Self-assembly of acetylcholinesterase on gold nanoparticles electrodeposited on graphite

Article abstract of DOI:10.2478/s11532-013-0307-3

The immobilisation of AChE enzyme through chemisorption on Au-modified graphite was examined with view of its prospective application in the design of membraneless electrochemical biosensors for the assay of enzyme inhibitors. The developed immobilisation protocol has been based on a two-stage procedure, comprising i) electrodeposition of gold nanostructures on spectroscopic graphite; followed by ii) chemisorption of the enzyme onto gold nanoparticles. Both the coverage of the electrode surface with Au nanostructures and the conditions for enzyme immobilisation were optimised. The proposed electrode architecture together with the specific type of enzyme immobilisation allow for a long-term retaining of the enzyme catalytic activity. The extent of inhibition of the immobilised acetylcholinesterase enzyme by the organophosphorous compound monocrotophos has been found to depend linearly on its concentration over the range from 50 to 400 nmol mL^-1 with sensitivity 77.2% inhibition per 1 μmol mL^-1 of monocrotophos. [Figure not available: see fulltext.]

Full text of DOI:10.2478/s11532-013-0307-3

Cent. Eur. J. Chem. • 11(11) • 2013 • 1740-1748
DOI: 10.2478/s11532-013-0307-3
Central European Journal of Chemistry
Self-assembly of acetylcholinesterase on gold
nanoparticles electrodeposited on graphite
Research Article
Nina Dimcheva1*, Elena Horozova1,
Yavor Ivanov2, Tzonka Godjevargova2#
1Department Physical Chemistry, Plovdiv University,
BG-4000 Plovdiv, Bulgaria
2Department Biotechnology, University “Prof. Dr Assen Zlatarov”,
BG – 8010, Burgas, Bulgaria
Received 30 April 2013; Accepted 22 June 2013
Abstract:
The immobilisation of AChE enzyme through chemisorption on Au-modified graphite was examined with view of its prospective
application in the design of membraneless electrochemical biosensors for the assay of enzyme inhibitors. The developed immobilisation
protocol has been based on a two-stage procedure, comprising i) electrodeposition of gold nanostructures on spectroscopic graphite;
followed by ii) chemisorption of the enzyme onto gold nanoparticles. Both the coverage of the electrode surface with Au nanostructures
and the conditions for enzyme immobilisation were optimised. The proposed electrode architecture together with the specific type
of enzyme immobilisation allow for a long-term retaining of the enzyme catalytic activity. The extent of inhibition of the immobilised
acetylcholinesterase enzyme by the organophosphorous compound monocrotophos has been found to depend linearly on its
-1
-1
concentration over the range from 50 to 400 nmol mL with sensitivity 77.2% inhibition per 1 μmol mL of monocrotophos.
Keywords: Electrodeposited Au-nanostructures • Self-assembly • Acetylcholinesterase • Acetylthiocholine • Inhibition
© Versita Sp. z o.o.
In most AChE biosensors, the pesticide assay is
based on the irreversible enzyme inhibition. Numerous
1. Introduction
Acetylcholinesterase (AChE) is a key enzyme in the immobilisation techniques have been proposed for
nervous system of mammalian organisms, responsible constructingAChE biosensors: direct physical adsorption
for transmitting nerve impulses to the cholinergic on a solid carrier [ ], encapsulation in a polymeric film
3-5
synapses, and can be easily inhibited by a variety [ ], immobilisation in porous membranes [ ], adsorption
6
7
of toxic compounds: heavy metals, nicotine and on nanoparticle modified matrices [8-13], binding
neuroparalytic gases, medications for the treatment through chemisorption [14-17] etc. However none of
of Alzheimer’s or Parkinson’s diseases, or pesticides them is considered universal for constructing AChE
(organophosphorous and carbamate). The accumulation biosensors. Enzyme immobilisation on nanoparticle
of organophosphorous and carbamate, being intensively modified electrodes, such as gold nanoparticles
used in modern agriculture to protect crops from insects, (Au NPs) [
8-13
], carbon nanotubes (CNTs) [
18-23
],
1
has to be carefully monitored [ ] since they are highly CdS – nanoparticles (CdS NPs) [ ] etc. allows for a
24
toxic for humans and domestic animals and therefore considerable improvement of both biosensor stability and
pose an environmental threat, thus motivating the sensitivity and lowers the limit of detection. Pesticides’
constantly growing scientific interest in developing assay with AChE biosensors commonly requires enzyme
electrochemical biosensors for quantitative analysis of reactivation, which is not always successful, and thus
pesticides [ ].
makes the re-use of the biosensor awkward. Hence,
2
* E-mail: nina.dimcheva@uni-plovdiv.net
# E-mail: godjevargova@yahoo.com
1740

N. Dimcheva et al.
AChE
AChE immobilisation on the single-use electrodes (e.g. acethylthiocholine + H O
→
2
AChE
screen- printed electrodes) has also been considered
thiocholine + acetic acid
(1)
→
[
].
25-27
A simple and versatile technique for enzyme
Both the non-enzymatic and the enzymatic
hydrolysis of acetylthiocholine to thiocholine depend on
the pH of the solution and were found to slow-down in
immobilisation based on gold-thiol chemistry is the
spontaneouschemisorptionofsulphur-containingorganic
compounds (cysteamine, mercaptobenzothiazole, etc.)
on the colloid gold modified electrode surfaces thus
forming a self-assembled monolayer, SAM, to which
the AChE enzyme is attached through intermolecular
basic media [ ].
29
Stage 2: Electrochemical oxidation of thiocholine
onto the electrode surface:
anodic oxidation→
complexes [
14-17,28
]. This immobilisation approach
2 thioholine
disulfide + 2H+ + 2e- (2)
provides conformational flexibility of the enzyme and
hence assures a more efficient biocatalytic process,
however enabling the formation of a thicker diffusion
layer that lowers electrode sensitivity. An even simpler
method for AChE immobilisation is to chemisorb it
directly on the surface of Au NPs without the need
for additional thiolated linkers [7,8,10,13], since
its protein shell contains terminal SH-groups. The
obvious challenge in this case is, however, to retain the
enzyme-functionalized NPs onto the electrode, which
is implemented by either their incorporation in a porous
The process takes place with the transfer of
one electron from the thiocholine and subsequent
dimerisation of the intermediate to disulfide [
When conventional electrodes are used
].
23,30
],
31-33
[
thiocholine electrooxidation proceeds at potentials
higher than 0.7 V (vs. SCE).
Stage 3: Upon the inhibition of the AChE enzyme,
the thiocholine oxidation current decreases. The extent
of enzyme inhibition is determined by the equation:
membrane [ ] or inclusion in a composite film cast on
7
,
(3)
the surface of the working electrode [
] thus yielding
8,10
a stable but thick catalytic layer that triggers additional
diffusion limitations.
where IS is the steady-state electrode response to the
control solution (at large thiocholine concentration
present); II is the enzyme electrode response at
the same thiocholine concentration plus a given
inhibitor concentration. In the present study the
organophosphorus compound monocrotophos was
used as a model AChE inhibitor.
In the present work, a two-step strategy for the
acetylcholinesterase immobilisation on spectroscopic
graphite is discussed: in the first stage gold nanoparticles
were grown directly onto the graphite surface through
electrochemical reduction of HAuCl4 and, in the second
stage, the AChE enzyme was directly chemisorbed
on them, forming a self-assembled monolayer. The
enzyme binds to the gold nanoparticles via its terminal
2. Experimental procedure
sulfur-containing groups of the protein shell [ ]. Direct
7
deposition of Au NPs onto the graphite surface followed
by the enzyme chemisorption represents a principally
novel approach for designing electrochemical AChE-
based biosensors, whose main advantages include:
i) enhanced enzyme load onto the electrode
surface with preserved conformational flexibility of the
immobilised biocatalyst that ensures retention of its
catalytic activity to a big extent (over one month); and
ii) minimal diffusional limitations on the substrate
access to the enzyme layer, warranting better sensitivity
(~20 times higher than the literature data) and faster
electrode response (at most 20 s).
The principle of detection of AChE enzyme inhibitors
using acetylthiocholine as the enzyme substrate is via
the following three stages:
Stage 1: AChE catalyses the hydrolysis of
acetylthiocholine to thiocholine and acetic acid at room
temperature:
2.1. Materials
Rods of spectroscopic graphite, type RWO (Ringsdorf
Werke, Bohn, Germany) with a diameter of 0.5 cm, fitted
into Teflon insulation, were used as working electrodes.
Acetylcholinesterase (AChE, EC 3.1.1.7) from an electric
eel with an activity of 425.94 U mg-1 (Fluka Biochemika);
acetylthiocholine iodide with purity of over 99% (Fluka);
KI (Valerus, Bulgaria) used as 2 mM aqueous solution,
HAuCl4 (Fisher) used as 50 mM solution in 0.1 M HCl;
КН2РО4 and К2НРО4 (Sigma-Aldrich) used for preparing
buffer solution were all of analytical grade and used as
received.
The solution of acetylthiocholine iodide, with an
initial concentration of 2×10-3 М was prepared just
before the experiment, and during the measurements
was stored in a cooling ice bath, at 1–2ºC, to avoid
its spontaneous hydrolysis; the organophosphorous
1741

Self-assembly of acetylcholinesterase on gold
nanoparticles electrodeposited on graphite
compound monocrotophos C7H14NO5P (Riedel-de- comparative experiments, Au NPs modified electrodes
Haën) used as an enzyme inhibitor was in the form without the enzyme were produced.
of aqueous solutions with concentrations from 0.05 to
0.9 μmol mL-1.
After measurements, the enzyme electrodes were
water rinsed and stored in 0.1 M phosphate buffer
Double-distilled water was used in all water and solution (рН = 7.6) in a refrigerator, and their activity was
buffer solutions.
monitored by periodically testing the enzyme electrode
response to acetylthiocholine.
2.2. Instrumentation
The enzyme electrodes with lost AChE activity were
A standard, three-electrode cell with a volume of regenerated by mechanical polishing on wet sand paper,
10-20 cm3 with working graphite electrode (with then ultrasonicated in bi-distilled water, followed by
modification), reference electrode - Ag/AgCl, 3M KCl electrodeposition ofAu NPs and enzyme immobilisation.
and a platinum wire as an auxiliary electrode, was used
in all electrochemical experiments. All the potentials 2.4. Measurement procedure
given in the paper were reported against Ag/AgCl, Thiocholine electrooxidation was examined by
3M KCl. The electrochemical measurements were cyclic voltammetry (CV) and constant – potential
performed with an electrochemical computer-controlled chronoamperometry. CVs were registered in 0.1 М
workstation Palm Sens, with PS Trace software (Palm phosphate buffer, pH= 7.6 at a scan rate of 10 mV s-1.
Instruments, The Netherlands). During amperometric The concentration dependencies of the current were
measurements, the solutions were stirred at 450 rpm measured on Au-modified graphite with or without
with a magnetic stirrer (IkaMag RCT, Ika, Germany). immobilised enzyme, as follows: the electrode was
The pH of the buffer solutions was controlled by a рН- poised at a constant potential, Е (typically 800 mV
meter HI 211 (Hanna Instruments, USA).
vs. Ag/AgCl, 3 M KCl) in the buffer as a background
The surface morphology of the modified electrodes electrolyte until a steady-state current response, І0 (µА)
was examined with a scanning electron microscope was established under constant stirring. Afterwards, an
(SEM), JEOL JSM-5500, operating at 10 kV for imaging. aliquot (20; 50; 200; 500 or 1000 µl) of the analyte stock
No conductive coatings or other treatment were applied solution (thiocholine: 2×10-3 М; or acetylthiocholine:
to the samples prior to SEM observations.
2×10-3 М) was added and the new value of the steady-
state electrode response IS (µA) was read. The time
for reaching a steady-state value of the current under
the given conditions did not exceed 0.5 minutes. All the
information needed for plotting the calibration graphs
was obtained from chronoamperometric records. The
enzyme inhibition by the organophosphorous insecticide
monocrotophos was studied by chronoamperometry
using aqueous solutions with concentrations from 0.05
to 0.9 μmol mL-1. Between measurements with different
inhibitor concentrations the electrode was reactivated
in a water solution of oxime (2- PAM, 5 mg mL-1). All
experimental results represent the mean value of at least
three measurements (n = 3 to 6) with the regression
analysis performed using MS EXCEL and Origin 7.5.
2.3. Electrode preparation, storage and
regeneration
Prior to use, the graphite electrodes were mechanically
cleaned by polishing on a fine wet sand-paper, with a
gradual decrease of the size of the abrasive particles
(Р800, Р1200, Р1500 and Р2000) and followed by
ultrasonication in bi-distilled water for 3 minutes each.
Gold nanoparticles were then deposited by a brief
electrolysis (1 or 10 seconds) at a constant potential
of -155 mV (vs. Ag/AgCl, 3M KCl) from 50 mM solution
of НАuCl4 dissolved in 0.1М НСl. After the deposition,
the surface of the electrode was washed with bi-distilled
water and cleaned electrochemically in 2 М H2SO4
by continuous cycling over the potential range from 0
to 1.7 V for at least 20 cycles until three consecutive
voltammograms overlapped.
3. Results and discussion
The immobilisation of the enzyme through a self-
assembly process was carried out on the gold-modified 3.1. Electrodeposition of Au NPs
graphite electrode, which was electrochemically One of the biggest challenges in developing the protocol
cleaned prior to enzyme immobilisation. The electrode for the AChE immobilisation on gold nanoparticles was
surface, rinsed with double distilled water was immersed the development of a simple, reliable and reproducible
into a small-volume container with enzyme solution procedure for their direct deposition onto the graphite
(1 mg mL-1) and stored in a refrigerator for 24 hours. surface. The deposition method had to meet the
following requirements posed by this particular practical
application: uniform distribution of the deposits over the
The non-adsorbed enzyme was removed by washing
the electrode with bi-distilled water and buffer. For
1742

N. Dimcheva et al.
Figure 1. CVs of graphite electrode in background electrolyte 0.1 M HCl (dash) and in 5 mM HAuCl4 present (solid).
(a)
(b)
Figure 2. Scanning electron microscopy (SEM) images of Au nano-structures deposited electrochemically on spectroscopic graphite for: a) 1 s;
and b) 10 s.
whole surface, small amount of deposited gold, good larger structures and multilayered formations, however
adhesion to the graphite and availability of sites for does not noticeably contribute to a greater surface
enzyme chemisorption.
coverage with Au nanoparticles.
Gold electrodeposition on graphite was carried
out in an acidic solution of tetrachloroaurate at 3.2. Electrochemical oxidation of thiocholine
-155 mV. The applied potential was more positive than The electrochemical oxidation of thiocholine – the
the potential at which its electrochemical reduction product of the enzymatic hydrolysis of acetylthiocholine
starts by 130-140 mV (Fig. 1), however, upon increasing (stage
the Au3+ concentration 10-fold, the coverage of the graphites, was studied by both CV and amperometry.
electrode surface with gold can be clearly observed
Voltammetric curves recorded in the presence of
2 of inhibitor detection) on gold-modified
by the naked eye even for deposition times as short thiocholine on the gold modified graphite electrodes for 1
as 1 second. The formation of uniformly distributed and 10 seconds are illustrated in Fig. 3. A pair of clearly
gold clusters that grow-up in 1 second to 3D- oblong expressed peaks is observable on the CVs with peak-
structures with an average particle size of ca. 200 to-peak separations of 120 mV and 90 mV, respectively,
nm (Fig. 2a) have been observed. By increasing the indicating quasi-reversible electrochemistry of the
duration of the electrolysis process to 10 seconds, larger process. These peaks, however, are not associated
structures were formed (Fig. 2b). A further increase in with the electrochemical oxidation of thiocholine, since
the time for deposition leads to the formation of even the constant potential amperometric measurements
1743

Self-assembly of acetylcholinesterase on gold
nanoparticles electrodeposited on graphite
Figure 3. CVs of Au-modified graphite electrodes in 1 mM thiocholine present; graphite with Au electrodeposited for 1 s (dash) and 10 s (solid);
0.1 M phosphate buffer, рН = 7.6; scan rate 10 mVs-1; room temperature.
Electrode response as a function of thiocholine concentration at potential 0.8 V (vs. Ag/AgCl); open circles: Au electrodeposited for
1 s (regression equation Y = 0.184 X; R2 = 0.992); closed circles: Au electrodeposited for 10 s (regression equation Y = 0.245 X;
R2 = 0.995); electrolyte: 0.1 M phosphate buffer, рН = 7.6; room temperature. Inset: Chronoamperometric record upon addition of
thiocholine; electrode: graphite with electrodeposited Au for 10 s; experimental conditions as stated above.
Figure 4.
indicated very weak electrode response at applied level. However, the electrooxidation of thiocholine on
potentials below 0.8 V. Although much cheaper than graphite modified with Au through electrodeposition
acetylthiocholine chloride, the use of acetylthiocholine for a duration twice as long (20 seconds) does not
iodide as the substrate for AChE causes undesired lead to an improved electrode sensitivity, despite the
electrooxidation of the iodide ions at the working amount of gold (determined from coulometric data)
potential, as it was shown earlier by Stoytcheva et al. being increased by 60% as compared to the 10 second
[34] and the quasi-reversible peaks observed on the modification. Obviously, as discussed above, the
CVs are most probably due to this process.
increased load of the modifier does not substantially
The dependence of the electrode response on contribute to the enhancement of the electrochemically
thiocholine concentration (Fig. 4) shows that the active electrode surface. Therefore, all further
increased duration of the Au electrodeposition, up to discussions will concern mainly the performance of
10 seconds, results in a 33% improvement in electrode the Au-electrodeposited on graphite for 10 seconds.
sensitivity: from 0.184 mA mM-1 (open circles) to
The effect of I- oxidation at 0.8 V and pH = 7.6 on
0.245 mA mM-1 (closed circles), keeping in the same Au NPs modified graphite for 10 seconds was examined
time strict linearity of the calibration plot and low noise using KI solution with the same concentrations as
1744

N. Dimcheva et al.
Figure 5.
a) Chronoamperometric record of the AChE-electrode responce upon addition of acetylthiocholine; b) Calibration plots of the same
enzyme electrode (AChE self-assembled on Au NPs modified graphite for 10 s); applied potential 0.8 V (vs. Ag/AgCl); 0.1 M phosphate
buffer, рН = 7.6; applied potential 0.8 V (vs. Ag/AgCl); room temperature. Y-error bars correspond to the 2.43 % standard deviation.
thiocholine iodide. Under our experimental conditions two clearly expressed peaks of oxygen desorption were
it has been found that the iodide electrooxidation observed on the CVs, suggesting that the surface oxides
contributes to the electrode response with 44.8% of the formed on Au NPs would allow the enzyme adsorption
signal, i.e., the partial rates of electrochemical oxidation on them only to an insignificant extent, or would even
of thiocholine and I- under the given conditions are block it. Alternatively, an enzyme layer was found to
almost the same.
form on the electrochemically cleaned electrodes, which
reaches maximal surface coverage within ~20 hours.
The degree to which this process took place was probed
by catalytic activity during acetylthiocholine hydrolysis,
3.3. Immobilisation of AChE through self-
assembly
The choice of electrode material and the immobilisation i.e., by the response of the enzyme electrode when
method [35] are of key importance for the design of aliquots of the substrate were added.
a reliable biosensing device, since these affect its
operational characteristics: sensitivity, response time, Fig. 5a demonstrates that the enzyme electrode
stability, reproducibility, etc. current increased stepwise upon the addition of
key element in this procedure was the acetylthiocholine aliquots, the enzymatic hydrolysis of
The chronoamperometric record, presented in
A
electrochemical cleaning of the modified surface, which generates thiocholine and acetic acid (Reaction 1)
preceding the chemisorption of the enzyme as it was followed by the electrochemical oxidation of thiocholine
found that the enzyme scarcely attaches to non-pre- (Reaction 2). The higher the substrate concentration,
treated electrodes. During the electrochemical treatment the higher the noise level observed. The increased
1745

Self-assembly of acetylcholinesterase on gold
nanoparticles electrodeposited on graphite
Figure 6. Remaining activity of the enzyme electrode as a function of the storage time. The Y-error bars correspond to the 2.43% standard
deviation.
noise is most probably due to the electrochemical that the electrode sensitivity will drop to ca. 0.115 µА
oxidation of iodide ions, but could also result from other µМ-1 since, as mentioned above, the contribution of the
side reactions (see e.g. Reaction 2).
electrooxidadation of iodide is considerable. Yet, the
The calibration graph (Fig.5b) of the produced anticipated electrode sensitivity will significantly exceed
AChE –electrode, shows a linear trend over the studied the literature data reported so far.
range of substrate concentrations – above 600 µМ,
3.4. Reproducibility and stability of AChE
with a regression correlation coefficient of 0.995 and a
slope of 0.209 µА µМ-1 (0.135 µА µМ-1 – for 1 second)
enzyme electrode
The inter-assay precision, or fabrication reproducibility,
was evaluated by determining the response to 50 μL
2 mM solution of ATCh of six different electrodes and
the relative standard deviation was found to be 2.43%.
The intra-assay precision of the sensors was assessed
by testing one enzyme electrode for six replicate
determinations and the RSD was calculated to be 1.12%
for an ATCh concentration of 50 μM. These results were
indicative of an acceptable reproducibility regarding
ATCh determination.
The stability of the enzyme electrode during long-
term storage was examined for over a month by testing
its activity with respect to its substrate ATCh. Fig. 6
shows the residual activity of the enzyme electrode
as a function of its storage time. The residual activity
was determined as the percentage of the measured
signal at a constant concentration of the substrate, to
the current at the same concentration, measured with
a freshly prepared AChE-electrode. As it is evident from
Fig. 6, the catalytic activity of the immobilized enzyme
decreases by half over the first 10 days, and over the
next 40 days it gradually goes down to 40% of the
initially measured signal with the same electrode.
representing the electrode sensitivity. A slight decrease
of the electrode sensitivities after enzyme chemisorption
is most probably associated with the coverage of
the electrode surface with a protein monolayer that
hampers the penetration of the enzymatically produced
thiocholine to its surface. The detection limit towards
acetylthiocholine was estimated to be 2.5 µМ at a signal
to noise ratio of 5. The enzyme electrode response
reached it steady-state value in at most 20 seconds.
The determined electrode sensitivity of 0.209 µА
µМ-1 towards acetylthiocholine is considerably improved
compared to the one reported in the previous studies
of the group (implemented with a biosensor with
AChE immobilised on a modified PAN membrane with
incorporated gold nanoparticles [7]), and much greater
than that reported in the literature for biosensors with
a similar method of enzyme immobilisation (AChE
chemisorbed on gold nanoparticles), viz.: about
18-24 times as high as the fabricated by Du and co-
workers [11] and approximately 48-64 times as high as
the reported by Shulga and co-authors [13] biosensor.
This finding is obviously due to the much greater
number of chemisorbed enzyme molecules resulting
from both the extended electrode surface area and
the enlarged number of available centres for enzyme 3.5. InhibitionstudiesofAChEenzymeelectrode
chemisorption owing to the electrochemical cleaning of In order to prove that the immobilised AChE enzyme
the gold surface. Indeed, upon exchanging the enzyme retains its enzymatic activity upon immobilisation to a
substrate to acetylthiocholine chloride it is expected large extent and the registered electrode response
1746

N. Dimcheva et al.
Figure 7. Linear dynamic range of the dependence of the extent of inhibition of AChE on monocrotophos concentration, at room temperature.
The error bars correspond to the standard deviation.
is due to the electrochemical oxidation of the operational characteristics at a working potential of
thiocholine produced by the enzymatic hydrolysis of 800 mV (vs. Ag/AgCl), as follows:
acetylthiocholine, the purposeful irreversible inhibition
• the detection limit of 2.5 µМ towards the enzyme
of the immobilised AChE with the organophosphorous substrate acetylthiocholine;
insecticide monocrotophos was performed. The
response of AChE-enzyme electrode to the enzyme
inhibitor was studied in an acetylthiocholine-rich buffer
• electrode sensitivity towardsATCh of 0.209 µА µМ-1 ;
• response time of 20-30 seconds;
• storage time up to 50 days within which time the
solution (рН =7.6) at a working potential of 0.8 V enzyme retains up to 40% of its initial activity;
(vs. Ag/AgCl) and room temperature. •degreeofinhibitionbytheinsecticidemonocrotophos
The effect of monocrotophos concentration on the that depends linearly on its concentration over the
degree of AChE inhibition is presented in Fig. 7. As range from 0.05 to 0.4 μmol mL-1 with a ‘sensitivity’
one can see, the linear part of the “calibration plot” of the inhibitor determination - 77.23% inhibition per
of the inhibition degree versus the monocrotophos 1 μmolmL-1 monocrotophos (R² = 0.985).
concentration, i.e., the range where the inhibition is
controlled by a first-order reaction, spans over the range principle opportunity to create
The results presented herein demonstrate the
cost-effective
a
from 0.05 µmol mL-1 to 0.4 µmol mL-1. Immobilised AChE electrochemical biosensor for the determination of
exhibited good initial sensitivity towards the pesticide, the enzyme inhibitor monocrotophos. Future work
up to a percentage inhibition of 30% at the upper concerning the increase of biosensor sensitivity
limit of the linearity range. The overall results outline towards the inhibitor, decrease of the noise level and
the good potential of the discussed immobilisation improvement of the detection limit, by e.g. replacing the
system for the construction of a biosensor for pesticide enzymesubstrateusedinthisworkwithacetylthiocholine
detection, since the inhibition pattern shows analogy to chloride, is planned. Another promising improvement to
a great extent with other biosensing systems reported be done is to decrease the operating potential so that
so far [7]. Further work is needed, however, in order to minimize the contribution of the side electrooxidative
to optimise the performance of the reported electrode reactions.
to design a biosensor for assaying the AChE inhibitor
monocrotophos.
Acknowledgements
4. Conclusions
Authors acknowledge the support from the Bulgarian
National Science Fund (grants DDVU – 02/38 and
A membrane-less amperometric enzyme electrode was DO 02 – 145), administered by the Bulgarian Ministry
developed with the AChE enzyme immobilised through of Education, Science and Youth. The technical help
chemisorption onto Au NPs modified graphite. The of the students Maria Chuleva and Boris Dzhuvinov is
electrode was characterized with respect to its basic appreciated.
1747

Self-assembly of acetylcholinesterase on gold
nanoparticles electrodeposited on graphite
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