Review

Journal of
Copyright 2016 American Scientific Publishers
Nanoscience and Nanotechnology
All rights reserved Vol. 16, 66486656, 2016
Printed in the United States of America www.aspbs.com/jnn

Recent Advances on Rapid Detection of Pesticides

Based on Enzyme Biosensor of Nanomaterials
Keke Liu1 , Hongming Dong1, and Yan Deng1
2

1
Economical Forest Cultivation and Utilization of 2011 Collaborative Innovation Center in Hunan Province,
Hunan Key Laboratory of Green Packaging and Application of Biological Nanotechnology,
Hunan University of Technology, Zhuzhou 412007, China
2
State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096, China

Recent research progress is reviewed in development of rapid detection of Organosphorous (OPs)

and carbamates (CBs) pesticide based on acetylcholinesterase (AChE) inhibition principle. Enzyme
biosensors have properties that can provide conveninent, rapid, sensitive and cheap on-field
detection with AChE. In recent years, effective monitoring of OPs and CBs pesticides has been
done successfully by using nanomaterials-based AChE sensors. More and more nanomaterials
such as modified carbon nanotubes, nanocomposite material, modified gold nanoparticles, quan-
tum dots(QDs) are applied to enzyme biosensors, the modified electrodes can promote significant
enhancements of determinations of pesticide residues.
Keywords:
Delivered by Ingenta to:(AchE),
Acetylcholinesterase West Virginia
Enzyme University/
Biosensor,Health Sci Ctr LIb
Pesticides, Nanomaterials,
Modified IP: 5.8.37.75 On: Mon, 20 Jun 2016 12:31:06
Electrode.
Copyright: American Scientific Publishers

CONTENTS human beings, such as memory decline, muscular paraly-

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Cholinesterase Biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. EC Biosensors Based on ChE . . . . . . . . . . . . . . . . . . . . . .
2.2. ECL Biosensors Based on ChE . . . . . . . . . . . . . . . . . . . . .
2.3. Piezoelectric Biosensors Based on ChE . . . . . . . . . . . . . . .
3. Conclusion and Future Propects . . . . . . . . . . . . . . . . . . . . . . . .
References and Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. INTRODUCTION
The chemical pesticides which were applied in prevent-
ing diseases and killing pests of crops such as vegeta-
bles, fruits are mainly organochlorine, organophosphorus
(OPs), carbamates (CBs) and pyrethroids. Organochlorine
has been prohibited to produce and use because it is ter-
atogenic, carcinogenic and mutagenic on human body.
OPs and CBs of the pesticides are specific inhibitors of
AChE which is an important enzyme in the synaptic clefts
of the central nervous system out of living organisms,1
have been widely used in agriculture due to its high
effectiveness for insect eradication and low price. As a
result of these pesticides abused, pesticides residues are
overproof in many crops, and jeopardize the safety of
Author to whom correspondence should be addressed.
6648 sis, convulsions, respiratory failure and even death.2 The
6650 toxicity of these pesticides arises from the formation of
6650
a stable complex through their reversible and irreversible
6653
6654 binding to the active site of AChE that is essential for
6654 nerve impulse responses.3 The enzyme inhibition mecha-
6654 nism is that OPs and CBs can react with OH bond on the
serine of AChE to inhibit the reaction with acetylcholine
chloride (Ach). Therefore, the threats ultimately pose the
need for the detection of OPs and CBs. More highly sen-
sitive methods have been developing for the determination
of OPs and CBs, such as gas-chromatography (GC),49
high performance liquid chromatography (HPLC), GC-
mass spectrometry (MC),1013 and HPLC-MC.14
15 These
methods can effectively detect the concentrations of dif-
ferent compounds and, at the same time, require com-
plicated sample pretreatments, highly trained technicians
and very expensive equipments. Moreover, they are rather
time-consuming, including field sample collection, solid-
phase extraction, analyzing the sample and dealing with
spectral data. Thus, methods of detection that are low-cost
and more user-friendly have been tried to investigate pes-
ticide residues. In recent years, enzyme biosensors on the
basis of the principle of specific enzyme activity inhibition
have been a hot research field in biosensor technology for

6648 J. Nanosci. Nanotechnol. 2016, Vol. 16, No. 7 1533-4880/2016/16/6648/009 doi:10.1166/jnn.2016.11392

Liu et al. Recent Advances on Rapid Detection of Pesticides Based on Enzyme Biosensor of Nanomaterials

rapid pesticide detection. In this manner, different kinds

of enzymes were investigated for their ability to detect
pesticides, such as AchE, butyrylcholinesterase (BuChE),
alkaline phosphatase.1619
In general, a enzyme-based biosensor is an analytical
device for detecting analytes that combine a sensitive
biological recognition element with a physicochemical
detector component (such as cholinesterase (ChE)). It con-
sists of a biological recognition element, a transducer
and a signal detector.20 According to the transducer types
depending on different principles, cholinesterase-based
biosensors can be classified as electrochemical (EC),2123
electrochemiluminescence (ECL)24
25 and piezoelectric26
biosensor, etc. Basic principle of these biosensors based
on enzyme is showed in Figure 1.
EC biosensor can directly convert a biological event to
an electronic signal such as the measurable current (amper-
ometric), altered conductive properties of a medium (con-
ductometric) between electrodes,20 and the most common
detection method are cyclic voltammetry (CV), square
wave voltammetry (SWV), electrochemical impedance
spectroscopy (EIS) and so on. ChE biosensors work on
Figure 1. Basic principle of enzyme-based biosensors.
inhibitory effects. Acetylthiocholine serves generally as
an electrochemical substrate. When the inhibitor such as
OPs and CBs is not present in the sample, the acetylth-
iocholine is converted into thiocholine and acetic acid.
However, if the analyte is present in the solution, the con-
centration of thiocholine is decreased or even null, this is
completely inhibits the conversion.27 Thiocholine is oxi-
dized using applied voltage. The anodic oxidation current
is inversely proportional to the concentration of inhibitor.
In other words, increased concentration of the inhibitor
leads to weak current signals. ECL belongs to one spe-
cial form of EC, which emits measurable luminescent sig-
nals by converting EC energy into radiative energy via an
EC reaction.20 Piezoelectric biosensor are a chemical sen-
sors based on mass deposited/adsorbed on the oscillating

Keke Liu completed her B.S. degree from Hengyang Normal University, China in 2013,
with specialization in the field of Chemistry, and currently she is pursuing her postgraduate
studies at the Hunan University of Technology, China in Biomedical Engineering.
Delivered by Ingenta to: West Virginia University/ Health Sci Ctr LIb
IP: 5.8.37.75 On: Mon, 20 Jun 2016 12:31:06
Copyright: American Scientific Publishers

Hongming Dong completed her B.S. degree from Yangtze University, China in 2013, with
specialization in the field of Bioengineering. Currently she is pursuing her M.S. degree in
Biomedical Engineering at the Hunan University of Technology, China.

Yan Deng was born in 1971. She obtained her M.Sc. degree from Hunan Normal Uni-
versity, China, and her Ph.D. degree from Southeast University, China. She is a associate
professor in Packing and Material School of Hunan University of Technology since 2007
and full professor now. Her research interest is focused on drug development, pharma-
cology, biosensors and controlled drug release. Currently, she particularly contributes her
research to the biosensors and controlled drug release. She published about 65 papers and
has 7 patents.

J. Nanosci. Nanotechnol. 16, 66486656, 2016 6649

Recent Advances on Rapid Detection of Pesticides Based on Enzyme Biosensor of Nanomaterials Liu et al.

piezoelectric quartz crystal surface, that resonates at a
fundamental frequency by the application of an external
alternating electric field. Using piezoelectric bioseneors,
mass changes occur simultaneously with the electrochem-
ical respone when a material is deposited on or lost from
the working electrode. ChE-based sensor increases the
mass deposition via the precipitation of enzymatic reaction
products.
EC, ECL and piezoelectric ChE-based bioseneors are
designed through immobilizing ChE on the working
electrode surface, respectively. The process affects the
performance of these biosensors in terms of sensitiv-
ity, stability, response time, and reproducibility for var-
ious interactions between the ChE and the electrode
materials or supports, which may be matrices, screen-
printed electrodes, semiconductors such as QDs and other
nanomaterials. These materials can be immobilized by
ways of physical adsorption, chemical covalent bonding,
electrodeposition, etc. With the development of nanotech-
nology, more and more nanomaterials such as modi-
fied carbon nanotubes, nanocomposite material, modified
gold nanoparticles, and QDs have been widely employed
in the electrode modification and electrochemical sensor
2835
development. It is that significantly improves the sen-
sitivity, detection limit and stability in OP and CB pes-
ticide determination. In this review, we have combined
with the latest research papers and summarized the use of
Delivered by Ingenta to: West Virginia University/ Health Sci Ctr LIb
biosensors for detection of OPs andIP: CBs based on various lization
5.8.37.75 On: Mon, 20
nanomaterials and different enzymes Copyright:
(AChE, ChE,American Scientific Publisherswith cysteamine (CA-NPG), secondly,
etc.), was
and discussed diffenct nanomaterials applied to AChE
biosensors for measurement of OP and CB compounds in
detail.
2. CHOLINESTERASE BIOSENSOR
In recent years, cholinesterase biosensor has been a
research hot-topic in measurement technology of pesticide
residues for diversification of research methods, high sen-
sitivity and fast response time, etc. Based on the difference
of transducers, cholinesterase biosensors can be divided
into EC, ECL and piezoelectric biosensors to be discussed
respectively.
2.1. EC Biosensors Based on ChE
The EC biosensor is based on the cholinesterase cat-
alytic substrate hydrolysis to generate electricity active
substance. Usually, there are two kinds of detection meth-
ods. One adopted electrochemical oxidation method to
directly detect the generated thiol choline.36
37 The other
one used choline oxidase to oxidize the generated choline,
and then adopted electrochemical method to measure O2
consumption or the generated H2 O2 in the process of the
catalytic oxidation. Based on modification materials of the
electrode surface, EC biosensors are discussed from car-
nanotubes (CNTs), gold nanoparticles (GNPs), nanocom-
posite material, etc.
2.1.1. Carbon Nanotubes (CNTs) Based on
ChE EC Biosensor
CNTs have been widely employed in electrode
modification owing to their good conductivity, high
mechanical strength, extremely miniaturized size and large
surface area.38 In fact, the CNTs and their applications
have received widespread attention.3942
Yang et al.43 developed a kind of multilayer films con-
taining multiwall carbon nanotubes (MWCNTs), chitosan
(CS) and AChE liposome bioreactor (ALB). The films
with the glassy carbon electrodes (GCE) can assemble dif-
ferent layers of multilayer films [(MWCNTs/ALB)n/GCE]
when GCE was alternately immersed in this solu-
tion. Among which six bilayers of multilayer films
based on resulting biosensors was the best. The
(MWCNTs/ALB)6 /GCE modified biosensor was applied to
detect dichlorvos concentration and exhibited a wide lin-
ear range (from 25 M to 1.75 M and from 2.00 M to
10.00 M) and a low detection limit (068 0076 g/L)
based on the inhibition of OPs on the AChE activity. The
Michalis-Menten constant was 0.28 mM for the enzymatic
reaction. The fabrication of the biosensor was simple, the
recovery was from 88% to 103%, the stability (30 d) was
acceptable and the response was fast (10 min).
Similarly, Ding et al. reported a highly sensitive EC
sensor for malathion detection.44 The biosensor was fab-
ricated firstly employing assembly of MWCNTs immobi-
on a 12:31:06
Jun 2016 nonporous gold film (NPG) electrode which
functionalized
AChE solution was dropped onto the modified electrode
to obtain the AChE-MWCNT-CA-NPG. The relative inhi-
bition of malathion based on inhibition principle of AChE
is linear at the concentration range 0.0010.5 g/mL, and
the detection limit is 0.5 ng/mL.
Kesik et al. employed a conducting polymer electropoly-
merized with functionalized MWCNTs modified graphite
electrode on which AChE was immobilized by covalent
linkage for organophosphorus pesticides detection.45 The
procedure for the construction of the proposed amperomet-
ric AChE biosensor was shown in Figure 2.
The conducting polymer, poly(4-(2,5-di(thiophen-2-yl)-
1H-pyrrol-1-yl)benzene amine) (poly(SNS-NH2 involves
the amino-group. This strategy can enhance electron trans-
fer rate and increase the stability of the enzyme electrode
by covalent immobilization between the enzyme molecules
and the modified electrodes for the strong and efficient
bonding of amide bonds. Xue et al.46 fabricated an amper-
ometric biosensor that employed silk fibroin (SF) and
AChE coated n the surface of GCE modified with MWC-
NTs which were immobilized between AChE and SF by
non-covalent adsorption or biocompatible interface. The
biosensor was applied to detect OPs and CBs, the range of
the concentration of methyl parathion was from 35 106
to 20 103 M with a detection limit of 50 107 M,
and that of carbaryl from 10 107 to 30 105 M with
a detection limit of 60 108 M.

6650 J. Nanosci. Nanotechnol. 16, 66486656, 2016

Liu et al. Recent Advances on Rapid Detection of Pesticides Based on Enzyme Biosensor of Nanomaterials

Figure 2. Schematic representation of the proposed biosensor.

However, Zhang et al.47 reported a novel bi-enzyme
biosensing system for assembly of unique multifunctional
nanointerfaces for discriminative detection of carbamates
(non-OP) and organophosphorus pesticides (OP), which
was layer-by-layer assembly electrostatically interacted
enzyme-coated MWCNT-AChE and MWCNT-OPH with
a set of cushioning bilayers consisting of MWCNT-DNA
and MWCNT-polyethyleneimine on GCE. The key of the
biosensor is that principle of detection of non-OP (such
as carbaryl) was the inhibition-based AChE reaction and
that for OP (for example Delivered by Ingenta
paraoxon) to: West Virginia
is the combination
IP: 5.8.37.75
of a direct catalytic OP hydrolysis reaction On: Mon, 20
and inhibition
Copyright:the
AchE-based assays. Under optimum conditions, American
linear Scientific
response range for carbaryl was 1080 M with dynamic
range 10100 mM, and that for paraoxon was 0.540 M
with dynamic range 0.550 mM. The limit of detection of
carbaryl and paraoxon was 10 M and 0.5 M, respec-
tively. Similarly, the linear range of direct detection of
paraoxon was 164 mM, and the detection limits was
1 mM. Besides, Khaled et al. fabricated a portable BuChE-
based biosensor for detection of ethion and its degradation
residues,48 which were incorporated with -cyclodextrin
ionphore on the basis of MWCNT-polyvinyl chloride com-
posite. The biosensor was applied to measure the relative
inhibition percentage of BuChE by analyzing the concen-
tration of ethion and its degradation products in range from
0 to 330 ng/mL. The biosensor exhibited good accuracy
and precision.
2.1.2. Gold, Ag Nanoparticles and Graphene Based on
ChE EC Biosensor
The same as CNTs, gold, Ag nanoparticles (Au, Ag NPs)
and graphene have also been widely used in biosensors for
improving the response, stability and sensitivity.22
36
4951 concentrations of chlorpyrifos and carbofuran were in the
With the application of Au NPs and CNTs,52
53 the effi-
ciency and the stability of sensors for detection of the pes-
ticides are greatly amplified, owing to that a nanoparticle
matrix can offer much more friendly environment to the
immobilized enzyme.
Liao et al. earlier developed an AChE liquid crys-
tal (LC) biosensor based on enzymatic growth of
GNPs for amplified detection of ACh and AChE
inhibitor for pesticide residue detection.54 Firstly,
a thiethoxysilybutylaldehyde/N ,N -dimethyl-noctadecyl(3-
aminopropyl)trimethoxysilyl chloride (TEA/DMOAP) film
was formed to functionalize the glass slide surface, and the
AChE was immobilized on the film by binding between
the amino groups of AChE and the aldehyde groups
of TEA/DMOAP. Finally, the AChE LC biosensor was
University/
obtained Health
through the Sci Ctrslide
glass LIb modified with AchE
Jun 2016
soaked in an 12:31:06
Au NPs growth solution. AChE catalyzes the
hydrolysisPublishers
of acetylthiocholine to form thiochline which
reduces AuCl 4 to Au NPs. AChE activity is inhibited and
decreases the catalytic growth of Au NPs in the presence
of thiochline or OPs, vice versa. The experiment results
show that the AChE LC biosensor was highly sensitive,
and displayed a detection limit of 0.3 nmol/L for OPs.
Dimcheva et al.55 fabricated a biosensor by self-
assembly of AChE and electrodepositing Au NPs on
graphite, which has found a concentration linear range
from 50 to 400 nmol/mL to detect monocrotophos. The
specific type of enzyme immobilization can lengthen a
retaining time of the AChE catalytic activity.
Recently, Sun et al. reported a highly sensitive AChE
biosensor based on hollow gold nanopheres (HGNs) for
pesticide detection.56 A bare GCE was modified with chi-
tosan and the HGNs which were achieved by using Co
nanoparticles (CoNP) (which can reduce HAuCl4 into
GNPs) as deoxidizer. Then, L-cysteine and HGNs were
assembled via AuS bond. Finally, AChE was immobi-
lized onto the modified GCE by covalent binding COOH
groups of L-cysteine. The results show that the detection
range from 0.1 g/L to 200 g/L and from 0.1 g/L
to 150 g/L with the detection limits of 0.08 g/L and
0.06 g/L, respectively.
The same as Au NPs, Ag NPs decorated with macro-
cyclic ligands were applied to modify GCE and achieved a

J. Nanosci. Nanotechnol. 16, 66486656, 2016 6651

Recent Advances on Rapid Detection of Pesticides Based on Enzyme Biosensor of Nanomaterials
highly sensitive AChE sensor, where AChE was immobi-
lized on the GCE by carbodiimide binding on carbon black
layer deposited.57 Under optimum conditions, the biosen-
sor detected malaoxon in the linear range 0.4 nM0.2 M
with a detection limit of 0.1 nM, detected paraoxon in
the linear range 0.2 nM0.2 M with a detection limit of
0.05 nM, detected carbofuran in the linear range 0.2 nM
2.0 M with a detection limit of 0.1 nM, and detected
aldicarb in the linear range 10 nM0.2 M with a detec-
tion limit of 10 nM.
Zhang et al.58 synthesized functionalized graphene oxide
nanomaterials (FGO) with affinity groups of AChE by
a diazonium reaction to introduce abundant carboxyl
groups. Then, AChE was immobilized on the function-
alized grapheme oxide by the specific binding between
function group and AChE tag group and the biosensor of
paraoxon detection was obtained. FGO with a large sur-
face area enhanced electrocatalytic activity of AChE and
increased anodic oxidation. The biosensor was applied to
detect paraoxon in the linear range 10 M1 mM with
a detection limit of 65 1010 M. This strategy provides
a platform to apply graphene oxide to fabricate biosensor
for pesticides detection.
2.1.3. Nanocomposite Material Based on
ChE EC Biosensor
With the deepening of the nanomaterial
Delivered research,to:some
by Ingenta
West
nanocomposites were prepared withIP: two5.8.37.75 On: Mon,
or many kinds
nanoparticles, which not only retain the Copyright: American Scientific
original character-
istics of the individual nanoparticles but also has other more
superior features. In recent years, the biosensors modified
based on several types of nanocomposites have received
extensive attention. The nanocomposites such as boronic
acid-functionalized Fe@Au magnetic nanoparticles,59 iron
oxide-chitosan nanocomposite,60 MWCNTs-SnO2 -chitosan
61
nanocomposite, polyaniline and MWCNTs coreshell
41
composite, 3-carboxyphenylboronic/reduced grapheme
oxide-gold nanocomposite28 were fabricated and applied
to modify AChE-based biosensors for detection of OPs
and SBs.
A sensitive amperometric AChE biosensor based on
nanocomposite of SnO2 , carboxylic graphene (CGR), and
nafion(NF)62 or platinum nanoparticles (PtNPs), CGR and
NF63 modified GCE has been fabricated for the detection
of pesticides. AChE was immobilized on SnO2 NPs-CGR-
NF or Pt-CGR-NF modified GCE using chitosan as cross-
linker and NF as a protective membrane. Appling the two
type biosensors to detect methyl parathion and carbofu-
ran, on the former the linear range of methyl parathion
is from 1013 M to 1010 M with the detection limit of
5 1014 M, and the linear range of carbofuran is from
1010 M to 108 M with the detection limit of 5 1013 M;
on the latter the linear range to detect methyl parathion
from 10 1013 M to 1 1010 M with the detection
limit of 5 1014 M, and the linear range for carbofuran
6652
Liu et al.
is from 10 1010 M to 1 108 M with the detection
limit of 5 1013 M. The result of using SnO2 NPs-CGR-
NF was in accordance with that of using Pt-CGR-NF. The
biosensors all exhibited good sensitivity, reproducibility,
acceptable stability for analysis of enzyme inhibitors as a
result of SnO2 NPs-CGR-NF and Pt-CGR-NF offering an
extremely hydrophilic surface for AChE adhesion.
Besides, nanocomposites like platinum-carbon aero-
gels (Pt-CAs)64 and gold nanoparticles-carbon aerogels
(AuNPs-CAs)65 were employed to modify boron-doped
diamond (BDD) electrode for AChE immobilization to
detect OPs. The biosensor modified with Pt-CAs is applied
to detect methamidophos and monocrotophos, the linear
concentration rang of inhibition rate is 1011 106 M,
and the detection limits are 31 1013 M and 27
1012 M, respectively. Similarly, the biosensor modified
with AuNP-CAs was utilized to detect chlorpyrifos and
methyl parathion, the linear concentration rang of inhibi-
tion rate is 1011 107 M with the detection limits 13
1013 M, and 1012 106 M with the detection limit of
49 1013 M, respectively. The reason is that the presence
of Pt-CAs composite on the electrode surface can improve
the reactive site and reduce the interfacial resistance, and
AuNP-CS nanohybrid has larger surface to improve the
adsorption of AChE and enhance its activity.
Reduced grapheme oxide sheets (rGO) with polypyrrole
new
(PPy)
Virginia in the introduction
University/ Health Sci of
CtrAuLIbNPs by electrochemical
of 20 Jun 2016technology
deposition 12:31:06 achieved a nanocomposite of Au-
PPy-rGO,Publishers
which showed excellent conductivity, high elec-
trocatalytic activity and specific affinity for thiocholine.66
(NH4 2 SiF6 by co-deposition with AChE was immobilized
on the Au-PPy-rGO nanocomposite after silica matrix
encapsulating AChE. Under the optimized condition, the
inhibition rate of paraoxon-ethyl was linearly proportional
to its concentration in the range of 10 103 5.0 M.
The detection limit was 0.5 nM. Similarly, Wu et al.67
employed rGO and NF hybrid nanocomposite (rGO-NF)
to modify electrode, and the fabricated biosensor was used
for the detection of dichlorvos in a linear concentration
range of 5.0100 ng/mL and 1.020 g/mL with low
detection limit of 2.0 ng/mL.
Zhao et al. fabricated OPs biosensor which employed
Prussian blue-chitosan (PB-CS) and reduced graphene
oxide (ERGO)-Au nanoparticles (AuNPs)--cyclodextrin
(-CD) on GCE for efficiently fixing AChE.68 The PB-
CS obviously improved the sensitivity of the biosensor
by shifting oxidation potential of TCh from 0.68 V to
0.2 V, the synergistic effect between AuNPs and ERGO
can remarkably enhanced the electrochemical oxidation
of TCh by significantly promoting the electron trans-
fer between GCE and PB. So, a high-performance and
advantageous platform was provided to integrate ERGO-
AuNPs--CD with PB-CS for sensing applications. When
detecting carbaryl and malathion, the modified electrode
showed wide linear ranges of 4.3100 103 pg/mL
J. Nanosci. Nanotechnol. 16, 66486656, 2016
Liu et al. Recent Advances on Rapid Detection of Pesticides Based on Enzyme Biosensor of Nanomaterials

and 7.982.00 103 pg/mL with low detection limits of form choline in the AChE catalysis. As shown in Figure 3,
1.15 pg/mL and 4.14 pg/mL, respectively. this sensing strategy is that ChOx in turn oxidizes cholin to
produce H2 O2 and betaine which can extinguish the pho-
2.1.4. Glutaraldehyde as Cross Linking Agent Based on toluminescence (PL) of SiQDs. However, the presence of
ChE EC Biosensor OPs inhibiting the activity of AChE leads to the decrease
Glutaraldehyde as a cross-linking agent is widely used in the created H2 O2 and the increase in the PL of SiQDs.
in enzyme immobilization. 69
70
Sharma et al. earlier The inhibition rate of pesticide to AChE activity was eval-
employed carboxylated single-walled carbon nanotube uated by surveying the increase in SiQDs PL. The lowest
(cSWCNT) to modified ferrule graphite electrode (FGE) detectable concentrations for parathion, diazinon, phorate,
and developed a highly sensitive amperometric biosen- and carbaryl reached 325 108 , 676 108 , 19 107 ,
sor for pesticide detection.71 AChE was immobilized on and 725 109 g/L, respectively, much lower than those
csWCNT-FGE using glutaraldehyde as a cross-linking previously reported.
agent. The experimental results show that the linear range Similarly, Meng et al. employed assembly of AchE,
for Sarin and diisopropylfluorophosphate were 2080 nM ChOx, ACh and QDs and developed a simple, sensitive,
and 2060 nM with a low detection limits of 1525 nM fast and convenient method for detection of dichlorvos in
81
and 2535 nM (3040% inhibition), respectively. real samples. Under the optimal experimental conditions,
72
Dutta and Puzari electro-immobilized AChE into a dichlorvos was detected in the linear range from 4.49 nM
thick conducting layer of polypyrrole using porcine skin to 6780 nM with the detection limit of 4.49 nM. The
gelatin and gluteraldehyde mixture cross-linking for sta- two type of biosensors will facilitate development of high-
bilizing the system. The biosensor was applied to detect throughput and rapid detection of pesticides in future.
ethylparaoxon and carbofuran and the detection limits Further, Guo et al. proposed a sensitive fluorescence
were 1.1 ppb and 0.12 ppb, respectively. The biosensor detection method of OPs using the inner filter effect
82
was fabricated with a easy and low-cost method, and pos- (IFE) of AuNPs on CdTe QDs. The presence of
sessed good stability and highly sensitivity. AuNPs remarkably quenched the fluorescence of CdTe
Arduini et al. have developed a mono-enzymatic AChE QDs via IFE. The catalysis of AChE to produce thio-
amperometric biosensor for pesticide detection. AChE73 cholin induced the aggregation of AuNPs, then their
was immobilized usingDelivered
glutaraldehyde as a cross characteristic absorption decreased,
link-Virginia University/ Health Sci Ctr LIb hence recovered IFE-
by Ingenta to: West
ing agent on a preformed cysteamine IP: 5.8.37.75 On: Mon, 20
self-assembled decreased
Jun 2016 fluorescence
12:31:06 of CdTe QDs. After the experi-
Copyright: American
monolayer (SAM) on gold-screen printed electrodes (Au- mental
Scientific conditions
Publishers were optimized, the limit of detection
SPEs). Under optimum conditions, the inhibition effect of for methamidophos is 2 g/kg. The linear range allowed
paraoxon is linearly related to the negative log of its con- a determination of methamidophos concentration from
centration in the 40 ppb range, and the detection limit of
2 ppb (10% of inhibition). The biosensor demonstrated to
be a useful analytical tool for OPs detection of drinking
water sample.

2.2. ECL Biosensors Based on ChE

The ECL biosensor is based on cholinesterase catalytic
hydrolyzing substrate to generate light reaction active sub-
stances. Quantum dots (QDs) and other nanomaterials are
the main modification materials of photochemical biosen-
sor, QDs itself have unique fluorescence properties on
account of the scale of the quantum confine effect,7476 and
new fluorescence nanocomposites were formed by com-
bination with other nanoparticles such as Au NPs and
some fluorochrome or QDs.77
78 Specially, silicon quan-
tum dots (SiQDs), whiec are nontoxic, abundant, inert, and
low-cost nanomaterials, have proved to be environmen-
tally friendly photoluminescence probes and have attracted
much interest.74
75
79 The materials possess desirable sur- Figure 3. Schematic illustration of the measuring principle of the pes-
face chemistry, physical properties, and optical proper- ticide biosensor. Inset: PL spectra of SiQDs mixtures, (a) only SiQDs
ties. Above this, bisoensor based on QDs and fluorescence and buffer, (b) SiQDs + 2 U/mL AChE + 0.6 U/mL ChOx + 1.0 mM
ACh + 8.0 105 g/L carbaryl, (c) SiQDs + 2 U/mL AChE + 0.6 U/mL
nanocomposites is widely applied in detection of OPs and ChOx + 1.0 mM ACh, and (d) SiQDs + 0.3 mM H2 O2 , respectively.
CBs. Yi et al. designed a novel label-free SiQDs-based Reprinted with permission from [80], Y. Yi et al., Anal. Chem. 85, 11464
sensor for ultrasensitive detection of pesticides.80 ACh can (2013). 2013 American Chemical Society.

J. Nanosci. Nanotechnol. 16, 66486656, 2016 6653

Recent Advances on Rapid Detection of Pesticides Based on Enzyme Biosensor of Nanomaterials Liu et al.

0.06 mg/kg to 0.78 mg/kg. The proposed assay was activity of AChE immobilized on one of the surfaces of the
superior to the method of GB/T 5009.199-2003. Simi- crystals, the extent and rate of the enzymatic reaction can
larly, Li et al. presented a sensitive photoelectrochem- be trailed in real time by surveying the frequency changes
ical (PEC) biosensor for detection of Ops,83 where connected with the mass changes at the crystal surface
the nanocomposite of CdSe@ZnS quantum dots (QDs) and lead to the accumulation of the enzymatic reaction
and graphene were deposited on the indium-doped tin product.8587 But, in recent years, piezoelectric biosensors
oxide (ITO) coated GE as a photoactive electrode. The have been paid widely attention by immunoreaction tech-
graphene/CdSe@ZnS/AChE electrode was achieved by the nique based on quartz crystal microbalance.8892
assembly of graphene/CdSe@ZnS nanocomposite systems
on ITO coated GE surface which was further modified
3. CONCLUSION AND FUTURE PROPECTS
with AChE. The biosensor was applied to detect dichlor-
This short review introduced the application of biosen-
vos and paraoxon, the linear concentration rang of inhibi-
12 6 sors based on enzyme for the detection of pesticides in
tion rate was 10 10 M, and the detectin limits was
12 14 recent years. With development and application of nano-
10 M and 10 M, respectively. The photoelectrochem-
materials, enzyme-biosensors showed high sensitivity, low
ical biosensor could be successfully detect OPs in real fruit
detect limits, super selectivity, and fast responses. Biosen-
samples.
sors based on ChE is an important research field from
Liu et al. have developed a dual readout biosensor based
comprehensive review presented above for detection of
on rhodamine B-covered Au NP(RB-AuNP) modified GC
OPs and CBs, and can be particularly applied to environ-
electrodes for detection of OPs and CBs.84 The presence
mental monitoring, human health concern and food indus-
of pesticides inhibited the activity of AChE and disturbed
tries. However, The technology of ChE sensors need to
the generation of thiocholine. Decrease in concentration of
improve in biological stability, precision, signal transduc-
thiocholine or no generation of thiocholine cause quench-
tion and cost-effctiveness. In terms of linear range and
ing of the fluorescence of RB and the RB-AuNP solutions
detection limit, the role of matrices has been described
turns red, the reasons is that thiocholine can not quench
for biosensing and the characteristics of various enzyme
the fluorescence of RB and change the color of RB-AuNP
biosensors. Nanoengineering and fabrication technology
solutions from red to blue simultaneously (Fig. 4). By
have an enormous impact on the pesticide biosensors from
using this dual-readout assay, the lowest
Delivered detection
by Ingenta concen-
to: West Virginia
theUniversity/ Health
comprehensive Sci presented
review Ctr LIb above. For the sensor
IP: 5.8.37.75
tration of carbaryl, diazinon, malathion, On:were
and phorate Mon, 20 Jun 2016 12:31:06
development, varieties of working electrodes can be fabri-
0.1, 0.1, 0.3, and 1 g/L, respectively.Copyright: American Scientific Publishers
cated for the improved detection of pesticide in different
samples.93
94
2.3. Piezoelectric Biosensors Based on ChE
Finally, we believe the development of cheap and handy
The basic thinking way to design a piezoelectric sensor is biosensors for detection of pesticide such as OPs and
based on the inhibitory effects of these substances on the CBs is needed very much. But many interfering com-
pounds hinder the sensitivity of the biosensor based on
enzymes in real samples. Recently, nanotecgnology has
been developed rapidly,95115 it can be anticipated that the
use of nanomaterials would improve further biosensing
and design of bioelectronic systems in the future.

Acknowledgments: We acknowledge the financial sup-

port from the National Natural Science Foundation of
China (61471168), the Economical Forest Cultivation and
Utilization of 2011 Collaborative Innovation Center in
Hunan Province [(20130) 448] and Open Research Fund
by State Key Laboratory of Bioelectronics, Southeast Uni-
versity, China (2013G10).

Figure 4. Schematic representation of the dual-readout biosensor. References and Notes

AChE can catalyze ATCh to produce thiocholine which cause RB-AuNP
solutions blue and fluorescence recovery of RB (the color of the stars
changed from gray to green). The presence of pesticides inhibit the activ-
ity of AChE and block generation of thiocholine, it results in the RB-
AuNP solutions remaining red and the fluorescence of RB quenched by
AuNPs. Reprinted with permission from [84], D. Liu et al., Anal. Chem.
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Received: 8 August 2014. Accepted: 8 December 2014.

6656 J. Nanosci. Nanotechnol. 16, 66486656, 2016