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Novel detection methods for markers of chemical and biological warfare

Dissertation
Author: Stephanie L. Youso
Abstract:
In this project, novel methods for the detection of biological markers for cyanide, as well as anthrax, were developed. Although the methods for the detection of these two warfare agents differ, both methods provide significant benefits to current technology. First, a new method for the determination of chronic cyanide exposure was developed, and its applicability was verified with the analysis of biological samples obtained from human smokers and non-smokers. Second, the development of a novel nanoelectrode sensor for DNA fragment detection is described. Exposure to cyanide can occur in a variety of ways, including exposure to smoke from cigarettes or fires, accidental exposure during industrial processes, and exposure from the use of cyanide as a poison or chemical warfare agent. Confirmation of cyanide exposure is difficult because, in vivo, cyanide quickly breaks down by a number of pathways, including the formation both free and protein-bound thiocyanate. A simple method was developed to confirm cyanide exposure by extraction of protein-bound thiocyanate moieties from cyanide-exposed plasma proteins and subsequent derivatization with pentafluorobenzyl bromide for GC-MS analysis. Thiocyanate levels as low as 2.5 ng mL -1 and cyanide exposure levels as low as 175 μg kg-1 were detected. This method was then used to analyze plasma from 25 smokers and 25 non-smokers for protein-bound thiocyanate. The amount of protein-bound thiocyanate found in the plasma of smokers, 1.35 ± 0.51 μM, was significantly elevated (p < 0.0001) when compared to non-smokers, 0.65 ± 0.34 μM. These results indicate the effectiveness of analyzing protein-bound thiocyanate in determining instances of chronic cyanide exposure with possible extension to confirmation of acute cyanide exposure. Biological agents not only pose a danger to society, but can also be difficult to detect in a timely manner, especially during the early stages of infection. One method to successfully determine biological warfare exposure is through the detection of a DNA sequence unique to the pathogen of interest. The objective of this project was to fabricate a novel nanoelectrode device capable of detecting a specific pathogen by DNA sequence. This device utilizes conformational changes of a probe DNA sequence upon hybridization when pathogen DNA is present. The change in DNA conformation causes a change in signal, which allows detection of a specific DNA sequence. The fabrication of the final nanoelectrode was completed using standard lithography and thin film deposition techniques. A ferrocene tag was attached to the probe ssDNA to increase its electrochemical signal and the attachment was confirmed by voltammetry. Results from the device indicate that it is comparable to nanoelectrodes previously reported in literature, with a target DNA detection limit of 10 nM.

Table of Contents

Page

Dedication

……………………………………………………………………...

iii

Abstract

……………………………………………………………………... iv

List of Abbreviations

… …………………………………………………………... xi

List of Tables

… …………………………………………………………………... x ii

List of Figures

……………………………………………………………………... x i ii

vii

I.

Chapter 1. Introduction

… …………………………………………….. ..........1

A.

Overall s ignificance

... …… ……………………………… .. …... ........1

B.

History of biological and chemical warfare

…... ........... . . ……… ........2

1.

Chemical w arfare a gents… …………………………………...2

2.

Biological w arfare a gents

… ………………………………....3

C.

Exposure to cyanide…………………………………………………..4

1.

Routes

of c yanide e xposur e ……………. …. ……………... .....4

2. Metabolism of cyanide…………. …………………………… . 5

3. Direct detection of cyanide………. …… ……… ……………..6

4. ATCA detection……………… …… …… ……… …………...

10

5. Thiocyanate detection………………… … …………… …...

11

D. Determination of biowarfare exposuree …………………………… .

14

1. Biosensors……………………………

…………………… .... 14

2. Fluorescence DNA analysis………………………………… 18

3. PCR……………………………………………………………20

4. Molecular beacon technology... … ...…………………………21

5. Electrochemical DNA detection………………………………23

viii

II.

Chapter II.

Methods…… ………………………………………………………. 2 5

A.

Precipitation of proteins from biological fluids… …………………….. 2 5

B.

G as chromatography - m ass spectrometry... …………………………… 2 7

C.

Derivatization techniques for gas chromatography. .. …………………. 28

D. Electrochemical analysis………………………………………………31

1.

Mass transpo rt considerations…………………………………

32

2.

Char ge transfer…………………………………………………37

3.

DNA - based electrode application...……………………………37

D.

Thin film fabrication …… ……………… ………… … …………….….. 40

1.

Substrate preparation

…………………………………………40

2.

Evaporative deposition…………………………………… ……42

3.

Patterning………………………………………………………43

4.

Scanning electron microscopy… . ……………………………. 46

ix

III.

Chapter 3 .

Determination of Cyanide Exposure by Gas Chromatography - Mass Spectrometry Analysis of Cyanide - Exposed Plasma Proteins ……………………… 48

A.

Introduction…………………………………………………… ……..48

B.

Materials………………………………………………………… …..51

C.

Methods… ………………………………………………………… … 5 2

D.

Results and Discussion… ………………………………………… …. 5 3

IV.

Chapter 4 .

The Analysis of Protein Bound Thiocyanate in Plasma of Smokers

and Non - smokers as a Marker of Chronic Cyanide Exposure ………………………

62

A.

Introduction…………………………………………………… ……. ..62

B.

Materials………………………………………………………… ……64

C.

Methods… ………………………………………………………….. . ..64

D.

Results

and Discussion

… ………………………………………… ….66

E.

Conclusions

…………………………………………………………...72

V.

Chapter 5 . The Development of a DNA - Based Nanoelectr ode Sensor for the Detection of

Anthrax

Bacteria ……………………………………………………… ..72

A.

Introduction…………………………………………………… …… ...72

B.

Materials………………………………………………………… ……74

C.

Methods

… ……………………………………………………………74

D.

Results and Discussion

… …………………………………………….81

E.

Conclusions

and future work ……… ……………………………… ….92

x

VI. Chapter 6 .

Overall

significance of research ………………………… …….

9 4

VIII. Bibliog rap hy

……………………………………………………………... …95

x i

LIST OF ABBREVIATIONS

ATCA: 2 - amino - 2 - thiazoline - 4 - carboxylic acid

CN: Cyanide

CV: Cyclic voltammetry

DCM: Dichloromethane

DNA: Deoxyribonucleic acid

DMSO: Dimethyl Sulfoxide

GCMS:

Gas chromatography - mass spectrometry

HCl: Hydrochloric Acid

M ALDI - TOF : Ma trix Assisted Laser Desorption Ionization - Time of Flight

MW: Molecular Weight

NaOH: Sodium Hydroxide

NHS: N - Hydroxys uccinimide

PFBBr: Pentafluorobenzyl bromide

SCN: Thiocyanate

ssDNA : single - stranded DNA

TBAS: Tetrabutylammonium sulfate

TCEP: Triscarboxyethyl phosphine

TEA A: Triethylammonium acetate

xii

LIST OF TABLES

Page

Table 1 : Plasma protein - bound thiocyanate concentrations for each group and sub - group of participants. ................................ ................................ ................................ ...................

69

Table 2 : Plasma protein - bound thiocyanate concentrations found for each group of smokers (grouped by of cigarettes per day)…………… ……………………………… … 71

xiii

LIST OF FIGURES

Page

Figure 1 . A diagram of the metabolic pathway of cyanide…… ……………………..6

Figure 2 . A molecular beacon attached to a gold working electrode. ……… ... . …….17

Figure 3 . Hairpin loop DNA is used as a molecular beacon to target a complimentary strand that is indicative of DNA strand of interest. ... …… …… ………………… ….22

Figure 4. A schemat ic of derivatization reactions.

... …………… …………… …….30

Figure 5 . An illustration of diffusion in an electrochemical system. ..…………….33

Figure 6 . A diagram of the migrational flux in an electrochemical system….… . ….34

Figure 7 . An illustration of the stagnant boundary layer that exists near the electrode surface. ………………………………………………………… ………. ………….35

Figure 8 . An example of the change in appearance of redox peaks wh en an analyte is tethered to the electrode surface. … …………………………….. ………………….38

Figure 9 . Evaporative deposition schematic showing the filament vaporizing the metal to allow the formation of a thin film on the substrate……….…… ………... ………….42

Figure 10 . Schem atic of lift off technique... ……………………… ………………..44

Figure 11 . Diagram of SEM, illustrating the path of electron beam to substrate, and the secondary electrons that are ejected from the sample into a collector and photomultipler for detection…….……………………………… …………… ……………... ……….46

Figure 12 . The reaction of cysteine residues with cyanide to form thiocyanate adducts on proteins and the subsequent removal of thiocyanate under basic conditions……………………….………………………………… …… …………….50

Figure 13 . Thiocyanate extracted fro m plasma proteins as a function of time (2 minutes to 6 hours) ……………………………………………………………… ……... …….. 55

Figure 14 . The concentration of thiocyanate released from plasma proteins as the pH of hydrolysis was varied from 8 to 11 …………………………………………… ……. .56

Figure 15 . The resulting thiocyanate calibration curve used for the analysis of human plasma samples

………….………….………………………………………… ……. .58

xiv

Figure 16 . The amount of thiocyanate extracted from plasma proteins as a function of the cyanide dose

……………… ... ……………………………………… … ….. ………….58

Figure 17 . The chromatograms of extracted thiocyanate from the plasma proteins

…. 61

Figure 18 . The c oncentration of protein - bound thiocyanate found in the plasma samples

of smokers compared to non - smokers. ……………………………………………...

.. 68

Figure 19 . Individual concentrations of protein - bound thiocyanate found in the plasma of male smokers (MS), male non - smokers (MNS), female smokers (FS), and female smokers

(FNS) .. ... ………………………………………………………... ……………. 69

Figure 20 . Individual concentrations of protein - b ound thiocyanate found in the plasma samples of male

and female smoking sub - groups.

... …………………………………. 71

Figure 21 . A

diagram of the final electrode structure, consisting of thin - film layers of chromium, gold, silicon dioxide, and platinum, layered

using e vaporative deposition..77

Figure 22 . An SEM image of the finger structures on the device at 80x magnification . 78

Figure 23 . The different conditions of the sensor.

In this schematic of the sensor’s surface, the gray ball represents the ferrocene moiety, attached to the hairpin loop DNA.

……………………………………………………………………………… ………… . 80

Figure 24 . The resulting H 1 - NMR spectroscopy

of the purified ferrocene succinimide ester.

………………….. ………………………………………………………………. 82

Figure 25 . The results of the MALDI - TOF mass spectrometry performed on the ferrocene tagged DNA ………… … ……………………………………………………………… 83

Figure 26 . Untagged DNA was analyzed by HPLC, as illustrated in (A). No significant abso rbance was observed at 440 nm. The untagged DNA eluted at approximately 7.0 minutes, as seen at 260 nm. In (B), HPLC analysis of ferrocene tagged DNA can be seen. Simultaneous absorbance at 260 nm and 440 nm is observed. ……………….. .99

Figure 27 . Analysi s of the electrolyte solution after the DNA was electrochemically removed from the working electrode.

…………… .. …………………………………. 87

Figure 28. The results from cyclic voltammetry indicate the improvement of the signal when the pH of the electrolyte solution was

increased to 9 (red) relative to the peak resulting when the pH of the electrolyte solution was neutral (blue)…………………88

xv

Figure 29. The observed CV when the ionic strength of the electrolyte was decreased to 0.1 M. When the strength of the electrolyte was

decreased to 0.1 M (red line) the resulting current signal is decreased, when compared to the original 1 M electrolyte concentration (blue)…………………………………………………………………89

Figure 30. An example of observed voltammetry results (anodic scan) of the ferrocene ta gged DNA attached to the final electrode device, when different concentrations of complementary DNA were introduced……………………………………………..91

1

I. Chapter 1. Introduction

A. Overall

Significance

Throughout history, chemical and biological weapons have been used to weaken enemies. By simply poisoning the enemy’s water supply, ancient Greeks and Romans attempted to gain an advantage during battle. During t he R enaissance in Europe, soldiers found that throwing arsenic powder into the enemy’s ship would induce asphyxiation to all that inhaled it

1 - 3 . Chemical and biological weapon concepts began to advance

and dev elop during World War I

when the German army began to develop strains of biological weapons, such as anthrax, glanders, and cholera for use in war. In 1925, the Geneva protocol was signed by 108 nations, who agreed to prohibit the use of chemical and biol ogical agents. However, by World War II, the Japanese had developed research facilities to cultivate

their understanding of biologic al agents

such as anthrax, syphilis, and plague. This was followed by British testing of anthrax bombs on Gruinard Island

1 ,

2

and also included the US developing their own research facilities.

Recent examples of chemical or biological warfare attacks are the 2001 attack on Tom

Daschle and others who

received letters in the mail laced with anthrax powder

3

and the

use of chemical warfare

agents during

the Iraq - Iran conflicts in the 1990s

4 ,

5 .

Currently ,

a ttack s,

as well as the dev elopment of additional agents ,

are

becoming

more sophisticated and developed. Therefore, q uick and efficient detection systems are necessary

to identify an exposure.

2

B. History of Chemical and Biological Warfare

1. Chemical war fare agents

A c hemical warfare agent (CWA) is any chemical or toxin that can be harnessed to damage an enemy’s forces. They are categorized into four different types, based on the mechanism of action. These categories are: blister agents, blood agents,

choking agents, and nerve agents

6 .

The first category, b lister agents ,

can cause severe chemical b urns to the skin or lung s

7 . Blister agents are more dense

than air and are readily absorbed through skin or mucosal membranes.

Examples of these agents include sulfur mustard and lewisite. B lood agents ,

including cyanide (the focus of this dissertation)

interfere with cellular respiration processes

and metabolic processes in the circulatory syste m.

By preventing a sufficient amount of oxygen to tissues, they eventua l l y cause

respiratory failure.

C hoking agents , including chlorine and phosgene,

damage lung tissue

and interfere

with the lung - blood barrier

8 . Due to their interference with respiratory activities, choking agents can induce asphyxia, causing chemical suffocation. Finally, nerve agents bind to acetocholinesterase ,

which then

prevent s

healthy interaction

between nerve

impulses in the central and p eripheral nervous system

9 ,

8 .

This leads to an accumulation of acetocholine , which triggers

an excited state, causing muscle exhaustion and eventual respiratory failure.

Nerve

agents include

the compounds:

sarin, soman, and VX

9 . Although all c onsidered CWA s,

the y

vary greatly in regards to their potency and danger .

The first recor ded use of a chemical weapon was during World War I on the battlefields of Europe

8 .

During the Cold War,

chemical weapons stock piles grew in the

3

Soviet Union, and at their peak ,

they accumulated almost twenty thousand tons of nerve and blister agents

10 . Since then, reports of

chemical warfare have surfaced during the Iraq - Iran conflict, as well as by Bosnian Serb forces during conflicts in 1995

5 ,

11 . Finally, in 19 93 , a treaty called the 1993 Chemical Weapons Convention was signed by more than 125 countries, agreeing to eliminate

chemical weapons

stockpiled in each country within ten years

12 .

2. Biological warfare a gents

Biological warfare agents (BWA s ) are defined

to be any bacteria, virus, or resulting toxin from a bacteria or virus,

that is easily spreadable and can infect and weaken or kill large groups of people

13 ,

5 . They are classified di fferently than chemical weapons .

I nstead of being organized by their mechanism of action, biological weapons are categorized

into three classes

by their potential for harm.

Category

A is considered the h ighest risk category as

they

can cause the most danger in a short time period

13 ,

14 . These BWAs are either easily spread from person to person or can

pose a significant danger when the exposure occurs.

Category B

agents are considered a moderate risk to people and are less

eas ily

spread

from person to person

13 ,

14 . Category C agents are

considered emerging pathogens that have the potential to cause threat

because they are widely available and could be engineered

in a laboratory

to become biological warfare agent s

13 ,

14 .

The use of biological warfare has been noted throughout history. For example,

ancient Roman battles, armies would dip their arrows in decaying carcasses of animals so as to incapacitate those on the receiving line of attack with infection

5 .

4

Due to the continuing threat of biological and chemical war fare attacks as well as increased public awareness of the potential for these attacks

15 , more efficient and sensitive methods of detection are required.

C.

E xposure to cyanide

1.

Routes of cyanide exposure

Outside of

chemical warfare attacks, there

are many other avenues of cyanide exposure. When cigarette smoke is inhaled, either by the smoker or through inhalation of second - hand smoke, a small dose of cyanide is also breathed into the lungs

16 .

Smoke from fire s , such as forest fires or house fires, contain s

many different products of combustion, including hydrogen cyanide gas

17 .

Further, f ires involving nitrogen -

containing compounds, such as acrylonitrile, can produce significant amounts of hydrogen cyanide during c ombustion. In 1984, Barillo et

al. found toxic levels of cyanide in the blood of 31 out of 384 victims of house fires

18 . Cyanide is also utilized in certain industries, such as gold and silver mining and metallurgy processes, and can cause chronic health problems for those workers who incur prolonged exposure ,

even to low levels

19 . Workers involved in other industries such as pe sticide production, nylon and other synthetic fabrics production, as well as pharmaceutical industries ,

also has

the potential for chronic cyanide exposure. Workers in this field are exposed to low levels of cyanide, which can induce health problems over time.

Chronic exposure

to cyanide

can induce serious health problems , includ ing

enlargement of the thyroid gland, and myelin deterioration

19

and t he Environmental Protection Agency (EPA), has a

chronic cyanide exposure

limit of 30 mg mL - 1

per day

20 .

5

2. Metabolism of cyanide

Due to significant and long - lasting dangers of cyanide exposure, methods to detect this exposure need to be timely,

specific , and sensitive . Cyanide is a very reactive nucleophile, and h as a half - life of less than one hour

when introduced into the body

21 . In vivo , cyanide readily reacts with sulfur - containing moieties, such as thiosulfate and the enzyme, rhodanese, to form its major metabol ite, thiocyanate (SCN - )

22 ,

23 ,

24 ,

25 ,

26 . Another metabolite 2 - amino - 2 - thiazoline - 4 - carboxylic acid (ATCA) can also be formed when cyanide reacts with cystine

27 .

A schematic

of

the major

cyanide metaboli c pathways

is provided in Figure 1. Figure 1 illustrates that 80% of cyanide is converted into thiocyanate by the reaction of sulfur containing compounds as the major metabolic pathway (bold). A

minor metabolic pathway is also shown, where cyanide reacts with cystine to produce ATCA.

6

Figure 1 . A diagram of the metabolic pathway of cyanide. The bold reaction illustrates the formation of thiocyanate, the major metabolic pathway of cyanide.

Currently, there are many methods of cyanide detection. These include methods of detecting cyanide directly, or by detecting one of its metaboli tes, including thiocyanate and ATCA. The following literature review will discuss significant accomplishments in the detection of cyanide as well as its metabolites, specifically, ATCA and thiocyanate.

3. Direct detection of c yanide

Direct detection

of

cyanide

in biological fluids

is

very challenging

because of its reactivity and volatility. Due to the reactivity of this analyte, it is difficult to quantitate it

successfully

after processing a biological

sample

28 ,

29 .

Cyanide also cannot be analyzed

7

directly

with traditional methods such as UV - vis spectroscopy or fluorescence spectroscopy. Other techniques such as mass spectrometry are difficult to apply to directly analyze cyanide because of its low molecular weight. However, there have been

successful dev elopments in the area of direct cyanide detection from biological fluids. Despite difficulties in the analysis of cyanide, t here are a myriad of methods developed to detect cyan ide from

biological fluids. T hese methods on a whole have illustrated success in this area, but also indicated the limitations that arise when analyzing cyanide directly. Although there are too many methods to allow each to be summarized, some significant

advances

are discussed

below .

Optical detection of cyanide has been examined to develop quick and efficient

detection systems. Typically , cyanide’s ability to bind to different metal complexes is exploited

in spectrophotometric detection methods . For example, in 2002, Kim et

al .

30

discussed the development of a metal complex that contained a zinc - porphyrin Lewis - acid binding site and a crown ether Lewis base moiety, where a color change from orange to green indicated the binding of cyanide. It was

proposed that this color change occurred

due to the ditopic binding of cyanide, rather than the monotopic binding of cyanide to o ther molecules such as sodium. This study provided a time efficient method of cyanide determination.

In 1980, Morgan and Way utilized the catalytic conversion of pyridoxal to 4 - pyridoxylactone for the detection of cyanide

31 . Cyanide interferes with this conversion, and because 4 - pyridoxylactone is fluorescent, a decrease in fluorescence was observed when cyanide was present. The application of this method to the analysis of blood from

8

cyanide exposed mice demonstrated its

applicability in determining cyanide exposure. After optimization of thi s method, detection limits of 100

n g/mL could be attained.

In another spectrophtometric technique, Laforge et al . 32

utilized the interaction between cyanide and hydroxocobalamine t o develop a relatively sensitive method for cyanide detection, with reported detecti on limit of approximately 70 n g/mL. The addition of hydroxocobalamin induced a color change when the complex was formed, with a maximum absorbance at approximately 350 nm.

The application of heat increased the intensity and the resulting sensitivity of this method for the detection of cyanide. This method demonstrated

a timely and

specific method to detect

cyanide.

In 2000, Cruz - Landeira et al. successfully attempted to improve the spectrophotometric analysis of cyanide with hydroxocobalamin by the purging of solutions with nitrogen gas

32 . This promoted the release of additional cyanide resulting in increased formation of the complex. This application decreased the

detection limit of this method to 28 ng / mL.

HPLC analysis of cyanide directly from biofluids has also been a significant area of research in this field. Due to the reactivity of cyanide, metabolites are quickly formed

21 ,

28 , resulting in large variability in the concentration of cyanide found in

samples.

D irect d etection of cyanide by traditional HPLC detectors can be difficult, as cyanide does not have

a chromophore or fluorophore. Therefore ,

successful analysis requires

converting or derivatizing the cyanide . In one such study, Cox et

al .

33

converted free cyanide into a nickel - cyanide complex by filtering

samples through a column containing Ni(OH) 2

to form

the complex

Ni(CN) 4 2 - . This complex could then be analyzed using an HPLC system with UV detection

at 268 nm. This method provided low standard

9

deviations and a detection limit of 0.08 mg/L. In another

study, Tracqui et

al .

34

employed microdiffusion techniques to complex cyanide with a mixture of tuarine and naphthalene - 2,3 - dicarboxaldehyde

(NDA)

to form a 1 - cyano - benzoisoindole derivative. This derivative could then be analyzed using HPLC coupled with a fluorescence detector. This method is selective and studies have shown there are little interfering compounds. It also demonstrates significan t sensitivity with a

detection limit of 5 ng/mL.

Another detection method applies the selectivity

of capillary electrophoresis coupled with fluorescence detection to detect cyanide from human blood

was developed by Chinaka et al .

in 2001

35 . Capillary electrophoresis was applied after derivatizing cyanide with 2,3 - naphthalenedialdehyde and taurine. After this separation was complete, fluorescen ce detection was employed with

a 418 nm excitation wavelength and

460 nm emission wavelength. This method was simple, selective, and resulted in a 0.1 ng/mL detection limit. Human blood was analyzed with this method to demonstrate its appli cability to biological samples . A concentration of 9.5 ng/mL of cyanide could be detected in the human sample. In 2005, Dumas et al .

36

coupled

h eadspace

(HS)

GC with

nitrogen - phosphorus detection to detect cyanide from biological samples . In this method an isotopic ally - labeled

internal standard of K 13 C 15 N

was used to obtain consistent results for the HS/GC analysis of samples . This reduced the large standard dev iation observed by Seto et al.

37

and produced

a detection limit of 8 ng/mL.

In addition to nitrogen - phosphorus d etection, electron capture detection (ECD) has been used with headspace gas chromatography. Zhang et al .

reported a method for cyanide determination in human tissue with ECD detection

38 . This mode of detection produced a detection limit of

10

approximately 0.5 ng/mL, demonstrating the selectivity that can be obtained with this detector. However, large variation was observed during the analysis of the different tissue samples obtained for this study and linearity was lost above 3 µg/mL.

4.

ATCA detection

Most

methods to detect ATCA involve HPLC, GC - MS, and UV - vis spectrometry , and generally exhibit low sensitivity when compared to other cyanide detection methods .

In 1995, Lundquist et

al

39

reported a novel HPLC method for analysis of ATCA. In this work, ATCA was converte d to N - carbamylcysteine and

analyzed using HP LC with a fluorescence detector

after several sample preparation steps had been completed. These sample preparation steps included the use

of

a cation exchange column to remove interfering substances. Secondly , intereferring disulfide molecules were removed by reduction to

a thiol group, followed by

adsor ption of

the resulting thiols to an organomercurial adsorbent to prevent any interference

during the analysis of ATCA . This method demonstrated sensitivity with a

detection limit of

43 ng/mL

ATCA and a standard deviation

of approximately 3.6%. This study also d emonstrated the significant stability of ATCA; stability over 3 months at 20

˚C was observed.

In 2005,

Logue et al .

27

reported the development of a sensitive method employing GC - MS to detect ATCA levels as low as 25 ng/mL. By working with synthetic urine and swine plasma, a sensitive method to detect ATCA in biological fluids was developed and optimized. In this method, samples were prepared for analysis using a mixed - mode cation exchange column. Next ,

the samples were ev aporated to dryness and derivatized with the silating agent N - methyl - N - trimethylsilyl - trifluoroacetamide (MSTFA) in

11

preparation for GC - MS analysis. To demonstrate the applicability of

this method, urine samples were obtained from human smokers and non - smokers and

analyzed for ATCA. Elevated levels of ATCA were seen in the urine of smokers relative to the levels found in the urine of non - smokers. The difference in ATCA concentrations found in the urine of smokers relative to n on - smokers was found to be significant at

a 99 .9 % confidence level. This study also indicated the effectiveness of ATCA as a biomarker for cyanide exposure, as the levels found in smokers were signif icantly elevated when compared to levels of non - smokers.

This results from the analysis of human urine also indicated that there is a large amount (85 ± 47 µM) of endogenous ATCA present, as seen in the levels of the non - smoking subjects.

A novel spectrophotometric analysis of

ATCA was reported by Baskin et

a l .

40

in 2006. By opening the thiazoline ring of ATCA, and reacting it with diphenylthiocarbazone, ATCA could be detected quickly at 625 nm. This method allowed for a sensitive and time efficient detection of ATCA as a marker f or cyanide exposure. Baskin et

Full document contains 121 pages
Abstract: In this project, novel methods for the detection of biological markers for cyanide, as well as anthrax, were developed. Although the methods for the detection of these two warfare agents differ, both methods provide significant benefits to current technology. First, a new method for the determination of chronic cyanide exposure was developed, and its applicability was verified with the analysis of biological samples obtained from human smokers and non-smokers. Second, the development of a novel nanoelectrode sensor for DNA fragment detection is described. Exposure to cyanide can occur in a variety of ways, including exposure to smoke from cigarettes or fires, accidental exposure during industrial processes, and exposure from the use of cyanide as a poison or chemical warfare agent. Confirmation of cyanide exposure is difficult because, in vivo, cyanide quickly breaks down by a number of pathways, including the formation both free and protein-bound thiocyanate. A simple method was developed to confirm cyanide exposure by extraction of protein-bound thiocyanate moieties from cyanide-exposed plasma proteins and subsequent derivatization with pentafluorobenzyl bromide for GC-MS analysis. Thiocyanate levels as low as 2.5 ng mL -1 and cyanide exposure levels as low as 175 μg kg-1 were detected. This method was then used to analyze plasma from 25 smokers and 25 non-smokers for protein-bound thiocyanate. The amount of protein-bound thiocyanate found in the plasma of smokers, 1.35 ± 0.51 μM, was significantly elevated (p < 0.0001) when compared to non-smokers, 0.65 ± 0.34 μM. These results indicate the effectiveness of analyzing protein-bound thiocyanate in determining instances of chronic cyanide exposure with possible extension to confirmation of acute cyanide exposure. Biological agents not only pose a danger to society, but can also be difficult to detect in a timely manner, especially during the early stages of infection. One method to successfully determine biological warfare exposure is through the detection of a DNA sequence unique to the pathogen of interest. The objective of this project was to fabricate a novel nanoelectrode device capable of detecting a specific pathogen by DNA sequence. This device utilizes conformational changes of a probe DNA sequence upon hybridization when pathogen DNA is present. The change in DNA conformation causes a change in signal, which allows detection of a specific DNA sequence. The fabrication of the final nanoelectrode was completed using standard lithography and thin film deposition techniques. A ferrocene tag was attached to the probe ssDNA to increase its electrochemical signal and the attachment was confirmed by voltammetry. Results from the device indicate that it is comparable to nanoelectrodes previously reported in literature, with a target DNA detection limit of 10 nM.