A nanocomposite of gold nanoparticles (AuNPs) decorating a magnetic Fe3O4 core was synthesized using cysteamine (SH–NH2) as linker, and characterized by TEM, XPS, UV and electrochemistry. Then a hydrolase biosensor, based on self-assembly of methyl parathion hydrolase (MPH) on the Fe3O4@Au nanocomposite, was developed for sensitive and selective detection of the organophosphorus pesticide (OP) methyl parathion. The magnetic nanocomposite provides an easy way to construct the enzyme biosensor by simply exerting an external magnetic field, and also provides a simple way to renew the electrode surface by removing the magnet. Unlike inhibition-based enzyme biosensors, the hydrolase is not poisoned by OPs and thus is reusable for continuous measurement. AuNPs not only provide a large surface area, high loading efficiency and fast electron transfer, but also stabilize the enzyme through electrostatic interactions. The MPH biosensor shows rapid response and high selectivity for detection of methyl parathion, with a linear range from 0.5 to 1000 ng mL−1 and a detection limit of 0.1 ng mL−1. It also shows acceptable reproducibility and stability. The simplicity and ease of operation of the proposed method has great potential for on-site detection of P–S containing pesticides and provides a promising strategy to construct a robust biosensor.

•Colloidal gold-based immunochromatographic test strip was developed for analysis of TCP.•The immunosensor was successfully used to detect human saliva sample without any pretreatment.•The signal was amplified by colloidal gold-tags which used for visible qualitative detection.A portable immunochromatographic strip-based biosensor for direct detection of trichloropyridinol (TCP), a specific biomarker of exposure to chlorpyrifos, in human saliva sample is presented. In this approach, a series of immunoreactions was performed on the test strip, where the targeted analytes (TCP) bound to the Au nanoparticles-labeled antibodies on the conjugate pad to form analyte–Au–antibody conjugates, and then free Au-labeled antibodies were captured by TCP–BSA in the test zone. Captured Au nanoparticles, increased with decreased levels of analytes, can be observed visibly without any equipment and later quantified by a colorimetric reader. Several experimental parameters were optimized including Au nanoparticle-to-TCP antibody coupling ratio, the amount of Au-labeled TCP antibody, immunoreaction time, the pretreatment of sample pad and the preparation of stock solution of Au–TCP antibody that realize sensitivity, selectivity and direct detection of TCP. Under optimal conditions, this biosensor displays a highly linear range of 0.625–20 ng/mL TCP, with a detection limit of 0.47 ng/mL. Moreover, the immunosensor was successfully used for direct analysis of human saliva sample without any pretreatment. These results demonstrate that this Au nanoparticles-based immunochromatographic test strip (ITS) provides a simple, accurate, and quantitative tool for TCP detection and holds a great promise for point-of-care and in-field analysis of other biomarkers.

Au–ZrO2–SiO2 nanocomposite spheres were synthesized and used as selective sorbents for the solid-phase extraction (SPE) of organophosphorous agents. A non-enzymatic electrochemical sensor based on a Au–ZrO2–SiO2 modified electrode was developed for the selective detection of organophosphorous pesticides (OPs). The Au–ZrO2–SiO2 nanocomposite spheres were synthesized by the hydrolysis and condensation of zirconium n-butoxide (TBOZ) on the surface of SiO2 spheres and then the introduction of gold nanoparticles on the surface. Transmission electron microscopy and X-ray photoelectron spectroscopy were performed to characterize the formation of the nanocomposite spheres. Fast extraction of OP was achieved by the Au–ZrO2–SiO2 modified electrode within 5 min via the specific affinity between zirconia and the phosphoric group. The assay yields a broad concentration range of paraoxon-ethyl from 1.0 to 500 ng mL−1 with a detection limit of 0.5 ng mL−1. This selective and sensitive method holds great promise for the enrichment and detection of OPs.

An integrated lateral flow test strip with an electrochemical sensor (LFTSES) device with rapid, selective, and sensitive response for quantification of exposure to organophosphorus (OP) pesticides and nerve agents has been developed. The principle of this approach is based on parallel measurements of postexposure and baseline acetylcholinesterase (AChE) enzyme activity, where reactivation of the phosphorylated AChE is exploited to enable measurement of the total amount of AChE (including inhibited and active) which is used as a baseline for calculation of AChE inhibition. Quantitative measurement of phosphorylated adduct (OP-AChE) was realized by subtracting the active AChE from the total amount of AChE. The proposed LFTSES device integrates immunochromatographic test strip technology with electrochemical measurement using a disposable screen printed electrode which is located under the test zone. It shows a linear response between AChE enzyme activity and enzyme concentration from 0.05 to 10 nM, with a detection limit of 0.02 nM. On the basis of this reactivation approach, the LFTSES device has been successfully applied for in vitro red blood cells inhibition studies using chlorpyrifos oxon as a model OP agent. This approach not only eliminates the difficulty in screening of low-dose OP exposure because of individual variation of normal AChE values but also avoids the problem in overlapping substrate specificity with cholinesterases and avoids potential interference from other electroactive species in biological samples. It is baseline free and thus provides a rapid, sensitive, selective, and inexpensive tool for in-field and point-of-care assessment of exposures to OP pesticides and nerve agents.

Acetylcholinesterase (AChE) enzyme activity in red blood cells (RBCs) is a useful biomarker for biomonitoring of exposures to organophosphorus (OP) pesticides and chemical nerve agents. In this paper, we reported a new method for AChE activity assay based on selective immuno-capture of AChE from biological samples followed by enzyme activity assay of captured AChE using a disposable electrochemical sensor. The electrochemical sensor is based on multiwalled carbon nanotubes–gold (MWCNTs–Au) nanocomposites modified screen printed carbon electrode (SPCE), which is used for the immobilization of AChE specific antibody. Upon the completion of immunoreaction, the target AChE (including active and inhibited) is captured onto the electrode surface and followed by an electrochemical detection of enzymatic activity in the presence of acetylthiocholine. A linear response is obtained over standard AChE concentration range from 0.1 to 10 nM. To demonstrate the capability of this new biomonitoring method, AChE solutions dosed with different concentrations of paraoxon were used to validate the new AChE assay method. AChE inhibition in OP dosed solutions was proportional to OP concentration from 0.2 to 50 nM. The new AChE activity assay method for biomonitoring of OP exposure was further validated with in vitro paraoxon-dosed RBC samples. The established electrochemical sensing platform for AChE activity assay not only avoids the problem of overlapping substrate specificity with esterases by using selective antibody, but also eliminates potential interference from other electroactive species in biological samples. It offers a new approach for sensitive, selective, and rapid AChE activity assay for biomonitoring of exposure to OPs.

This paper described the preparation, characterization, and electrochemical properties of a graphene-ZrO2 nanocomposite (GZN) and its application for both the enrichment and detection of methyl parathion (MP). GZN was fabricated using electrochemical deposition and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS), which showed the successful formation of nanocomposites. Due to the strong affinity to the phosphoric group and the fast electron-transfer kinetics of GZN, both the extraction and electrochemical detection of organophosphorus (OP) agents at the same GZN modified electrochemical sensor was possible. The combination of solid-phase extraction and stripping voltammetric analysis allowed fast, sensitive, and selective determination of MP in garlic samples. The stripping response was highly linear over the MP concentrations ranging from 0.5 ng mL−1 to 100 ng mL−1, with a detection limit of 0.1 ng mL−1. This new nanocomposite-based electrochemical sensor provides an opportunity to develop a field-deployable, sensitive, and quantitative method for monitoring exposure to OPs.

A multiplexed electrochemical immunoassay integrating enzyme amplification and electric field-driven strategy was developed for fast and sensitive quantification of phosphorylated p53 at Ser392 (phospho-p53392), Ser15 (phospho-p5315), Ser46 (phospho-p5346), and total p53 simultaneously. The disposable sensor array has four spatially separated working electrodes, and each of them is modified with different capture antibody, which enables simultaneous immunoassay to be conducted without cross-talk between adjacent electrodes. The enhanced sensitivity was achieved by a multienzyme amplification strategy using gold nanorods (AuNRs) as nanocarrier for coimmobilization of horseradish peroxidase (HRP) and detection antibody (Ab2) at a high ratio of HRP/Ab2, which produced an amplified electrocatalytic response by the reduction of HRP oxidized thionine in the presence of hydrogen peroxide. The immunoreaction processes were accelerated by applying +0.4 V for 3 min and then −0.2 V for 1.5 min; thus, the whole sandwich immunoreactions could be completed in less than 5 min. Under optimal conditions, this method could simultaneously detect phospho-p53392, phospho-p5315, phospho-p5346, and total p53 ranging from 0.01 to 20 nM, 0.05 to 20 nM, 0.1 to 50 nM, and 0.05 to 20 nM with detection limits of 5 pM, 20 pM, 30 pM, and 10 pM, respectively. Accurate determinations of these proteins in human plasma samples were demonstrated by comparison to the standard ELISA method. The disposable immunosensor array shows excellent promise for clinical screening of phosphorylated proteins and convenient point-of-care diagnostics.

We reported a graphene-based immunosensor for electrochemical quantification of phosphorylated p53 on serine 15 (phospho-p5315), a potential biomarker of gamma-radiation exposure. The principle is based on sandwich immunoassay and the resulting immunocomplex is formed among phospho-p53 capture antibody, phospho-p5315 antigen, biotinylated phospho-p5315 detection antibody and horseradish peroxidase (HRP)-labeled streptavidin. The introduced HRP results in an electrocatalytic response to reduction of hydrogen peroxide in the presence of thionine. Graphene served as sensor platform not only promotes electron transfer, but also increases the surface area to introduce a large amount of capture antibody, thus increasing the detection sensitivity. The experimental conditions including blocking agent, immunoreaction time and substrate concentration have been optimized. Under the optimum conditions, the increase of response current is proportional to the phospho-p5315 concentration in the range of 0.2–10 ng mL−1, with the detection limit of 0.1 ng mL−1. The developed immunosensor exhibits acceptable stability and reproducibility and the assay results for phospho-p5315 are in good correlation with the known values. This easily fabricated immunosensor provides a new promising tool for analysis of phospho-p5315 and other phosphorylated proteins.Graphical abstract. A new graphene-based immunosensor for electrochemical quantification of phosphorylated p53 on serine 15 (phospho-p5315) was proposed. This easily fabricated immunosensor provides a potential tool for analysis of other biomarkers.Highlights► A new graphene-based immunosensor for electrochemical quantification of phosphorylated p53 on serine 15 (phospho-p5315) was proposed. ► Graphene served as sensor platform greatly increased detection sensitivity. ► This easily fabricated immunosensor provides a potential tool for analysis of other biomarkers.

A new sandwich-like electrochemical immunosensor has been developed for quantification of organophosphorylated acetylcholinesterase (OP-AChE), an exposure biomarker of organophosphate pesticides and nerve agents. Zirconia nanoparticles (ZrO2 NPs) were anchored on a screen printed electrode (SPE) to preferably capture OP-AChE adducts by metal chelation with phospho-moieties, which was selectively recognized by lead phosphate-apoferritin labeled anti-AChE antibody (LPA–anti-AChE). The sandwich-like immunoreactions were performed among ZrO2 NPs, OP-AChE and LPA–anti-AChE to form ZrO2/OP-AChE/LPA–anti-AChE complex and the released lead ions were detected on a disposable SPE. The binding affinity was investigated by both square wave voltammetry (SWV) and quartz crystal microbalance (QCM) measurements. The proposed immunosensor yielded a linear response current over a broad OP-AChE concentrations range from 0.05 nM to 10 nM, with detection limit of 0.02 nM, which has enough sensitivity for monitoring of low-dose exposure to OPs. This method avoids the drawback of unavailability of commercial OP-specific antibody as well as amplifies detection signal by using apoferritin encoded metallic phosphate nanoparticle tags. This nanoparticle-based immunosensor offers a new method for rapid, sensitive, selective and inexpensive quantification of phosphorylated adducts for monitoring of OP pesticides and nerve agents exposures.

Acetylcholinesterase (AChE) activity is a well established biomarker for biomonitoring of exposures to organophosphates (OPs) pesticides and chemical nerve agents. In this work, we described a novel electrochemical oxidative desorption-process of thiocholine, the product of enzymatic reaction, for rapid and highly sensitive determination of AChE activity in human serum. This principle is based on self-assembling of produced thiocholine onto core–shell Fe3O4/Au nanoparticles (Fe3O4/AuNPs) magnetic nanocomposites and its oxidation at electrode surface. Fe3O4 magnetic core is not only used for magnetic separation from sample solutions, but also carrying more AuNPs due to its large surface-to-volume ratio. The core–shell Fe3O4/AuNPs nanocomposites were characterized by UV–Vis spectroscopy, field-emission scanning electron microscopy (FE-SEM) and electrochemical measurements. A linear relationship was obtained between the AChE activity and its concentration from 0.05 to 5.0 mU mL−1 with a detection limit of 0.02 mU mL−1. The method showed good results for characterization of AChE spiked human serum and detection of OP exposures from 0.05 to 20 nM, with detection limit of 0.02 nM. This new oxidative desorption assay thus provides a sensitive and quantitative tool for biomonitoring of the exposure to OP pesticides and nerve agents.

A simple method to immobilize acetylcholinesterase (AChE) on polypyrrole (PPy) and polyaniline (PANI) copolymer doped with multi-walled carbon nanotubes (MWCNTs) was proposed. The synthesized PAn-PPy-MWCNTs copolymer presented a porous and homogeneous morphology which provided an ideal size to entrap enzyme molecules. Due to the biocompatible microenvironment provided by the copolymer network, the obtained composite was devised for AChE attachment, resulting in a stable AChE biosensor for screening of organophosphates (OPs) exposure. MWCNTs promoted electron-transfer reactions at a lower potential and catalyzed the electro-oxidation of thiocholine, thus increasing detection sensitivity. Based on the inhibition of OPs on the AChE activity, using malathion as a model compound, the inhibition of malathion was proportional to its concentration ranging from 0.01 to 0.5 μg/mL and from 1 to 25 μg/mL, with a detection limit of 1.0 ng/mL. The developed biosensor exhibited good reproducibility and acceptable stability, thus providing a new promising tool for analysis of enzyme inhibitors.

An amperometric biosensor for highly selective and sensitive determination of methyl parathion (MP) was developed based on dual-signal amplification: (1) a large amount of introduced enzyme on the electrode surface and (2) synergistic effects of nanoparticles towards enzymatic catalysis. The fabrication process includes (1) electrochemical deposition of gold nanoparticles by a multi-potential step technique at multiwalled carbon nanotube (MWCNT) film pre-cast on a glassy carbon electrode and (2) immobilization of methyl parathion degrading enzyme (MPDE) onto a modified electrode through CdTe quantum dots (CdTe QDs) covalent attachment. The introduced MWCNT and gold nanoparticles significantly increased the surface area and exhibited synergistic effects towards enzymatic catalysis. CdTe QDs are further used as carriers to load a large amount of enzyme. As a result of these two important enhancement factors, the proposed biosensor exhibited extremely sensitive, perfectly selective, and rapid response to methyl parathion in the absence of a mediator. The detection limit was 1.0 ng/mL. Moreover, since MPDE hydrolyzes pesticides containing the P–S bond, it showed high selectivity for detecting MP and many interfering compounds, such as carbamate pesticides. Other organophosphorous pesticides and oxygen-containing inorganic ions (SO42−, NO3−) did not interfere with the determination. The proposed MPDE biosensor presents good reproducibility and stability, and the MPDE is not poisoned by organophosphate pesticides. Unlike cholinesterase-based biosensor, the MPDE biosensor can be potentially reused and is suitable for continuous monitoring.

This work described a highly sensitive amperometric biosensor for organophosphates (OPs) pesticides based on immobilization of acetylcholinesterase (AChE) on multiwall carbon nanotubes (MWCNTs)-β-cyclodextrin (β-CD) composite modified glassy carbon electrode. The synthesized composite through polymer wrapping strategy has been characterized by attenuated total reflection Fourier-transform infrared spectra (ATR-FTIR) measurement and scanning electron microscopy (SEM) images. Due to the good dispersibility and porous structures of MWCNTs-β-CD composite, the resulting surface provided a favorable microenvironment for acetylcholinesterase biosensor fabrication and maintained the bioactivity of AChE for screening of OPs exposure. MWCNTs promoted electron-transfer reactions at a lower potential and catalyzed the electro-oxidation of thiocholine, thus increasing detection sensitivity. Based on the inhibition of OPs on the AChE activity, using dimethoate as a model compound, the inhibition of dimethoate was proportional to its concentration ranging from 0.01 to 2.44 and 2.44 to 10.00 μM, with a detection limit was 2 nM (S/N = 3). The developed biosensor exhibited good reproducibility and acceptable stability, thus providing a new promising tool for analysis of enzyme inhibitors.

A facile, one-step synthesis of nanocomposites using multiwalled carbon nanotube coating gold nanoparticles (MWCNTs-Au) was presented. Scanning electron microscopy and UV–vis spectroscopy confirmed that more than 97% of gold nanoparticles have been loaded on the surface of carbon nanotubes without congregation. The formed MWCNTs-Au nanocomposites offered an extremely hydrophilic surface for biomolecule adhesion, leading to a stable acetylcholinesterase (AChE) biosensor. Due to the excellent conductivity of the nanocomposites, the immobilized AChE showed favorable affinity to acetylthiocholine (ATCl) and could catalyze the hydrolysis of ATCl with a Kmapp value of 268 μM to form thiocholine, which was then oxidized to produce a detectable and fast response. Based on the inhibition of organophosphates (OPs) on the enzymatic activity of AChE, the magnitude of peak current from thiocholine on the biosensor is a simple and effective way to biomonitoring of OPs exposure. Using malathion as a model compound, the inhibition of malathion was proportional to its concentration ranging from 1.0 to 1000 ng mL−1 and from 2 to 15 μg mL−1, with a detection limit 0.6 ng mL−1. The developed biosensor exhibited good reproducibility and acceptable stability, thus providing a new promising tool for analysis of enzyme inhibitors.

Thin film of a molecular imprinted polymer based on electropolymerization method with sensitive and selective binding sites for dimethoate was developed. This film was cast on gold electrode by electrochemical polymerization in solution of o-phenylenediamine and template dimethoate via cyclic voltammetry scans and further deposition of Ag nanoparticles. The surface plasmon resonance and cyclic voltammetric signals were also recorded simultaneously during the electropolymerization, controlling the thickness of the polymer film to be 25 nm. The imprinted film showed high selectivity towards to dimethoate. The recognition between the imprinted sensor and target molecule was observed by measuring the variation amperometric response of the oxidation–reduction probe, K3Fe(CN)6, on electrode. Under the optimal experimental conditions, the peak currents were proportional to the concentrations of dimethoate in two ranges, from 1.0 to 1000 ng mL−1 and from 1.0 to 50 μg mL−1, with the detection limit of 0.5 ng mL−1. Due to the high affinity, selectivity and stability the imprinted sensor provides a simple detection platform for organophosphate compounds.

Bismuth-film electrode (BiFE) was prepared by ex situ depositing bismuth onto glassy carbon electrode for the detection of organophosphate (OP) pesticides, using methyl parathion (MP) as a model analyte. The sufficiently wide negative potential window available made the BiFE a potentially suitable electrode for application in the field of cathodic electrochemical detection of OPs. The BiFE showed similar or even favorable behavior compared to that of mercury and bare electrode. Operational parameters, including the deposition potential and time of bismuth film, pH of the analytic solution and concentration of surfactant, cetyltrimethyl ammonium bromide (CTAB) have been optimized. Under the optimum experimental conditions, the cathodic voltammetric response was proportional to the concentration of MP range from 3.0 to 100 ng mL−1, with a correlation coefficient of 0.9981. The detection limit was 1.2 ng mL−1 (threefold standard deviation of a blank). Due to the easy preparation and regeneration of BiFE together with its good reproducibility and stability, the combination of the promising BiFE and cathodic voltammetric performances opens new opportunity for fast, simple, and sensitive analysis of OP compounds in samples.

A sensitive electrochemical stripping voltammetric method for analyzing organophosphate (OP) compounds was developed based on solid-phase extraction (SPE) at zirconia (ZrO2) nanoparticles modified electrode. ZrO2 nanoparticles were proved as a new sorbent for SPE of OP pesticides. Because of the strong affinity of ZrO2 for the phosphoric group, nitroaromatic OPs can strongly bind to the ZrO2 nanoparticle surface. The combination of SPE with square-wave voltammetry (SWV) provided a fast, sensitive, and selective electrochemical method for nitroaromatic OP compounds using methyl parathion (MP) as a model. The stripping response was highly linear over the MP range of 0.003–2.0 μg/mL, with a detection limit of 0.001 μg/mL. The fast extraction ability of ZrO2 nanoparticles makes it promising sorbent for various solid-phase extractions.

Multiwalled carbon nanotube (MWCNT) was developed as a new sorbent for solid-phase extraction (SPE) of organophosphate (OP) pesticides. A combination of SPE with square-wave voltammetric (SWV) analysis resulted in a fast, sensitive, and selective electrochemical method for determination of OP pesticide using methyl parathion (MP) as a representative. Because of the strong affinity of MWCNT for phosphoric group, nitroaromatic OP compounds can strongly bind to the MWCNT surface. The macroporosity and heterogeneity of MWCNT allow extracting a large amount of MP less than 5 min. The stripping response was highly linear over the MP range of 0.05–2.0 μg/mL, with a detection limit of 0.005 μg/mL. The determination of MP in garlic samples showed acceptable accuracy. The fast extraction ability of MWCNT makes it promising sorbent for various solid-phase extractions.

A simple method to immobilize acetylcholinesterase (AChE) on the controllable adsorption of multiwalled carbon nanotubes (MWCNTs) onto an alkanethiol self-assembled monolayer (C6H13SH SAM) modified Au electrode was proposed. The surface coverage of the MWCNTs was readily controlled by adjusting the immersion time for the adsorption of the MWCNTs. Atomic force microscopy (AFM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to monitor these controllable fabrication processes. The MWCNTs adsorbed onto the SAM surface substantially restores the heterogeneous electron transfer between the bare Au electrode and the redox system in the solution phase that is almost totally blocked by the SAM of C6H13SH, and as a result, the prepared MWCNT-SAM-modified electrode possesses good electrode reactivity without a remarkable barrier to heterogeneous electron transfer. Due to the inherent conductive properties of MWCNTs, the immobilized AChE exhibited high affinity to its substrate and produced a detectable and fast response. Thus, a sensitive, efficient and stable amperometric sensor for quantitative determination of carbaryl was developed. The inhibition of carbaryl was proportional to its concentration ranging from 0.001 to 1 μg mL−1 and 2 to 15 μg mL−1, with a detection limit of 0.6 ng mL−1. The determination of carbaryl in garlic samples showed acceptable accuracy, which provided a new promising tool for analysis of enzyme inhibitors.

In this paper, a simple method for immobilization of acetylcholinesterase (AChE) on cysteamine self assembled monolayers modified gold electrode coupled with CdTe quantum dots (QDs) was proposed and thus a sensitive, fast and stable amperometric biosensor for quantitative determination of carbaryl was developed. The fabrication procedure was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and fluorescence spectrum. The nanoparticles of CdTe QDs led to an increased effective surface area for immobilization of enzyme and their electrocatalytic activity promoted electron transfer reactions and catalyzed the electro-oxidation of thiocholine, thus amplifying the detection sensitivity. Due to the notable decrease in voltammetric signal of the immobilized AChE, a simple method for determination of carbaryl was established. The inhibition of carbaryl was proportional to its concentration in two ranges, from 1 to 50 ng mL−1 and from 50 to 500 ng mL−1, with a detection limit of 0.6 ng mL−1. The constructed biosensor which processes prominent characteristics and performance, such as good precision and reproducibility, acceptable stability and accuracy, fast response and low detection limit, has potential application in the characterization of enzyme inhibitors and detection of toxic compounds against to enzyme.

One-step electrochemical deposition of gold nanoparticles (AuNPs) in chitosan hydrogel onto a planar gold electrode was used to create a favorable surface for the attachment of the enzyme acetylcholinesterase (AChE). AChE exhibited high affinity to its substrate of acetylthiocholine chloride (ATCl) from calculated Michealis–Menten constant (Kmapp) of 0.309 mM. Thiocholine, the product through AChE-catalyzed hydrolysis of ATCl, could be chemisorbed on the surface with an applied potential of −0.7 V and then desorbed in KOH solution, giving a measurable peak current. When enzymatic activity was inhibited by organophosphate pesticides, the amount of chemisorpted thiocholine on the interface was anticipated to decrease. As a result, a simple method for rapid determination of malathion was established based on the chemisorption/desorption process of thiocholine used as an indicator. Under the optimal conditions, the decrease in response was proportional to the concentration of malathion from 0.1 to 20 ng mL−1, with detection limit of 0.03 ng mL−1 (0.1 nM). The method based on one-step electrochemical deposition and chemisorption/desorption process of thiocholin has potential application in high-sensitive pesticide determinations.

Gold nanoparticles (AuNPs) were synthesized in situ and electrodeposited onto Au substrate. The AuNPs modified interface facilitates electron transfer across self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid (MUA). After activation of surface carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, the interface displayed good stability for immobilization of biomolecules. These modification processes were characterized by contact angle measurement, cyclic voltammetry and electrochemical impedance spectra. The immobilized acetylcholinesterase (AChE), as a model, showed excellent activity to its substrate, leading to a stable AChE biosensor. Under the optimal experimental conditions, the inhibition of malathion on AChE biosensor was proportional to its concentration in two ranges, from 0.001 to 0.1 μg mL−1 and from 0.1 to 25 μg mL−1, with detection limit of 0.001 μg mL−1. The simple method showed good reproducibility and acceptable stability, which had potential application in biosensor design.

A simple method has been devised for immobilization of acetylcholinesterase (AChE) covalent bonding to a multiwall carbon nanotube (MWNT)-cross-linked cellulose acetate composite on a screen-printed carbon electrode (SPCE) and a sensitive and disposable amperometric sensor for rapid determination of carbaryl pesticide is proposed. The immobilized enzyme was preserved on this film because of the excellent biocompatibility and non-toxicity of cellulose acetate. Based on the inherent conductive properties of the MWNT, the immobilized AChE had greater affinity for ATCl and excellent catalytic effect in the hydrolysis of ATCl. MWNT improved the interface enzymatic hydrolysis reaction and increased the amperometric response of the sensor. Under optimum conditions, the inhibition of carbaryl on AChE increased linearly with the increasing concentration of carbaryl in two ranges, from 0.01 to 0.5 μg mL−1 and from 2 to 20 μg mL−1, with the correlation coefficients of 0.9985 and 0.9977, respectively. The detection limit was 0.004 μg mL−1 taken as the concentration equivalent to 10% decrease in signal. The sensor showed acceptable stability, accuracy and could be fabricated in batches, thus it is economic and portable. This type of disposable enzyme-based amperometric sensor has extensive application potential in environmental monitoring of pesticides.

Based on the change in electrochemical behavior of enzymatic activity induced by pesticide, a novel electrochemical method for investigation of pesticide sensitivity using acetylcholinesterase (AChE) biosensor was developed. The sol–gel-derived silicate network assembling gold nanoparticles (AuNPs-SiSG) provided a biocompatible microenvironment around the enzyme molecule to stabilize its biological activity and prevented them from leaking out of the interface. The composite was characterized using atomic force microscopy and proved to be chemically clean, porous and homogeneous. AuNPs promoted a conductive pathway for electron transfer and improved electrochemical reactions at a lower potential. Typical pesticides such as monocrotophos, methyl parathion and carbaryl were selected for pesticide sensitivity tests. Due to the inhibitions of pesticides, the electrochemical responses of substrate on AChE-sensors decreased greatly. The inhibition curves showed good correspondence with the results by UV spectrophotometry assay. The proposed electrochemical pesticide sensitivity test exhibited high sensitivity, desirable accuracy, low cost and simplified procedures. This method could be developed as a conventional method to select efficient enzyme inhibitors and investigate toxic compounds against to enzyme.

CdTe quantum dots (QDs)-based electrochemical sensor for recognition of neutravidin, as a model protein, using anodic stripping voltammetry at electrodeposited bismuth film is presented. This biosensor involves the immobilization of the captured QDs conjugates which was dissolved with 1 M HCl solution to release cadmium ions and metal components were quantified by anodic stripping voltammetry after a 3-min accumulation at −1.2 V on bismuth-film electrode (BiFE) of the biotin, served as recognition element, onto the gold surface in connection with a cysteamine self-assembled monolayer. The modification procedure was characterized by electrochemical impedance spectroscopy and atomic force microscopy. We exploit QDs as labels for amplifying signal output and monitoring the extent of competition process between CdTe-labeled neutravidin and the target neutravidin for the limited binding sites on biotin. As expected for the competitive mechanism, the recognition event thus yields distinct cadmium stripping voltammetric current peak, whose response decreases upon increasing the level of target neutravidin concentrations. Under optimal conditions, the voltammetric response is highly linear over the range of 0.5–100 ng L−1 neutravidin and the limit of detection is estimated to be 0.3 ng L−1 (5 nM). Unlike earlier two-step sandwich bioassays, the present protocol relies on a one-step competitive assay, which is more accurate and sensitive, showing great promise for rapid, simple and cost-effective analysis of protein.

In this paper, a novel acetylcholinesterase (AChE) biosensor was constructed by modifying glassy carbon electrode with CdTe quantum dots (QDs) and excellent conductive gold nanoparticles (GNPs) though chitosan microspheres to immobilize AChE. Since GNPs have shown widespread use particularly for constructing electrochemical biosensors through their high electron-transfer ability, the combined AChE exhibited high affinity to its substrate and thus a sensitive, fast and cheap method for determination of monocrotophos. The combination of CdTe QDs and GNPs promoted electron transfer and catalyzed the electro-oxidation of thiocholine, thus amplifying the detection sensitivity. This novel biosensing platform based on CdTe QDs–GNPs composite responded even more sensitively than that on CdTe QDs or GNPs alone because of the presence of synergistic effects in CdTe-GNPs film. The inhibition of monocrotophos was proportional to its concentration in two ranges, from 1 to 1000 ng mL−1 and from 2 to 15 μg mL−1, with a detection limit of 0.3 ng mL−1. The proposed biosensor showed good precision and reproducibility, acceptable stability and accuracy in garlic samples analysis.

Based on the change in electrochemical behavior of enzymatic activity induced by medicines related to Alzheimer’s disease (AD), a simple electrochemical method has been developed for investigation of medicine sensitivity using acetylcholinesterase (AChE) biosensor. The sol–gel-derived silicate network incorporating gold nanoparticles (AuNPs-SiSG) provided a biocompatible microenvironment around the enzyme molecule to stabilize its biological activity and prevent them from leaking out of the interface. AuNPs provided a conductive pathway for electron transfer and improved electrochemical reactions at a lower potential. Typical medicines for treatment of AD such as galantamine and neostigmine were selected for medicine sensitivity test. Due to the inhibitions of medicines, the electrochemical responses of substrate on AChE-sensor decreased greatly. The inhibition curves were similar to Michaelis–Menten and the Michaelis–Menten constants (Km) were calculated to be 0.14 μM and 0.19 μM, respectively. The inhibition curves showed good correspondence with the results by UV spectrophotometry assay. Ninety-six percent reactivation of the inhibited AChE could be regenerated for using pralidoxime iodide within 8 min. The proposed electrochemical drug sensitivity test exhibited high sensitivity, low cost and simplified procedures, which provided a new promising tool for investigation of drug sensitivity.

A novel interface embedded in situ gold nanoparticles (GNPs) in chitosan hydrogel was constructed by one-step electrochemical deposition in solution containing tetrachloroauric (III) acid and chitosan. This deposited interface possessed excellent biocompatibility and good stability by characterization of attenuated total reflection Fourier-transform infrared spectra (ATR-FTIR), atomic force microscopy (AFM) and UV–vis spectra. The immobilized AChE, as a model, showed excellent activity to its substrate and provided a quantitative measurement of organophosphate pesticides involved in the inhibition action. Operational parameters, including the deposition time, tetrachloroauric (III) acid concentration have been optimized. Under the optimal electrodeposition, an amperometric sensor for the fast determination of malathion and monocrotophos, respectively was developed 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.

Based on the change in electrochemical behavior of enzymatic activity induced by pesticide, a novel electrochemical method has been devised for investigation of pesticide sensitivity using acetylcholinesterase (AChE) biosensor. Because of the excellent biocompatibility and good stability of chitosan matrix, it prevented leakage of the AChE from electrode. Multiwall carbon nanotube (MWNT) promoted electron transfer reaction at a lower potential and catalyzed the electro-oxidation of thiocholine, thus amplifying the sensitivity and amperometric response of the biosensor. Four pesticides of carbaryl, malathion, dimethoate and monocrotophos were selected to discuss their inhibition efficiencies to AChE. The inhibition curves were similar to Michealis–Menten and the Michealis–Menten constants (Km) were calculated to be 0.96 μM, 1.78 μM, 1.97 μM and 4.28 μM, respectively. Ninety-five percent reactivation of the inhibited AChE could be regenerated using pralidoxime iodide within 8 min. The proposed electrochemical pesticide sensitivity test exhibited high sensitivity, low cost and simplified procedures, which is a promising new tool for comparison of pesticide sensitivity and for selection of the most efficient enzyme inhibitors.

A sensitive and stable amperometric sensor has been devised for rapid determination of triazophos based on efficient immobilization of acetylcholinesterase (AChE) on silica sol–gel (SiSG) film assembling multiwall carbon nanotubes (MWNTs). The sol–gel matrix provided a biocompatible microenvironment around the enzyme and efficiently prevented leakage of the enzyme from the film. In the presence of acetylthiocholine chloride (ATCl) as a substrate, MWNTs promoted electron transfer reactions at a lower potential and catalyzed electrochemical oxidation of enzymatically formed thiocholine, thus increasing detection sensitivity. Based on the inhibition of organophosphorous compound on the enzymatic activity of AChE, using triazophos as a model compound, the effects of pH, temperature, and MWNTs contents were explored. Under optimum conditions, the inhibition of triazophos was proportional to its concentration from 0.02 μM to 1 μM and from 5 μM to 30 μM, with a detection limit of 0.005 μM. The determination of triazophos in garlic samples showed acceptable accuracy. Fabrication reproducibility of the sensor was good and stability was acceptable. The sensor is a promising new tool for pesticide analysis.

A simple method to immobilize acetylcholinesterase (AChE) on silica sol–gel (SiSG) film assembling gold nanoparticles (AuNPs) was proposed, thus a sensitive, fast and stable amperometric sensor for quantitative determination of organophosphorous insecticide was developed. The large quantities of hydroxyl groups in the sol–gel composite provided a biocompatible microenvironment around enzyme molecule and stabilized its biological activity to a large extent. The immobilized AChE could catalyze the hydrolysis of acetylthiocholine chloride (ATCl) with a Kmapp value of 450 μM to form thiocholine, which was then oxidized to produce detectable single with a linear range of 10–1000 μM. AuNPs catalyzed the electro-oxidation of thiocholine, thus increasing detection sensitivity. Based on the inhibition of organophosphorous insecticide on the enzymatic activity of AChE, using monocrotophos as a model compound, the conditions for detection of the insecticide were optimized. The inhibition of monocrotophos was proportional to its concentration ranging from 0.001 to 1 μg/ml and 2 to 15 μg/ml, with the correlation coefficients of 0.9930 and 0.9985, respectively. The detection limit was 0.6 ng/ml at a 10% inhibition. The developed biosensor exhibited good reproducibility and acceptable stability, thus providing a new promising tool for analysis of enzyme inhibitors.

A reagentless immunosensor for rapid determination of carbohydrate antigen 19-9 (CA19-9) in human serum was proposed. This strategy was based on the immobilization of antibody in colloidal gold nanoparticle modified carbon paste electrode and the direct electrochemistry of horseradish peroxidase (HRP) that was labeled to a CA19-9 antibody. The nanoparticles were efficient for preserving the activity of immobilized biomolecules. Thus, the immobilized HRP displayed its direct electrochemistry with a rate constant of 1.02 s−1. The incubation of the immunosensor in phosphate buffer solution (PBS) including CA19-9 antigen leading to the formation of antigen–antibody complex, which made the block of electron transfer of HRP toward electrode and resulted in significant peak current decrease of HRP. Under the optimal conditions, the current decrease was proportional to CA19-9 concentrations ranging from 2 to 30 U/ml with a detection limit of 1.37 U/ml at a current decrease by 10%. The immunosensor showed an acceptable accuracy compared with those obtained from immunoradiometric assays, with intra-assay coefficient of 7.3 and 6.9% at CA19-9 concentrations of 5 and 15 U/ml, respectively, and inter-assay coefficient of 9.6% at a CA19-9 concentration of 20 U/ml. The storage stability was acceptable in a pH 7.0 PBS at 4 °C for 10 days. This method avoids the addition of electron transfer mediator, thus simplifies the immunoassay procedure and decreases the analytical time. It provides a new promising platform for clinical immunoassay.

A sensitive, fast and cheap sensor for quantitative determination of carbaryl pesticide using amperometric acetylcholinesterase (AChE) sensor based on electrochemically deposited chitosan was reported. From a mildly acidic chitosan solution, a chitosan film is electrochemically deposited on Au electrode surface via a negative voltage bias, leading to a stable AChE sensor. The characteristics of the deposited layer were observed to be dependent upon the deposition time, pH, and the chitosan concentration. Fourier-transform infrared spectra proved that the immobilized enzyme could preserve their native structure due to the excellent biocompatibility and non-toxicity of chitosan. Under the optimal experimental conditions, the carbaryl inhibition on AChE-CHIT/Au was proportional to its concentration in two ranges, from 0.005 to 0.1 μg/ml and 0.5 to 5 μg/ml, with the correlation coefficients of 0.9966 and 0.9982, respectively. The detection limit was 0.003 μg/ml taken as the concentration equivalent to a 10% decrease in signal. The determination of carbaryl in garlic samples obtained from export of farm base showed acceptable accuracy. The developed sensor exhibited good fabrication reproducibility and acceptable stability, which provided a new promising tool for pesticide analysis.

This paper described the preparation, characterization, and electrochemical properties of a graphene-ZrO2 nanocomposite (GZN) and its application for both the enrichment and detection of methyl parathion (MP). GZN was fabricated using electrochemical deposition and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS), which showed the successful formation of nanocomposites. Due to the strong affinity to the phosphoric group and the fast electron-transfer kinetics of GZN, both the extraction and electrochemical detection of organophosphorus (OP) agents at the same GZN modified electrochemical sensor was possible. The combination of solid-phase extraction and stripping voltammetric analysis allowed fast, sensitive, and selective determination of MP in garlic samples. The stripping response was highly linear over the MP concentrations ranging from 0.5 ng mL−1 to 100 ng mL−1, with a detection limit of 0.1 ng mL−1. This new nanocomposite-based electrochemical sensor provides an opportunity to develop a field-deployable, sensitive, and quantitative method for monitoring exposure to OPs.

Au–ZrO2–SiO2 nanocomposite spheres were synthesized and used as selective sorbents for the solid-phase extraction (SPE) of organophosphorous agents. A non-enzymatic electrochemical sensor based on a Au–ZrO2–SiO2 modified electrode was developed for the selective detection of organophosphorous pesticides (OPs). The Au–ZrO2–SiO2 nanocomposite spheres were synthesized by the hydrolysis and condensation of zirconium n-butoxide (TBOZ) on the surface of SiO2 spheres and then the introduction of gold nanoparticles on the surface. Transmission electron microscopy and X-ray photoelectron spectroscopy were performed to characterize the formation of the nanocomposite spheres. Fast extraction of OP was achieved by the Au–ZrO2–SiO2 modified electrode within 5 min via the specific affinity between zirconia and the phosphoric group. The assay yields a broad concentration range of paraoxon-ethyl from 1.0 to 500 ng mL−1 with a detection limit of 0.5 ng mL−1. This selective and sensitive method holds great promise for the enrichment and detection of OPs.