Download - Dounin Vladimir v 201011 MSc Thesis
Innovative Approaches for the Electrochemical
Detection of Acetylcholinesterase Inhibitors
Vladimir Dounin
A thesis submitted in conformity with the requirements for
the degree of Master of Science
Graduate Department of Chemistry
University of Toronto
© Copyright by Vladimir Dounin (2010)
ii
Innovative Approaches for the Electrochemical Detection of
Acetylcholinesterase Inhibitors
Vladimir Dounin
Master of Science
Department of Chemistry University of Toronto
2010
Abstract
This document describes research conducted during 2009-2010 in the Kerman
Group laboratory at the University of Toronto Scarborough to investigate the
application of electrochemical techniques for the detection of acetylcholinesterase
inhibitors in aqueous samples. Two main projects were completed and are
discussed herein. The first project demonstrated that the new unmodified, nano-
structured gold disposable electrochemical printed (DEP) chips produced by
BioDevice Technology can compete with surface-modified electrode configurations
to detect trace concentrations of insecticides. This was achieved through the
measurement of acetylcholinesterase-catalyzed production of thiocholine after
incubation of the enzyme with low concentrations of paraoxon (10 ppb) and
carbofuran (8 ppb). The second project featured the novel application of a glassy
carbon (GC) electrode to monitor the changes in availability of Thioflavin T (ThT) for
oxidation at the electrode surface, which is non-linearly modulated by the presence
of acetylcholinesterase and the enzyme’s pre-treatment with trace concentrations of
paraoxon and carbachol.
iii
Acknowledgments
I would like to thank my Supervisor, Professor Kagan Kerman, for all of his support
and guidance throughout this past year of exciting research and for many good
memories. My time as a Masters student with Professor Kerman has proved to be
an invaluable experience where I have learned a great deal not only about research
in electrochemistry but also about the interface of research with industry, academic
careers, education, and everyday life in this world around us.
None of my graduate study experience with the Department of Chemistry would
have taken place without the recommendation of Dr. Svetlana Mikhaylichenko, who
has been one of the most influential people in my life and a great source of advice,
encouragement, and a great role model since my undergraduate years.
I would also like to extend my thanks to all of the students in the Kerman group –
particularly those who I have had the privilege of working with the most: Anthony,
Andrea, Christopher, Tiffiny, and Vinci – for being key players in keeping a very
productive working environment in the group that is full of lively discussions,
inspiring ideas, and much-needed and well-timed humour. I have been fortunate to
be associated with such a talented group of people and I am certain that this talent
will lead them to many great accomplishments.
Furthermore, I would like to thank the administrative staff in the University of
Toronto’s Department of Chemistry at the St. George Campus, the Department of
Physical and Environmental Sciences at the Scarborough Campus, and of course
the School of Graduate Studies for their patience and support throughout my time as
a graduate student. Finally, a grateful thank-you to Professor Ulrich Krull - the
second reader of this document. Professor Krull’s teachings in his Chemical Sensors
course have really put into perspective the many possibilities that exist (and have
yet to be discovered) for the construction of a sensor and the logical steps that can
be taken in the process.
I wish everybody the best in moving forward from here to greater achievements and
successes in all future enterprises.
iv
Table of Contents
Abstract ii
Acknowledgments iii
List of Figures v
Commonly Used Abbreviations vii
1. Introduction 1
1.1 Towards the Detection of Acetylcholinesterase Inhibitors 1
1.2 Biological Sensing 2
1.3 Acetylcholinesterase Inhibitors: The Target Analytes 3
1.4 The Biological Recognition Element 6
1.4.1 Acetylcholinesterase 6
1.4.2 Operation of the Biological Recognition Element 8
1.5 Literature Review of Acetylcholinesterase Inhibitor Detection 11
1.6 Electrochemical Transduction: Voltammetry 19
1.6.1 Electrode Materials: Manufacturing Process,
Characteristics, and Uses
22
1.7 Literature Review: Electrochemical Transduction for Detection of
AChE Inhibitors
25
1.8 Contributions of this Research Work 29
2. Results and Discussion 31
2.1 Project #1: Gold disposable electrochemical printed chips for the
analysis of acetylcholinesterase inhibition using differential pulse
voltammetry
31
2.2 Project #2: Electrochemical Detection of Thioflavin T’s Interaction
with the Acetylcholinesterase Peripheral Binding Site: Application to
the Detection of Acetylcholinesterase Inhibitors
44
2.3 Concluding Remarks and Future Directions 55
3. Experimental Description and Supporting Material 58
3.1 Project #1: DEP gold chips for the analysis of AChE inhibition
using DP voltammetry
58
3.2 Project #2: Electrochemical Detection of ThT’s Interaction with the
AChE Peripheral Binding Site: Application to the Detection of AChE
Inhibitors
61
4. Literature Cited 73
v
List of Figures
CHAPTER 1
Figure 1.1 Illustration of a general biosensor flowchart 2
Figure 1.2 Chemical structures for organophosphate and carbamate AChE
inhibitors
4
Figure 1.3 Mechanisms of AChE inhibition by paraoxon and carbofuran 5
Figure 1.4 Chemical formula for the AChE-catalyzed breakdown of ACh 6
Figure 1.5 An illustration of the AChE enzyme structure for visualization 7
Figure 1.6 General reaction scheme of OPH function of an organophosphate 8
Figure 1.7 Diffusion versus kinetic controlled conditions for enzyme function 10
Figure 1.8 An illustration of the screen-printing process to make disposable
electrochemically printed chips
23
Figure 1.9 An illustration of masks that can be used in the screen-printing
process
24
CHAPTER 2
Figure 2.1 Photograph and diagram of BioDevice Technology gold DEP chip 31
Figure 2.2 DP voltammograms of 2.1 mM ATCh on gold DEP chip 34
Figure 2.3 DP voltammograms of a solution of 0.5 nM AChE and 2.1 mM
ATCh on a gold DEP chip after a 10 min incubation
34
Figure 2.4 Comparison of DP voltammograms after ATCh incubation with
AChE that has or has not been pre-treated with 50 ppb carbofuran
36
Figure 2.5 Calibration plots for paraoxon and carbofuran obtained for the
ATCh-AChE biosensor using the gold DEP chips
37
Figure 2.6 Background DP voltammogram measurements for milk and river
water real samples
38
Figure 2.7 Comparison DP voltammograms of AChE and ATCh after a 10 min
incubation in real samples
39
Figure 2.8 Illustration of a proposed biosensor design featuring AChE
attached to magnetic microbeads
43
Figure 2.9 Chemical structure of ThT 44
Figure 2.10 Experimental setup of the three-electrode system used to conduct
experiments with ThT
45
Figure 2.11 DP voltammogram of 280 nM ThT on GC electrode 45
vi
Figure 2.12 Calibration plot of ThT oxidation peak current concentration
dependence
46
Figure 2.13 Calibration plot of 280 nM ThT oxidation peak current pH
dependence
48
Figure 2.14 Calibration plot of 280 nM ThT oxidation peak current dependence
on AChE concentrations
49
Figure 2.15 Comparison of ThT and BTA-1 chemical structures 50
Figure 2.16 Sample DP voltammograms illustrating changes in 860 nM BTA-1
oxidation peak currents in the presence of AChE and paraoxon
51
Figure 2.17 Calibration plots for the ThT-based AChE inhibitor biosensor and
DP voltammograms illustrating changes occurring to ThT oxidation
peak currents during measurements
52
Figure 2.18 Illustration of ThT oxidation at the GC electrode surface while in the
presence of AChE and inhibitor molecules
53
CHAPTER 3
Figure 3.1 Flowchart representation of procedural steps completed during
measurements for the gold DEP chip research
60
Figure 3.2 Comparison of DP voltammograms for 280 nM ThT taken directly in
solution or after a wash step in PBS buffer
64
Figure 3.3 Comparison of DP voltammograms for a solution of 280 nM ThT
and 12.5 nM AChE taken directly in solution or after a wash step in
PBS buffer
64
Figure 3.4 Comparison of CVs for 20 mM ferri-ferrocyanide on a GC electrode
in PBS buffer and in solution with 12.5 nM AChE
66
Figure 3.5 Comparison of CVs for 20 mM ferri-ferrocyanide on a GC electrode
in solutions with varying AChE concentrations
67
Figure 3.6 Comparison of CVs for 20 mM ferri-ferrocyanide on a GC electrode
in PBS buffer versus a solution of 100 ppm carbachol
69
Figure 3.7 Comparison of CVs for 20 mM ferri-ferrocyanide on a GC electrode
in a solution of 12.5 nM AChE versus a solution of 12.5 nM AChE
with 100 ppm carbachol
70
Figure 3.8 Comparison of CVs for 20 mM ferri-ferrocyanide on a GC electrode
in a solution of 200 nM AChE versus a solution of 200 nM AChE
with 280 nM ThT
71
vii
Commonly Used Abbreviations
ACh: Acetylcholine
AChE: Acetylcholinesterase
ATCh: Acetylthiocholine
BTA-1: Benzothiazole-1
Ch: Choline
CNT: Carbon nanotubes
CV: Cyclic voltammetry
DEP chips: Disposable electrochemical printed chips
DPV: Differential pulse voltammetry
FET: Field effect transistor
GC electrode: Glassy carbon electrode
NMR: Nuclear magnetic resonance
OPH: Organophosphorous hydrolase
PBS: Phosphate buffer solution (saline)
TCh: Thiocholine
ThT: Thioflavin T
1
1. INTRODUCTION
1.1 Towards the Detection of Acetylcholinesterase Inhibitors
The acetylcholine (ACh) neurotransmitter performs very important functions in the
peripheral and central nervous systems. The presence of the ACh neurotransmitter
is primarily regulated by the acetylcholinesterase (AChE) enzyme, whose native
function is to cleave ACh into choline and acetate. AChE inhibitors disrupt ACh
regulation and consequently promote elevated levels of ACh in nervous and muscle
tissues with complex physiological effects1-3. Some of these effects are understood
well enough to warrant medicinal uses of AChE inhibitors such as in Alzheimer’s
disease treatments. Critical elevation of ACh produces a fatal disruption of the
nervous system. This fact has led to the successful introduction of AChE inhibitors to
maximize crop yields in agriculture by killing insects and other pests that destroy
crops. The use of AChE inhibitors in warfare has also occurred such as with the
infamous Sarin gas, which was dispersed by a radical religious cult to target
innocent people in the 1995 “Sarin Subway Incident” in Tokyo4. Furthermore, poor
control of AChE inhibitors used in agriculture has resulted in many accidental
poisonings and the introduction of these chemicals into water and food resources.
The effects of consuming AChE inhibitors at sub-critical concentrations over
prolonged periods of time are currently poorly understood. Existing data, particularly
on organophosphate AChE inhibitors, suggests that there is a range of sensitive
non-AChE targets in living systems5 in addition to AChE and that the
phosphorylation of these additional targets leads to detrimental effects in the fields
of development and behaviour6. Regulatory bodies in Europe and North America
have limited the maximum allowable concentration of many AChE inhibitors in food
and water to be at most in the mid-ppb range and usually at 20 ppb or lower7-9.
Early efforts towards the detection of AChE inhibitors were traditionally
accomplished through the chromatographic separation of samples followed by
analysis through nuclear magnetic resonance and mass spectrometry10. These
approaches provide accurate determination of AChE inhibitor identities and can
quantify their sample concentrations precisely. However, these traditional
2
approaches take hours to complete, need sample preparation steps, and require a
skilled operator to run the appropriate test procedures11. In modern times, alternative
detection methods continue to be developed in the form of biosensors that are
relatively cost-effective, sensitive, and offer rapid analysis of samples for AChE
inhibitors with a minimal amount of required training of personnel. This chapter will
provide an overview of biological sensing for these inhibitors before proceeding on
to the details of the featured research completed in the Kerman Group laboratory in
2009-2010.
1.2 Biological Sensing
The term ‘biosensor’ describes a device designed for the detection of a particular
analyte through a measurable interaction of the analyte with a biological recognition
element. The biological recognition element can consist of structures that are
relevant to living organisms, ranging from entire cells to complicated proteins or
even to something as small as a short oligonucleotide. The interaction of analyte
with the biological recognition element can be detected through a suitable
transducer, which is a device integrated into the biosensor that can respond to the
biological interaction events in the form of a change in some measurable quantity,
usually presenting itself as an electrical signal 12. The signal from the transducer can
then be amplified and processed for interpretation by the end-user in analog or
digital formats13.
Figure 1.1. A general flowchart representation of biosensor operation.
The choice of the biological recognition element and transducer ultimately
determines the capacity of the assembled device for biological sensing in terms of
3
the simplicity of use, assembly and operation costs, portability, and the overall
performance of measurements in selectivity, sensitivity, speed, and stability.
The characteristics of the biological recognition element are most important in
determining the biosensor’s performance in selectivity – the capacity to detect target
analytes without significant signal interference from non-target analytes – and
specificity – the capacity to only detect the target analyte in the presence of non-
target analytes13. The detection occurs either through a selective binding event,
such as an interaction of an antigen with an antibody, or through a selective reaction
such as that observed for enzymes and their substrates12. Sensitivity and sensor
measurement speed are usually inversely related due to the limiting rates of the
interactions taking place at the biological recognition layer. Finally, the biological
recognition element may undergo degradation and structural changes over time,
which affects stability and reusability. Immobilization of the biological component is
possible using a variety of approaches, including gel entrapment, adsorption,
membrane confinement, and chemical functionalization13. Meanwhile, the
technology that is used for the transducer limits the sensitivity and portability of the
biosensor. Technological improvements in transducer development can improve
sensitivity and allow for smaller sensor size for better portability. The transducer also
affects the speed of measurements made with the biosensor since some transducer
technologies take longer to work than others.
1.3 Acetylcholinesterase Inhibitors: The Target Analytes
AChE inhibitors are chemical or biological compounds that can interact with the
AChE enzyme to inhibit its function of breaking down ACh. A variety of chemical
structures show AChE-inhibiting activity. Several different categories of AChE
inhibitors exist, of which the most popular are the carbamates and
organophosphates for their applications in agriculture. The carbamate category
includes a variety of compounds that contain the carbamate ester functional group.
4
In contrast, organophosphate AChE inhibitors are ester derivatives of phosphoric
acid. The chemical structures of these two categories of inhibitors are shown below.
Figure 1.2. General chemical structures for the most popular categories of AChE inhibitors,
namely organophosphates (left) and carbamates (right).
The routes of entry for AChE inhibitors are through ingestion, absorption, and
respiration. The absorption of these compounds into the body results in the targeting
of AChE enzymes that exist in the muscle, blood, and nervous system14. As the
AChE inhibitor encounters an AChE enzyme, it usually interacts with the enzyme’s
catalytic site, which is located at the bottom of a deep and narrow gorge15. This
interaction can be irreversible with the covalent modification of a serine residue in
the catalytic site or can be reversible if the interaction is based on temporary affinity
binding. For example, the carbamate class of AChE inhibitors reacts with the serine
residue through a carbamylation reaction, which transfers the methylcarbamoyl ester
group to the serine. In contrast, the organophosphate class of AChE inhibitors
undergo a phosphorylation reaction with the same serine residue, leaving a
phosphate group attached.
Phosphorylation of the serine residue is not readily reversible without an antidote
such as 2-pralidoxime (2-PAM) and becomes permanent within 10 h of exposure as
“aging” – the process of dealkylation at the attached organophosphate’s R groups –
takes place16. In this process, the alkoxy-O-P bonds of the attached
organophosphate are broken and replaced with weaker hydroxy leaving groups
either through a general acid catalyzed nucleophilic substitution reaction or assisted
by stabilization of leaving groups by amino acids within the catalytic site of the
enzyme17. In contrast to what happens with organophosphates, a carbomylated
5
serine is very unstable and undergoes hydrolysis in a matter of hours to regenerate
the serine residue18.
Figure 1.3. Mechanisms involved in the action of paraoxon (an organophosphate AChE
inhibitor) and carbofuran (a carbamate AChE inhibitor) on AChE’s active site serine residue.
Apart from carbamates and organophosphates, other types of AChE inhibitors
include phenanthrene, piperidine, and indanone. Their mechanisms of action are
affinity-based with ionic and hydrogen bond interactions between functional groups
and amino acid residues in the catalytic site of AChE. In addition to the catalytic site,
the peripheral binding site exists near the entrance to the gorge that leads to the
catalytic site. It is another possible target for AChE inhibitors that function by
blocking the entrance of ACh to the enzyme’s catalytic site. Inhibitors that interact
with the peripheral site include small molecules such as propidium and also peptide
toxins like fasciculin15. Certain compounds have also been specifically designed for
medicinal purposes to interact weakly with both the catalytic site and peripheral
binding site, such as Donepezil for Alzheimer’s disease treatment.
6
The toxicity of AChE inhibitors to an organism varies depending on several factors
including the chemical structure of the inhibitor and the species variant of the AChE
enzyme exposed to the inhibitor. In general, AChE inhibitors that target the catalytic
site have a toxicity that is primarily determined by the conformational freedom of the
leaving group that is removed during the alkylation step and also by the inhibitor’s
hydrophobicity. These properties determine the accessibility of the catalytic site to
the inhibitor, since the catalytic gorge consists largely of hydrophobic amino acid
residues19. Variations in the DNA sequence encoding for the AChE enzyme between
different species can also make particular AChE enzymes more susceptible to
certain AChE inhibitors than others. This has been exploited for the production of
AChE mutants that can be applied for the purpose of AChE inhibitor detection with
some degree of selectivity for particular inhibitor structures.
1.4 The Biological Recognition Element
1.4.1 Acetylcholinesterase
The AChE enzyme has an asymmetric, usually globular ellipsoidal protein structure
that consists of a large central α/β-sheet core with 8 β-sheets that are connected to
one another by α-helices, which designates it structurally as a α/β-fold enzyme20.
This central core is surrounded by another 15 α-helices21. The primary function of
AChE is to break down the neurotransmitter ACh into choline (Ch) and acetate at
cholinergic synapses as indicated in the following equation
Figure 1.4. The AChE-catalyzed cleavage reaction of acetylcholine (ACh) into choline (Ch)
and acetate.
7
The AChE enzyme appears most abundantly in a tetrameric form with average
dimensions of 25 x 18 x 1.6 nm (~720’000 Å3)21. Each enzyme has an active site
volume of ~300 Å3 at the bottom of a ~20 Å deep hydrophobic gorge. The gorge is
lined with mostly aromatic amino acid residues along with a few acidic residues,
which are known to affect the affinity of AChE enzymes from different species to
their substrates and inhibitors20. Near the edge of this hydrophobic gorge lies an
anionic peripheral binding site, whose amino acid residues form an electric field that
attracts the cationic acetylcholine substrate into the gorge and down towards the
active site with the help of dipole-dipole interactions with the aromatic amino acid
residues20.
Figure 1.5. An illustration of the AChE enzyme’s structure for visualization purposes, with
the green area representing the active site and a yellow molecule shown occupying the
peripheral binding site. X-ray diffraction results showing the interaction of Thioflavin T with
electric eel AChE are available on the Protein Data Bank at the DOI: 10.2210/pdb2j3q/pdb
The AChE enzyme is classified as a serine hydrolase, with a catalytic triad present
at its active site that consists of serine, histidine, and glutamate amino acid residues.
The latter acidic residue is usually found to be aspartate in most serine hydrolase
enzymes. The aspartate residue stabilizes the histidine residue’s intermediate
imidazolium cation, which isolates the choline group of the natural substrate20. This
is followed by a hydrolysis reaction that cleaves off acetate. In addition to AChE’s
native function in modulating ACh concentrations in the nervous system, its other
functions include neuritogenesis, synaptogenesis, and amyloid-β complex
formation22.
8
1.4.2 Operation of the Biological Recognition Element
The biological recognition element of a biological sensor can contain sugars,
proteins (including enzymes such as AChE), nucleic acids, receptors, or entire cells.
The mode of action is classified as being either catalytic or affinity-based23. Catalytic
recognition is made possible by the selective affinity of enzymes for their substrates.
When the enzymes are activated or inhibited, the quantity of product made over time
changes and the difference can be measured24. Furthermore, enzymes can be
involved in transforming the target analyte as a substrate into a different measurable
product. An example of catalytic recognition applied for AChE inhibitor detection is
the use of organophosphorous hydrolase (OPH), an enzyme that breaks P-O bonds
of organophosphate compounds to make alcohol and acid products25. These
products can then be detected with a variety of transducers.
P
O
O
O
O
+ H2O +R3
OHR3
R1
R2
P
HO
O
O
O
R1
R2
OPH
Figure 1.6. General reaction scheme of an organophosphate compound with the OPH
enzyme.
Affinity-based recognition involves the irreversible and non-catalytic binding of a
target species to the biological recognition element23. Affinity-based recognition of
AChE inhibitors has traditionally been achieved with immunoassays by using
antibodies that have affinity for certain inhibitor compounds. In the first research
project described in this document, catalytic-based recognition was applied for the
detection of AChE inhibitors using the AChE enzyme.
In order to understand the processes taking place at the catalytic-based biological
recognition element, enzymes are modelled with fundamental enzyme kinetics and
Michaelis-Menten kinetics in mind. The observed initial rate of reaction depends on
the concentration of the substrate (S), enzyme (E), and the Michaelis (KM) and
9
dissociation (kd) constants respectively. The constants reflect the relative rates of
substrate-enzyme association/dissociation and product formation.
As product P is created through the enzyme catalyzed reaction, there is an initial
linear relationship between the change in product concentration d[P] and time.
However, as [S] decreases and [P] increases, the rate of enzyme activity decreases
due to a lack of saturation of the enzyme by substrate and/or competitive inhibition
due to the affinity of the product to the enzyme’s active site. Therefore, depending
on how an enzyme-based biosensor is calibrated in terms of incubation times with
the substrate, different types of responses can be obtained for the same measured
phenomenon. This is complicated when AChE is the chosen enzyme since AChE
inhibitors may also be substrates of AChE. Furthermore, some AChE inhibitors
permanently inhibit the enzyme (organophosphates), some do so temporarily
(carbamates), and yet others simply compete with ACh for the catalytic site.
There are also some subtle details about the structure and function of enzymes that
affect biosensor measurements. Very low concentrations of inhibitors can
sometimes tend to activate instead of inhibit AChE. This is likely due to interactions
of the inhibitors with the peripheral binding site of AChE, causing conformational
changes that improve accessibility of the active site. These interactions also appear
to be time-dependent, with prolonged peripheral site binding leading to decreased
accessibility of substrates to the active site26. The effectiveness of AChE inhibitors
decreases with increasing concentrations of the inhibitors due to the development of
steric blockades around the entrance to the enzyme active site27. These facts
explain the wide variety of possible output responses, linear and non-linear,
10
obtained from different biosensor designs that use various concentrations of enzyme
and substrate along with different incubation times.
Realizing the need for a standardized approach to achieving linear sensor
responses in the design and calibration of AChE-based AChE inhibitor biosensors,
Zhang et al.28 recommended that AChE and its substrate should exist at
concentrations that ensure the rate of product formation is not contingent on
substrate concentration. In this case, kinetically controlled conditions would be
maintained. The substrate should saturate the enzyme (i.e. [S] >> KM) to ensure that
it operates with zero-order kinetics29. In contrast, the incubation of AChE with
inhibitors in the experimental sample should take place under diffusion controlled
conditions, so that the enzymes are not saturated by the inhibitors at any time. If
saturation of AChE occurs during this step, the rate of inhibition is no longer linearly
dependent on inhibitor concentration since there is competition between inhibitor
molecules for access to the enzyme active site.
Figure 1.7. Schematic of diffusion controlled conditions (a), where diffusion of substrate to the enzyme determines the rate of reaction, unlike kinetic controlled conditions (b), where the enzymes are saturated and working as fast as they can. Sometimes, in kinetically controlled conditions, steric hindrance of the substrates near the active site can slow the exit of the enzyme product, thus decreasing the aggregate rate of reaction.
The suggested standardized approach by Zhang et al.28 may not necessarily work
for all different types of enzymes that may appear at the biological recognition
11
element. In general, enzyme interactions with the target analyte that is being
quantified should take place under diffusion controlled conditions to ensure linearity
in sensor response. Saturation of the enzyme by the analyte creates a situation
where not all of the analyte molecules have an opportunity to interact with the
enzyme during the incubation step. However, when the enzyme-catalyzed reaction
is used strictly to amplify the biosensor signal, kinetic controls are necessary to
ensure that the enzymes are consistently creating products for maximum biosensor
signal response. Without kinetic control, inhibited enzymes may not even have any
significant impact on overall product formation during the incubation step with their
substrate. Under kinetic controls, all of the enzymes work throughout the substrate
incubation step and a decrease in product due to enzyme inhibition will be more
visible. In practice, saturation of the enzyme at the biological recognition layer leads
to kinetic control but, if too much substrate is present, reaction rates decrease due to
the presence of steric blockades at the enzyme active sites.
1.5 Literature Review of Acetylcholinesterase Inhibitor Detection
The use of NMR and mass spectrometry is a well-established analytical approach
for the detection and identification of AChE inhibitors. However, detection
techniques which are faster, cheaper, and portable have been developed over the
past thirty years. In the early-to-mid 1980s, immunoassays had come into popularity
and some antibodies had been developed for certain AChE inhibitors, such as
soman and paraoxon, either directly or as a part of haptens when the inhibitor
molecules are too small to be recognized by antibodies on their own10, 30. Using a
competitive inhibition enzyme immunoassay format, Hunter et al.10 showed that it
was possible to detect paraoxon in low-ppb ranges both in solution and in serum.
Detection usually involves the enzyme-linked immunosorbent assay format or an
assay featuring fluorescent- or chemiluminescent-tagged antibodies31. Currently,
research is ongoing to produce recombinant antibodies for a variety of small
molecules such as AChE inhibitors, although the overall number of useful antibodies
remains low to this day. Unfortunately, the impressive detection abilities of
12
immunoassays also usually require multiple preparation steps and a long incubation
times that require multiple hours to complete11. However, it has been recently shown
that immunoassays can actually be used to detect AChE inhibitors in as little as 10
min and in a single step but with somewhat higher detection limits nearing 250 ppb.
This was accomplished by Zhou et al.32 in the form of a gold
immunochromatographic assay using carbofuran monoclonal antibodies labelled
with colloidal gold particles. Although this detection limit is not as low as other more
sophisticated sensing platforms, this immunoassay approach is sufficient to test for
toxic levels of many AChE inhibitors. It would also be useful to test for regulatory
compliance in the United States, where AChE inhibitor concentrations are regulated
to be in the mid-ppb to low-ppm ranges9. Further developments of this type of
immunochromatographic technique may also improve detection limits in the near
future, such as with silver enhancement of the gold nanocolloid, which allows for
double labelling of the same antibody31. Thus, the application of rapid
immunoassays for AChE detection is not an idea that should be readily dismissed.
There remains an overall scarcity of useful antibodies that respond to AChE
inhibitors even to this day. Once this scarcity is addressed, the usefulness of
immunoassay techniques will be better recognized.
The slow detection times of traditional immunoassay techniques existing in the
1980s drew attention to the exploration of other biological recognition and
transduction approaches for the detection of pesticides including AChE inhibitors. At
about the same time as immunoassays were being developed for this purpose,
research groups were beginning to apply the AChE and organophosphate hydrolase
(OPH) enzymes as the biological recognition elements coupled with a variety of
common transducers in optical, electrochemical, and mass-sensitive sensor
designs. OPH is an enzyme that only breaks down organophosphate compounds
into an alcohol and an acid, both of which are more useful for transduction purposes
than the original triester compounds themselves25. In the literature, there are many
peer-reviewed articles that document research on AChE inhibitor detection featuring
AChE and OPH. Furthermore, excellent efforts have been made to develop methods
13
of preserving enzyme activity over time so that the potential biosensors would have
a substantial shelf life on the order of months to years.
Sensors featuring optical transduction mostly rely on the interaction of an indicator
or sensor surface either directly with the analyte or indirectly with other species in
the sensor environment that can report on analyte concentrations. Sensing occurs
when resulting changes in absorbance or fluorescence are detected. Optical
transduction systems are very diverse and include many flavours of absorbance,
bioluminescence, chemiluminescence, evanescence and fluorescence33. In 2005,
White and Harmon demonstrated that portable and rapid optical solid-state detection
of organophosphates was feasible using OPH immobilized on glass microscope
slides with detection limits in the ppt-range and detection times of 10 seconds34. This
technique featured monitoring the absorbance of copper metalloporphyrins that
interact with the OPH enzyme and get displaced by trace concentrations of the
organophosphate substrates. A similar concept using AChE was applied by
Nagatani et al.35 where 5,5 dithiobis-2-nitrobenzoic acid was converted through a
reaction with thiocholine into 5 -mercapto-2-nitrobenzoic acid, where the latter
chemical compound was detected optically as a yellow dye that absorbed light at
410 nm. In the presence of AChE inhibitors, AChE would produce fewer thiocholine
molecules from its cleavage of acetylthiocholine (ATCh) substrate, producing lower
concentrations of the yellow dye. This approach allowed for visual discrimination
between 0.1 and 0.2 ppm DZN-oxon and down to the low-ppb range with a hand-
held photometer. More recently in 2007, Vamvakaki and Chaniotakis36 applied
liposomes to trap AChE and a pH-sensitive fluorescent dye called pyranine while
allowing the transport of ACh and AChE inhibitors through porins in the liposome. In
the presence of AChE inhibitors, AChE activity decreased inside the liposomes and
pyranine fluorescence decreased. Using this approach, detection limits in the mid-
ppq range were established with measurement times in as little as 5 min. Then, in
2009, Dale and Rebek37 achieved millisecond detection times without a biological
recognition element, opting instead to use oxime ring chemistry to detect
organophosphate nerve gas agents at ppm levels and lower. The presence of
organophosphates produces a ring-closing reaction that red shifts oxime
14
fluorescence maxima by 30 nm. This sensor has excellent potential to be used for
real-time personnel monitoring in hazardous conditions where AChE inhibitors may
be present.
The use of semiconductor nanoparticles (quantum dots) for AChE inhibitor detection
has also just recently been realized as biosensor research continued to develop
through 2003. Quantum dots are desirable for use in optical detection systems due
to their resistance to photo-bleaching, their wide excitation wavelength ranges, and
narrow size-dependent emission wavelengths that allow for multiplexing applications
when compared to conventional fluorescent dyes31. An excellent review of quantum
dots and their applications in optical detection systems can be found in the cited
literature12, 38. Using quantum dots functionalized with OPH, Constantine et al.39
found that paraoxon’s binding with OPH resulted in changes in quantum dot
photoluminescence. This type of biosensor yielded detection limits in the low ppb-
range for paraoxon almost instantly upon introduction of the sample to the quantum
dots as the OPH underwent conformational changes. However, more development
is required to improve selectivity of this type of sensor by experimenting with
different quantum dot coatings, possibly changing the structural properties of OPH
or replacing OPH with other biomolecules that interact with AChE inhibitors31.
However, quantum dots are definitely not limited to use with optical transduction,
since they are also applicable in electronic transduction systems due to their
capacity for electron exchange. Quantum dots have also been shown to be useful in
photoelectrochemical transduction designs in AChE inhibitor biosensors. Pardo-
Yissar et al.40 used AChE-derivatized quantum dots that were then covalently linked
to a gold electrode surface to create a photoelectrochemically active biosensor that
responds to thiocholine. Thiocholine interacts with the quantum dots, enabling them
to produce a stable photocurrent upon excitation by wavelengths of light in their
absorption band. In the presence of AChE inhibitors, tested in the ppm-range for this
sensor, less thiocholine was produced and the photocurrent decreased significantly.
Although the detection limit was not reported in this study, it is nevertheless a very
unique application of quantum dots and nanomaterials towards the detection of
AChE inhibitors.
15
Continuing on the topic of nanomaterials, the growth processes of nanoparticles
have also been utilized in biosensor designs for the detection of AChE inhibitors.
When AChE breaks ATCh into thiocholine, the latter product’s presence apparently
serves to slow the growth of gold-silver nanoparticles originating from the deposition
of silver on seed gold nanoparticles in the presence of a reducing agent such as
ascorbic acid. Thiocholine binds to the seed gold nanoparticle surface and blocks
the access of silver atoms to the gold nanoparticle surfaces. In the presence of
AChE inhibitors, less thiocholine is produced by AChE and an increase in the rate of
nanoparticle growth is seen. Virel et al.41 applied this observation to make a
biosensor using a colorimetric assay measuring absorbance at 400 nm for the gold-
silver nanoparticle plasmon band. AChE inhibitors such as paraoxon were tested
and detected in low ppb-levels in a matter of 5-10 min. A similar system in which
osmium complexes are made to play a role in gold nanoparticle growth was realized
by Xiao et al.42. When AChE cleaved ACh into Ch, this allowed for the reduction of
an oxidized osmium complex through the concurrent oxidation of Ch into betaine.
The reduced osmium complex promotes nanoparticle growth. The presence of
AChE inhibitors decreased the production of choline, the necessary reducing agent
to regenerate the required osmium complex, subsequently slowing nanoparticle
growth42.
Interesting work has also been conducted using carbon nanotubes (CNTs) for
application in biosensor design. CNTs represent a nanomaterial with many possible
applications in sensor design due to their large surface area, electrical conductivity,
stability, self-assembly, and capacity for surface modifications. CNTs have mostly
found applications in sensors using electric and electrochemical transduction
schemes, the latter of which will be introduced later in this document after a primer
introduction to electrochemical transduction. As of yet, there are very few notable
cases that are not related to electrochemical transduction where CNTs were applied
for the detection of AChE inhibitors, such as with the sensor developed by Ishii et
al.43 that features afield effect transistor (FET)- based AChE inhibitor biosensor
using AChE immobilized on carbon nanotubes. This biosensor exploits the fact that
CNTs are useful as semiconductor materials. When AChE inhibitors bind to the
16
immobilized enzymes on the CNT surface, the effective potential at the surface of
the CNTs is changed and can be detected through the source-drain current in the
microampere range. With an incubation time of 10 min, the sensor could achieve
detection limits of 200 ppq, which makes it one of the most sensitive devices for
measuring the presence of the AChE inhibitors acephate and fenitrothion.
Mass-sensitive transduction schemes have also been applied for the purpose of
AChE inhibitor detection. These include piezoelectric transducers and surface
plasmon resonance transducers (both the standard and localized varieties).
Piezoelectric transducers feature materials, such as quartz, which respond to
mechanical stresses by producing an electric potential that can be detected,
amplified and analyzed as a quantitative signal. A good example of an AChE
inhibitor biosensor using a piezoelectric transducer is the precipitation biosensor
reported by Kim et al.44 in 2007. A quartz crystal microbalance was used with a gold
modified quartz surface, which allowed for the immobilization of AChE in close
proximity to the surface via a sulphur-tagged linker. The substrate used was 3-
indolyl acetate, which is cleaved into 3-hydroxyindole by AChE. This product
oxidizes into a blue precipitate that settles onto the quartz surface and thus creates
a shift in the resonance frequency detected on the microbalance. Using this sensor
configuration, Kim et al.44 achieved mid-ppt to low-ppb detection limits for carbofuran
and EPN in 10 min. In contrast to piezoelectric transduction, surface plasmon
resonance (SPR) relies on the sensitivity of surface plasmons to changes in the
refractive index of bulk solution (or air) within nanometers of the metal surface45.
Using SPR, Rajan et al.46 developed a flow-based sensor that detected binding
events of an AChE inhibitor with AChE that was immobilized on the silver core of a
plastic-cladded silica optical fiber. In the presence of acetylcholine substrate, the
introduction of chlorphyrifos changed the refraction index in the vicinity of the silver
core and thus led to changes in the SPR wavelength. This effect was believed to be
occurring due to expulsion of acetylcholine from the AChE active site. This approach
led to detection limits in the low-ppb range in less than 10 min. Using a similar flow-
based SPR approach and similar measurement timelines, Mauriz et al.47 obtained
17
ppt-range detection limits for chlorphyrifos by replacing AChE with anti-chlorphyrifos
antibodies.
In closing of this literature review on AChE inhibitor biosensor designs, it is worth
mentioning the application of photothermal transduction for the purpose of detecting
AChE inhibitors due to the distinct characteristics of the transduction method, which
measures temperature changes in samples. Using an argon ion laser, Pogacnik et
al.48 irradiated thiocholine and measured the temperature change of the sample to
give an indication of existing AChE activity versus control experiments. The
presence of AChE inhibitors in a sample would decrease AChE activity. This would
in turn lead to lower amounts of thiocholine in solutions and lower detected changes
in temperature versus controls. With this approach, it was possible to achieve
detection of low-ppb ranges of AChE inhibitors in 6 min with excellent matching to
real sample concentrations detected by GC-MS measurements. Although the
thermal lens spectrometer required for these measurements is quite bulky, there is
definitely room for miniaturization as current portable models come in the size of a
suitcase (15 cm x 10 cm x 3 cm) containing all of the components including laser
diodes and bioanalytical column49. This type of biosensor would be most applicable
to work with samples that do not require substantial preparation steps, such as river
water, since foodstuffs typically require lengthy procedures including pureeing and
centrifugation prior to injection into the bioanalytical column of the biosensor.
It is evident from this brief literature review that many different biosensor designs
exist for the purpose of detecting AChE inhibitors. These rely on a variety of different
transduction schemes but many share the common biological recognition element
that is the AChE enzyme or OPH enzyme. These enzymes allow for limited
specificity in a biosensor given that OPH responds to organophosphates whereas
AChE is inhibited by AChE inhibitors. However, the enzymes are also affected by
their solvents, pH, ionic strength, and proteases existing in solution that degrade the
enzyme protein structure. This is critical when such biosensors are tested using real
samples as it can lead to questions about the reliability of the devices for on-field
use. It is not surprising then that very few (~20%) peer-reviewed academic papers
18
describing biosensors for AChE inhibitors include any real sample analyses in their
research reports50. Furthermore, where the enzymes can be demonstrated to work
as intended in real samples, they cannot distinguish between different
organophosphates or AChE inhibitors in any given unknown sample. The activity of
OPH only indicates the presence of any solvated organophosphate substrates
whereas loss of AChE activity only indicates the presence of some kind of AChE
inhibitor. Thus, in real samples where the identity of AChE inhibitors present in
solution is unknown, the sensor output has very limited value beyond indicating that
some species are present that interact with the enzyme at the biological recognition
element. Composite samples containing more than one type of AChE inhibitor would
require additional modifications to a biosensor in order for the device to provide
meaningful information about the individual components. Some attempts have been
made to achieve this through favourable modification of enzyme conformation.
Using directed evolution and amino acid substitutions, it is possible to isolate AChE
mutants that are more or less sensitive to particular AChE inhibitor species. For
example, Bachmann et al.51 created genetically engineered variants of the
Drosophila melanogaster AChE enzyme, which they subsequently applied in the
development of an artificial neural network (ANN) that could identify the component
concentrations of composite solutions of paraoxon and carbofuran. The ANN
processed collected readings from four different D. melanogaster AChE enzymes to
predict the individual concentrations of paraoxon and carbofuran in each sample
solution. The group succeeded in establishing a detection range between 0-5 ppb
for each compound with ~10% prediction error.
In the research described within this document, electrochemical transduction was
utilized along with AChE as the biological recognition element. This research
direction is not meant to reflect a particular bias for electrochemical techniques or
the biological recognition element, since the variety of options reported in this
literature review have all shown very impressive capabilities for AChE inhibitor
detection. It serves as a contribution to our current understanding of electrode
surface modifications and explores a new way of monitoring AChE inhibitors through
19
the oxidation of molecules that weakly intercalate with AChE. This is described in
further detail in Section 1.8.
1.6 Electrochemical Transduction: Voltammetry
This primer on electrochemical transduction was assembled based on the reference
material presented in an excellent introductory book to electrochemistry techniques
called Analytical Electrochemistry by Joseph Wang 52. Readers who wish to become
more familiarized with electrochemistry as it applies to chemical and biological
sensors are encouraged to consult this resource in the cited literature. In addition,
the electrochemistry scholar seeking a thorough understanding of electrochemical
techniques may refer to Alan J. Bard’s and Larry R. Faulkner’s book Electrochemical
Methods: Fundamentals and Applications53. The use of electrochemical transduction
offers the benefits of low costs, short measurement times, and an excellent potential
for the miniaturization and portability of the final assembled biosensor24.
Electrochemical transduction is not affected by the turbidity of sample solutions,
which is a major problem in the application of optical transduction platforms to such
samples54. Furthermore, the transduction process is relatively simple compared to
other technologies. In addition to the sample being analyzed, the components
required for electrochemical transduction include an electrode system, a voltage
source, and a potentiostat to collect electrical measurements. The electrode system
usually consists of three electrodes: working, reference, and counter. There are
many forms of electrode systems that are available in a variety of shapes and sizes
of which two were used in the research described herein: rod-shaped individual
electrodes and screen-printed electrodes.
Electrochemical transduction involves the monitoring of redox (reduction-oxidation)
reactions at the working electrode surface under various conditions of applied
potential from the voltage source. In general, a redox reaction at the electrode
surface involves the transfer of electrons to and from the members of the redox
species at a particular value of applied potential as described by the Nernst
equation,
where
electrons involved in the redox reaction per molecule, F is
is the universal gas constant, T is the Kelvin scale temperature, C
oxidized analyte concentration and C
The current observed at the applied potential depends on the flu
(O) or reduced (R)
where D represents the diffusion coefficient for the analyte (in cm
of the redox active species, V(x,t) is the hydrodynamic velocity in the x
C(x,t) is the concentration
added to negate flux due to a potential gradient and the sample solution is not
stirred during
After addressing the
diffusional flux, current can eventually be expressed through the Cottrell equation
(given here for a planar
where
applied past and above the value of E
become depleted and a diffusion layer
called
on the x
exists within 59 mV of E
solution over the voltage scanning period
where Eo is the standard redox potential for the redox reaction,
electrons involved in the redox reaction per molecule, F is
is the universal gas constant, T is the Kelvin scale temperature, C
oxidized analyte concentration and C
The current observed at the applied potential depends on the flu
(O) or reduced (R)
where D represents the diffusion coefficient for the analyte (in cm
of the redox active species, V(x,t) is the hydrodynamic velocity in the x
represents the concentration gradient,
C(x,t) is the concentration
added to negate flux due to a potential gradient and the sample solution is not
stirred during the
After addressing the
diffusional flux, current can eventually be expressed through the Cottrell equation
en here for a planar
here A is the area of a planar electrode (in cm
applied past and above the value of E
become depleted and a diffusion layer
called a voltammogram
on the x-axis. The result of the sweep is a
exists within 59 mV of E
olution over the voltage scanning period
is the standard redox potential for the redox reaction,
electrons involved in the redox reaction per molecule, F is
is the universal gas constant, T is the Kelvin scale temperature, C
oxidized analyte concentration and C
The current observed at the applied potential depends on the flu
(O) or reduced (R) analyte to the electrode surface,
where D represents the diffusion coefficient for the analyte (in cm
of the redox active species, V(x,t) is the hydrodynamic velocity in the x
represents the concentration gradient,
C(x,t) is the concentration
added to negate flux due to a potential gradient and the sample solution is not
the measurement
After addressing the mathematical expression for the
diffusional flux, current can eventually be expressed through the Cottrell equation
en here for a planar electrode):
A is the area of a planar electrode (in cm
applied past and above the value of E
become depleted and a diffusion layer
voltammogram can be plotted with current
The result of the sweep is a
exists within 59 mV of Eo, demonstrating the oxidation of reduced species existing in
olution over the voltage scanning period
is the standard redox potential for the redox reaction,
electrons involved in the redox reaction per molecule, F is
is the universal gas constant, T is the Kelvin scale temperature, C
oxidized analyte concentration and C
The current observed at the applied potential depends on the flu
analyte to the electrode surface,
where D represents the diffusion coefficient for the analyte (in cm
of the redox active species, V(x,t) is the hydrodynamic velocity in the x
represents the concentration gradient,
C(x,t) is the concentration of O or R at a particular position and time. If excess salt is
added to negate flux due to a potential gradient and the sample solution is not
measurement, only diffusion plays a role in determining the flux.
mathematical expression for the
diffusional flux, current can eventually be expressed through the Cottrell equation
electrode):
A is the area of a planar electrode (in cm
applied past and above the value of E
become depleted and a diffusion layer
can be plotted with current
The result of the sweep is a
, demonstrating the oxidation of reduced species existing in
olution over the voltage scanning period
is the standard redox potential for the redox reaction,
electrons involved in the redox reaction per molecule, F is
is the universal gas constant, T is the Kelvin scale temperature, C
oxidized analyte concentration and CR is the initial reduced analyte concentration.
The current observed at the applied potential depends on the flu
analyte to the electrode surface,
where D represents the diffusion coefficient for the analyte (in cm
of the redox active species, V(x,t) is the hydrodynamic velocity in the x
represents the concentration gradient,
at a particular position and time. If excess salt is
added to negate flux due to a potential gradient and the sample solution is not
, only diffusion plays a role in determining the flux.
mathematical expression for the
diffusional flux, current can eventually be expressed through the Cottrell equation
A is the area of a planar electrode (in cm
applied past and above the value of Eo, the reduced species at the electrode surface
become depleted and a diffusion layer of redox
can be plotted with current
The result of the sweep is a voltammogram with a current
, demonstrating the oxidation of reduced species existing in
olution over the voltage scanning period. At a constant applied potential or with a
is the standard redox potential for the redox reaction,
electrons involved in the redox reaction per molecule, F is
is the universal gas constant, T is the Kelvin scale temperature, C
is the initial reduced analyte concentration.
The current observed at the applied potential depends on the flu
analyte to the electrode surface,
where D represents the diffusion coefficient for the analyte (in cm
of the redox active species, V(x,t) is the hydrodynamic velocity in the x
represents the concentration gradient, is the potential gradient, and
at a particular position and time. If excess salt is
added to negate flux due to a potential gradient and the sample solution is not
, only diffusion plays a role in determining the flux.
mathematical expression for the
diffusional flux, current can eventually be expressed through the Cottrell equation
A is the area of a planar electrode (in cm2). When a pote
, the reduced species at the electrode surface
redox species is
can be plotted with current (I) on the y
voltammogram with a current
, demonstrating the oxidation of reduced species existing in
. At a constant applied potential or with a
is the standard redox potential for the redox reaction, n is the number of
electrons involved in the redox reaction per molecule, F is the Faraday constant,
is the universal gas constant, T is the Kelvin scale temperature, C
is the initial reduced analyte concentration.
The current observed at the applied potential depends on the flux (J)
where D represents the diffusion coefficient for the analyte (in cm2/s), z is the charge
of the redox active species, V(x,t) is the hydrodynamic velocity in the x
is the potential gradient, and
at a particular position and time. If excess salt is
added to negate flux due to a potential gradient and the sample solution is not
, only diffusion plays a role in determining the flux.
mathematical expression for the time-dependence of the
diffusional flux, current can eventually be expressed through the Cottrell equation
When a pote
, the reduced species at the electrode surface
species is established.
on the y-axis and potential
voltammogram with a current
, demonstrating the oxidation of reduced species existing in
. At a constant applied potential or with a
n is the number of
the Faraday constant,
is the universal gas constant, T is the Kelvin scale temperature, CO is the initial
is the initial reduced analyte concentration.
(J) of the oxidized
/s), z is the charge
of the redox active species, V(x,t) is the hydrodynamic velocity in the x-direction,
is the potential gradient, and
at a particular position and time. If excess salt is
added to negate flux due to a potential gradient and the sample solution is not
, only diffusion plays a role in determining the flux.
dependence of the
diffusional flux, current can eventually be expressed through the Cottrell equation
When a potential sweep is
, the reduced species at the electrode surface
established. A graph
axis and potential
voltammogram with a current peak that
, demonstrating the oxidation of reduced species existing in
. At a constant applied potential or with a
20
n is the number of
the Faraday constant, R
is the initial
is the initial reduced analyte concentration.
oxidized
/s), z is the charge
direction,
is the potential gradient, and
at a particular position and time. If excess salt is
added to negate flux due to a potential gradient and the sample solution is not
, only diffusion plays a role in determining the flux.
dependence of the
diffusional flux, current can eventually be expressed through the Cottrell equation
ntial sweep is
, the reduced species at the electrode surface
A graph
axis and potential (V)
peak that
, demonstrating the oxidation of reduced species existing in
. At a constant applied potential or with a
21
linear potential sweep, the area of this peak can be used to quantify the
concentration of the analyte in solution. However, in many applied electrochemical
experiments, including the ones performed in the described research, non-linear
potential sweeps were applied to produce peaks that cannot be theoretically linked
to analyte concentration. However, this is a trade-off for sharper and more distinct
peaks than seen in linear potential sweeps. This is the case for the technique of
differential pulse voltammetry (DP Voltammetry), which involves the application of
pulsed potentials superimposed on a linear potential sweep. Current is recorded just
before and right after the pulse is applied so as to allow background charging
processes at the electrode surface that are unrelated to the presence of analyte in
solution to be completed first, thus decreasing measurement noise. The resulting
voltammogram yields peaks whose height rather than area is directly proportional to
analyte concentration,
where σ is and ∆E is the pulse amplitude. The peak potential, Ep, is related
to the polarographic half-wave potential E0.5 by
Inorganic compounds, such as the model ferri-ferrocyanide redox couple, are found
to yield reproducible oxidation and reduction peaks theoretically separated by 59
mV. With cyclic, linear potential sweeps, the peak currents appear proportional to
the square-root of the scan rate (in V/s). The application of potential sweeps,
whether linear or not, to organic molecules usually yields peaks that are not
reversible. An oxidation sweep on organic molecules usually yields peaks that do
not have a symmetrical matching peak in a subsequent reduction sweep and vice
versa. This property usually extends for most biological molecules including
proteins, sugars, fats, and nucleic acids.
22
1.6.1 Electrode Materials: Manufacturing Process, Characteristics,
and Uses
The electrochemistry research that is described in this document was conducted
using disposable electrochemically printed chips from BioDevice Technology and a
renewable glassy carbon electrode from CH Instruments. Each type of electrode has
a distinct manufacturing process and physical characteristics.
The glassy carbon electrode is made through a process called carbonization.
Starting from a pre-moulded phenolic polymer resin (phenol-formaldehyde), the
material is exposed to high temperatures of above 300 °C and up to 1200 °C in an
inert atmosphere over a long period of time55. The process must occur gradually in
order to allow for gaseous oxygen, nitrogen, hydrogen, and other species to slowly
diffuse to the surface in order to avoid the formation of defects (cavities) in the
glassy carbon material56. The result is the very smooth (glassy) carbon surface that
defines this type of electrode. The microscopic structure of the surface consists of
many layers of cross-linked graphite-like sheets arranged as tangled ribbons55.
Although not as conductive as metal electrodes, carbon-based electrodes are
relatively inexpensive and are frequently used in electrochemical studies to take
advantage of their large potential window and the many possibilities of implementing
surface chemistry55. Glassy carbon is very pure, conductive, and impermeable to
gas and chemical reactions57. Upon oxidation, the surface does end up containing
some redox active groups with the chemical adsorption of oxygen and these include
carbonyls and quinone/hydroquinone56. The physical characteristics of glassy
carbon electrodes require a compromise for its use. The glassy carbon surface is
easily renewable after the completion of a measurement through the application of a
polishing step with alumina powder to remove adsorbed species. However, the
glassy carbon surface is not perfect due to the formation of defects in the
carbonization process. There may be small cavities left by escaping gas molecules
that will thereafter harbour adsorbed species such that they cannot be removed
through polishing and may create interfering signals in measurements. Furthermore,
the glassy carbon surface is also very easy to scratch. The scratch would then serve
23
as a defect. The removal of scratches is possible but requires extensive polishing
steps with several different grades of emery paper and alumina powder56.
The advent of screen-printing techniques ushered in the era of disposable
electrochemically printed chips. These printed chips can be produced to include all
three electrodes on one chip or only the working electrode if it is so desired. The
manufacturing procedure is completed in a sequence of steps which include the
application of screens/masks on a plastic substrate. Each mask is patterned to allow
for the deposition of an ink on a specific region of the electrode. This allows for
conductive elements and insulating elements to be placed precisely where needed
in order to make the final product.
Figure 1.8. An illustration of the screen-printing process to make disposable
electrochemically printed chips. Grooves in the mask allow for the squeegee to move the ink
onto precise locations on the plastic substrate.
Usually, entire sheets of electrodes are made simultaneously with screen-printing.
First, a conductive ink, such as silver, gold, or platinum, is used to define the
electrode connectors from the surfaces of the working, counter, and reference
electrodes. Next, a graphite ink is applied with a mask that permits for the deposition
of the working electrode surface58. The graphite ink may then be modified with
biomolecules, metals, nanoparticles, and a variety of other functionalization options
that can improve the detection capability of the finished screen printed electrode. For
24
example, metal nanoparticle electro-deposition is possible on a carbon paste screen
printed electrode surface at -0.4 V by covering the working electrode in a highly
acidic metal chloride solution of gold and platinum59. Finally, an insulating ink may
be applied with a mask that helps to prevent short-circuits between the conductive
leads traveling from the different electrodes when an aqueous sample is applied on
their surface.
Figure 1.9. An illustration of a possible sequence of mask steps to create a screen-printed
electrode. In order, (1) deposits the counter electrode and its lead, (2) deposits the working
electrode and its lead, (3) deposits the reference electrode and its lead, and (4) deposits an
insulating hydrophobic layer across all three of the electrode leads.
The benefits of disposable electrochemically printed chips include their low cost and
disposability, which avoids the possibility of electrode surface contamination
between measurements. The accuracy of the modern screen-printing process,
which is automated and subjected to quality control procedures, means that the
electrodes that are prepared on a single sheet are very similar and should allow for
very reproducible measurements.
25
1.7 Literature Review: Electrochemical Transduction for Detection
of AChE Inhibitors
The detection of AChE inhibitors using electrochemical techniques mostly appears
in academic literature in the form of amperometric and voltammetric measurements
of products from reactions catalyzed by OPH or AChE. Potentiometric,
conductimetric, and impedimetric electrochemical detection of AChE inhibitors has
also occasionally been reported. Potentiometric detection of AChE inhibitors has
traditionally been focused on the measurement of pH changes that result from the
activity of the AChE enzyme. When AChE is inhibited, fewer acetate molecules are
produced from the breakdown of ACh and thus the pH of the solution remains more
acidic (or less basic) when compared to a sample containing uninhibited AChE60.
Not much appears to have changed with potentiometric detection methods of AChE
inhibitors since 1977. In 1998, Mulchandani et al.61 immobilized cell membrane OPH
enzyme-expressing Escherichia coli cells on the glass surface of a pH electrode, an
exquisite step up from previous designs that immobilized the enzyme onto the
electrode surface via covalent bonding and cross-linking. The bacterial cell
immobilization effectively bypassed the necessity to express, purify, collect, and
immobilize the OPH enzyme – all of which are tedious, costly, and time consuming
steps. OPH produces two protons for each organophosphate molecule that is
cleaved by the enzyme and the acidification of the test solution is detectable by the
pH electrode down to the mid-ppb range for several organophosphate compounds
including paraoxon and methyl parathion62. The advent of nanoscale potentiometric
sensors designed to work with metal amplification labels may yet revive the use of
potentiometric detection for AChE inhibitors by greatly improving detection limits63,
but thus far such a system has yet to be designed and reported.
Conductimetric detection of AChE inhibitors is very rarely reported and involves the
measurement of solution conductivity and changes in this property as it reflects the
target analyte. Using AChE as the biological recognition element, Suwansa-ard et
al.64 showed that the performance of conductimetric detectors for AChE inhibitors
26
was comparable to potentiometric detectors. As AChE cleaves ACh into Ch and
acetate, the acetate anions alter the conductivity of the solution. Using
conductimetry and potentiometry, the group obtained the same detection limits and
linear range for carbaryl and carbofuran in the mid-to-low ppb range respectively.
However, conductimetric detection proved to be less sensitive than potentiometric
detection in the defined linear range for each carbamate AChE inhibitor. Another
electrochemical technique seldom seen applied for the detection of AChE inhibitors
is electrochemical impedance spectroscopy (EIS). This technique measures
changes in impedance at the surface of the electrode by means of interpreting the
response of the electrode system to applied alternating voltage at different
frequencies. The current response to the applied voltage will be a sinusoidal wave
that has a phase difference from the voltage sinusoid. The ratio of voltage to current
represents the impedance65. The impedance depends on events that occur at the
electrode surface, such as molecular interactions, adsorption, and redox reactions.
Diffusion and electrode kinetics also play a role in determining the impedance
response. Most commonly, a Nyquist plot is made up of the impedance values for
each scanned frequency with the imaginary component of impedance on the y-axis
and real component on the x-axis. Briefly described, the shape of the Nyquist plot,
usually a semi-circle with a tail, provides information about the state of the electrode
surface. EIS is a very versatile tool for studying events at electrode surfaces, but
perhaps the technique is too complicated for easy integration into a biosensor
design for AChE inhibitor detection when compared to other competing
electrochemical techniques. Instead, voltammetric and amperometric designs are
much more prevalently found in peer-reviewed publications.
Both amperometric and voltammetric measurement systems are easily applied to
AChE inhibitor detection. Amperometric detection involves applying a constant
potential over time that will be sufficient to oxidize or reduce a reporter species in
solution. The current recorded from the redox reaction of the reporter species should
give some indication about the concentration of AChE inhibitors in the sample. Thus,
amperometry allows for real-time measurements of samples as they come into
contact with the electrode surface. Voltammetric detection, in contrast to
27
amperometry, allows the user to scan through an applied potential range in order to
create a voltammogram that shows the peak potential and peak current of the
reporter species undergoing a redox reaction at the electrode surface. Although
voltammetric detection does not offer real-time monitoring of samples, it is not a far
stretch to collect multiple measurements over a short period of time through an
automated process which would provide a pseudo-real-time measurement system.
The advantage of voltammetry over amperometry is the additional information
provided about both the peak potential and peak shape, either of which can change
depending on measurement conditions. With amperometry, only current is
measured without the additional information obtained in voltammetry. If the peak
potential changes, the amperometric current measurements continue to be recorded
at an off-peak potential unless there is an in-built correction system in place to
prevent this from happening. However, amperometry is generally known to be the
more sensitive technique over voltammetry.
The performance of AChE inhibitor sensors featuring amperometric transduction is
impressive. In 1991, Kulys66 reported a simple electrochemically-printed sensor with
a TCNQ-modified graphite surface. TCNQ serves as a mediator that is reduced by
choline or thiocholine and whose oxidation signal intensity can thereafter be
interpreted as a measurement of acetylcholinesterase activity. Butyrylcholinesterase
enzyme was immobilized on the electrode surface and was exposed to S-
butyrylthiocholine as the chosen substrate. It was possible to record measurements
at 100 mV (vs. Ag/AgCl reference) and to detect a decrease in thiocholine
production in the presence of 60 ppb paraoxon concentrations and higher. Schulze
et al.67 demonstrated a 500 ppt detection limit for paraoxon by comparing
amperometric data of thiocholine produced by immobilized acetylcholinesterase
mutants on graphite screen printed electrodes in the presence and absence of
incubation with the insecticide. Laschi et al.68 showed in 2007 that screen-printed
cobalt(II) phthalocyanine- modified carbon electrodes could also be used to measure
thiocholine production by AChE at an applied potential of 100 mV. Using this
technique, a detection limit of 110 ppt was reached for carbofuran with a
measurement time of 15 min. Real-time inhibition measurements have also been
28
demonstrated using an amperometry-based system with gelatin-immobilized
acetylcholinesterase electrodes by Pohanka et al.69 with reported achievable real-
time detection of 200 ppb injected paraoxon with no prior electrode incubation over 1
min of measurement. Marinov et al.70 recently published their research in 2010
where they immobilized AChE on a Poly(acrylonitrile-methylmethacrylate-
sodiumvinylsulfonate) membrane (PAN membrane) loaded with gold nanoparticles.
The PAN membrane protected the enzyme from degradation and prevented
biological fouling of the platinum working electrode surface during measurements of
ATCh at an applied potential of 0.8 V. Between measurements, the PAN membrane
was replaced. This approach was reported to have yielded a detection limit for
paraoxon in the low-ppq range after a 20 min incubation, which is very surprising
considering the high applied potential which should oxidize the ATCh substrate,
thiocholine product, and the enzyme itself.
Although not as commonly seen as amperometry-based detection, voltammetry is a
competitive approach to AChE inhibitor detection. The core design of the biosensor
utilizing voltammetry is usually identical to that of one using the amperometric
detection technique in that an enzyme product’s redox activity is still monitored to
yield information about the activity of the enzyme at the biological recognition
element. Qiu et al.71 applied square wave voltammetry to measure choline
production via 2,6-dichloroindophenol as the redox indicator and reached low ppb
detection levels of parathion-methyl. By immobilizing AChE on TCNQ-modified
screen printed electrodes, Arvinte et al.72 were able to apply DP voltammetry to
detect methyl paraoxon at concentrations approaching 1 ppb. Hernandez et al. used
a similar approach with DP voltammetry using the TCNQ oxidation signal to report a
20 ppt detection limit for carbofuran with a 10 min incubation, which exceeded the
performance of a similar amperometric sensor described previously by Laschi et
al29.
29
1.8 Contributions of this Research Work
The first project was conducted to determine whether two components could be
applied successfully to electrochemically detect common AChE inhibitors. The first
component was the BioDevice Technology unmodified disposable gold
electrochemically printed chip, which would be used to oxidize thiocholine produced
from the AChE-catalyzed breakdown of acetylthiocholine substrate. This chip is
unique in that the gold nanostructures were electrodeposited onto the carbon paste
surface by the developers and no further modifications were performed by our
group. In the cited literature, the gold working electrodes used by various groups
were prepared through the surface immobilization of colloidal gold nanoparticles 70
and featured additional surface modifications of the working electrode with
mediators and the AChE enzyme 66-72. The second component was the AChE
enzyme itself, which was the F345Y mutant selected from a group of N. brasiliensis
enzymes donated to Professor Kerman by his colleague, Professor Bachmann. The
hypothesis was that we could obtain competitive detection limits since we would
utilize the full surface area of the gold nanostructured disposable electrodes in the
absence of any surface modifications, which might block thiocholine’s access to the
electrode surface. Using differential pulse voltammetry, the results showed low-ppb
detection limits of 8 ppb for carbofuran and 10 ppb for paraoxon in 20 mM Tris 100
mM NaCl buffer solution. We also spiked real samples of Highland Creek river water
and 6% milk with 50 ppb and 35 ppb paraoxon respectively to show that real
aqueous samples could be tested successfully with the sensor design. The real
samples conformed to the calibration curve obtained in the Tris buffer. However, in
the process, we discovered that our AChE enzyme worked better in the Highland
Creek river water sample than it did in Tris, which was likely due to the higher ionic
strength in the river sample than in the Tris buffer. This reinforced the importance of
conducting baseline AChE activity tests in each unique real sample composition
prior to operating the sensor in the same type of sample that also contains AChE
inhibitors.
30
The second project was aimed to translate fluorescence results that used Thioflavin
T (ThT), a fluorescent dye, to indirectly detect AChE inhibitors. ThT intercalates
weakly with AChE, which increases its fluorescence. In the presence of AChE
inhibitors, this intercalation is disrupted and the fluorescence of ThT decreases as a
result. We proceeded to examine this intercalation electrochemically through the
oxidation of ThT on a glassy carbon electrode surface. Electrochemical analysis
permits the use of very small concentrations of reagents. In this case, we used 280
nM ThT and 12.5 nM AChE, compared to micromolar amounts that are used in
fluorescence spectroscopy studies. We found that ThT oxidation decreases when
AChE is present on the electrode surface beyond the drop that is expected from
surface fouling. The results suggest that ThT is attaching to the surface of the AChE
enzymes that exist on the electrode surface beyond a 1:1 ratio, suggesting that
interactions occur between ThT and AChE that may remove ThT from solution but
not increase the dye’s fluorescence. We also found that ThT oxidation increases if
the AChE is pre-treated with carbachol or paraoxon, which are believed to disrupt
ThT’s intercalation with AChE. Interestingly, the calibration plots for each AChE
inhibitor were complex, with multiple maxima. The difference in ThT oxidation was
used to detect the presence of carbachol and paraoxon at concentrations down to
10 ppb.
31
2. RESULTS AND DISCUSSION
2.1 Project #1: Gold disposable electrochemical printed chips for
the analysis of acetylcholinesterase inhibition using differential
pulse voltammetry
The first research project discussed in this paper featured gold disposable
electrochemically printed (DEP) chips. These chips were used in a traditional
electrochemical biosensor configuration where AChE was the biological recognition
element and ATCh was the substrate. The enzyme-catalyzed breakdown product,
thiocholine, was oxidized directly on the electrode surface after an incubation step of
the substrate in solution with AChE. The oxidation reaction of thiocholine at a gold
working electrode surface takes advantage of gold-sulphur affinity that facilitates the
measurement of oxidation peak currents superior to that obtainable from carbon-
based electrode surfaces. The activity of AChE in solution was monitored by
comparing thiocholine oxidation currents between inhibitor-containing experimental
samples and control measurements.
Figure 2.1. Illustrations of the gold DEP chips used for the first research project. (a) A gold
DEP chip after sample-loading with 12 µL of solution. (b) A diagram of the gold DEP chip’s
surface features. The actual surface area of the working electrode on the chips was
approximately 3 mm2.
a. b.
32
The major differences between the biosensor reported in this project and other
previously published reports using AChE and ATCh were: 1. The application of new
nanostructured, unmodified gold DEP chips to take advantage of gold-sulphur
chemistry for improved biosensor signal measurements, 2. A lack of enzyme
immobilization on the electrode surface in order to maximize the available surface
area for thiocholine oxidation, and 3. the use of a mutant AChE enzyme at the
biological recognition element.
At the time when the decision was made to undertake this project, the gold DEP
chips had been made available from Bio Device Technology (Japan) compliments of
Professor E. Tamiya (Japan Advanced Institute of Science and Technology).
Furthermore, several Nippostrongylus brasiliensis AChE mutants were also
available at the time, having been kindly donated by Professor T. T. Bachmann
(University of Edinburgh). These mutants were expressed by Schulze et al.67 in an
effort to find AChE enzymes that would show greater sensitivity to particular AChE
inhibitors. Schulze et al.67 had evaluated each of the mutants by their ki constants (in
M-1min-1) relative to that of the wild-type N. brasiliensis AChE enzyme in response to
a variety of AChE inhibitors as shown in the following equation
where E is the concentration of active enzyme after incubation with the chosen
inhibitor, E0 is the initial concentration of the enzyme before incubation, t is the
elapsed time (chosen to be 1 min), and [Inhibitor] is the concentration of the chosen
inhibitor in solution. Higher ki values obtained in that experiment indicated that a
lower concentration of inhibitor was needed to inhibit the enzyme in question by
some arbitrary amount indicated by the natural logarithm ratio on the left side of the
equation. The ratio of constant’s values of any particular N. brasiliensis AChE
mutant (referred to here as ki*) to that of the wild-type AChE (ki) for a chosen AChE
inhibitor indicated whether the mutation made the resulting enzyme more vulnerable
or more protected against the inhibitor.
33
The F345Y mutant (phenylalanine switched to tyrosine at position 345), which was
used for our research project featuring gold DEPs, showed a lower value for its ki*
constant (less sensitivity) in response to both carbofuran and paraoxon compared to
the wild-type N. brasiliensis AChE. The F345Y mutation occurs at the choline
binding site of the enzyme. Although the consequences of this mutation on overall
enzyme structure were not clear, modifications to the choline binding site will affect
the docking ability of substrates and inhibitors at the catalytic triad. The ki*of F345Y
versus the wild-type enzyme was in the same order of magnitude for both of these
AChE inhibitors (1 <ki/ki*< 10), although paraoxon’s measured ki
* was approximately
1-2 times smaller than the ki* for carbofuran.
It was anticipated that lower paraoxon concentrations than carbofuran could be
needed to inhibit the F345Y AChE. This would already have been the case even
without using the mutant AChE since the ki values for the wild-type N. brasiliensis
AChE were reported to be 1.0x106 M-1min-1 for paraoxon and 4.4x105M-1min-1 for
carbofuran. Without the mutation, N. brasiliensis AChE is roughly twice as sensitive
to paraoxon compared to carbofuran. With F345Y, the difference in the enzyme’s
sensitivity between the two inhibitors is diminished almost to unity according to the
measurements made by Schulze et al.67 in 2005.
Electrochemistry of Acetylthiocholine
The electrochemical properties of ATCh were examined as the second step in this
project after determining the Tris buffer baseline control voltammogram, which
showed no significant contaminants that oxidized to produce current beyond the low
nanoampere range. The ATCh molecule has a distinct oxidation voltammogram
featuring a set of peaks at 0.44 V and 0.57 V. These appear to be the primary
diagnostic peaks that are indicative of the ATCh concentration present in solution.
ATCh stocks that have spent a considerable amount of time in storage will show
lower peak currents at these potential values compared to new ATCh stocks since
the molecule is hygroscopic and undergoes hydrolysis over time. Experimentally,
34
hydrolysis only became noticeable after many hours when ATCh was prepared in a
pH 7 buffered solution. In addition to the peaks at 0.44 V and 0.57 V, there is one
more peak that appeared at 0.75 V but usually fluctuated unpredictably with each
different measurement.
Figure 2.2. Three DP voltammograms of 2.1 mM ATCh measured on new gold DEP chips.
Figure 2.3. Three DP voltammograms of the 0.5 nM AChE – 2.1 mM ATCh solution, each
taken after a 10 min incubation period on a new gold DEP chip.
35
When AChE is added into solution with 2.1 mM ATCh and incubated (in this case a
concentration of ~0.5 nM was used for AChE, which does not contribute any
significant oxidation current on the voltammogram), the thiocholine product can be
seen having been oxidized at ~0.25 V on the voltammogram. This peak grows
predictably with increased incubation times, but the other peaks were not found to
be as diagnostically useful.
The general trends that were noticed between the substrate and product
voltammograms was the increase of the oxidation peak current at 0.44 V, the
disappearance of the peak at 0.57 V, and a slight but unstable decrease in the
current intensity of the peak at 0.75 V. These results suggest that the peak at 0.57 V
is likely representative of the ATCh substrate or at least one of its oxidation
processes. The peak at 0.44 V may represent the oxidation current from both ATCh
and the thiocholine product, likely related to the gold-sulphur interaction since this
peak shows a major difference in current intensity between carbon and gold DEP
chips. The gold DEP chips show 3-4 times greater oxidation peak currents at 0.44 V
compared to carbon DEP chips. It is possible that the peak at 0.57 V gets
superimposed at the 0.44 V position. This could be confirmed by oxidizing pure
thiocholine at a concentration that produces a peak of matching intensity at 0.25 V
and then observing the difference in the voltammogram at the 0.44 V position.
Finally, the peak at 0.75 V varies dramatically in peak currents recorded between
DEP chips, suggesting an oxidation process whose likelihood in this experiment
depends too heavily on variability in the chip surfaces. Additional investigation of this
peak using renewable GC or gold working electrodes may yield a better idea of what
this peak represents and any possibility of using it for detection purposes. In the
interest of saving time on measurements, the scanning range was limited to run from
0.1 to 0.6 V.
The thiocholine oxidation peak current at 0.25 V increases in magnitude with
increasing incubation times of the substrate but reaches a maximum either due to
competitive inhibition of the enzyme by the product or due to the depletion of the
substrate. If the AChE is first incubated in solution with an AChE inhibitor prior to
36
incubation with the ATCh substrate, the oxidation peak current of thiocholine at 0.25
V is measured to have a lower magnitude compared to controls with uninhibited
AChE. Furthermore, the peak at 0.44 V appeared to change in shape, again hinting
at a possibility that an oxidation potential overlap of substrate and product exists.
Figure 2.4. A comparison of DP voltammograms showing thiocholine production in a control
measurement (top, black line) with AChE versus AChE treated with 50 ppb carbofuran
(lower, gray line).
The different oxidation peak currents were then used to quantify the AChE enzyme
activity after incubation with various concentrations of the AChE inhibitors paraoxon
and carbofuran.
Biosensor Performance
Experimental results showed that it was possible to get good detection limits for both
paraoxon and carbofuran even when using an AChE mutant (F345Y) that is less
sensitive to both of these inhibitors than enzymes commonly found in such sensors.
Carbofuran was detected at 8 ppb and paraoxon at 10 ppb. The calibration plots that
were constructed from experimental data showed that F345Y was still slightly more
37
sensitive to paraoxon than to carbofuran, with 80 ppb of paraoxon leading to
(80±10)% inhibition versus (50±10)% for the same concentration of carbofuran.
Figure 2.5. The calibration plots of the featured biosensor on the gold DEP chips in
response to carbofuran and paraoxon concentrations in Tris buffer. On the right, in the
paraoxon calibration plot, real samples can be seen as the diamond data series appearing
at ~35 ppb (milk sample) and ~50 ppb (river sample) respectively.
Both calibration plots showed an overall logarithmic response tendency of the
average AChE activity with increasing concentrations, although linear regions were
seen between 10 and 40 ppb for both inhibitors. The logarithmic shape indicates that
the enzymes in solution are being saturated by inhibitors at the higher tested
concentrations during the incubation step. The shape of the calibration curve is
tuneable through the modification of the incubation step length and the
concentration of AChE. Using lower AChE concentrations potentially leads to better
detection limits and saturation of the enzymes at lower inhibitor concentrations.
However, under these conditions less substrate is broken down during a fixed
incubation time and thus less product can be oxidized, which decreases sensor
signal intensity. This has to be compensated by increasing the length of the
incubation step with the substrate. The concentration of the substrate should be
sufficiently in excess to maintain kinetic control and to ensure that product inhibition
of the enzyme is not significant during the incubation step with the substrate. In the
scenario where kinetic control is not achieved, the sensor will have poor
discrimination between concentration values at the lower end of the calibration plot
38
for an inhibitor. If product inhibition of AChE is significant, substrate cleavage will be
halted and the maximum current intensity that is generated during the oxidation of
the electrode will not be realized, which is detrimental to the sensor’s overall signal
to noise ratio.
During the collection of data for this research project, it was found that the solvent
used to dissolve the AChE inhibitor can play a major role in the overall activity of the
AChE enzymes. Carbofuran dissolves very poorly in aqueous solution, which is why
2% DMSO was used to aid in solubility. Higher concentrations of DMSO decrease
AChE activity substantially, such that 20% DMSO results in almost complete
enzyme inhibition in the featured biosensor design. However, other solvent effects
vary and lower concentrations of certain solvents have also been reported to
enhance AChE performance 73. Regardless of the solvent used, it is important to run
inhibitor-free controls using exactly the same solution composition as the sample.
Therefore, all carbofuran measurements of thiocholine production were compared
against carbofuran-free controls with AChE and ATCh in Tris buffer with 2% DMSO.
Figure 2.6. Background DP voltammogram measurements for 6% milk in Tris (dashed line)
and river water in Tris (solid line). Note that the current is in nano-amperes compared to the
micro-ampere values seen in the measurements of 2.1 mM ATCh.
The sensor was challenged with two real samples – a 6% milk sample spiked with
35 ppb paraoxon and a river sample with 50 ppb paraoxon. The milk sample was
39
made by diluting with Tris buffer from 20% cream stock, while the river sample was
diluted the same way. Background voltammogram measurements were taken for
each real sample before the addition of paraoxon and the results showed negligible
amounts of oxidation products in the low nanoampere current ranges.
When the spiked real samples were tested, these samples produced output data
that fit reasonably on the paraoxon calibration curve for the sensor. Interestingly, the
milk sample led to a greater degree of inhibition on average than did the river
sample even with a lower paraoxon concentration. This observation illustrates the
challenge of applying such sensors to real sample analyses, since the operation of
the sensor can be quite unpredictable without careful consideration of the
experimental conditions imposed by the real sample components. Furthermore, both
real samples produced measurements of baseline AChE activity (i.e. in clean, Tris
buffered milk and river water solutions) that was elevated beyond the activity of
AChE in Tris buffer controls.
Figure 2.7. A sample collection of DP voltammogram measurements after the 10 min
AChE-substrate incubation step in Tris buffer (dashed line), river water (black line), and 6%
milk (gray line).
The river sample yielded the greatest AChE production of thiocholine that was
measured to be 30% greater than Tris buffer controls. The milk sample allowed
40
AChE to produce only marginally less thiocholine with the same incubation length,
but the results were more variable possibly due to interference from other proteins
present in the milk. Furthermore, the thiocholine peak recorded in milk was much
sharper around 0.3 V. Additional testing is needed to determine why AChE
performance improves when Tris buffer is mixed with milk or river samples. Intuition
suggests that the increased ionic strength in such real samples plays a major role in
the increase of activity. It has been well-known for quite some time that lower ionic
strength promotes aggregation of AChE enzymes, which may reduce their
performance due to decreased accessibility of substrate to the enzyme active
sites74, 75. However, a more plausible explanation may simply be the effect of ionic
strength on the active site’s catalytic triad, where lower ionic strength destabilizes
the interaction between glutamate and histidine residues76. This leads to poorer
affinity of the substrate at the active site and thus decreased enzyme performance
compared to real samples with higher ionic strengths.
Finally, it is important to discuss the impact of experimental error sources on the
performance of this biosensor. Although the detection limits were stated as being 8
ppb for carbofuran and 10 ppb for paraoxon, it was actually possible to detect
paraoxon as low as 500 ppt and carbofuran at 2 ppb. However, the variability in
measured thiocholine oxidation at these concentrations meant that the error bars for
the data sets overlapped with the relevant control values. Standard deviations of
~10-15% were seen in most measurement sets for any particular inhibitor
concentration. This appears to be the norm for published studies of these types of
biosensors that use AChE as the biological recognition element. For example,
Laschi et al.68 showed standard deviations of ~8% for most of their measurements
with a similar biosensor configuration testing for carbofuran concentrations. With on-
field application of these types of sensors, these error bars will determine the
confidence of an operator in the inhibitor concentration value that is obtained from
any recorded measurement. With large error bars (e.g. my 40 ppb carbofuran
measurement) it becomes difficult to rely on the sensor to distinguish between
significant and insignificant concentrations of AChE inhibitors in a given sample. In
the calibration curves collected for this research project, the error bars of several
41
different measurements overlapped, especially for carbofuran. In retrospect, it would
have been more appropriate to collect measurements of inhibitor concentrations in
magnitude increments (i.e. 10xM) to obtain a data set that includes more signals that
do not have overlapping error bars. Further, more rigorous measurement conditions
could be implemented with more sophisticated experimental settings. These would
include auto-pipetting and controlled mixing of the samples throughout all incubation
steps to ensure that human error in the manipulation of instrumentation did not
contribute towards the standard deviation of measurement sets. The use of a
Faradaic cage enclosure around the DEP chips during electrochemical
measurements would also serve to decrease the influence of background
electromagnetic noise that may have contributed to fluctuations in sensor readings.
With the implementation of these changes, it should be possible to lower the
detection limits and show the true capabilities of this type of sensor.
Summary
This project demonstrated the viability of applying nanostructured gold DEP chips to
AChE inhibitor detection without any additional surface modifications. By keeping
AChE dissolved in solution rather than immobilized on the electrode surface, signal
loss due to the blocking of the electrode surface by the enzyme is avoided.
Furthermore, since the gold surface is permanently modified by the sulphur-gold
interaction with thiocholine, immobilization of the enzyme on the gold surface would
prove relatively costly since the electrode has to be replaced between each
measurement. Immobilization itself is a time-consuming and costly process, so
wasting the immobilized enzyme is not desirable. In order to re-use AChE in this
configuration, it is definitely feasible to apply magnetic separation techniques to
retrieve AChE enzymes from solution between measurements. This technique was
demonstrated by Sole et al.77 where AChE was attached to magnetic microbeads by
means of a bioengineered linker that was introduced into the enzyme at a location
away from the active site. The magnetic microbeads were then retrieved through the
application of a magnetic field during wash steps. This approach provides superior
42
limits of detection in biosensors of AChE inhibitors over those that use traditional
enzyme immobilization on electrode surfaces. When Istanboulie et al.78 bound AChE
to magnetic microbeads using Ni-Histidine affinity, they obtained detection limits 100
times better than those achieved in an identical biosensor that had AChE
immobilized on the electrode surface using a traditional Azide Unit water soluble
polymer (AWP).
It is not implied here that enzyme immobilization is not important in biosensor
designs. Immobilization serves to keep the enzyme from being degraded by
proteases that may appear in solution and from natural unfolding processes that
occur over time if enzyme movement is not restricted. Rather, what is being
contested here is the benefit of enzyme immobilization on the electrode surface. The
active electrode surface area should be optimized and the enzyme can still be
protected from degradation through immobilization on other surfaces that are not
involved in transduction. Furthermore, it is not necessary and arguably not even
desirable for AChE or other enzymes to be present in solution when measurements
are being collected. Hernandez et al.29 reported a comparison of a similar biosensor
using solvated and electrode surface-immobilized AChE. Although it is sometimes
argued that the closer proximity of the enzyme’s products to the electrode surface
may improve product oxidation at the electrode surface, the experimental results of
Hernandez et al. showed poorer sensitivity overall in the design featuring the
electrode surface-immobilized enzyme. On the contrary, the enzymes may adsorb
on the electrode surface, blocking the oxidation of products in the vicinity of the
working electrode, or the enzymes may become degraded when a potential
difference is applied across the electrode. Instead, with a neat sensor design using
magnetic microbeads, the incubation steps with the sample and substrate can take
place in separate solutions. Thereafter, the substrate solution, having reacted with
the enzyme, can be injected onto the electrode surface while AChE remains
magnetically separated from this entire process. This proposed design is illustrated
Figure 2.8:
43
Figure 2.8. Proposed design and illustration for an AChE inhibitor biosensor with AChE
fixed in solution attached to magnetic microbeads. Here, in the diagrams, I is an inhibitor
molecule in the sample, S is acetylthiocholine, and P is thiocholine. After the second
magnetic separation, only the remaining substrate and product are oxidized on the electrode
surface without the AChE enzymes in solution.
With the completion of this research project, it is my perspective that the optimal
biosensor assembly for this purpose is one of a lab-on-a-chip biosensor containing a
wash solution chamber and a substrate solution chamber connected to separate
compartments containing the enzyme and the electrode using microfluidic
technology. However, this type of catalytic-mode biosensor may find competition
from alternative biosensor designs that utilize newly discovered properties of
indicator compounds, which can report indirectly on the presence of AChE inhibitors.
One such indicator compound is Thioflavin T, which was at the focus of my second
research project due to its reported interactions with the peripheral binding site of
the AChE enzyme.
44
2.2 Project #2: Electrochemical Detection of Thioflavin T’s
Interaction with the Acetylcholinesterase Peripheral Binding Site:
Application to the Detection of Acetylcholinesterase Inhibitors
Thioflavin T (ThT) is a positively charged benzothiozole fluorescent dye that is well-
known from the research of amyloid-β aggregation in Alzheimer’s disease. The dye
is incorporated into the β-sheet structure of the amyloid peptides, which alters the
ThT molecular conformation to increase measured fluorescence 79. ThT is also
known to interact with the β-sheet structure of serum albumins80. In 2001, it was
shown that ThT also has a measurable interaction with the AChE enzyme structure.
In the pioneering work of the Rosenberry group using fluorescence spectroscopy, it
was demonstrated that ThT interacts with the AChE peripheral binding site81. This
interaction does not serve to inhibit the function of the AChE enzyme. Furthermore,
Rosenberry et al.82 showed that when inhibitor molecules interacted with the AChE
active site, ThT’s fluorescence decreased. For instance, the fluorescence signal of 1
µM ThT was quenched when exposed to 76 nM AChE that was pre-treated with 0.5
- 60 mM carbachol, a common carbamate pesticide83. This indicated that binding of
inhibitors at the active site modifies ThT’s binding or conformation at the peripheral
binding site of AChE83-89. The second research project which was started near the
end of 2009 set the goal of translating Rosenberry’s research with ThT and AChE
using electrochemical techniques in place of fluorescence spectroscopy.
Figure 2.9. A representation of the chemical structure of ThT (4-(3,6-dimethyl-1,3-
benzothiazol-3-ium-2-yl)-N,N-dimethylaniline chloride).
45
Figure 2.10. An illustration of the three-electrode system used to take measurements for the
ThT-based research project. A 6 mL sample was loaded into a sample container which
could also accommodate the Ag/AgCl glass reference electrode (R), GC working electrode
(W) and the platinum counter electrode (C). The apparatus was placed on a magnetic stir
plate, which was used to stir the sample solution using a magnetic stir bar prior to
measurement collection.
Figure 2.11. DP voltammogram of a 280 nM ThT solution in PBS pH 7.4 on a GC electrode.
The peak appearing near 1.2 V is not diagnostically useful since it is unstable and is near
the upper limit of the GC electrode potential window.
46
ThT Electrochemical Profile
ThT’s electrochemical properties were not well-known when the project was started.
The analysis of ThT using DP voltammetry in a three-electrode system indicated that
ThT undergoes oxidation near 0.9 V on a glassy carbon (GC) electrode surface at
pH 7.4 in a phosphate-buffered solution (PBS). Furthermore, ThT appears to
degrade when in solution over a period of 24 h, leading to substantially decreased
oxidation peak currents unless a new stock solution is prepared every day for each
new experiment. Unlike the DEP chips that were used in the previous research
project, the GC three-electrode system was reusable and required polishing of the
GC working electrode between measurements. It was seen that the ThT oxidation
current intensity increased linearly with increasing ThT concentration between 40
and 400 nM. Higher concentrations than 400 nM are detectable but are not
recommended for three-electrode systems such as the one used for this project
since the glass surface of the Ag/AgCl reference electrode has a significant affinity
for the ThT molecules. With higher ThT concentrations, the reference electrode
quickly acquires a yellow colour as ThT binds to its surface.
Figure 2.12.Calibration plot for the concentration dependence of ThT’s oxidation peak
current with DP voltammetry.
47
Once this exposure to high concentrations of ThT occurs, the reference electrode
begins to contribute to unstable measurements and yields a completely modified
ThT calibration curve if lower concentrations of ThT are then re-tested. The fouled
reference electrode seems to decrease the overall sensitivity of electrochemical
detection – i.e. the slope of the new calibration curve is less steep than the original
calibration curve obtained before the reference electrode becomes fouled. The
affinity of ThT for the reference electrode caused many problems at the onset of this
research project, since it was necessary to find a ThT concentration that had the
highest possible oxidation peak current without causing problems to measurement
reproducibility. After a substantial amount of testing, the ThT concentration was
selected at 280 nM to achieve a reproducible and stable detection platform. In
hindsight, if this research project were to ever be repeated, I would strongly
recommend that the scientist responsible for the project use DEP chips instead of
the three-electrode system. This may allow for the use of higher ThT concentrations
without fear of contaminating the reference electrode.
Apart from a potentially problematic reference electrode, a time-dependence was
seen in the observed ThT oxidation peak current. Oxidation current increased with
increasing incubation time in a predictable manner up to 5 min. If longer incubation
times were applied, the measurement of ThT oxidation current would sporadically
change from current values similar to shorter incubations down to almost no current
at all. This may have occurred due to unexpected events taking place at the open-
circuit potential or possible fouling of the reference electrode with the longer
incubation times. For this reason, incubation was limited to 4 min at open-circuit
potential and the electrode system integrity was checked every three measurements
against a known stock concentration of ThT to ensure that it was still possible to
produce the same oxidation peak current as obtained with the very first
measurement of that solution in the day. This setup proved to be stable for the
purposes of this research project.
In addition to determining the ThT calibration curve with the GC three-electrode
system, it was observed that ThT’s oxidation signal is also pH-dependent.
48
Continuing with a concentration of 280 nM ThT, the optimal oxidation peak current
occurred between pH 7-7.4, with the current falling off on either side of this range.
Changing the pH of the solution also led to changes in the ThT oxidation potential,
with higher pH values reducing the oxidation potential towards 0.8 V, whereas more
acidic pH values increased oxidation potential towards 0.95 V.
Figure 2.13. The pH dependence of 280 nM ThT oxidation peak current between pH 4 and
9. A maximum occurs near pH 7, with the oxidation current dropping off on either side.
Interaction of ThT with AChE
With the electrochemical properties of ThT having been defined for the given
electrochemical detection platform, the interaction of ThT with AChE was probed.
Using electric eel AChE, it was seen that the ThT oxidation peak current dropped
substantially when the enzyme was present in solution. A calibration plot was made
to determine the dependence of 280 nM ThT oxidation peak current on AChE
concentration. The dependence was found to be inversely exponential, with a more-
or-less linear decrease between 0 and 12.5 nM AChE followed by an insignificant
decrease in ThT oxidation current when enzyme concentration was increased to 25
nM. Surprisingly, a particularly reproducible ThT oxidation peak current was
measured with 12.5 nM AChE.
49
Figure 2.14. Calibration plot of 280 nM ThT oxidation peak current as a function of AChE
concentration.
With enzyme economy and reproducibility in mind, 12.5 nM was chosen for the
AChE concentration used in this research project. Previous work done by
Rosenberry et al.82 suggested a 1:1 stoichiometric ratio for the interaction of ThT
and AChE. However, the electrochemical data collected during this project did not
appear to reflect the ratio determined by Rosenberry et al.82 from fluorescent data.
On average, a 12.5 nM AChE solution decreased the oxidation peak current of 280
nM ThT by almost 3-fold. Control measurements were performed to explore the
possible reasons for this observation. The ThT oxidation peak current drops
regardless of whether the measurement is taken with AChE in solution together with
ThT or if the GC working electrode is first incubated in AChE solution and then
washed and transferred into the ThT solution. This indicates that the observed
current decrease is due to events that are occurring with AChE that adsorbs at the
electrode surface. To determine whether this was simply a matter of electrode
surface fouling by the AChE enzyme, the GC electrode was studied through control
experiments designed using cyclic voltammetry and the ferri-ferrocyanide redox
couple.
50
Investigation of the Surface Fouling Hypothesis
The ferri-ferrocyanide redox couple experiences reversible redox events at the GC
electrode surface as iron is repeatedly oxidized and reduced. This was observed
with a 20 mM ferri-ferrocyanide solution. Upon incubation of the GC electrode with
AChE and subsequent washing and exposure of the electrode to the ferri-
ferrocyanide redox cycle, it was seen that AChE had aggregated on the electrode
surface. The current intensity of ferri-ferrocyanide redox began at almost the same
value as the control for the first scan, but the current progressively decreased in the
four subsequent scans. Increasing the AChE concentration yielded a lower current
intensity for the first scan and subsequent scans showed even lower currents.
However, even with an AChE concentration that was more than 50 times larger than
what was used in our DP voltammetry experiments, the ferri-ferrocyanide redox
current intensity had only dropped by ~50%. This observation suggested that
electrode surface fouling was not primarily responsible for the drop in ThT oxidation
signal observed when the working electrode was incubated with AChE.
Figure 2.15. (left) The structure of ThT and (right) the structure of BTA-1, the neutral
derivative of ThT that lacks a methyl substituent on each nitrogen and in the benzothiozole
benzene.
Electrochemical Profile and Interactions of BTA-1
Additional experiments were attempted using BTA-1 in place of ThT. BTA-1 is a very
similar compound to ThT except for BTA-1’s neutral charge and fewer methyl
substituents on the structure. BTA-1 is a more expensive compound that turns out to
be much less stable in an aqueous solution at pH 7.4 than ThT. As a result, BTA-1
solutions had to be made hourly during experiments with the compound.
Furthermore, BTA-1 appears to be less electrochemically active, so a concentration
51
of 860 nM had to be used to achieve comparable oxidation peak currents. When
BTA-1 was placed in solution with AChE and oxidized, a similar drop in oxidation
peak current was seen. However, the pre-treatment of AChE by inhibitors did not
produce any significant regenerative effects on the oxidation peak current of BTA-1
on the GC electrode surface. The lack of an effect on BTA-1’s oxidation at the
electrode surface in the presence of AChE inhibitors disproves the electrode surface
fouling hypothesis. Instead, the experimental results with BTA-1 show that the
charged state of ThT and possibly the methyl substituents contribute to some sort of
unstable interaction with AChE that BTA-1 does not participate in.
Figure 2.16. Sample DP voltammograms showing (a) the oxidation of 860 nM BTA-1, (b)
860 nM BTA-1 with 20 nM AChE and 4 ppm paraoxon, and (c) 860 nM BTA-1 with only 20
nM AChE. The differences between the oxidation peak currents of (b) and (c) were not
found to be significant over several measurements.
If surface fouling by AChE is not the major reason for the observed drop in ThT
oxidation peak current, it is possible that the enzymes that are adsorbed on the GC
electrode surface may be capturing more ThT molecules besides the one that is
known to interact with the peripheral binding site. Fluorescence spectroscopy
studies by Rosenberry et al.82 had indicated a 1:1 stoichiometric ratio for the
interaction of a single ThT molecule with the AChE peripheral binding site of a single
enzyme. This interaction is detected as an increase in the measured fluorescence
levels of ThT when the molecule undergoes a favourable conformational change
during its interaction with the
molecules may also be interacting with the enzyme without experiencing the same
type of conformational cha
electrochemical measurements but not through fluorescence measurements. In this
scenario, not only would the AChE enzymes on the electrode surface be capturing
ThT at the peripheral binding site but also other ThT
the enzyme topology. This hypothesis would need to be investigated using
fluorescence microscopy or atomic force microscopy to visualize the distribution of
ThT on the GC electrode surface after incubation with AChE.
Figure
A maximum oxidation peak current is observed at a paraoxon concentration of 5 ppm. The
bottom graph shows the sensor response at lower paraoxon concentrations. Concentrations
of par
electrode surface. (
showing three maxima ~ 10 ppb, 10 ppm, and 30 ppm respectively. (
voltammogram o
and 7 ppm paraoxon, and (c) 280 nM
A
during its interaction with the
molecules may also be interacting with the enzyme without experiencing the same
type of conformational cha
electrochemical measurements but not through fluorescence measurements. In this
scenario, not only would the AChE enzymes on the electrode surface be capturing
ThT at the peripheral binding site but also other ThT
the enzyme topology. This hypothesis would need to be investigated using
fluorescence microscopy or atomic force microscopy to visualize the distribution of
ThT on the GC electrode surface after incubation with AChE.
Figure 2.17. (A)
A maximum oxidation peak current is observed at a paraoxon concentration of 5 ppm. The
bottom graph shows the sensor response at lower paraoxon concentrations. Concentrations
of paraoxon approaching and exceeding 20 ppm quench the oxidation of ThT at the
electrode surface. (
showing three maxima ~ 10 ppb, 10 ppm, and 30 ppm respectively. (
voltammogram o
and 7 ppm paraoxon, and (c) 280 nM
during its interaction with the
molecules may also be interacting with the enzyme without experiencing the same
type of conformational cha
electrochemical measurements but not through fluorescence measurements. In this
scenario, not only would the AChE enzymes on the electrode surface be capturing
ThT at the peripheral binding site but also other ThT
the enzyme topology. This hypothesis would need to be investigated using
fluorescence microscopy or atomic force microscopy to visualize the distribution of
ThT on the GC electrode surface after incubation with AChE.
Calibration plots of the ThT
A maximum oxidation peak current is observed at a paraoxon concentration of 5 ppm. The
bottom graph shows the sensor response at lower paraoxon concentrations. Concentrations
aoxon approaching and exceeding 20 ppm quench the oxidation of ThT at the
electrode surface. (B) Calibration plot of the same biosensor for carbachol
showing three maxima ~ 10 ppb, 10 ppm, and 30 ppm respectively. (
voltammogram of (a) 280 nM
and 7 ppm paraoxon, and (c) 280 nM
during its interaction with the AChE
molecules may also be interacting with the enzyme without experiencing the same
type of conformational change. This could be what was detected using
electrochemical measurements but not through fluorescence measurements. In this
scenario, not only would the AChE enzymes on the electrode surface be capturing
ThT at the peripheral binding site but also other ThT
the enzyme topology. This hypothesis would need to be investigated using
fluorescence microscopy or atomic force microscopy to visualize the distribution of
ThT on the GC electrode surface after incubation with AChE.
Calibration plots of the ThT
A maximum oxidation peak current is observed at a paraoxon concentration of 5 ppm. The
bottom graph shows the sensor response at lower paraoxon concentrations. Concentrations
aoxon approaching and exceeding 20 ppm quench the oxidation of ThT at the
) Calibration plot of the same biosensor for carbachol
showing three maxima ~ 10 ppb, 10 ppm, and 30 ppm respectively. (
f (a) 280 nM ThT oxidation, (b) 280 nM
and 7 ppm paraoxon, and (c) 280 nM ThT oxidation with only 12.5 nM AChE.
AChE peripheral site. However,
molecules may also be interacting with the enzyme without experiencing the same
nge. This could be what was detected using
electrochemical measurements but not through fluorescence measurements. In this
scenario, not only would the AChE enzymes on the electrode surface be capturing
ThT at the peripheral binding site but also other ThT
the enzyme topology. This hypothesis would need to be investigated using
fluorescence microscopy or atomic force microscopy to visualize the distribution of
ThT on the GC electrode surface after incubation with AChE.
Calibration plots of the ThT-based AChE
A maximum oxidation peak current is observed at a paraoxon concentration of 5 ppm. The
bottom graph shows the sensor response at lower paraoxon concentrations. Concentrations
aoxon approaching and exceeding 20 ppm quench the oxidation of ThT at the
) Calibration plot of the same biosensor for carbachol
showing three maxima ~ 10 ppb, 10 ppm, and 30 ppm respectively. (
ThT oxidation, (b) 280 nM
ThT oxidation with only 12.5 nM AChE.
B
C
peripheral site. However,
molecules may also be interacting with the enzyme without experiencing the same
nge. This could be what was detected using
electrochemical measurements but not through fluorescence measurements. In this
scenario, not only would the AChE enzymes on the electrode surface be capturing
ThT at the peripheral binding site but also other ThT molecules at other locations on
the enzyme topology. This hypothesis would need to be investigated using
fluorescence microscopy or atomic force microscopy to visualize the distribution of
ThT on the GC electrode surface after incubation with AChE.
based AChE-inhibitor biosensor for paraoxon.
A maximum oxidation peak current is observed at a paraoxon concentration of 5 ppm. The
bottom graph shows the sensor response at lower paraoxon concentrations. Concentrations
aoxon approaching and exceeding 20 ppm quench the oxidation of ThT at the
) Calibration plot of the same biosensor for carbachol
showing three maxima ~ 10 ppb, 10 ppm, and 30 ppm respectively. (
ThT oxidation, (b) 280 nM ThT oxidation with 12.5 nM
ThT oxidation with only 12.5 nM AChE.
peripheral site. However,
molecules may also be interacting with the enzyme without experiencing the same
nge. This could be what was detected using
electrochemical measurements but not through fluorescence measurements. In this
scenario, not only would the AChE enzymes on the electrode surface be capturing
molecules at other locations on
the enzyme topology. This hypothesis would need to be investigated using
fluorescence microscopy or atomic force microscopy to visualize the distribution of
ThT on the GC electrode surface after incubation with AChE.
inhibitor biosensor for paraoxon.
A maximum oxidation peak current is observed at a paraoxon concentration of 5 ppm. The
bottom graph shows the sensor response at lower paraoxon concentrations. Concentrations
aoxon approaching and exceeding 20 ppm quench the oxidation of ThT at the
) Calibration plot of the same biosensor for carbachol
showing three maxima ~ 10 ppb, 10 ppm, and 30 ppm respectively. (C
ThT oxidation with 12.5 nM
ThT oxidation with only 12.5 nM AChE.
peripheral site. However, additional ThT
molecules may also be interacting with the enzyme without experiencing the same
nge. This could be what was detected using
electrochemical measurements but not through fluorescence measurements. In this
scenario, not only would the AChE enzymes on the electrode surface be capturing
molecules at other locations on
the enzyme topology. This hypothesis would need to be investigated using
fluorescence microscopy or atomic force microscopy to visualize the distribution of
inhibitor biosensor for paraoxon.
A maximum oxidation peak current is observed at a paraoxon concentration of 5 ppm. The
bottom graph shows the sensor response at lower paraoxon concentrations. Concentrations
aoxon approaching and exceeding 20 ppm quench the oxidation of ThT at the
) Calibration plot of the same biosensor for carbachol – this time
C) A sample DP
ThT oxidation with 12.5 nM AChE
ThT oxidation with only 12.5 nM AChE.
52
additional ThT
molecules may also be interacting with the enzyme without experiencing the same
nge. This could be what was detected using
electrochemical measurements but not through fluorescence measurements. In this
scenario, not only would the AChE enzymes on the electrode surface be capturing
molecules at other locations on
the enzyme topology. This hypothesis would need to be investigated using
fluorescence microscopy or atomic force microscopy to visualize the distribution of
inhibitor biosensor for paraoxon.
A maximum oxidation peak current is observed at a paraoxon concentration of 5 ppm. The
bottom graph shows the sensor response at lower paraoxon concentrations. Concentrations
aoxon approaching and exceeding 20 ppm quench the oxidation of ThT at the
this time
) A sample DP
AChE
Figure 2.18.
when ThT is exposed to
AChE that was pre
disrupts the docking of the ThT molecule(s) on the enzyme.
AChE and
It was observed that the incubation of AChE with carbachol and paraoxon before
exposure to ThT produces a greater ThT oxidation p
control with untreated AChE. However, the calibra
not linear with 10
concentrations. There appears to be a complex relationship
concentration used to treat AChE and the resulting ThT oxidation peak current after
incubation with the treated AChE. For carbachol,
peak current
30 ppm. Paraoxon produced
led to values below the AChE controls at concentrations beyond 20 ppm
ppm, paraoxon had almost completely
was performed that confirmed paraoxon to decrease ThT oxidation peak current in
the absence of AChE at concentrations approaching and exceeding 20 ppm. This
suggests the possibility of a simple ThT sensor for
The same control test ap
peak current
There was a marked difference in ThT oxidation peak current obtained f
AChE controls and 10 ppb concentrations for either inhibitor. The discrimination of
these concentrations from baseline AChE controls already illustrate very impressive
Figure 2.18. An illustration of the events that may be taking place at the electrode surface
when ThT is exposed to
AChE that was pre
disrupts the docking of the ThT molecule(s) on the enzyme.
AChE and Pre-
It was observed that the incubation of AChE with carbachol and paraoxon before
exposure to ThT produces a greater ThT oxidation p
control with untreated AChE. However, the calibra
not linear with 10
concentrations. There appears to be a complex relationship
concentration used to treat AChE and the resulting ThT oxidation peak current after
incubation with the treated AChE. For carbachol,
peak current were
30 ppm. Paraoxon produced
led to values below the AChE controls at concentrations beyond 20 ppm
ppm, paraoxon had almost completely
was performed that confirmed paraoxon to decrease ThT oxidation peak current in
the absence of AChE at concentrations approaching and exceeding 20 ppm. This
suggests the possibility of a simple ThT sensor for
The same control test ap
peak current even with carbachol concentrations
There was a marked difference in ThT oxidation peak current obtained f
AChE controls and 10 ppb concentrations for either inhibitor. The discrimination of
these concentrations from baseline AChE controls already illustrate very impressive
An illustration of the events that may be taking place at the electrode surface
when ThT is exposed to (A)
AChE that was pre-treated with inhibitors. The presence of inhibitors in solution with
disrupts the docking of the ThT molecule(s) on the enzyme.
-Exposure to Inhibitors: Effects on ThT Oxidation
It was observed that the incubation of AChE with carbachol and paraoxon before
exposure to ThT produces a greater ThT oxidation p
control with untreated AChE. However, the calibra
not linear with 10-30% increases in current versus AChE controls at various inhibitor
concentrations. There appears to be a complex relationship
concentration used to treat AChE and the resulting ThT oxidation peak current after
incubation with the treated AChE. For carbachol,
were obtained with the inhibitor’s concentration at 10 ppb, 10 ppm and
30 ppm. Paraoxon produced
led to values below the AChE controls at concentrations beyond 20 ppm
ppm, paraoxon had almost completely
was performed that confirmed paraoxon to decrease ThT oxidation peak current in
the absence of AChE at concentrations approaching and exceeding 20 ppm. This
suggests the possibility of a simple ThT sensor for
The same control test ap
even with carbachol concentrations
There was a marked difference in ThT oxidation peak current obtained f
AChE controls and 10 ppb concentrations for either inhibitor. The discrimination of
these concentrations from baseline AChE controls already illustrate very impressive
An illustration of the events that may be taking place at the electrode surface
AChE that has not been pre
treated with inhibitors. The presence of inhibitors in solution with
disrupts the docking of the ThT molecule(s) on the enzyme.
xposure to Inhibitors: Effects on ThT Oxidation
It was observed that the incubation of AChE with carbachol and paraoxon before
exposure to ThT produces a greater ThT oxidation p
control with untreated AChE. However, the calibra
30% increases in current versus AChE controls at various inhibitor
concentrations. There appears to be a complex relationship
concentration used to treat AChE and the resulting ThT oxidation peak current after
incubation with the treated AChE. For carbachol,
obtained with the inhibitor’s concentration at 10 ppb, 10 ppm and
30 ppm. Paraoxon produced a maximum ThT oxidation peak current at 7 ppm
led to values below the AChE controls at concentrations beyond 20 ppm
ppm, paraoxon had almost completely
was performed that confirmed paraoxon to decrease ThT oxidation peak current in
the absence of AChE at concentrations approaching and exceeding 20 ppm. This
suggests the possibility of a simple ThT sensor for
The same control test applied with carbachol showed no
even with carbachol concentrations
There was a marked difference in ThT oxidation peak current obtained f
AChE controls and 10 ppb concentrations for either inhibitor. The discrimination of
these concentrations from baseline AChE controls already illustrate very impressive
An illustration of the events that may be taking place at the electrode surface
AChE that has not been pre
treated with inhibitors. The presence of inhibitors in solution with
disrupts the docking of the ThT molecule(s) on the enzyme.
xposure to Inhibitors: Effects on ThT Oxidation
It was observed that the incubation of AChE with carbachol and paraoxon before
exposure to ThT produces a greater ThT oxidation p
control with untreated AChE. However, the calibra
30% increases in current versus AChE controls at various inhibitor
concentrations. There appears to be a complex relationship
concentration used to treat AChE and the resulting ThT oxidation peak current after
incubation with the treated AChE. For carbachol,
obtained with the inhibitor’s concentration at 10 ppb, 10 ppm and
maximum ThT oxidation peak current at 7 ppm
led to values below the AChE controls at concentrations beyond 20 ppm
ppm, paraoxon had almost completely quenched the oxidation of ThT.
was performed that confirmed paraoxon to decrease ThT oxidation peak current in
the absence of AChE at concentrations approaching and exceeding 20 ppm. This
suggests the possibility of a simple ThT sensor for
plied with carbachol showed no
even with carbachol concentrations
There was a marked difference in ThT oxidation peak current obtained f
AChE controls and 10 ppb concentrations for either inhibitor. The discrimination of
these concentrations from baseline AChE controls already illustrate very impressive
An illustration of the events that may be taking place at the electrode surface
AChE that has not been pre-treated with any inhibitors and (B)
treated with inhibitors. The presence of inhibitors in solution with
disrupts the docking of the ThT molecule(s) on the enzyme.
xposure to Inhibitors: Effects on ThT Oxidation
It was observed that the incubation of AChE with carbachol and paraoxon before
exposure to ThT produces a greater ThT oxidation peak current compared to the
control with untreated AChE. However, the calibration plots for both inhibitors are
30% increases in current versus AChE controls at various inhibitor
concentrations. There appears to be a complex relationship
concentration used to treat AChE and the resulting ThT oxidation peak current after
incubation with the treated AChE. For carbachol, three maxima for
obtained with the inhibitor’s concentration at 10 ppb, 10 ppm and
maximum ThT oxidation peak current at 7 ppm
led to values below the AChE controls at concentrations beyond 20 ppm
quenched the oxidation of ThT.
was performed that confirmed paraoxon to decrease ThT oxidation peak current in
the absence of AChE at concentrations approaching and exceeding 20 ppm. This
suggests the possibility of a simple ThT sensor for toxic concentrations of paraoxon.
plied with carbachol showed no
even with carbachol concentrations of 100 ppm
There was a marked difference in ThT oxidation peak current obtained f
AChE controls and 10 ppb concentrations for either inhibitor. The discrimination of
these concentrations from baseline AChE controls already illustrate very impressive
An illustration of the events that may be taking place at the electrode surface
treated with any inhibitors and (B)
treated with inhibitors. The presence of inhibitors in solution with
xposure to Inhibitors: Effects on ThT Oxidation
It was observed that the incubation of AChE with carbachol and paraoxon before
eak current compared to the
tion plots for both inhibitors are
30% increases in current versus AChE controls at various inhibitor
concentrations. There appears to be a complex relationship between the inhibitor
concentration used to treat AChE and the resulting ThT oxidation peak current after
maxima for
obtained with the inhibitor’s concentration at 10 ppb, 10 ppm and
maximum ThT oxidation peak current at 7 ppm
led to values below the AChE controls at concentrations beyond 20 ppm
quenched the oxidation of ThT.
was performed that confirmed paraoxon to decrease ThT oxidation peak current in
the absence of AChE at concentrations approaching and exceeding 20 ppm. This
toxic concentrations of paraoxon.
plied with carbachol showed no effect on ThT oxidation
100 ppm and greater
There was a marked difference in ThT oxidation peak current obtained f
AChE controls and 10 ppb concentrations for either inhibitor. The discrimination of
these concentrations from baseline AChE controls already illustrate very impressive
An illustration of the events that may be taking place at the electrode surface
treated with any inhibitors and (B)
treated with inhibitors. The presence of inhibitors in solution with AChE
It was observed that the incubation of AChE with carbachol and paraoxon before
eak current compared to the
tion plots for both inhibitors are
30% increases in current versus AChE controls at various inhibitor
between the inhibitor
concentration used to treat AChE and the resulting ThT oxidation peak current after
maxima for ThT oxidation
obtained with the inhibitor’s concentration at 10 ppb, 10 ppm and
maximum ThT oxidation peak current at 7 ppm
led to values below the AChE controls at concentrations beyond 20 ppm. At 100
quenched the oxidation of ThT. A control test
was performed that confirmed paraoxon to decrease ThT oxidation peak current in
the absence of AChE at concentrations approaching and exceeding 20 ppm. This
toxic concentrations of paraoxon.
effect on ThT oxidation
and greater.
There was a marked difference in ThT oxidation peak current obtained from the
AChE controls and 10 ppb concentrations for either inhibitor. The discrimination of
these concentrations from baseline AChE controls already illustrate very impressive
53
An illustration of the events that may be taking place at the electrode surface
treated with any inhibitors and (B)
AChE
It was observed that the incubation of AChE with carbachol and paraoxon before
eak current compared to the
tion plots for both inhibitors are
30% increases in current versus AChE controls at various inhibitor
between the inhibitor
concentration used to treat AChE and the resulting ThT oxidation peak current after
ThT oxidation
obtained with the inhibitor’s concentration at 10 ppb, 10 ppm and
maximum ThT oxidation peak current at 7 ppm but
At 100
A control test
was performed that confirmed paraoxon to decrease ThT oxidation peak current in
the absence of AChE at concentrations approaching and exceeding 20 ppm. This
toxic concentrations of paraoxon.
effect on ThT oxidation
rom the
AChE controls and 10 ppb concentrations for either inhibitor. The discrimination of
these concentrations from baseline AChE controls already illustrate very impressive
54
detection capabilities, since regulatory standards require that most AChE inhibitors
be present at 20 ppb and preferably lower in foodstuffs and water8, 9. No lower
concentrations were tested during this research project since the purpose was to
establish that electrochemical monitoring of ThT oxidation in the presence of AChE
could be used to detect AChE inhibitors. Due to time and resource constraints, the
measurement of the true detection limits for this sensor configuration was left for
future experimentation. Complete calibration plots could be obtained using DEP
chips and square-wave voltammetry, which would allow for much faster and reliable
data collection.
Summary
Thus, in this research project, it was shown that the electrochemical oxidation signal
of ThT and BTA-1 decreases in the presence of AChE. Incubation of AChE with
AChE inhibitors, carbachol and paraoxon, prior to incubation with ThT but not BTA-1
led to the collection of DP voltammograms that displayed unique patterns of current
signal intensity above the baseline controls obtained with AChE but below control
measurements with ThT oxidation alone. These results imply that in addition to
fluorescent detection, ThT’s oxidation signal may be useful for the detection of
interactions between insecticides and AChE using electrochemical techniques.
Electrochemical detection of these interactions is preferable since fewer amounts of
reagents are used and greater sensitivity is achievable than with fluorescence
studies of the same phenomenon. This featured electrochemical research may be a
promising tool that can be used to probe AChE interactions with a variety of small
molecule ligands.
55
2.3 CONCLUDING REMARKS AND FUTURE DIRECTIONS
The research projects completed during 2009 and 2010 have produced interesting
results and ample opportunities and ideas for future research. First, a biosensor was
developed using gold DEP chips that could achieve competitive detection limits even
with an AChE enzyme at the biological recognition element that was 5-10 times less
sensitive to the tested inhibitors compared to the wild-type form. Second, a novel
electrochemical detection system for AChE inhibitors was demonstrated through the
monitoring of ThT oxidation peak currents in the presence of AChE exposed to
different inhibitor concentrations. The ThT oxidation peak currents increased when
the dye was incubated with inhibitor-pre-treated AChE. This project produced
unusual results with calibration plots that were not linear but still clearly distinct from
the baseline controls measured in the absence of inhibitors. Thus, such a sensor
would work as a “there or not” detector within the concentration ranges tested for
carbachol and paraoxon. It was shown that this system has the capacity to detect
concentrations of carbachol and paraoxon as low as 10 ppb and possibly lower with
further testing.
Several questions remain that can be answered in future research efforts. With the
first project featuring gold DEP chips, some questions that would be interesting to
see answered include:
1. What kind of performance would be seen in this biosensor with the use of
different mutant strains of the N. brasiliensis AChE enzymes and different
AChE inhibitors? The F345Y variant was less sensitive than the wild-type
AChE for both inhibitors that were tested. However, there exist different
mutants with much higher sensitivities and different activities. Beyond N.
brasiliensis, there exist much more advanced genetically engineered
AChE enzymes that work much faster on their substrates and it would be
interesting to study their application in this type of biosensor. The genetic
engineering of AChE enzymes for the purpose of using them in AChE
inhibitor biosensors involves a trade-off between enzyme activity and
inhibitor sensitivity. With a better understanding of protein folding, it may
56
eventually be possible to engineer complex AChE derivatives that are
specific for chosen inhibitors and are still able to rapidly convert substrate.
2. What is the meaning of the differences in thiocholine oxidation peak
shapes on the voltammograms obtained from real samples such as
solutions with milk versus Tris buffer? In DP voltammetry, peak area does
not have a theoretical analytical value. However, consistent differences in
peak shape under controlled conditions suggests that a diagnostic value
may still exist.
3. How would the biosensor perform if the AChE enzymes were linked to
magnetic beads? Istamboulie et al.78 utilized a TCNQ-modified carbon
paste electrode for their sensor featuring AChE attached to magnetic
beads. It would be interesting to see a comparison of the carbon paste
electrode to the gold DEP chips used in this particular research project.
Furthermore, the use of OPH enzyme attached to magnetic microbeads in
a step-wise sensor design would allow for the conservation of AChE
enzymes if a sample measurement shows OPH activity.
With the second project featuring ThT oxidation as an indicator of AChE
contamination by inhibitors, there is much research needed to explain unusual
observations:
1. The events taking place at the electrode surface that lead to decreased
ThT oxidation peak currents in the presence of AChE remain unresolved.
Likewise, it is also unclear why the ThT oxidation current increases if the
AChE is pre-treated with AChE inhibitors but not with sucrose or ATCh.
Fluorescence microscopy or atomic force microscopy should give
additional insight into this mystery.
2. The separation of AChE from the sample mixture after incubation with
ThT may also clarify the situation taking place at the electrode surface.
This can be compared to results with AChE immobilized on the electrode
surface.
57
3. The biosensor’s response was studied for paraoxon and carbachol. It
would be interesting to see calibration plots of the sensor’s response to
other AChE inhibitors. The existing calibration plots for paraoxon and
carbachol are complex. Explaining the shapes will require more
electrochemical data from different inhibitors together with microscopy
images.
4. ThT is a sulphur-containing compound. Thus far, a GC electrode was
used for the described research. Other types of electrode surfaces should
also be tested, particularly gold and platinum, both of which can benefit
from interacting with the sulphur group.
Without question, the research described in this document has provided me with
much insight and intrigue into the use of AChE in biological sensing. In my opinion,
the most fruitful future directions for research with AChE would be to probe the
enzyme’s interactions with other biological molecules of interest, such as amyloid-β
which has relevance in Alzheimer’s disease. It was previously discussed that AChE
is involved in complex-formation with amyloid-β and other important processes in
living organisms in addition to its traditional role as a regulator of the acetylcholine
neurotransmitter22. The use of unconventional electrochemical detection systems
with sensitive dyes such as ThT or Congo red is not very well-reported at this time
and this research niche could be effectively filled by scientists who are willing to
venture into such unconventional approaches to yield exciting innovative biosensor
designs.
58
3. EXPERIMENTAL DESCRIPTION AND SUPPORTING
MATERIAL
3.1 Project #1: DEP gold chips for the analysis of AChE inhibition
using DP voltammetry
Reagents
Except when stated otherwise, all samples were prepared using a Tris buffer
solution consisting of 20 mM Tris and 100 mM NaCl at pH 7. Paraoxon stock
solutions were prepared by diluting PS-610 Paraoxon oil (1.274 g/mL) purchased
from Sigma-Aldrich in the Tris buffer solution. Carbofuran (Sigma-Aldrich) stock
solutions required 1 part of DMSO per 50 parts of the Tris buffer to assist with
solubility. The F345Y N. brasiliensis AChE mutant was selected since its sensitivity
to carbofuran and paraoxon was comparable based on data from previous studies67.
The substrate, acetylthiocholine iodide (ATCh), was obtained from Sigma-Aldrich.
For the real samples, Highland Creek water that was collected near the University of
Toronto Scarborough and 18% cream (Sealtest Dairy Creamer™) were diluted 3-
fold with Tris buffer. This yielded 1:3 river water in Tris and 6% cream in Tris
(referred to as 6% milk).
Measurement Procedure
Initially, equal volumes (30 µL) of enzyme and either paraoxon or carbofuran
solutions were mixed together and allowed to incubate for 30 min as previously done
by Schulze et al.67. The enzyme-insecticide solutions were briefly mixed through
medium-speed vortexing every 10 min during the incubation. Upon completion of the
incubation time with the insecticide sample, another equal volume (30 µL) of
substrate solution was injected into the enzyme-insecticide solution for the substrate
incubation step. For the studies featuring constant enzyme concentration of 323
mU/mL (~5x10-10 M), the final substrate concentration for ATCh was set at 2.1 mM.
A voltammogram was collected upon completion of a subsequent 10 min incubation
period with the substrate, during which the sample was vortexed every 2 min.
59
The Eco Chemie µAutolab Type III FRA2 Potentiostat/Galvanostat, purchased from
Metrohm Autolab, was used to collect electrochemical data from disposable
electrochemical printed (DEP) chips (Fig. 1A). The DEP chips (DEP-ER-N) were
kindly donated by Professor Eiichi Tamiya from Osaka University and BioDevice
Technology, Japan. The counter electrode and the connecting wires of these DEP
chips were printed using carbon ink. The reference electrode contained Ag/AgCl
paste. The hydrophobic coating between the electrical connectors and the working
area of the chip prevented the contact of liquids with the electrical connections of the
potentiostat. The overall dimensions of the chip were 12.5 mm (Length) x 4 mm
(Width) x 0.3 mm (Thickness).
For each measurement, a 12 µL drop of sample was placed evenly over the working
electrode surface of a new DEP chip. DP voltammetry measurements were taken at
a modulation amplitude of 50 mV, step potential of 5 mV, modulation time of 50 ms,
interval time of 500 ms, and equilibration time of 5 s. The scan range was set
between – 0.1 V and 0.9 V. The data was processed using a moving average
baseline correction with minimum peak height of 0.003 prior to signal peak analysis.
The oxidation peak current of thiocholine was recorded at ~0.3 V.
Inhibition Calculations
For the evaluation of the enzyme inhibition, the TCh peak current at ~0.3 V was
used to detect inhibition by carbofuran and paraoxon. The following expression for
enzyme inhibition was applied:
where Itreatment is the insecticide-containing sample’s TCh current peak amplitude,
while Icontrol refers to the corresponding control sample in the absence of the
insecticide. The detection limit was defined as the smallest concentration of
insecticide in the treatment sample that resulted in a 20% average inhibition of
AChE (I20). Each concentration was evaluated with 3 DEP chips for the Itreatment value
60
and compared to control values (Icontrol) collected through another 3 DEP chips. A
summary of the procedure is shown in Figure 24.
Figure 3.1. The flowchart of the procedure used to collect measurements for the gold DEP
chip research project. Note that three measurements were taken on three new DEP chips
for each insecticide concentration and for each control value.
61
3.2 Project #2: Electrochemical Detection of ThT’s Interaction with
the AChE Peripheral Binding Site: Application to the Detection of
AChE Inhibitors
Reagents
All reagent solutions were prepared in a 20 mM phosphate 100 mM NaCl buffer
solution at pH 7.4 unless otherwise specified. Purified 18.2 MΩ water from the PAL
Cascada LS purification system was used to make all aqueous solutions. Thioflavin
T (T3516) and BTA-1 (B9934) were obtained from Sigma Aldrich. Electric eel AChE
(C3389), paraoxon (PS610), and carbachol (C2409) were all obtained from Sigma.
Sample Preparation Steps and DP Voltammetry
A conventional three-electrode system from CH Instruments, consisting of a CH104
GC working electrode (GCE), CH111 Ag/AgCl reference electrode, and CHI115
platinum wire counter electrode, was connected to an Eco Chemie µAutolab Type III
FRA2 Potentiostat/Galvanostat (Metrohm International). The General Purpose
Electrochemical System (GPES) software by Eco Chemie was used as the data
collection interface.
Prior to each oxidation voltammogram measurement, a 6 mL sample solution (280
nM ThT together with 12.5 nM AChE and/or variable concentrations of paraoxon or
carbachol) was prepared and lightly stirred for 1 min on a magnetic stir plate. This
was followed by the introduction of the electrode system and incubation with stirring
for an additional 4 min at the open-circuit potential (OCP). At this point, the stirrer
was turned off. The sample was then oxidized on the GCE from 0-1.2 V using
differential pulse voltammetry (DP Voltammetry) at a modulation amplitude of 50
mV, step potential of 5 mV, modulation time of 50 ms, interval time of 500 ms, and
equilibration time of 10 s. The oxidation voltammogram was processed using the
GPES moving average baseline correction tool with a selected minimum peak height
of 0.003 prior to peak analysis using the peak search function. Between
measurements, the GCE surface was renewed via polishing on a polishing pad for 1
62
min in a suspension of 0.05 micron Gamma Alumina Powder from CH Instruments
and 18.2 MΩ Cascada water. On average, each data point consists of three to four
measurements (n ≥ 3).
Calibration Plot Construction for ThT Concentration Dependence
Five concentrations of ThT between 40 and 400 nM were oxidized on the GCE, with
the peak current measured at 0.87 V to make the calibration plot for ThT
concentration dependence. A linear response was measured in this concentration
range for ThT oxidation. Measurements of higher concentrations were attempted,
but this altered the values obtained for the recorded linear range when those
concentrations were re-tested. A yellow discoloration or the reference electrode was
clearly visible after these measurements at higher ThT concentrations were
collected. Polishing and/or replacement of the GC working electrode did not have a
significant effect on the measurements. Replacement of the glass Ag/AgCl reference
electrode returned measurements to normal expected readings. As a result, higher
concentrations were avoided in subsequent experimentation steps.
Calibration Plot Construction for pH Dependence of 280 nM ThT Peak
Oxidation Current
Solutions of 280 nM ThT were prepared in buffer solutions set at pH 4, 5, 6.5, 6.8, 7,
7.4 and 9. Of these pH values, a 20 mM acetic acid / sodium acetate buffer was
used to prepare the pH 4 and 5 buffer, 20 mM PBS buffer was used for pH 6.5, 6.7,
7 and 7.4 buffer, and finally 20 mM Tris was used for the pH 9 buffer. Each buffer
solution also contained 100 mM NaCl electrolyte as with the PBS buffer mentioned
earlier. Measurements were taken as described previously (n≥3).
Calibration Plot Construction for 280 nM ThT Peak Oxidation Current
Dependence on AChE Concentration
Five AChE concentrations were tested for their effect on the 280 nM ThT peak
oxidation current. AChE stock solutions were prepared via serial 2x dilutions starting
63
from 25 nM and proceeding through 12.5 nM, 6.25 nM, 3.125 nM, and 1.563 nM
respectively. Measurements were then collected as previously described (n≥3).
Control Experiments
Impact of Paraoxon and Carbachol Concentrations on ThT Oxidation Signal
Intensity
In this experiment, 280 nM ThT was incubated for 5 min with varying concentrations
of paraoxon and carbachol before DP voltammetry measurement. No AChE was
added at any point during this control experiment. Carbachol showed no significant
effect on the ThT oxidation signal even at concentrations as high as 100 ppm.
Paraoxon showed no significant effects on the ThT signal when present at low
concentrations approaching 20 ppm. At concentrations higher than 20 ppm,
paraoxon had the effect of decreasing the intensity of the ThT signal substantially.
This explains the drop in ThT oxidation when AChE is pre-treated with higher
paraoxon concentrations. The inhibition of ThT’s oxidation signal by ppm levels of
paraoxon is worth investigating in the future for a simple detection method of toxic
levels of this insecticide.
ThT Adsorption and Oxidation Signal Intensity
The GC electrode was incubated for four min in a 280 nM ThT solution. The GC
electrode was then removed from the 280 nM ThT solution and rinsed with PBS
buffer. The GC electrode was then placed in a sample of clean PBS buffer
solution. The DP voltammogram was immediately collected to see how much of
the ThT signal was due to ThT aggregation at the GC electrode surface. The ThT
peak intensity of the DP voltammetry measurement in clean buffer solution was
80% as big as the measurement taken in ThT solution. This shows that there is a
high level of ThT adsorption taking place at the GC electrode prior to a
measurement being made. Therefore, this control experiment shows that most of
the recorded current is due to the adsorption of ThT on the electrode surface.
64
Figure 3.2. DP voltammetry oxidation signal taken in 280 nM ThT solution (a) and in
PBS buffer after rinsing the GC electrode (b).
When 12.5 nM AChE and 280 nM ThT are tested in the same way, the same
results are found. The DP voltammetry measurement taken in a clean PBS
solution after prior GC electrode incubation with ThT and AChE produces a ThT
oxidation peak that is 80% as big as the measurement recorded in solution with
ThT and AChE.
Figure 3.3. DP voltammetry oxidation signal taken in 280 nM ThT and 12.5 nM AChE
solution (a) and in PBS buffer after rinsing the GC electrode (b).
These results suggest that most of the ThT oxidation signal that is observed is
65
determined by ThT’s adsorption behaviour at the surface of the electrode during
the 4 min incubation step. The same proportion (80%) of ThT adsorbs to the
electrode surface when AChE is present in solution.
Investigation of Surface Fouling by AChE
It is possible that the observed drop in ThT oxidation signal in the presence of
AChE is due to surface fouling of the GC electrode by AChE. AChE would block
ThT’s access to the electrode surface for oxidation. Several control experiments
were designed and tested to investigate this possibility.
1. Separate GC electrode incubation steps
In this experiment, the GC electrode was incubated first in 12.5 nM AChE
solution for 4 min. The electrode was then removed and rinsed with PBS
buffer solution. This was followed by an incubation of the GC electrode for
another 4 min in 280 nM ThT solution. The DP voltammetry measurement
was then taken in the 280 nM ThT solution. This produced similar results
as seen with DP voltammetry measurements taken in a solution of 280 nM
ThT and 12.5 nM AChE.
These results indicated that the drop in ThT oxidation signal intensity does
not require AChE to be in solution with ThT. The signal decrease is
presumably seen with AChE adsorbed on the surface of the GC electrode.
The question that followed was whether AChE blocked the access of ThT
to the GC electrode surface, thus leading to the measured reduction in ThT
oxidation peak current.
2. Ferri-Ferrocyanide Cyclic Voltammograms
Cyclic voltammograms (CVs) with ferri-ferrocyanide allow for the
measurement of the Fe2+/3+ redox processes taking place at the electrode
surface. The measurements are often used to characterise modified
electrode surfaces, where a drop in ferri-ferro redox activity would indicate
66
the presence of adsorbed or bound species on the electrode surface. The
CV measurements taken in the following control experiments used 5
standard linear scans from -0.5to 1.25 V, with characteristic Fe2+/3+ redox
occuring at ~ 0.23 V and 0.27 V respectively.
i. Testing ferri-ferrocyanide accessibility to the GC electrode
surface after incubation with 12.5 nM AChE.
The GC electrode was incubated for 4 min in 12.5 nM AChE
solution. It was then washed with PBS bufer solution and placed into
20 mM ferri-ferrocyanide soluion. CVs were measured and showed
the first scan to be very close to the CV measurements in clean 20
mM ferri-ferrocyanide solution. Scans 2-5 showed a progressive
decrease in redox couple intensity, with the fifth scan showing a
20% drop in intensity compared to scan 1.
Figure 3.4. CVs of 20 mM ferri-ferrocyanide with a 4 min pre-incubation of the GC
electrode in PBS buffer solution (black) and 12.5 nM AChE solution (red).
ii. Testing ferri-ferrocyanide accessibility to the GC electrode
surface after incubation with 200 nM AChE.
The same experiment was repeated but with 200 nM AChE. The
67
same pattern was observed, but with an overall 40% drop in the
redox couple intensity after 5 scans compared to the 20% drop seen
with 12.5 nM AChE in the previous experiment..
iii. Testing ferri-ferrocyanide accessibility to the GC electrode
surface after incubation with 600 nM AChE.
When this experiment was repeated with 600 nM AChE, there was a
50% drop in the redox couple intensity after 5 scans. In addition, the
first scan now showed a 20% drop in redox couple intensity
compared to the control measurements made on a clean GC
electrode in 20 mM ferri-ferrocyanide solution.
Figure 3.5. CVs of 20 mM ferri-ferrocyanide after 4 min pre-incubations of the GC
electrode in 12.5 nM AChE (black), 200 nM AChE (red), and 600 nM AChE (blue).
Even with 600 nM AChE, the GC surface is not saturated with the enzyme at the time
of measurement.
In summary, the first CV scan showed that the ferri-ferrocyanide molecules
had easy access to the GC electrode at the experimental AChE
concentrations. Only when the AChE concentration was ~60 times higher
(600 nM vs 12.5 nM) was there a substantial change in the first CV scan
intensity when compared to clean GC electrode controls in 20 mM ferri-
68
ferrocyanide solution. Furthermore, even after 5 scans, the ferri-
ferrocyanide species could still undergo their redox processes with 50% of
the initial signal magnitudes even at 600 nM AChE. This shows that the
electrode surface is not saturated at this concentration or at the
experimental 12.5 nM AChE conditions.
These results suggest that, with our experimental conditions of 12.5 nM
AChE, the enzyme does not block a substantial portion of the electrode
area to justify the observed ~60% drop in ThT oxidation signal intensity.
Instead, the ThT molecules may be preferably interacting with AChE
before oxidizing on the surface. The native tetrameric form of AChE may
be creating a surface modification that effectively captures ThT from
solution near the electrode surface.
iv. Testing ferri-ferrocyanide accessibility to the GC electrode
surface after incubation with 100 ppm carbachol.
In this experiment, the GC electrode was incubated in 100 ppm
carbachol for 4 min. The electrode was rinsed in PBS buffer solution
and then placed into 20 mM ferri-ferrocyanide solution, where CV
measurements were taken. The incubation with carbachol showed
no effect on the redox processes of ferri-ferrocyanide in any of the 5
scans even at this high concentration, indicating that carbachol does
not adsorb significantly to the electrode surface.
69
Figure 3.6. CVs of 20 mM ferri-ferrocyanide after a 4 min pre-treatment of the GC
electrode in PBS buffer solution (black) and 100 ppm carbachol solution (red).
v. Testing ferri-ferrocyanide accessibility to the GC electrode
surface after incubation with 100 ppm carbachol-pre-treated
12.5 nM AChE.
The same procedure was followed as in (iv) to see the effects of
incubating the GC electrode in a solution of 12.5 nM AChE and 100
ppm carbachol. The CVs that were measured were not substantially
different from the CVs collected in (i) for clean 12.5 nM AChE.
70
Figure 3.7. CVs of 20 mM ferri-ferrocyanide after a 4 min pre-incubation of the GC
electrode in 12.5 nM AChE (black) and 12.5 nM AChE with 100 ppm carbachol (red).
No significant differences are observed in any of the 5 scans.
These results suggest that the changes in ThT signal intensity
observed in our studies in the presence of insecticides are not a
result of the acetylcholinesterase inhibitors somehow detaching the
enzyme from the electrode surface. The electrode surface is
essentially the same whether carbachol is present or not.
vi. Testing ferri-ferrocyanide accessibility to the GC electrode
surface after incubation with 200 nM AChE followed by another
incubation with 280 nM ThT.
This experiment featured the incubation steps described in (ii) but
also included an extra step where the GC electrode was washed
with PBS buffer solution and then incubated for another 4 min in 280
nM ThT solution. The GC electrode was then washed again with
clean PBS buffer solution and placed in 20 mM ferri-ferrocyanide
solution for CV measurements. The ferri-ferrocyanide CVs obtained
in (i) showed redox couple current intensities that were 10% greater
71
than those obtained after incubation with 280 nM ThT.
Figure 3.8. CVs of 20 mM ferri-ferrocyanide after a 4 min pre-incubation of the GC
electrode in 200 nM AChE solution (black) compared with the same pre-incubation in
200 nM AChE followed by another 4 min incubation in 280 nM ThT (red). The ThT
adsorbs to the GC electrode surface and blocks ferri-ferrocyanide redox processes by
an additional ~10% over AChE.
These results indicate that the ferri- and ferro-cyanide molecules are
still easily able to reach the electrode surface even in the presence
of adsorbed species of both 280 nM ThT and 200 nM AChE on the
GC electrode surface. Experimentally, 12.5 nM AChE was used, so
there was more opportunity for ThT to reach the GC electrode
surface.
Overall, the ferri-ferrocyanide CV control experiments suggest that surface
fouling by AChE is not the primary reason behind the measured drop in ThT
oxidation at the GC electrode surface.
Determining the Selectivity of the Biosensor for AChE Inhibitors
The ThT oxidation peak current increases when AChE is pre-treated with varying
72
concentrations of carbachol and paraoxon (the latter below 20 ppm). As a
selectivity control, ThT oxidation was monitored when AChE was pre-treated with
ATCh (the substrate used in the first research project) and sucrose. ATCh binds
to the active site of AChE, but its presence in solution at low concentrations
(<<0.1 mM) did not affect the pure 280 nM ThT oxidation or the decrease in ThT
oxidation that is seen when AChE is in solution. At higher concentrations of
ATCh, the compound’s oxidation dwarfs the oxidation signal from 280 nM ThT.
Likewise, tests with 20 mM sucrose showed no impact on ThT’s oxidation signal
in the presence and absence of 12.5 nM AChE. Thus, the ThT-based biosensor
appears to show selectivity for the presence of paraoxon and carbachol and not
for sucrose or ATCh.
73
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