One-step electrochemically deposited gold nanoparticles interface grafted with avidin for acetylcholinesterase biosensor design

Article abstract of DOI:10.1166/jnn.2010.2476

In this study, an interface embedded in situ gold nanoparticles (AuNPs) and biotin in chitosan hydro-gel was constructed by one-step electrochemical deposition in solution containing tetrachloroauric (III) acid, biotin and chitosan. This deposited interface acts as biosensing platforms and provides specific binding sites for avidin, which are further capable of attaching any biotinylated bimolec-ular for biosensor design. Atomic force microscopy (AFM), A.C. impedance and surface plasmon resonance were used to characterize this interface. The immobilized acetylcholinesterase (AChE), as a model, showed excellent activity to its substrate and provided a quantitative measurement of organophosphate pesticide. Under the optimal experimental conditions, the inhibition of dimethoate was proportional to its concentrations in the range of 0.05 to 15 μg mL^-1 with detection limit of 0.001 μg mL ^-1. The simple method showed good fabrication reproducibility and acceptable stability, which provided a new avenue for electrochemical biosensor design.

Full text of DOI:10.1166/jnn.2010.2476

Copyright © 2010 American Scientific Publishers
All rights reserved
Printed in the United States of America
Journal of
Nanoscience and Nanotechnology
Vol. 10, 5685–5691, 2010
One-Step Electrochemically Deposited Gold
Nanoparticles Interface Grafted with Avidin for
Acetylcholinesterase Biosensor Design
Weiying Zhang, Jiawang Ding, Yuehua Qin, Deli Liu∗, and Dan Du∗
Key Laboratory of Pesticide and Chemical Biology of Ministry of Education,
Central China Normal University, Wuhan 430079, P. R. China
In this study, an interface embedded in situ gold nanoparticles (AuNPs) and biotin in chitosan hydro-
gel was constructed by one-step electrochemical deposition in solution containing tetrachloroauric
(III) acid, biotin and chitosan. This deposited interface acts as biosensing platforms and provides
specific binding sites for avidin, which are further capable of attaching any biotinylated bimolec-
ular for biosensor design. Atomic force microscopy (AFM), A.C. impedance and surface plasmon
resonance were used to characterize this interface. The immobilized acetylcholinesterase (AChE),
as a model, showed excellent activity to its substrate and provided a quantitative measurement of
organophosphate pesticide. Under the optimal experimental conditions, the inhibition of dimethoate
was proportional to its concentrations in the range of 0.05 to 15 ꢀg mL−1 with detection limit of
0.001 ꢀg mL−1. The simple method showed good fabrication reproducibility and acceptable stability,
which provided a new avenue for electrochemical biosensor design.
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Keywords: Electrodeposition, Gold Nanoparticles, Biotin Labeled Acetylcholinesterase,
Copyright: American Scientific Publishers
Dimethoate, Biosensor.
1. INTRODUCTION
controllable thickness.14 Chitosan as a kind of matrix
for biomolecules possess attractive properties including
excellent film-forming ability, high permeability toward
water, good adhesion, non-toxicity, and biocompatibility.15
It is one of preferred materials in biosensor design. Previ-
ous study indicated that chitosan could be electrodeposited
onto gold surface at an initial potential of −3.0 V.16–18 The
deposition procedure of chitosan is based on a local elec-
trochemically induced pH modulation caused by the oxida-
tion or reduction of water which can be easily confined to
a defined volume at the electrode surface by proper adjust-
ment of the nature, strength, and capacity of the buffer in
the electrolyte solution.19 Incorporation of gold nanopar-
ticles (AuNPs) into polymer matrices has also attracted
increasing interest in improving the stability and biocom-
patibility of interface and enhancing the surface capabil-
ity for immobilization of biomolecules due to the superior
Surfaces grafted with biomolecules have received exten-
sive attention for possible applications in advanced
technologies including drug deliver,1 bioseparation,2
microreactor,3 and biosensor fabrication.4–6 The devel-
opment of procedures allowing the spatially controlled
deposition of biomolecules is especially crucial for tailor-
ing molecular recognition events at surfaces in biosens-
ing as well as for investigating biological interactions.7ꢀ8
Usual methods including direct physical adsorption,9
encapsulation,10 cross-linking,11 and covalent binding12
have been widely used in biosensor design. In order
to create molecular interactions with exigent control of
biomolecules at the surface, many new methods have been
developed.
Electrochemical deposition has been reported recently
as an effective method for immobilization of biomolecules
due to the formation of sol–gel films.13 Compared
with the traditional process, electrochemical deposition
is simple, the performance conditions are moderate, and
it is suitable for selective deposition of films with
properties of nanoparticles.20ꢀ21 AuNPs embedded in chi-
22–24
tosan composite film has been reported
and the inter-
face embedded AuNPs in situ has also been constructed
by one-step electrochemical deposition.25
Avidin is a glycoprotein (molecular weight, 68 000)
found in egg white and is known to contain four identi-
cal binding sites to biotin. The binding constant between
∗Authors to whom correspondence should be addressed.
J. Nanosci. Nanotechnol. 2010, Vol. 10, No. 9
1533-4880/2010/10/5685/007
doi:10.1166/jnn.2010.2476
5685

One-Step Electrochemically Deposited AuNPs Interface Grafted with Avidin for AChE Biosensor Design
avidin and biotin has been reported to be ca. 1015 M−1 26
2.3. Enzyme Labeling
Zhang et al.
.
In this article, the strong affinity between biotin and avidin
was used for biosensor fabrication.
AChE was labeled to NHS-LC-biotin (AChE-biotin) as
following: 0.45 mg NHS-LC-biotin was dissolved in 1 mL
of 0.1 M PBS (pH 7.0) containing 0.15 mg AChE.
Acetylcholinesterase, an enzyme that plays a key role as
a neurotransmitter in the central nervous system, was cho-
sen for its high biological (Alzheimer disease27ꢁ and ana-
lytical interest (development of pesticide biosensors.28ꢀ29
In this study, an interface of biotin in chitosan hydrogel
embedded AuNPs in situ was constructed by one-step elec-
trochemical deposition. This deposited interface provided
specific binding sites for avidin, which was further capa-
ble of attaching any biotinylated bimolecular. The immo-
bilized AChE, as a model, showed excellent activity to
its substrate and provided a quantitative measurement of
organophosphate pesticide.
ꢀ
After incubation at 37 C for 3 h, thꢀe excess NHS-LC-
biotin was removed via dialysis at 4 C for 2 days with
0.1 M PBS. The phosphate buffer was replaced every
8 h. The dialysis cassette (Pierce) with molecular weight
cutoff of 5000 was used throughout. The activity of the
enzyme after biotinylation was determined by the result
of acetylthiocholine hydrolysis to thiocholine and the oxi-
dation of 5, 5,-bithiobis-2-nitrobenzoate (DTNB) to yield
the yellow 5-thio-2-nitrobenzoic acid, which was detected
spectrophotometrically.31
2.4. Biosensor Design
2. MATERIALS AND METHODS
Gold disk electrode (1.0 mm in diameter) was used for
the electrochemical measurement. Before modification, the
gold electrode was abraded with fine SiC paper, lightly
polished with 0.05 ꢂm alumina, followed by sonication in
ethanol and double distilled water for 10 min, respectively.
For electrochemical deposition, a clean gold electrode
was immersed in 2.0 mL 0.025% (w/v) chitosan solution
including 0.01% (v/v) HAuCl4 · 4H2O and 0.18 mg/mL
NHS-LC-biotin and an initial potential −3ꢃ0 V was
2.1. Reagents
Acetylcholinesterase (Type C3389, 500 U/mg from electric
eel), acetylthiocholine chloride (ATCl), Sulfosuccinimidyl-
6-(biotinamido) hexanoate (NHS-LC-biotin), and avidin
(from egg white) were purchased from Sigma-Aldrich
(St. Louis, MO, USA) and used as received. HAuCl4 ·
4H O (Au% > 48%) was obtained from TreeChem.co
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2
applied on the electrode. The electrodeposition was per-
IP: 12.170.246.82 On: Tue, 20 Oct 2015 13:14:57
(Shanghai, China). 5, 5,-bithiobis-2-nitrobenzoate (DTNB)
was obtained from Alfa Aesar, Dimethoate was obtained
from AccuStandard (New Haven, CT, USA). Chitosan
(95% deacetylation), phosphate buffer solution (PBS, pH
7.0) and other reagents used were of analytical reagent
grade. All aqueous solution was prepared with double dis-
tilled water.
Copyright: American Scientific Publishers
formed on a CHI 660 C electrochemical workstation. At
this potential, H+ was reduced to H2 at the cathode and
released, and the pH near the cathode surface gradu-
ally increased. When the pH was higher than 6.3, chi-
tosan became insoluble.32 After being immersed in PBS
7.0 for one minute to reduce nonspecific interaction, the
deposited electrode (biotin-CS-AuNPs/Au) was dried in
air at room temperature for about 1 h. After that, the
electrode was immersed into 0.01 mg/mL avidin solution
for about 30 min and washed by double distilled water.
For biosensor design, the electrode was then incubated in
AChE-biotin solution for another 2 h to obtain the
AChE-biotin-avidin-biotin-CS-AuNPs/Au.
2.2. Instrumentation
Electrochemical measurements were performed with a
CHI 660 c electrochemical workstation. A conventional
three-electrode electrochemical cell was used consisting
one of the above electrodes, a Pt-wire auxiliary electrode,
and an Ag/AgCl reference electrode. The A.C. impedance
experiment was carried out in 5 mM Fe(CN)46−/3− with
frequencies ranging from 100 kHz to 0.1 Hz with an
amplitude of 5 mV. UV spectra were recorded using
a UV-2501 spectrophotometer (Shimazu, Kyoto, Japan).
Atomic force microscopy (AFM) experiments were per-
formed on SPA-300 HV atomic force microscopy with a
SPI 3800 controller (Seiko, Japan). The diameter size of
gold nanoparticles was measured using Malvern Nano-ZS
ZEN3600 (Malvern, UK).
2.5. Electrochemical Detection of Pesticide
For the measurements of dimethoate, the pretreated AChE-
biotin-avidin -biotin-CS-AuNPs/Au was first immersed in
the PBS solution containing different concentrations of
standard dimethoate solution for 12 min, and then trans-
ferred to the electrochemical cell of 1.0 mL pH 7.0 PBS
containing 0.3 mM ATCl to study the electrochemical
response by cyclic voltammetry (CV). The inhibition of
dimethoate was calculated as follows:
Surface plasmon resonance (SPR) measurements were
conducted using a single-channel Autolab SPRINGLE
instrument (Eco Chemie, Netherlands). The configuration
of this equipment is described elsewhere.30
Inhibition (%) = 100% × (iPꢀcontrol – iPꢀexpꢁ/iPꢀcontrol
Where iPꢀcontrol and iPꢀexp were the peak currents of ATCl
on AChE-biotin-avidin-biotin-CS-AuNPs/Au without and
with pesticide inhibition, respectively.
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Zhang et al.
One-Step Electrochemically Deposited AuNPs Interface Grafted with Avidin for AChE Biosensor Design
3. RESULTS AND DISCUSSION
524 nm, which was the characteristic absorbance of unag-
gregated colloidal gold nanoparticles.25 The laser light
scattering measurement has been proven very sensitive
to the size, and spatial distribution of metallic nanoparti-
cles. The diameter size of AuNPs was further measured
using Malvern Nano-ZS ZEN3600. The average diameter
of AuNPs was estimated to be 33 nm (Fig. 2). No aggre-
gation could be identified even after 3 months.
3.1. UV-Vis Spectrum
The position of Soret absorption band can provide detailed
information about conformation of protein.33 Figure 1
shows the UV-vis spectra of biotin, AChE and AChE-
biotin. No Soret band was observed in NHS-LC-biotin
(curve (a)). However, solution-phase AChE displayed a
narrow Soret band at 406 nm (curve (b)). After AChE was
labeled to NHS-LC-biotin, an obvious Soret band without
shift compared to AChE was clearly seen (curve (c)), indi-
cating that AChE was labeled onto NHS-LC-biotin and no
significant denaturation occurred.
Inset in Figure 1 further illustrated the photograph
images of the AChE before (2) and after (1) biotinylation
in the presence of 5, 5,-bithiobis-2-nitrobenzoate (DTNB)
and ATCl. As compared with control experiment (3), the
solution was orange. It can be concluded that the enzyme
maintains its activity. The activity of the enzyme after
biotinylation was determined by acetylthiocholine hydrol-
ysis to thiocholine and the oxidation of DTNB to yield
the yellow 5-thio-2-nitrobenzoic acid, which was detected
spectrophotometrically at 412 nm.31 The enzyme after
biotinylation maintained 80.2% of its original activity. On
the basis of the previous results, we might conclude that
the as-prepared AChE-biotin, as a model, could be prelim-
inarily applied for further construction of biosensor.
3.3. AFM Characterization
AFM measurement is an effective method to observe
surface topography and confirm the fabrication pro-
cess. As seen from Figure 3(a), the pretreated bare Au
slice displayed an even surface with height less than
1 nm. When the Au slice was immersed in deposi-
tion solution containing chitosan, HAuCl4 · 4H2O and
NHS-LC-biotin for 2 min, the deposited biotin-CS-AuNPs
interface showed a uniform distribution and the embed-
ded biotin in chitosan hydrogel could be clearly seen
(Fig. 3(b)). Upon incubation of avidin, Figure 3(c) clearly
illustrated that a homogeneous dispersion of avidin was
achieved on the biotin-CS-AuNPs interface due to the
strong affinity between biotin and avidin. The thickness
of all the layers increased to ∼5 nm. After biotiny-
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lated AChE molecules were further immobilized onto the
IP: 12.170.246.82 On: Tue, 20 Oct 2015 13:14:57
avidin-biotin-CS-AuNPs film, the image showed the pres-
Copyright: American Scientific Publishers
ence of the large agglomerate of AChE molecules on the
modified gold surface (Fig. 3(d)) and the thickness of all
the layers increased to 11 nm. The surface morphology
could be clearly distinguished from that in Figure 3(c). On
the base of AFM results, we conclude that AChE has been
successfully immobilized on the electrode surface and thus
the biosensor has been fabricated.
3.2. AuNPs Characterization
Our previous report has shown that the freshly pre-
pared AuNPs showed a maximum absorbance (ꢄmaxꢁ at
c
80
60
40
20
0
b
a
360
380
400
420
440
Wavelength (nm)
0
30
Fig. 1. UV spectra of (a) biotin, (b) AChE and (c) biotin-AChE. Inset:
photograph images of the AChE before (2) and after (1) biotinylation and
control (3) in the absence of 5, 5,-bithiobis-2-nitrobenzoate (DTNB) and
ATCl.
60
120
150
90
180
d (nm)
Fig. 2. Diameter size of gold nanoparticles.
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One-Step Electrochemically Deposited AuNPs Interface Grafted with Avidin for AChE Biosensor Design
Zhang et al.
(b)
(curve (a)), biotin-CS-AuNPs/Au (curve (b)), avidin-
biotin-CS-AuNPs/Au (curve (c)) and AChE-biotin-avidin-
biotin-CS-AuNPs/Au (curve (d)) were 83 ꢅ, 150 ꢅ,
175 ꢅ and 411 ꢅ, respectively. These results showed that:
(1) the interface with a biotin moiety can tailor molec-
ular recognition events at surfaces through biotin-avidin
interaction.
(a)
(2) After the formation of AChE-biotin layer, a barrier
to electrochemical process was introduced. The significant
change occurred at AChE-biotin-avidin-biotin-CS-AuNPs/
Au might be related to the interaction between biotin-
avidin.
(d)
(c)
In order to confirm the significant effect of AuNPs in the
rate of electron transfer at the interface, the impedance
of biotin-CS/Au was further measured to be 476 ꢅ
(curve (e)). Comparing with that of biotin-CS-AuNPs/Au
(curve (b)), the charge-transfer resistance (Rctꢁ at the inter-
face of biotin-CS/Au was dramatically increased. This
demonstrates that AuNPs immobilized on the electrode
played an important role similar to an electron-conducting
tunnel, making electron transfer to the electrode sur-
face easier. These results were further confirmed by CV
measurements.
Fig. 3. AFM images of (a) bare Au slice, (b) biotin-CS-AuNPs
deposited on Au slice, (c) avidin-biotin-CS-AuNPs on Au slice and
(d) AChE-biotin-avidin-biotin-CS-AuNPs on Au slice.
3.5. Monitoring Bio-Specific Interactions by SPR
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SPR has emerged as a powerful tool for real-time monitor-
3.4. AC Impedance Measurements
IP: 12.170.246.82 On: Tue, 20 Oct 2015 13:14:57
ing small changes in a solid/liquid interface. As shown in
Copyright: American Scientific Publishers
Figure 5, after washing with PBS for 100 sec (curve (a)),
biotin-CS-AuNPs was immobilized on gold chip. The
increases of signal, curves (d) and (f) indicated the bind-
ing of avidin to biotin-CS-AuNPs/Au and the binding
of AChE-biotin to biotin-avidin-biotin-CS-AuNPs, respec-
tively. Obviously, when AChE-biotin was introduced, the
An equivalent circuit Rs(Rct CPE) was used to model
the impedance data (inset in Fig. 4), thus enabling
the extraction of electrical parameters, such as resis-
tance, from the impedance spectra.34 As seen from
the electrochemical impedance spectra, the electron
transfer resistances of Fe(CN)46−/3− at bare electrode
Fig. 5. SPR responses to sequential reaction steps between (b) biotin-
CS-AuNPs and Au, (d) avidin and biotin-CS-AuNPs, (f) AChE-biotin
and avidin-biotin-CS-AuNPs. Curves (a), (c), (e) and (g) are washing
steps.
Fig. 4. Impedance measurements of (a) bare Au, (b) biotin-CS-
AuNPs/Au, (c) avidin-biotin-CS-AuNPs/Au, (d) AChE-biotin-avidin-
biotin-CS-AuNPs/Au and (e) biotin-CS/Au in 5 mM Fe(CN)64−/3−
.
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Zhang et al.
One-Step Electrochemically Deposited AuNPs Interface Grafted with Avidin for AChE Biosensor Design
saturated SPR angle shift of step (f) (ꢆ angle = 307 mdeg)
was larger than that of step (d) (ꢆ angle = 94 mdeg). From
different changes of SPR angles, the binding content of
AChE-biotin to avidin was 3.26-fold higher than that of
avidin to biotin-CS-AuNPs. The different SPR angle shift
might be related to the larger Mw of AChE. It also might
be related to biotin-avidin interaction since more than one
unit AChE-biotin could bind to an avidin. In order to
reduce nonspecific interaction between biotin and avidin,
washing steps (curve (a), (c), (e) and (g)) were carried out
after every binding step.
performed on AChE-biotin-avidin-biotin-CS/Au without
AuNPs at the same conditions. A small oxidation peak of
ATCl was seen on the AChE-biotin-avidin-biotin-CS/Au
(curve (e)) and the peak potential was much higher than
that on AChE-biotin-avidin-biotin-CS/Au. Apparently, a
well-defined oxidation peak and negative direction-shifted
oxidation potential was presented at AChE-biotin-avidin-
biotin-CS/Au (curve (d)). This result indicated that the
AuNPs played an important role in catalyzing the oxidative
reaction of thiocholine. The surface assembling AuNPs
provided a conductive pathway to electron transfer and
thus promoted electron transfer reactions at a lower poten-
tial. Thus the AChE-biotin-avidin-biotin-CS-AuNPs/Au
was used in following experiments.
3.6. Cyclic Voltammetric Behavior of
AChE-Biotin-Avidin-Biotin-CS-AuNPs/Au
As shown from Figure 6, no peak was observed
at avidin-biotin-CS-AuNPs/Au (curve (a)) and AChE-
biotin-avidin-biotin-CS-AuNPs/Au (curve (b)) in pH
7.0 PBS. When 0.3 mM ATCl was added into PBS,
the cyclic voltammograms of AChE-biotin-avidin-biotin-
CS-AuNPs/Au showed an irreversible oxidation peak
at 884 mV (curve (d)), while no detectable signal
was observed at avidin-biotin-CS-AuNPs/Au (curve (c)).
Obviously this peak came from the oxidation of thio-
choline, hydrolysis product of ATCl, which was cat-
alyzed by immobilized AChE. Thus the immobilized
3.7. Effect of Incubation Time on Response of
AChE-Biotin-Avidin-Biotin-CS-AuNPs/Au
After biosensor was immersed in 10 ꢂg mL−1 dimethoate
for different time, the current of ATCl decreased dras-
tically. The decrease in peak current was related to the
increase of incubation time (Fig. 7). This was because
dimethoate as one of the organophosphate pesticide
involved in the inhibition action to AChE, thus reduced
the enzymatic activity to its substrate. Dimethoate dis-
played an increasing inhibition on AChE with immersing
time. When the immersing time was longer than 12 min
the curve trended to a stable value, indicating the binding
interaction with active target groups in enzyme could reach
saturation. This change tendency of peak current reflected
the alteration of enzymatic activity, which resulted in the
change of the interactions with its substrate. However, the
maximum value of inhibition of dimethoate was not 100%,
which was likely to attribute to the binding equilibrium
between pesticide and binding sites in enzyme.
AChE through tailoring molecular recognition at surfaces
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of constructed biosensor exhibited a fast response and
Copyright: American Scientific Publishers
high affinity to its substrate. The produced current by
thiocholine was used as a quantitative measurement of
the enzyme activity, which could reflect the biological
effect of organophosphate pesticides involved in the inhi-
bition action.35 To further demonstrate the peaks in rela-
tion to the actions of AuNPs, a comparable assay was
Fig. 6. Cyclic voltammograms of (a) bare Au, (b) AChE-avidin-
biotin-CS-AuNPs/Au in 7.0 PBS, (c) avidin-biotin-CS-AuNPs/Au, (d)
AChE-avidin-biotin-CS-AuNPs/Au and (e) AChE-avidin-biotin-CS/Au in
pH 7.0 PBS containing 0.3 mM ATCl.
Fig. 7. Effect of inhibition time on amperometric response after
immersion of AChE-biotin-avidin-biotin-CS-AuNPs/Au in 10 ꢂg mL−1
dimethoate solution.
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One-Step Electrochemically Deposited AuNPs Interface Grafted with Avidin for AChE Biosensor Design
Zhang et al.
4. CONCLUSIONS
In summary, a novel interface embedded in situ AuNPs
and biotin in chitosan hydrogel was constructed by one-
step electrochemical deposition. This deposited interface
provided specific binding sites for avidin, which was fur-
ther capable of attaching the AChE-biotin for biosen-
sor design. The CV, EIS, SPR and AFM have been
used as powerful tools to characterize the fabrication pro-
cesses. Several advantages of the proposed method should
be highlighted. First, gold nanoparticles promote elec-
tron transfer and favor the interface enzymatic hydrol-
ysis reaction to form electroactive substance, resulting
in an improvement of the detection sensitivity. Second,
the immobilized AChE, as an enzyme model, showed
excellent activity to its substrate and provided a quanti-
tative measurement of organophosphate pesticide. Third,
this method for biosensor design was simple, fast and
“green,” which has potential application for the fabrica-
tion of amperometric biosensors. Fourth, we are currently
facing the disadvantage of the low selectivity of AChE
because the degree of inhibition on AChE by organophos-
phate pesticide is almost the same. However this does
not necessarily compromise its value extended toward the
preparation of other electrochemical sensors based on this
mode. The ease of performance and the cost effectiveness
Fig. 8. Cyclic voltammograms of AChE-avidin-biotin-CS-AuNPs/Au in
pH 7.0 PBS containing 0.3 mM ATCl after immersed in dimethoate solu-
tion with different concentrations of (a) 0, (b) 0.01, (c) 1, (d) 5 and
(e) 8 ꢂg mL−1. Inset: Calibration curve for dimethoate determinations.
3.8. Calibration Curve for Dimethoate
Determination
Due to the notable change in voltammetric signal of
the AChE-biotin-avidin-biotin-CS-AuNPs/Au, a simple
of the system for organophosphate detection show the high
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method for determination of dimethoate was established.
potentiality in pesticide analysis.
Copyright: American Scientific Publishers
With increasing the concentrations of dimethoate the pro-
duced currents of ATCl on AChE-biotin-avidin-biotin-CS-
AuNPs/Au decreased. Under the optimal experimental
conditions, the inhibition of dimethoate on AChE-biotin-
avidin-biotin-CS-AuNPs/Au was proportional to its con-
centrations in the range of 0.05 to 15 ꢂg/mL with the
correlation coefficients of 0.9990 (Fig. 8). The detection
limit was 0.001 ꢂg/mL.
Acknowledgments: The authors are gratefully
acknowledging the financial support of the National
Natural Science Foundation of China (No. 20705010,
30771429) and the Research Fund for the Doctoral
Program of Higher Education (No. 20070511015).
References and Notes
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3.9. Reproducibility and Stability of
AChE-Biotin-Avidin-Biotin-CS-AuNPs/Au
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The inter-assay precision was estimated by determining
the response of 0.3 mM ATCl at five different elec-
trodes, which were immersed in 0.1 ꢂg/mL and 2.0 ꢂg/mL
dimethoate for 12 min, respectively. The coefficient of
variation was calculated to be 4.6% and 2.7%, respectively,
indicating acceptable fabrication reproducibility. The intra-
assay precision of the sensors was evaluated by assaying
one enzyme electrode for five replicate determinations,
and the RSD was 3.9% at the ATCl concentration of
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When the enzyme electrode was not in use, it was stored
at 4 ꢀC in dry condition. No obvious decrease in the
response to ATCl was observed in the first 5-day storage.
After two weeks, the sensor retains 85% of initial current
response to ATCl.
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Received: 12 June 2009. Accepted: 13 July 2009.
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