Fluorescent assay for acetylcholinesterase activity and inhibitor screening based on lanthanide organic/inorganic hybrid materials.

It is of great significance for the clinical diagnosis of Alzheimer's disease (AD) to achieve the on-site activity evaluation of acetylcholinesterase (AChE), the hydrolase of acetylcholine (ACh). Herein, we have developed a biosensing method endowed with considerable superiority based on the organic-inorganic hybrid composite Eu(DPA)3@Lap with excellent stability and fluorescent properties for this purpose by loading Eu3+ ions and 2,6-dipicolinic acid (DPA) into LAPONITE® (Lap). Through the comprehensive consideration of the specific hydrolysis of acetylthiocholine (ATCh) into thiocholine (TCh) by AChE, the high binding affinity of TCh to copper ion (Cu2+), and the selective fluorescence quenching ability of Cu2+, a simple Eu(DPA)3@Lap-based assay was developed to realize the rapid and convenient evaluation of AChE activity. Owning to the facile signal on-off-on response mode with a clear PET-based sensing mechanism, our assay presents favorable selectivity and sensitivity (LOD of 0.5 mU mL-1). Furthermore, the fluorescent assay was successfully applied for assessing AChE activity in human serum samples and screening potential AChE inhibitors, showing potential for application in the early diagnosis and drug screening of AD, as a new development path of AD therapy.

Identification, Baculoviral Expression, and Biochemical Characterization of a Novel Cholinesterase of Amblyomma americanum (Acari: Ixodidae).

A cDNA encoding a novel cholinesterase (ChE, EC 3.1.1.8) from the larvae of Amblyomma americanum (Linnaeus) was identified, sequenced, and expressed in Sf21 insect cell culture using the baculoviral expression vector pBlueBac4.5/V5-His. The open reading frame (1746 nucleotides) of the cDNA encoded 581 amino acids beginning with the initiation codon. Identical cDNA sequences were amplified from the total RNA of adult tick synganglion and salivary gland, strongly suggesting expression in both tick synganglion and saliva. The recombinant enzyme (rAaChE1) was highly sensitive to eserine and BW284c51, relatively insensitive to tetraisopropyl pyrophosphoramide (iso-OMPA) and ethopropazine, and hydrolyzed butyrylthiocholine (BuTCh) 5.7 times as fast as acetylthiocholine (ATCh) at 120 µM, with calculated KM values for acetylthiocholine (ATCh) and butyrylthiocholine of 6.39 µM and 14.18 µM, respectively. The recombinant enzyme was highly sensitive to inhibition by malaoxon, paraoxon, and coroxon in either substrate. Western blots using polyclonal rabbit antibody produced by immunization with a peptide specific for rAaChE1 exhibited reactivity in salivary and synganglial extract blots, indicating the presence of AaChE1 antigenic protein. Total cholinesterase activities of synganglial or salivary gland extracts from adult ticks exhibited biochemical properties very different from the expressed rAaACh1 enzyme, evidencing the substantial presence of additional cholinesterase activities in tick synganglion and saliva. The biological function of AaChE1 remains to be elucidated, but its presence in tick saliva is suggestive of functions in hydrolysis of cholinergic substrates present in the large blood mean and potential involvement in the modulation of host immune responses to tick feeding and introduced pathogens.

Electrochemical and fluorescent dual-mode sensor of acetylcholinesterase activity and inhibition based on MnO2@PD-coated surface.

We developed an electrochemical and fluorescent dual-mode sensor for assessing acetylcholinesterase (AChE) activity and inhibition by taking advantage of the high redox sensitivity of surface-coated mesoporous MnO2@polymer dot (MnO2@PD) towards AChE. The following phenomena constitute the basis of the detection mechanism: fluorescence resonance energy transfer (FRET) effect between MnO2 and PD; catalytic hydrolysis of acetylthiocholine (ATCh) to thiocholine (TCh) by AChE expressed by PC-12 cells, inducing fluorescence restoration and change in the conductivity of the system due to MnO2 decomposition; the presence of the inhibitor neostigmine preventing the conversion of ATCh to TCh. The surface-coated biosensor presents both fluorescence-based and electrochemical approaches for effectively monitoring AChE activity and inhibition. The fluorescence approach is based on the fluorescent "on/off" property of the system caused by MnO2 breakdown after interaction with TCh and the subsequent release of PDs. The conductivity of the coated electrode decreased dramatically as AChE concentration increased, resulting in electrochemical sensing of AChE activity and inhibition screening. Real-time wireless sensing can be conducted using a smartphone to monitor the resistance change, investigating the potential use of MnO2@PD nanocomposites in biological studies, and offering a real-time redox-fluorescent test for AChE activity monitoring and inhibitor screening.

Conducting polymer-based electrochemical biosensor for the detection of acetylthiocholine and pesticide via acetylcholinesterase.

A voltammetric biosensor for acetylthiocholine (ATCh) and paraoxon detection was successfully developed. To achieve this goal, polypyrrole (PPy) was synthesized onto the platinum (Pt) electrode surface in 0.30 M oxalic acid solution containing 25 mM pyrrole. PPy-coated Pt (Pt/PPy) electrode surface was covered with chitosan (Chi) (Pt/PPy/Chi). The acetylcholinesterase (AChE) enzyme was immobilized on the Pt/PPy/Chi electrode surface to build a voltammetric biosensor (Pt/PPy/Chi/AChE). The storage stability of the biosensor was determined to be 72% even after 60 days. The operational stability was determined to be 94% after 20 consecutive measurements. For the biosensor, the linear range was determined to be 30-50 µM for ATCh and 0.46-1.84 nM for paraoxon. The limit of detection (LOD) was determined to be 0.45 µM for ATCh and 0.17 nM for paraoxon.

Novel Insights into the Thioesterolytic Activity of N-Substituted Pyridinium-4-oximes.

The pyridinium oximes are known esterolytic agents, usually classified in the literature as catalysts, which mimic the catalytic mode of hydrolases. Herein, we combined kinetic and computational studies of the pyridinium-4-oxime-mediated acetylthiocholine (AcSCh+) hydrolysis to provide novel insights into their potential catalytic activity. The N-methyl- and N-benzylpyridinium-4-oximes have been tested as oximolytic agents toward the AcSCh+, while the newly synthesized O-acetyl-N-methylpyridinium-4-oxime iodide was employed for studying the consecutive hydrolytic reaction. The relevance of the AcSCh+ hydrolysis as a competitive reaction to AcSCh+ oximolysis was also investigated. The reactions were independently studied spectrophotometrically and rate constants, koxime, kw and kOH, were evaluated over a convenient pH-range at I = 0.1 M and 25 °C. The catalytic action of pyridinium-4-oximes comprises two successive stages, acetylation (oximolysis) and deacetylation stage (pyridinium-4-oxime-ester hydrolysis), the latter being crucial for understanding the whole catalytic cycle. The complete mechanism is presented by the free energy reaction profiles obtained with (CPCM)/M06-2X/6-311++G(2df,2pd)//(CPCM)/M06-2X/6-31+G(d) computational model. The comparison of the observed rates of AcSCh+ oximolytic cleavage and both competitive AcSCh+ and consecutive pyridinium-4-oxime-ester hydrolytic cleavage revealed that the pyridinium-4-oximes cannot be classified as non-enzyme catalyst of the AcSCh+ hydrolysis but as the very effective esterolytic agents.

A comparative study of acetylcholinesterase and general-esterase activity assays using different substrates, in vitro and in vivo exposures and model organisms.

Acetylcholinesterase (AChE) and general-esterase (GE) activities are important to understand detoxification processes of xenobiotics. The assays to quantify them have employed different substrates, inhibitors, types of experiments (in vitro and in vivo) and model organisms. The aim of this work was to give a systematic overview of the effect of the above factors on the outcome of AChE and GE activity measurements. We showed that AChE activity could be measured with the substrate acetylthiocholine iodide (AChI) but not with acetylcholine bromide (AChB) and only in in vitro assays. For GE activity, Michaelis-Menten kinetics differed between the substrates 4-methylumbellifery butyrate (4-MUB) and 1-naphtyl acetate (1-NA) in the measurements of in vitro activity, but their inhibition curves and IC50 values for the general inhibitor tetraisopropyl pyrophosphoramide (iso-OMPA) were similar, confirming that both substrates targeted the same group of enzymes. The GE substrate 4-MUB was applicable both in vitro and in vivo, while 1-NA was only applicable in vitro due to its high acute toxicity. When comparing the zooplankton crustacean Daphnia magna and the sediment dwelling Chironomus riparius, the latter had a four-fold higher maximal AChE activity (Vmax) and a higher susceptibility to the AChE inhibitor BW284c51 (four-fold lower 50% inhibitory concentration, IC50), but a lower maximal GE activity and lower susceptibility to iso-OMPA (higher IC50), indicating significant species differences between in C. riparius and D. magna. We conclude that both choice of substrate and exposure method matters for the outcome of esterase assays and that esterase compositions between species may vary significantly.

A New Tellurium- and Selenoorganic Compound as an Inhibitor of Acetylcholinesterase in Brain.

We studied the effect of a new cyanine dye containing selenium and tellurium on acetylcholinesterase activity in synaptic membrane in rat brain. The cyanine dye dose-dependently inhibits activity of this enzyme, and the concentration of half-maximal inhibition of acetylcholinesterase activity was 20.46 µM. The cyanine dye instantly inhibits the enzyme; the degree of inhibition depends on acetylthiocholine concentration: the lower is acetylthiocholine concentration, the higher is the degree of inhibition. On the Lineweaver-Burk plot, the concentration dependence curves of acetylcholinesterase with and without cyanine dye intersect in one point on the abscissa axis. In this case, the cyanine dye reduces the maximum inhibition rate (Vmax) and does not affect Michaelis constant (Km). The calculated inhibition constant Ki for the cyanine dye is 7.74 µM. Thus, the cyanine dye is a non-competitive inhibitor of acetylcholinesterase.

Oxidase-Like Fe-N-C Single-Atom Nanozymes for the Detection of Acetylcholinesterase Activity.

Single-atom catalysts (SACs) have attracted extensive attention in the catalysis field because of their remarkable catalytic activity, gratifying stability, excellent selectivity, and 100% atom utilization. With atomically dispersed metal active sites, Fe-N-C SACs can mimic oxidase by activating O2 into reactive oxygen species, O2- • radicals. Taking advantages of this property, single-atom nanozymes (SAzymes) can become a great impetus to develop novel biosensors. Herein, the performance of Fe-N-C SACs as oxidase-like nanozymes is explored. Besides, the Fe-N-C SAzymes are applied in biosensor areas to evaluate the activity of acetylcholinesterase based on the inhibition toward nanozyme activity by thiols. Moreover, this SAzymes-based biosensor is further used for monitoring the amounts of organophosphorus compounds.

Single-particle enumeration-based ultrasensitive enzyme activity quantification with fluorescent polymer nanoparticles.

Acetylcholinesterase (AChE) plays a vital role in nerve conduction through rapidly hydrolyzing the neurotransmitter acetylcholine (ACh) and is correlated with Alzheimer's disease. In this work, a label-free single-particle enumeration (SPE) method for the quantitative detection of acetylcholinesterase (AChE) activity is developed. The design is based on the fluorescence resonance energy transfer (FRET) between fluorescent conjugated polymer nanoparticles (FCPNPs) and MnO2 nanosheets. The fluorescence of FCPNPs can be effectively quenched by MnO2 nanosheets via hydrogen bonding interaction. In the presence of acetylcholinesterase (AChE), acetylthiocholine (ATCh) could be hydrolyzed to thiocholine (TCh), which can reduce MnO2 to Mn2+ and trigger the decomposition of MnO2 nanosheets. As a result, the fluorescence of FCPNPs is restored. Taking advantage of the superior brightness and stable fluorescence emission from individual FCPNPs, the accurate quantification of AChE is achieved by statistically counting the fluorescent particles on the glass slide surface. A linear range from 5 to 1600 µU mL-1 is obtained for AChE assay and the limit-of-detection (LOD) is 1.02 µU mL-1, which is far below the spectroscopic measurements in bulk solution. In the human serum sample, satisfactory recovery efficiencies are determined in a range of 91.0%-103.0%. Furthermore, pesticide carbaryl as an inhibitor of AChE activity was detected. The LOD is 1.12 pg mL-1 with linear responses ranging from 5 to 300 pg mL-1, which demonstrates the feasibility of this approach for AChE inhibitor screening. As a consequence, the label-free SPE-based method affords a promising platform for the sensitive detection of target molecules in the future.

Bio-inspired nanozyme: a hydratase mimic in a zeolitic imidazolate framework.

Nanozymes provide comparative advantages over natural enzymes and conventional artificial enzymes for catalytic reactions. However, nanozymes are only suitable for limited types of reactions, whose catalytic principles are not yet fully revealed. Herein, a new nanozyme based on a bionic zeolitic imidazolate framework is proposed. Zeolitic imidazolate framework-8 (ZIF-8) possesses a similar geometric structure to that of the active center of human carbonic anhydrase II (hCAII) and exhibits catalytic performance analogous to that of the hCAII. The less imidazolate coordinated zinc cations on the external surface of ZIF-8 can act as Lewis acid sites, lowering the pKa of Zn-bound H2O molecules from 14 to 8.4, which facilitates the deprotonation of H2O molecules and generation of zinc-bound hydroxide nucleophiles. The esterase-like ZIF-8 nanozyme shows a similar affinity to p-nitrophenyl acetate compared with hCAII. The ZIF-8 nanozyme also promotes CO2 hydration and acetylthiocholine hydrolysis reaction, and a series of ZIFs are also found with intrinsic enzyme-like activities due to similar compositions and spatial structures. These results imply that the bionic nanoparticles can be developed to fabricate a new generation of nanozymes by mimicking the active sites of natural enzymes.

Interactions of human butyrylcholinesterase with phenylvalerate and acetylthiocholine as substrates and inhibitors: kinetic and molecular modeling approaches.

Phenyl valerate (PV) is a substrate for measuring the PVase activity of neuropathy target esterase (NTE), a key molecular event of organophosphorus-induced delayed neuropathy. A protein with PVase activity in chicken (model for delayed neurotoxicity) was identified as butyrylcholinesterase (BChE). Purified human butyrylcholinesterase (hBChE) showed PVase activity with a similar sensitivity to inhibitors as its cholinesterase (ChE) activity. Further kinetic and theoretical molecular simulation studies were performed. The kinetics did not fit classic competition models among substrates. Partially mixed inhibition was the best-fitting model to acetylthiocholine (AtCh) interacting with PVase activity. ChE activity showed substrate activation, and non-competitive inhibition was the best-fitting model to PV interacting with the non-activated enzyme and partial non-competitive inhibition was the best fitted model for PV interacting with the activated enzyme by excess of AtCh. The kinetic results suggest that other sites could be involved in those activities. From the theoretical docking analysis, we deduced other more favorable sites for binding PV related with Asn289 residue, situated far from the catalytic site ("PV-site"). Both substrates acethylcholine (ACh) and PV presented similar docking values in both the PV-site and catalytic site pockets, which explained some of the observed substrate interactions. Molecular dynamic simulations based on the theoretical structure of crystallized hBChE were performed. Molecular modeling studies suggested that PV has a higher potential for non-competitive inhibition, being also able to inhibit the hydrolysis of ACh through interactions with the PV-site. Further theoretical studies also suggested that PV could yet be able to promote competitive inhibition. We concluded that the kinetic and theoretical studies did not fit the simple classic competition among substrates, but were compatible with the interaction with two different binding sites.

Characterization of butyrylcholinesterase from porcine milk.

Human butyrylcholinesterase (HuBChE) is under development for use as a pretreatment antidote against nerve agent toxicity. Animals are used to evaluate the efficacy of HuBChE for protection against organophosphorus nerve agents. Pharmacokinetic studies of HuBChE in minipigs showed a mean residence time of 267 h, similar to the half-life of HuBChE in humans, suggesting a high degree of similarity between BChE from 2 sources. Our aim was to compare the biochemical properties of PoBChE purified from porcine milk to HuBChE purified from human plasma. PoBChE hydrolyzed acetylthiocholine slightly faster than butyrylthiocholine, but was sensitive to BChE-specific inhibitors. PoBChE was 50-fold less sensitive to inhibition by DFP than HuBChE and 5-fold slower to reactivate in the presence of 2-PAM. The amino acid sequence of PoBChE determined by liquid chromatography tandem mass spectrometry was 91% identical to HuBChE. Monoclonal antibodies 11D8, mAb2, and 3E8 (HAH 002) recognized both PoBChE and HuBChE. Assembly of 4 identical subunits into tetramers occurred by noncovalent interaction with polyproline-rich peptides in PoBChE as well as in HuBChE, though the set of polyproline-rich peptides in milk-derived PoBChE was different from the set in plasma-derived HuBChE tetramers. It was concluded that the esterase isolated from porcine milk is PoBChE.

Paper-based amperometric sensor for determination of acetylcholinesterase using screen-printed graphene electrode.

A simple paper-based sensor with electrochemical detection was first developed for rapid, selective, and sensitive determination of acetylcholinesterase (AChE). The screen-printed graphene electrode was used as working electrode providing sensitivity for the sensor. The amperometric detection of AChE is based on the determination of thiocholine (TCh) produced from hydrolysis of acetylthiocholine chloride (ATCh) by AChE. To detect TCh, the ATCh immobilized sheet was stacked onto the detection sheet using double adhesive tape, then samples of AChE were dropped on the back side of an ATCh immobilized sheet with only 1min of incubation time. To avoid interference, glutathione (GSH), the potential of 0.5V vs. Ag/AgCl was applied onto a graphene electrode and the current, which depends on AChE concentration, was measured. Under optimized conditions, the limit of detection (LOD) from the experiment of AChE determination was 0.1U/mL with AChE concentration in range of 0.1-15U/mL. The data correlated well with the data obtained using spectrophotometric method. The developed sensor had been successfully applied to detect AChE in blood samples.

Acetylcholine-hydrolyzing activities in soluble brain fraction: Characterization with reversible and irreversible inhibitors.

Some effects of organophosphorus compounds (OPs) esters cannot be explained through actions on currently recognized targets acetylcholinesterase or neuropathy target esterase (NTE). In soluble chicken brain fraction, three components (Eα, Eß and Eγ) of pheny lvalerate esterase activity (PVase) were kinetically discriminated and their relationship with acetylcholine-hydrolyzing activity (cholinesterase activity) were studied in previous works. In this work, four enzymatic components (CS1, CS2, CS3 and CS4) of cholinesterase activity have been discriminated in soluble fraction, according to their sensitivity to irreversible inhibitors mipafox, paraoxon, PMSF and iso-OMPA and to reversible inhibitors ethopropazine and BW284C51. Cholinesterase component CS1 can be related to the Eα component of PVase activity and identified as butyrylcholinesterase (BuChE). No association and similarities can be stablished among the other PVase component (Eß and Eγ) with the other cholinesterase components (CS2, CS3, CS4). The kinetic analysis has allowed us to stablish a method for discriminating the enzymatic component based on a simple test with two inhibitors. It can be used as biomarker in toxicological studies and for monitoring these cholinesterase components during isolation and molecular identification processes, which will allow OP toxicity to be understood by a multi-target approach.

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.

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.

Enzymatic reaction modulated gold nanorod end-to-end self-assembly for ultrahigh sensitively colorimetric sensing of cholinesterase and organophosphate pesticides in human blood.

We present herein the first reported self-assembly modulation of gold nanorods (AuNRs) by enzymatic reaction, which is further employed for colorimetric assays of cholinesterase (ChE) and organophosphate pesticides (OPs) in human blood. ChE catalyzes its substrate (acetylthiocholine) and produces thiocholine and acetate acid. The resulting thiols then react with the tips of the AuNRs by S-Au conjunction and prevent subsequent cysteine-induced AuNR end-to-end (EE) self-assembly. Correspondingly, the AuNR surface plasmon resonance is regulated, which results in a distinctly ratiometric signal output. Under optimal conditions, the linear range is 0.042 to 8.4 µU/mL, and the detection limit is as low as 0.018 µU/mL. As ChE is incubated with OPs, the enzymatic activity is inhibited. So, the cysteine-induced assembly is observed again. On the basis of this principle, OPs can be well determined ranging from 0.12 to 40 pM with a 0.039 pM detection limit. To our knowledge, the present quasi pU/mL level sensitivity for ChE and the quasi femtomolar level sensitivity for OPs are at least 500 and 7000 times lower than those of previous colorimetric methods, respectively. The ultrahigh sensitivity results from (1) the rational choice of anisotropic AuNRs as building blocks and reporters and (2) the specific structure of the enzymatic thiocholine. Because of ultrahigh sensitivity, serum samples are allowed to be extremely diluted in the assay. Accordingly, various nonspecific interactions, even from glutathione/cysteine, are well avoided. So, both ChE and OPs in human blood can be directly assayed without any prepurification, indicating the simplicity and practical promise of the proposed method.

In situ induced metal-enhanced fluorescence: a new strategy for biosensing the total acetylcholinesterase activity in sub-microliter human whole blood.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities (i.e., total AChE) in human blood are biomarkers for theranostic monitoring of organophosphate neurotoxin-poisoned patients. We developed an ultra-sensitive method to detect the total AChE activity in sub-microliter human whole blood based on in situ induced metal-enhanced fluorescence (MEF). Both AChE and BChE can catalyze the hydrolysis of the acetylthiocholine (ATCh) substrate and produce positively-charged thiocholine (TCh). TCh can reverse the negatively-charged surface of core-shell Ag@SiO2 nanoparticles (NPs). The negatively-charged fluorescent dye (8-hydroxypyrene-1,3,6-trisulfonic acid, HPTS) is then confined to the surface of Ag@SiO2 NPs and generates an enhanced fluorescence signal in situ. Changes in the surface charge of Ag@SiO2 NPs are monitored by Zeta potential, and the MEF effect is confirmed by the measurements of fluorescence time decay. AChE activity has a dynamic range of 0 U/mL to 0.005 U/mL and a detection limit of 0.05 mU/mL. The total AChE activity in the sub-microliter human whole blood could be determined; the results were further validated. Therefore, combining the AChE catalytic reaction with MEF provides a simple, ultra-sensitive, and cost-effective "in situ MEF" approach to determine the total AChE activity in human whole blood sample down to sub-microliters without matrix interferences. The strategy also allows potential usage in other tissues and other fields.

Resurfaced fluorescent protein as a sensing platform for label-free detection of copper(II) ion and acetylcholinesterase activity.

Protein engineering by resurfacing is an efficient approach to provide new molecular toolkits for biotechnology and bioanalytical chemistry. H39GFP is a new variant of green fluorescent protein (GFP) containing 39 histidine residues in the primary sequence that was developed by protein resurfacing. Herein, taking H39GFP as the signal reporter, a label-free fluorometric sensor for Cu(2+) sensing was developed based on the unique multivalent metal ion-binding property of H39GFP and fluorescence quenching effect of Cu(2+) by electron transfer. The high affinity of H39GFP with Cu(2+) (Kd, 16.2 nM) leads to rapid detection of Cu(2+) in 5 min with a low detection limit (50 nM). Using acetylthiocholine (ATCh) as the substrate, this H39GFP/Cu(2+) complex-based sensor was further applied for the turn-on fluorescence detection of acetylcholinesterase (AChE) activity. The assay was based on the reaction between Cu(2+) and thiocholine, the hydrolysis product of ATCh by AChE. The proposed sensor is highly sensitive (limit of detection (LOD) = 0.015 mU mL(-1)) and is feasible for screening inhibitors of AChE. Furthermore, the practicability of this method was demonstrated by the detection of pesticide residue (carbaryl) in real food samples. Hence, the successful applications of H39GFP in the detection of metal ion and enzyme activity present the prospect of resurfaced proteins as versatile biosensing platforms.