Acetylthiocholine Research Articles

Rapid detection of organophosphorus pesticide residue on Prussian blue modified dual-channel screen-printed electrodes combing with portable potentiostat

A novel electrochemical method for the rapid detection of organophosphorus pesticide residues was realized on a dual-channel screen-printed electrode (DSPE) that was integrated with a portable smartphone-controlled potentiostat. The two carbon working channels of DSPE were first modified by electrodepositing of Prussian blue. The channels were then modified with acetylcholinesterase (AChE) via Nafion. The inhibition ratio of AChE was detected by comparing the electrical current of acetylthiocholine (ATCh) that was catalyzed by the enzyme electrodes with (channel 1) and without (channel 2) organophosphorus pesticide. Inhibition ratios were related with the negative logarithm of the organophosphorus pesticide (trichlorfon, oxamyl, and isocarbophos) concentrations at optimum experimental conditions (pH 6.9 of electrolyte, 0.2V working potential, 2.5μL AChE modification amount, and 15min inhibition time). The linear equations were I%=32.30lgC+253.3 (R=0.9750) for isocarbophos, I%=35.99lgC+270.1 (R=0.9668) for chlorpyrifos, and I%=33.70lgC+250.5 (R=0.9606) for trichlorfon. The detection limits were calculated as 10−7g/mL. Given that the inhibition ratios were only related with pesticide concentration and not with pesticide species, the proposed electrodes and electrometer can rapidly detect universal organophosphorus pesticides and assess pesticide pollution.

Carbon quantum dots as fluorescence resonance energy transfer sensors for organophosphate pesticides determination

Carbon quantum dots (CQDs) obtained from natural organics attract significant attention due to the abundance of carbon sources, varieties of heteroatom doping (such as N, S, P) and good biocompatibility of precursor. In this study, tunable fluorescence emission CQDs originated from chlorophyll were synthesized and characterized. The fluorescence emission can be effectively quenched by gold nanoparticles (Au NPs) via fluorescence resonance energy transfer (FRET). Thiocholine, which was produced from acetylthiocholine (ATC) by the hydrolysis of butyrylcholinesterase (BChE), could cause the aggregation of Au NPs and the corresponding recovery of FRET-quenched fluorescence emission. The catalytic activity of BChE could be irreversibly inhibited by organophosphorus pesticides (OPs), thus, the recovery effect was reduced. By evaluating the fluorescence emission intensity of CQDs, a FRET-based sensing platform for OPs determination was established. Paraoxon was studied as an example of OPs. The sensing platform displayed a linear relationship with the logarithm of the paraoxon concentrations in the range of 0.05–50μgL−1 and the limit of detection (LOD) was 0.05μgL−1. Real sample study in tap and river water revealed that this sensing platform was repeatable and accurate. The results indicate that the OP sensor is promising for applications in food safety and environmental monitoring.

Highly sensitive GQDs-MnO2 based assay with turn-on fluorescence for monitoring cerebrospinal acetylcholinesterase fluctuation: A biomarker for organophosphorus pesticides poisoning and management

In this study, we demonstrated an assay with turn-on fluorescence for monitoring cerebrospinal acetylcholinesterase (AChE) fluctuation as a biomarker for organophosphorus pesticides (OPs) poisoning and management based on single layer MnO2 nanosheets with graphene quantum dots (GQDs) as signal readout. Initially, the fluorescence of GQDs was quenched by MnO2 nanosheets mainly due to the inner filter effect (IFE). However, with the presence of reductive thiocholine (TCh), the enzymatic product, hydrolyzed from acetylthiocholine (ATCh) by AChE, the redox reaction between MnO2 and TCh occurred, leading to the destruction of the MnO2 nanosheets, and thereby IFE was diminished gradually. As a consequence, the turn-on fluorescence of GQDs with the changes in the spectrum of the dispersion constituted a new mechanism for sensing of cerebrospinal AChE. With the method developed here, we could monitor cerebrospinal AChE fluctuation of rats exposed to OPs before and after therapy, and could thereby open up the pathway to a new sensing platform for better understanding the mechanism of brain dysfunctions associate with OPs poisoning.

Microcalorimetric Study of Acetylcholine and Acetylthiocholine Hydrolysis by Acetylcholinesterase

Acetylcholinesterase (AChE) is an important enzyme responsible for the cleavage of acetylcholine. Studies of the activity of this enzyme use an artificial substrate, acetylthiocholine, because a product of its catalysis, thiocholine, readily generates a light absorbing product upon reaction with Elman’s reagent 5,5’-dithiobis-(2-nitrobenzoic acid (DTNB). The hydrolysis of acetylcholine cannot be assayed with this method. The isothermal titration calorimetry can assay the hydrolysis of both substrates, without requiring additional reagents other than the enzyme and the substrate. To compare kinetic values obtained in the hydrolysis of acetylcholine (ACh) and acetylthiocholine (ATCh), with carbaryl acting as inhibitor, a calorimetric technique was used to evaluate kinetic properties of the two reactions. This method can show the hydrolysis of both substrates by the heat exchange that occurs during catalysis. In addition, it allowed the assessment of the AChE inhibition by carbaryl, a common insecticide. The results show a similarity between values obtained with both substrates, which are slightly higher for acetylcholine, the enzyme natural substrate. Enzymatic parameters values from ATCh and ACh were similar to each other and inhibitory constants using carbaryl were also similar, displaying that any approach to ACh is feasible using ATCh. The results obtained from ITC show the precision achieved by the calorimetric method.

Assessment of acetylcholinesterase (AChE) from Oreochromis mossambicus (Cuvier, 1831) as a source of enzyme for insecticides detection

In this work we assess the potential of acetylcholinesterase (AChE) from Oreochromis mossambicus (Toman) as a sensitive test for the presence of insecticides. The partial purification and characterization of a soluble AChE from Oreochromis mossambicus brain tissues using affinity chromatography gel (procainamide–Sephacryl S-1000) showed that the partially purified AChE was most active on acetylthiocholine (ATC) but had low activities on propionylthiocholine (PTC) and butyrylthiocholine (BTC), indicating that the partially purified fraction was predominantly AChE. Soluble AChE was partially purified 9.27-fold with a 91.12% yield. The partially purified AChE displayed the highest activity on ATC at pH 7 and at 30oC using 0.1 M Tris buffer. The enzyme exhibited Michaelis-Menten kinetic constants, Km, for ATC, BTC and PTC at 36, 77 and 250 µM, respectively, and the maximum velocities, Vmax, were 18.75, 0.12 and 0.05 µmol/min/mg protein, respectively. Moreover, the AChE from Oreochromis mossambicus presented comparable sensitivity to carbamates and organophosphates insecticides than that from Electrophorus electricus and many other fish AChE by comparing half maximal inhibitory concentration values. Therefore, the enzyme is a valuable source for insecticides detection in Malaysian waters at lower cost.

Ultrasensitive electrochemiluminescence biosensor for organophosphate pesticides detection based on carboxylated graphitic carbon nitride-poly(ethylenimine) and acetylcholinesterase

A signal on electrochemiluminescence (ECL) biosensor was designed for sensitive organophosphate pesticides (OPs) detection based on a novel composite of carboxylated graphitic carbon nitride-poly(ethylenimine) (C-g-C3N4-PEI) and acetylcholinesterase (AChE). The C-g-C3N4-PEI nanocomposite which was prepared through covalent bonding between the COOH of C-g-C3N4 and the NH2 of PEI exhibited significantly enhanced ECL efficiency and stability. K2S2O8 as the coreactant of C-g-C3N4-PEI could be consumed by thiocholine, produced by the hydrolysis of acetylthiocholine (ATCl) in the presence of AChE. Since OPs are one of AChE inhibiter, the consumption of coreactant K2S2O8 decreased with the increasing concentration of OPs, thus enhancing ECL signal. Under the optimum conditions, the proposed biosensor for OPs detection (using ethyl paraoxon as a model OPs) exhibited a wide linear ranging from 1.0 pM to 5.0μM with a low detection limit of 0.3 pM. The novel strategy has the advantages of fine practicality, good stability and reproducibility, which might provide a new promise for OPs detection in real-life samples.

Prussian blue nanocubes peroxidase mimetic-based colorimetric assay for screening acetylcholinesterase activity and its inhibitor

In this paper, a novel, convenient and sensitive colorimetric assay for acetylcholinesterase (AChE) activity and its inhibitor screening is successfully proposed based on the peroxidase-like activity of Prussian blue nanocubes (PB NCs). PB NCs can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 to develop a blue color and an absorption peak centered at 652nm. AChE mediates the hydrolysis of acetylthiocholine (ATCh) to yield a reducing agent thiocholine (TCh) that cause the reduction of oxidized TMB and it can also chelate with Fe3+, both of which result in a blue color fading and a decrease of the absorbance. The generation of TCh is inhibited and the absorbance intensity is recovered after the introduction of AChE inhibitor. Consequently, the assay is also utilized in AChE inhibitor screening. The PB NCs-H2O2-TMB based platform is highly sensitive for AChE activity sensing with a low detection limit of 0.04mU/mL. In addition, this assay has great potential in discriminatively determining AChE over other enzymes. The proposed method is fairly novel, simple and sensitive, which may pave the way for the detection of other hydrolytic enzyme activities with properly selected substrates.

Development of a Flow Injection Biosensor System Enables Glucose and Cortisol Simultaneous Measurement for the Evaluation of Fish Stress

The continued growth of the aquaculture industry has led fish farmers to increase production capacity. Increasing production capacity requires high-density aquaculture and excess feeding, which are stressful to the fish and negatively affect fish health and immunity. Further, the accumulation of residual feed and excretions deteriorates the water quality of the aquaculture environment. Although medicine can be administered to the fish for disease prevention, there are concerns about the effects of the medicine that remain in the fish, which may be harmful to the consumer. Thus, a rapid and convenient, non-scheduled fish stress check would be an ideal solution. To evaluate fish stress, people always focus on the change of some plasma indicator, such as cortisol and glucose. Fish respond to stressors by the release of stress hormones such as cortisol. These stress hormones singly and in combination increase glucose production in fish. Because glucose is an important energy substrate for several tissues, the higher glucose production is thought to assist the animal in metabolically coping with the increased energy demand caused by stress. Currently, blood tests are performed to evaluate fish stress states. Conventional methods, such as enzyme-linked immunosorbent assay or colorimetric method, are both very time-consuming and costly, however, making it impractical to perform regular stress evaluations. On the other hand, cortisol, which is secreted rapidly after exposure to a stressor, is not easy to be detected. In addition, though glucose changes are easy to monitor, it may also arise after the feeding. Therefore, rather than measure both cortisol and glucose separately, simultaneous detection of both cortisol and glucose will enhance our understanding of fish stress conditions. In this study, we developed a flow injection biosensor system for measuring fish glucose and cortisol simultaneously. System schematic diagram was shown in the supplement image. For the glucose measurement, it was based on the decrease of the oxygen concentration detected by optical probe due to a glucose oxidation reaction. While, the measurement of cortisol based on competitive immunologic reactions, magnetic separation, and an electrochemical measurement. An anti-cortisol antibody was immobilised on magnetic beads and injected into the system. At first, the glucose concentration was detected first from the change on oxygen decrease by optical probe which immobilized glucose oxidase on its surface. After that, a specific quantity of acetylcholinesterase-labelled cortisol (cort-AChE) was injected into the flow system and the sample will go through a reaction coil as a competitive reaction unit. After reacting in the reaction coil, the sample was separated magnetically using a neodymium magnet. The cort-AChE was detached from the magnetic beads and transferred into the enzyme reaction unit with acetylthiocholine (ATCh). ATCh was hydrolysed by the cort-AChE to produce thiocholine. The thiocholine was quantified downstream by electrochemical detection using a Pt-Ir electrode. We also found that the performance of the proposed biosensor system was optimised under the following conditions: pH 7.5, temperature 25°C, flow rate 0.16 ml min− 1. After the understanding the optimal parameters, we performed the measurement on both glucose and cortisol. The fluorescence intensity was well correlated with the concentration of the cortisol standard solution (range: 0–30 mg dL− 1, R= 0.9785). While the output current was well correlated with the concentration of the cortisol standard solution (range: 0.06–40 ng mL− 1, R= 0.9812). The results obtained using the proposed flow method were compared with those obtained using conventional method (correlation coefficient was 0.9869 for glucose and 0.9848 for cortisol). These findings suggest that the proposed system can be used to analyse glucose and cortisol in fish plasma samples simultaneously and help us to understand the relationship between the stress hormone and glucose change. Figure 1

Glutathione regulation-based dual-functional upconversion sensing-platform for acetylcholinesterase activity and cadmium ions

A dual-functional platform for the sensing of acetylcholinesterase (AChE) activity and cadmium ions (Cd2+) was developed based on the fluorescence resonance energy transfer (FRET) between NaYF4:Yb,Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs) via glutathione regulation. The detection mechanism is based on the fact that AuNPs can quench the fluorescence of UCNPs. AChE catalyzes the hydrolysis of acetylthiocholine (ATC) into thiocholine which reacts with AuNPs by S-Au conjunction and results the aggregation of AuNPs and change in fluorescence of UCNPs. Therefore, the AChE activity can be detected through the changes of the color of solution and fluorescence recovery of UCNPs. However, the presence of glutathione (GSH) can protect AuNPs from aggregation and enlarge the inter-particle distance between AuNPs and UCNPs. When Cd2+ is added into the stable mixture of AuNPs, GSH and AChE/ATC, Cd2+ could interact with GSH to form a spherical shaped (GSH)4Cd complex, which decreases the free GSH on the surface of AuNPs to weaken the stability of AuNPs and lead to the easily aggregation of them in the system. The aggregated-AuNPs are released from the surface of UCNPs, which results in the fluorescence of UCNPs gradually recovered. Under the optimized conditions, the detection limits of AChE activity and Cd2+ are estimated to be 0.015mU/mL and 0.2µM, respectively. The small molecules regulated dual-functional platform based on UCNPs/AuNPs is a simple, label-free method and can be applied for the turn-on fluorescence detection of AChE activity in human serum and Cd2+ in real water samples. The present work demonstrates a general strategy for the design of small molecules regulated multifunctional platform and will be expanded for different areas in the future.

Electrochemical biosensing of carbaryl based on acetylcholinesterase immobilized onto electrochemically inducing porous graphene oxide network

This work describes a sensitive electrochemical biosensor for detection of carbamate pesticides based on immobilization of acetylcholinesterase (AChE) on the electrochemically inducing porous graphene oxide network (e-pGON) which is prepared by scanning the GO modified electrode using successive cyclic voltammetry method. The e-pGON effectively promotes the electron transfer rate and facilitates the access of substrates to the active centers. The as-prepared biosensor shows high affinity to acetylthiocholine (ATCl) with a Michaelis-Menten constant value of 0.45mmolL−1. Under optimum conditions, the inhibition of carbaryl is proportional to its concentration ranging from 0.3 to 6.1ng/mL. The detection limit is 0.15ng/mL. The developed biosensor exhibits good performance such as reproducibility and stability, thus providing a promising tool for the analysis of enzyme inhibitors.

Polyacrylic acid-coated cerium oxide nanoparticles: An oxidase mimic applied for colorimetric assay to organophosphorus pesticides

It is important and urgent to develop reliable and highly sensitive methods that can provide on-site and rapid detection of extensively used organophosphorus pesticides (OPs) for their neurotoxicity. In this study, we developed a novel colorimetric assay for the detection of OPs based on polyacrylic acid-coated cerium oxide nanoparticles (PAA-CeO2) as an oxidase mimic and OPs as inhibitors to suppress the activity of acetylcholinesterase (AChE). Firstly, highly dispersed PAA-CeO2 was prepared in aqueous solution, which could catalyze the oxidation of TMB to produce a color reaction from colorless to blue. And the enzyme of AChE was used to catalyze the substrate of acetylthiocholine (ATCh) to produce thiocholine (TCh). As a thiol-containing compound with reducibility, TCh can decrease the oxidation of TMB catalyzed by PAA-CeO2. Upon incubated with OPs, the enzymatic activity of AChE was inhibited to produce less TCh, resulting in more TMB catalytically oxidized by PAA-CeO2 to show an increasing blue color. The two representative OPs, dichlorvos and methyl-paraoxon, were tested using our proposed assay. The novel assay showed notable color change in a concentration-dependent manner, and as low as 8.62 ppb dichlorvos and 26.73 ppb methyl-paraoxon can be readily detected. Therefore, taking advantage of such oxidase-like activity of PAA-CeO2, our proposed colorimetric assay can potentially be a screening tool for the precise and rapid evaluation of the neurotoxicity of a wealth of OPs.

Optical Detection of Enzymatic Activity and Inhibitors on Non-Covalently Functionalized Fluorescent Graphene Oxide.

It has been of great interest to measure the activity of acetylcholinesterase (AChE) and its inhibitor, as AChE is known to accelerate the aggregation of the amyloid beta peptides that underlie Alzheimer's disease. Herein, we report the development of graphene oxide (GO) fluorescence-based biosensors for the detection of AChE activity and AChE inhibitors. To this end, GO was non-covalently functionalized with phenoxy-modified dextran (PhO-dex-GO) through hydrophobic interaction; the resulting GO showed excellent colloidal stability and intense fluorescence in various aqueous solutions as compared to pristine GO and the GO covalently functionalized with dextran. The fluorescence of PhO-dex-GO remarkably increased as AChE catalyzed the hydrolysis of acetylthiocholine (ATCh) to give thiocholine and acetic acid. It was found that the turn-on fluorescence response of PhO-dex-GO to AChE activity was induced by protonation of carboxyl groups on it from the product of the enzymatic hydrolysis reaction, acetic acid. On the basis of its turn-on fluorescence response, PhO-dex-GO was able to report kinetic and thermodynamic parameters involving a maximum velocity, a Michaelis constant, and an inhibition dissociation constant for AChE activity and inhibition. These parameters enable us to determine the activity of AChE and the efficiency of the inhibitor.

Realization of a graphene gate field effect transistor for electrochemical detection and biosensors

A study of 4H-SiC annealing temperature was performed using a rf-induction furnace in order to determined the optimal conditions to obtain suitable graphene layers. The graphene layer has been identified and confirmed by electrical measurements and physico-chemical analyses using Atomic Force Microscopy, X-Ray Photoelectron Spectroscopy and Infra-Red reflection measurements. GFET (Graphene Field-Effect Transistors) planar structure has been fabricated, with source and drain contacts formed directly on the graphene by lift-off. For the insulating gate, SiO2 layers have been deposited by sputtering. The graphene layers have been investigated by electrochemical detection with Fe3(CN)6, Ru(NH3)6 and Catechol. Graphene electrodes have also been tested as transducers for biosensors, with acetylthiocholine (AChE), allowing this electrode to detect inhibitors of neurotransmitters. This study demonstrates the ability to include epitaxially grown graphene at the surface of SiC as a channel for the development of transistors, and it shows the efficiency of this layer playing the role of electrode in electrochemistry and that of transducer in biosensing. In addition, these results provide a very promising perspective for the elaboration of ISFETs with a similar graphene synthesis.

Simultaneous Photoelectrochemical Immunoassay of Dual Cardiac Markers Using Specific Enzyme Tags: A Proof of Principle for Multiplexed Bioanalysis.

In this Letter, on the basis of the CdS quantum dots functionalized TiO2 nanotubes electrode, we proposed a simultaneous photoelectrochemical (PEC) immunoassay of dual cardiac markers using specific enzyme tags of alkaline phosphatase (ALP) and acetylcholine esterase (AChE). ALP and AChE were integrated into the PEC system through the sandwich immunobinding and could specifically catalyze the hydrolysis of ascorbic acid 2-phosphate (AAP) or the acetylthiocholine (ATC) to in situ generate ascorbic acid (AA) or thiocholine (TC) for sacrificial electron donating. These two enzymes were thus used to differentiate the signals of two cardiac targets in connection with the sandwich immunorecognition and PEC responses to the corresponding electron donors. This strategy demonstrates a proof of principle for the successful integration of dual enzyme tags with PEC immunoassay that can potentially provide a general format for multiplexed PEC bioanalysis.

An Acetylcholinesterase Biosensor Based on Graphene/Polyaniline Composite Film for Detection of Pesticides

AbstractThis work reports a sensitive amperometric biosensor for organophosphate pesticides (OPs) fabricated through modifying glassy carbon electrode with acetylcholinesterase (AChE) immobilized on graphene/polyaniline (G/PANI) composite film. The obtained G/PANI composite films show large specific area, high conductivity, good biocompatibility, and fast redox properties and have perfect layered and encapsulated structures. The as‐prepared biosensor shows high affinity to acetylthiocholine (ATCl) with a Michaelis‐Menten constant value of 0.20 mmol/L. Furthermore, based on the inhibition of the enzymatic activity (immobilized AChE) caused by the model compound of carbaryl (one kind of pesticides), it is found that the inhibition activity of carbaryl is proportional to its concentration ranging from 38 to 194 ng·mL−1. The developed biosensor shows a detection limit of 20 ng·mL−1 (S/N=3) for OPs detection and exhibits good performance such as good reproducibility and acceptable stability, which makes it possible to provide a new and promising tool for the analysis of enzyme inhibitors.

Colorimetric assay for acetylcholinesterase and inhibitor screening based on the Ag [I] ion–3,3′,5,5′-tetramethylbenzidine (TMB)

A novel colorimetric method for the detection of acetylcholinesterase (AChE) and its inhibitor by taking utilization of Ag [I] ion–3,3′,5,5′-tetramethylbenzidine (TMB) detection system has been proposed. Ag [I] ion could oxidize TMB to induce a blue color and an absorption peak centered at 652nm. In the presence of AChE, acetylthiocholine (ACh) was hydrolyzed to thiocholine. Ag [I] ion could interact with thiocholine and thiocholine can reduce the oxidized TMB (oxTMB) to TMB, both of which suppressed the oxidation capacity of Ag [I] ion, resulting in a blue color fading and a decrease of the absorbance at 652nm. The developed assay is highly sensitive with a low detection limit of 4.3μU/mL. Moreover, the method was also applied in AChE inhibitor sensing. The obtained assay is fairly simple, inexpensive and sensitive without the utilization of nanomaterials, which may be not only used for the sensing of other hydrolytic enzyme activities with properly selected substrates, but also used for the screening of potential inhibitor drugs.

A simple electrochemical biosensor based on AuNPs/MPS/Au electrode sensing layer for monitoring carbamate pesticides in real samples

A simple electrochemical biosensor for quantitative determination of carbamate pesticide was developed based on a sensing interface of citrate-capped gold nanoparticles (AuNPs)/(3-mercaptopropyl)-trimethoxysilane (MPS)/gold electrode (Au). The biosensor was fabricated by firstly assembling three-dimensional (3D) MPS networks on Au electrode and subsequently assembling citrate-capped AuNPs on 3D MPS network via AuS bond. The interface of AuNPs/MPS/Au was negatively charged originating from the citrate coated on AuNPs that would repulse the negatively charged ferricyanide ([Fe(CN)6]3−/4−) to produce a negative response. In the presence of acetylcholinesterase (AChE) and acetylthiocholine (ATCl), the AChE catalyzes the hydrolysis of ATCl into positively charged thiocholine which would replace the citrate on AuNPs through the strong AuS bond and convert the negative charged surface to be positively charged. The resulted positively charged AuNPs/MPS/Au then attracted the [Fe(CN)6]3−/4− to produce a positive response. Based on the inhibition of carbamate pesticides on the activity of AChE, the pesticide could be quantitatively determined at a very low potential. The linear range was from 0.003 to 2.00μM. The sensing platform was also proved to be suitable for carbamate pesticides detection in practical sample.

Acetylcholinesterase inhibition-based ultrasensitive fluorometric detection of malathion using unmodified silver nanoparticles

Abstract An ultrasensitive sensor was developed for the detection of an organophosphorus compound using unmodified silver nanoparticles (AgNPs). The hydrolysis of acetylthiocholine (ATCh) by acetylcholinesterase (AChE) generates a thiol-containing compound, thiocholine, which influences the AgNP aggregation. Herein, we describe an indirect enzyme-based detection of malathion with AChE and ATCh in the presence of AgNPs using the fluorometric method. The addition of malathion to the system decreased the enzyme hydrolysis and thiocholine generation, and thus was able to quench the AgNPs, with a corresponding decrease in the emission intensity at 423 nm ( λ ex = 340 nm). The proposed system showed linearity in the range, 2–100 fM, with an excellent limit of detection of 0.556 fM by fluorescence spectroscopy. The aggregation and disaggregation of AgNPs were also supported by transmission electron microscopy and dynamic light scattering analyses. The potential application of the system was demonstrated by determination of malathion in real samples like agricultural runoff water, lake water, cabbage, and apple samples using fluorometric method, and the obtained results were further validated with HPLC.

Microfluidic Device for Coulometric Detection of Organophosphate Pesticides

A microdevice for coulometric detection of organophosphate pesticides (OPs) was fabricated based on the measurement of the inhibition of an enzyme, acetylcholinesterase (AChE), by OPs. Thiocholine (TCh) produced in the enzymatic reaction of AChE with acetylthiocholine (ATCh) as a substrate was oxidized on a microelectrode array formed in a main flow channel. Volumes of plugs of necessary solutions were measured using a structure consisting of a row of rhombuses formed in an auxiliary flow channel. The plugs were merged and solution components were mixed at a T-junction formed with the main and auxiliary flow channels. A linear relationship was confirmed between the generated charge and the logarithm of the OP (malathion) concentration in a concentration range between 10(-6) and 10(-3) M with a correlation coefficient of 0.951. The lower limit of detection was 412 nM.

Gold nanoclusters–Cu2+ ensemble-based fluorescence turn-on and real-time assay for acetylcholinesterase activity and inhibitor screening

Based on the specific binding of Cu(2+) ions to the 11-mercaptoundecanoic acid (11-MUA)-protected AuNCs with intense orange-red emission, we have proposed and constructed a novel fluorescent nanomaterials-metal ions ensemble at a nonfluorescence off-state. Subsequently, an AuNCs@11-MUA-Cu(2+) ensemble-based fluorescent chemosensor, which is amenable to convenient, sensitive, selective, turn-on and real-time assay of acetylcholinesterase (AChE), could be developed by using acetylthiocholine (ATCh) as the substrate. Herein, the sensing ensemble solution exhibits a marvelous fluorescent enhancement in the presence of AChE and ATCh, where AChE hydrolyzes its active substrate ATCh into thiocholine (TCh), and then TCh captures Cu(2+) from the ensemble, accompanied by the conversion from fluorescence off-state to on-state of the AuNCs. The AChE activity could be detected less than 0.05 mU/mL within a good linear range from 0.05 to 2.5 mU/mL. Our proposed fluorescence assay can be utilized to evaluate the AChE activity quantitatively in real biological sample, and furthermore to screen the inhibitor of AChE. As far as we know, the present study has reported the first analytical proposal for sensing AChE activity in real time by using a fluorescent nanomaterials-Cu(2+) ensemble or focusing on the Cu(2+)-triggered fluorescence quenching/recovery. This strategy paves a new avenue for exploring the biosensing applications of fluorescent AuNCs, and presents the prospect of AuNCs@11-MUA-Cu(2+) ensemble as versatile enzyme activity assay platforms by means of other appropriate substrates/analytes.