ranges from Cdl = 10 – 30 µF/cm2 for several

metal-solution systems.[32][47] When the capacitance values are normalized to the

nominal surface area of the electrode cards (0.0314 cm2) the results from the

non-activated cards conform to the literature value and range from 23 – 33

µF/cm2. It also appears that repeat measurements on a single non-activated card

are quite reproducible (RSD = 2.4%). Measurements between non-activated

cards are more variable, which could stem from the low level of signal used for

these calculations. The measurements between activated cards estimate the

capacitance from 37 – 48 µF/cm2 and the measurements within one multiply-

activated card estimate the capacitance from 48 – 106 µF/cm2, where the value

78

increases with each consecutive activation/measurement. Since Cdl is directly

proportional to active surface area the values described here would indicate that

surface area is changing upon reuse and activation. This suggests that reuse of

SPCE cards results in a change of signal, particularly when activation is part of

the protocol, and is in contrast to Vanysek et al. and Wang et al. Vanysek et al.

suggested SPCE were stable over 6000 cycles from -0.1 V to 0.5 V (vs. Ag/AgCl)

when measuring ferricyanide, though the electrodes were not activated in that

study.[31] Wang et al. concluded that the nucleic acid signal was reproducible

over 14 repetitive measurements after a +1.8 V (vs. Ag/AgCl) activation, a short

+0.5 V (vs. Ag/AgCl) accumulation, and stripping analysis for each

measurement.[37]

3.5.2 Exchange Current Density and Activation Coefficient

The measurements using the [Ru(NH3)6]3+/2+ analyte also consisted of a

non-activated and activated set of electrodes, a new and reused set, and

provided four measurements for each category. Figure 23 shows the resulting

CVs (cycle 10). Figure 23 A displays the data for the measurements on different

cards. Figure 23 B displays the data for repeat measurements on the same card;

two cards were used, one of which was activated before each measurement.

79

FIGURE 23. (A) Measurements of eight different cards, four of which were activated, run in 0.0018

M [Ru(NH3)6]3+/2+

/0.02 M Tris-HCl from -0.5 V to +0.3 V (vs. Ag/AgCl). (B) Four consecutive

measurements of two cards, one card was activated prior to each measurement. Gray curves are

from cards that were activated, black curves are from cards that were not activated.

Reproducibility between cards that have not undergone successive

activations is consistent with that observed in the performance studies described

in Section 3.1 and is evident in Figure 23 A. Figure 23 A also shows that the

background current has changed post-activation but this does not appear to

affect the calculated peak current. Though the portion of the curve in which the

baseline is anchored has shifted, the maximum current values of the peaks have

also shifted. The RSD of all eight peak current values for the cathodic curve is a

little less than 2%. The increase in the background current following each peak

would not be a concern since the peak is still distinguishable from the baseline.

Figure 23 B shows that reproducibility of the cathodic ip on the same card

is consistent with that in the performance study for the card that was not

activated (RSD = 1.7%). However, the same cannot be said for the card that was

reused and successively activated four times (i.e. prior to each run). In this case,

the background current increased with each consecutive measurement, and

80

while the maximum peak current value also increased, it was not proportional to

the background current (RSD = 4.1%, greater than that expected for within-

electrode variation). It is therefore expected that continued activation and cycling

may cause the background current to eventually mask the cathodic peak in the

location shown by the black arrow. These results likely indicate a change in the

surface area.

A Tafel slope analysis was performed on each voltammogram to

determine the activation coefficient (α) and exchange current density (i0). Only

the electrode cards that had been activated provided data suitable for a Tafel

analysis via linear regression. Non-activated data did not possess a linear region

on which to perform a linear regression, indicating that other complex processes

were affecting the system. As a result, the activation coefficient and exchange

current density could not be calculated for that data via a high-field analysis.

Exchange current density was determinable for these cards using a low-field

analysis.

A second method based on a chi-squared best fit was able to calculate the

constants from the non-activated electrodes, as an alternative to linear

regression of the ln(i), curves. The program determines goodness of fit by

using non-linear chi squared (Χ2) minimization, estimates new parameter values

using the Marquardt algorithm, and iterates until the fit with the observed data is

optimized.[71] This method was used as an alternative method to calculate the

81

activation coefficient and exchange current density for comparison purposes to

the constants calculated via linear regression.

Exchange current density is representative of the intrinsic reaction rate

when the system is at equilibrium. It describes the kinetics of a system without

the application of potential forcing a reaction to occur and cannot be directly

measured. However it can be approximated using the Butler-Volmer equation

(Equation 9).[32] The exchange current density was calculated using data in the

high-field and low-field regions. The high-field method analyzes a portion of the

curve where faradaic current has already started passing through the circuit and

generally occurs at high overpotential. The low-field method analyzes the portion

of the curve that is approximately ± 5 mV about = 0 mV. The relationship

between overpotential and current density will be linear in these regions and

parameters of interest can be determined via linear regression. Figure 24 shows

the values of exchange current density calculated from a linear regression of the

high- and low-field regions of data (white and gray data points). Error bars

represent the propagated standard error from the calculations.

82

FIGURE 24. Exchange current density data obtained from the cathodic curve of 0.0018 M

[Ru(NH3)6]3+/2+

. Runs 1 – 4 represent between-electrodes data, Runs 4 – 7 represent within-

electrode data. HFC = High Field Cathodic, LFC = Low Field Cathodic, Linear Reg = Linear

Regression, Χ 2 = Chi-Squared Minimization.

Since all measurements were recorded in the same sample, any

differences in the value of i0 would be a consequence of electrode surface

changes. As evident in Figure 24, activation significantly affects exchange

current density. Not only were the values calculated via linear regression nearly

half that of non-activated electrode cards, there is also more variation between

measurements (RSDHFC = 19.8% between cards, 28.7% within a card; and

RSDLFC = 26.6% between cards, 48.3% within a card). The non-activated

83

measurements had small variation between cards and within a card (RSDLFC =

1.3% and 1.7%, respectively).

An analysis using the Χ2 minimization technique was performed on the

cathodic sweep of the tenth CV curve and the corresponding values for

exchange current density are shown in Figure 24 (black data points). A benefit to

using this technique is that the algorithm doesn’t require a fully developed linear

region to calculate the parameters of interest.[72] When the analysis is performed

using the traditional high-field analysis, the linear region must be well-defined.

The Χ2 minimization technique calculated larger values of i0 than the traditional

analysis however the same trend of a decrease in i0 with activation is obvious for

both calculations. For example, the exchange current density decays between

Runs 4 – 7 (within a card) for the high field (HFC) linear regression and Χ2

minimization estimations from activated cards. Additionally, the variation is

comparable between the two methods; for activated cards the RSD is 27.1%

(between cards, Runs 1 – 4) and 45.5% (within a card, Runs 4 – 7), for non-

activated cards the RSD is 2.6% (between) and 3.6% (within). Since the Χ2

minimization method can be used for both activated and non-activated

electrodes, and estimated reliable values for i0, the method was considered

validated for the purposes of this study.

The activation coefficient, which is related to the free energy of activation

of the system, can also be determined from the high-field region. Figure 25

displays the data for α obtained by calculations using traditional linear regression

84

and by Χ2 minimization. The commonly used value of α for a single electron

transfer at room temperature is 0.5[32] and is indicated by the gray dashed

horizontal line in Figure 25 A.

FIGURE 25. (A) Activation coefficient data obtained from the cathodic curve of 0.0018 M

[Ru(NH3)6]3+/2+

. Runs 1 – 4 represent between-electrodes data, Runs 4 – 7 represent within-

electrode data. Linear Reg = Linear Regression, Χ 2 = Chi-Squared Minimization. The gray

dashed line represents the theoretical value of α. (B) Tafel plots; gray lines represent non-

activated cards, black lines represent activated cards, solid lines ( ) represent runs on new

electrodes, and dashed lines (- - -) represent repeated runs on one electrode.

The value for α approached 0.5 as the electrode card was successively

activated (Runs 4 – 7) and the appropriate portion of the ln(i) vs. curve became

more linear (Figure 25 B). This resulted in more reproducible results as the linear

range became more easily definable. n contrast, the Χ2 minimization method

resulted in smaller variation between cards (Runs 1 – 4), indicating that it is

reliable. Additionally, as shown previously, although the absolute values of the

activation coefficients were affected by analysis method, trending was not. As a

85

result, Χ2 minimization was deemed an appropriate analytical tool for determining

i0 and α. Figures 24 and 25 A show that activation impacts both the exchange

current density and the activation coefficient.

3.5.3 Real Surface Area

Once Cdl, i0 and α were determined, the active working electrode surface

area was calculated using the double-layer capacitance model and the quasi-

reversible Randles-Sevčik equation, as per Equations 12 and 16 respectively.

For the quasi-reversible Randles-Sevčik equation, the surface area was

calculated using the theoretical value for activity coefficient, α 0.5, as well as

the experimentally determined values. Figure 26 summarizes the data calculated

using the double-layer capacitance model and Figure 27 summarizes the data

calculated using the quasi-reversible Randles-Sevčik equation.

86

FIGURE 26. Active surface area (SA) calculated using Equation 12, and the measurements taken

in blank buffer.

In Figure 26, the consecutive measurements described by the circles

(within a card, activated) exhibit a steep upward trend and all lie above the line

demarking the nominal surface area. The triangles (between cards, activated)

and a couple of the squares (within a card, non-activated) also lie above this line,

while the diamonds (between cards, non-activated) were calculated as less than

the nominal surface area.

87

FIGURE 27. Active surface area (SA) calculated using Equations 10 and 13 (Tafel analysis and

Randles-Sevčik equation), and the measurements taken in 0.0018 M [Ru(NH3)6]3+/2+

in 0.02 M

Tris-HCl. (A) displays the data from cards that were used once, (B) displays the data from the

cards that were reused.

In Figure 27, gray data points represent calculations using α 0.5, and

black data points represent calculations using α as determined by Tafel analysis

with the Χ2 minimization technique. The different approximations for activity

coefficient create a large level of difference in absolute quantities of the resulting

electroactive surface area. What is more notable, however, is the trend observed

for each data set. The non-activated data (diamonds, squares) and the between-

electrodes activated data (triangles) remain fairly consistent in their calculation of

surface area while the within-electrode activated data sets (circles) exhibit an

upward trend (connected via lines).

88

A majority of the estimates of real surface area are less than the nominal

surface area of 0.0314 cm2. This result would support the theory that some

organic binder used in the printing process is left behind and effectively

‘insulates’ the graphite from electron transfer. n this case it would also indicate

that the method for cleaning a new electrode card, by cycling over a large

potential range for multiple cycles in blank buffer, is not effective. Another

property that may affect effective surface area is the ratio of basal-plane and

edge-plane sites on the graphite particles that comprise the carbon electrode.

The literature reports that edge-plane sites typically exhibit higher rates of

faradaic electron transfer.[21][28][30][36] Pine SPCE may contain a particular ratio of

edge-plane sites to basal-plane sites that will ultimately limit the amount of

surface area available for effective current transfer. Despite the fact that a rough

surface typically indicates a greater real surface area than nominal area, the

working electrodes may only have the capacity for active sites at a fraction of the

nominal surface area. The data in Figure 27, where active surface area was

calculated using the faradaic current generated by the redox couple hexaammine

ruthenium, appear to support this theory. Areas calculated at values larger than

the nominal value could be explained by the removal of binder and exposure of

additional edge-plane sites on the graphite particles through repetitive activation.

The smallest area calculated was 0.021 cm2 and is approximately two-

thirds of the nominal surface area. This corroborates the active electrode surface

areas calculated by others who have obtained ratios of effective surface area-to-

89

nominal area between 39% and 79% on other commercial SPCE when using the

standard Randles-Sevčik equation.[28] The equation that uses double-layer

capacitance systematically produced a larger calculated surface area when

compared to the equation that uses peak current.

Part of this study aimed to determine the effect of reuse of the electrode

cards from a quantitative standpoint. For this, comparison of the trends in data

sets is more important than the absolute values of surface area. In Figures 26

and 27 B, data points from cards that were reused are displayed from left-to-right

as consecutive measurements. The sets of interest are marked with squares

(non-activated) and circles (activated). The non-activated data do not display any

particular trend aside from fair consistency of calculated surface area using both

methods (RSDs < 2.4%). As described above, the activated electrodes show an

upward trend with each measurement and more variation (4.1% < RSDs <

24.2%). This result supports the theory that activation affects the surface while

non-activated cards appear relatively reproducible. Specifically, it is the reuse

used in conjunction with activation that caused large changes in active surface

area. That is, in Figure 27, only the reused and activated data displays the

upward trend.

Activation is now a common practice when using SPCE because it has

been shown to greatly improve the electron transfer kinetics of the surface and

the amenability of the surface to adsorbed species, which was a primary concern

in this research. Wang has performed a significant amount of work using this

90

activation method on SPCE,[37][38][55][73] as well as others who have used similar

methods.[29][56][63][74-77] While it has useful effects on adsorption, activation

changes the electrode surface such that it might affect the development of a

precise and reliable quantitation curve.

Figure 26 shows how the effective surface area increased with each

consecutive activation, resulting in a proportional increase in double-layer

capacitance. The activation procedure theoretically also removes any binder that

may be on the surface, thus increasing the effective area[63] and is known to

enhance the background current of CVs.[34][55][63] The EDX data show both

activated and non-activated electrodes had significant levels of chlorine, however

whether there was a post-activation decrease in the amount of chlorine could not

be determined. If the binder, signified by the presence of chlorine, is the cause of

electrode differences then pre-processing methods which go beyond typical and

traditional methods are required to stabilize the SPCE over long periods of time

or over multiple runs.

The capacitance data are consistent with the literature. For example, Cui

et al. observed that activated, bare carbon electrodes exhibit a capacitance of

between 20 and 70 µF/cm2. Their study analyzed the capacitance after activation

at multiple potentials and for varying lengths of time. A potential of +1.6 V (vs.

Ag/AgCl), applied for 0.5 min caused a capacitance of 23.05 µF/cm2 and when

applied for 2 min caused a capacitance of 38.38 µF/cm2. A potential of +2.0 V

(vs. Ag/AgCl), when applied for 0.5 min, caused a capacitance of 24.20 µF/cm2

91

and when applied for 2 min resulted in a capacitance of 41.40 µF/cm2. These

values were obtained from electrochemical impedance spectra. Capacitance

data shown in Table 6 conform to this range for the between-electrodes,

activated measurements but it must also be noted that the non-activated data,

between- and within-electrode measurements also conform. Cui et al. further

explain that prolonged application of potential (i.e. +1.2 V vs. Ag/AgCl for more

than 5 min) will cause ‘excessive’ removal of organic binders. This lends

evidence to why multiple activations and measurements on the same electrode

card further increased the capacitance, however even after 20 min at +2.0 V (vs.

Ag/AgCl) Cui et al. noted that the capacitance did not exceed 89 µF/cm2.[63] In

this research, the card that was repeatedly activated had a capacitance of

approximately 106 µF/cm2, and this was after successive 1 min activations

between runs at +1.7 V (vs. Ag/AgCl).

Another study analyzed the double-layer capacitance after a pretreatment

step. Su et al. observed an increase in capacitance from 0.7 µF to 3.9 µF after

cycling a SPCE from -0.6 V to +1.6 V (vs. Ag/AgCl) at 0.1 V/s for 40 cycles in 0.1

M PBS.[21] The nominal surface area of the SPCE was 0.2 cm2, which would

normalize the capacitances to 3.5 µF/cm2 before cleaning and 19.5 µF/cm2 after

cleaning. The value for post-cleaned electrodes is slightly less than that obtained

with the post-cleaned, non-activated Pine SPCE cards utilized in this study,

which ranged from 23.1 – 31.1 µF/cm2.

92

Figure 27 shows that when using faradaic current to calculate the active

surface area, the measured area was consistently lower than the nominal value,

except for the final activation of the reused electrode card. When α was

determined experimentally and used in the calculations, the resulting real area

was less than that when a value of α 0.5 was used, which is a generally

accepted and common assumption.[32]

3.6 Recommendations of Carbon Electrodes for Forensic Biosensors

The experiment described in the last section has confirmed results

obtained earlier that the electrode cards may, in some cases, be reused without

incurring large amounts of variation. When activation is a part of the protocol,

however, the electrodes change to a point where reproducibility is poor and

reuse is not recommended. Additionally, increased background current may

cause problems in determining signal from direct electron transfer. This research

has indicated that Pine SPCE may not be a useful choice of electrode to create a

biosensor. Carbon’s inability to create a monolayer recognition layer, low

selectivity and the need to activate the carbon to increase its sensitivity makes it

a less desirable candidate than noble metals, like gold, for forensic applications.

4.0 CONCLUSION

This research aimed to develop a method to quantify genomic DNA using

carbon screen-printed electrodes and cyclic voltammetry. The simplest

technique, the DNA stick design, was not sensitive enough to directly detect DNA

93

in solution and may be limited by diffusion effects of large genomic molecules.

Further, electrostatic adsorption appears unsuccessful for loading

oligonucleotides onto carbon because complex surface processes may be

hindering immobilization and specialized pre-processing techniques would need

to be developed to make SPCE a viable alternative for quantitative DNA analysis.

Pine carbon SPE performed quite well when used with simple and short

experiments. They can be reused without introducing a significant amount of

variation to the signal. However, the protocol used for creating a biosensor is

more complex than short experiments that measure one analyte in solution, and

the application of various steps with differing parameters (scan rate, scan range,

time, etc.) changed the electrode surfaces. In particular, activation at high

overpotentials was shown to change the electrode surface to such an extent that

bias was introduced into the resulting measurements. This step is employed in

many experiments involving carbon surfaces and biochemical molecules

because it improves probe loading. Unfortunately it also introduces confounding

variables, such as increasing background signal from the solvent (charging

current) that affect resolution and sensitivity of the assay.

Forensic DNA quantification is used with samples of very low

concentration and this type of assay is required to be highly sensitive to small

changes in concentration and have a large dynamic range. The current

technique, qPCR, fits these requirements but concentration is determined by an

external calibration curve that is subject to pipetting discrepancies and instrument

94

performance, and is prone to error due to the exponential growth of the signal.

The biosensor technique provides an alternative to qPCR and also has the

potential to determine quality of a forensic DNA sample, which will aid in

determining the appropriate downstream processing to maximize the information

obtained. Towards the development of a biosensor, Pine carbon screen-printed

electrodes are not a suitable choice for the foundation of the recognition layer.

5.0 FUTURE RESEARCH

Since carbon SPE have been shown to be sub-optimal for forensic

biosensor development, gold SPE are currently being studied. Unlike carbon,

gold SPE can be cleaned by harsher methods such as mechanical abrasion with

alumina slurries, sonication in deionized water, and cycling in sulfuric acid.

Additionally, the surface chemistry with gold electrodes provides more

confidence in the orientation of the probe oligonucleotide and its accessibility to

target molecules. The probe molecule is modified with a sulfur derivative,

typically attached via a short hydrocarbon chain.

Current work involves a TP X locus oligonucleotide with a 5’ thiol C6

attachment and a 3’ Methylene Blue attachment, 5’-S-C6-

CGGGAAGGGAACAGGACTAAG-MB-3’ (Biosearch Technologies, nc., Novato,

CA). Thiolated oligonucleotides tend to form dimers because disulfide bonds are

more stable than hydrogen-sulfur bonds. As a result the disulfide bonds must be

reduced to expose the individual sulfur atoms for attachment to the gold

electrode surface. Dithiothreitol (DTT) is a common reducing agent, the

95

mechanism of which was described by W. W. Cleland.[78] The disadvantage to

using DTT is that it must be removed prior to applying the probe to the electrode

surface, as it also contains sulfur atoms, by a process such as ethanol

precipitation. An alternative reducing agent is tris(2-carboxyethyl)phosphine

(TCEP), which can be left in the probe solution during immobilization on the

electrode. The complete reduction of the probe dimers is indicated by a color

change of the solution from blue to clear because the Methylene Blue is also

reduced to its colorless form, Leuco-Methylene Blue.

Once successful reduction is accomplished the next goal will be to

determine the suitability of gold SPE as a foundation for the recognition layer by

creating a uniform mixed monolayer on the working electrode surface. A mixed

monolayer is created by incubating the electrode with the probe and a short

thiolated hydrocarbon. The thiolated hydrocarbon is often mercaptohexanol and

helps stabilize the monolayer.[42] It is theorized that the mixed monolayer may

also provide barrier characteristics to background current caused by the solvent.

Minimization of the background current may increase resolution of peaks caused

by a hybridization event (the signal of interest). Barrier or not, the ability to

reproduce the recognition monolayer between electrode cards will be an

important preliminary step towards making a biosensor applicable for forensic

DNA quantification.

96

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7.0 CURRICULUM VITAE

CANDACE RENEE CHURINSKY

Year of Birth: 1988

769 Samuel Chase Ln.

West Melbourne, FL 32904

(303)476-8640

cchurinsky@gmail.com

EDUCATION

Colorado School of Mines Golden, CO 12/2010

B.S. Chemical and Biochemical Engineering

PROFESSIONAL EXPERIENCE

Genetic Services, Inc. Cambridge, MA 09/2011-05/2012

Laboratory Technician

Maintained Drosophila stock lines, QC for phenotypic contamination

Colorado Bureau of Investigation Lakewood, CO 06/2010-02/2011

Laboratory Intern, Biological Sciences Unit

Developed and performed a research project to determine the length of time that

male DNA persists in the female vagina after a sexual assault. Validated the

sensitivity of a room-temperature phenolphthalin blood-testing kit on swatches of

diluted sheep’s blood to determine viability at crime scenes.

STUDENT ASSOCIATIONS

American Institute of Chemical Engineers (AIChE) 08/2008-07/2010

Society of Women Engineers (SWE) 08/2006-07/2009

RELEVANT SKILLS

DNA extraction techniques: Organic, Differential, Chelex®

Instruments: AB 7500 RT-PCR System, AB 3130 Genetic Analyzer, Gamry

Potentiostat

Software: Gene Mapper, Osiris, Echem Analyst

Biological screening methods: phenolphthalin test for blood, acid phosphatase

overlay test for semen, spermatozoa search with KPIC stain and microscopy