Thiocholine-Mediated One-Pot Peptide Ligation and Desulfurization.

Thiol catalysts are essential in native chemical ligation (NCL) to increase the reaction efficiency. In this paper, we report the use of thiocholine in chemical protein synthesis, including NCL-based peptide ligation and metal-free desulfurization. Evaluation of thiocholine peptide thioester in terms of NCL and hydrolysis kinetics revealed its practical utility, which was comparable to that of other alkyl thioesters. Importantly, thiocholine showed better reactivity as a thiol additive in desulfurization, which is often used in chemical protein synthesis to convert Cys residues to more abundant Ala residues. Finally, we achieved chemical synthesis of two differently methylated histone H3 proteins via one-pot NCL and desulfurization with thiocholine.

Label-Free and Ultrasensitive Detection of Butyrylcholinesterase and Organophosphorus Pesticides by Mn(II)-Based Electron Spin Resonance Spectroscopy with a Zero Background Signal.

Mn(II)-based electron spin resonance (ESR) spectroscopy was used for detecting butyrylcholinesterase (BChE) and organophosphorus pesticides (OPs). MnO2 nanosheets were synthesized with manganese chloride and hydrogen peroxide. With the catalysis of BChE, S-butyrylthiocholine iodide (BTCh) was hydrolyzed into thiocholine which has a reducing -SH group. In the presence of thiocholine, MnO2 nanosheets were broken down and Mn(IV) in MnO2 nanosheets was reduced into Mn(II). Mn2+ is a paramagnetic ion and gives a good ESR signal. In contrast, MnO2 nanosheets have no ESR signal and need not be separated from Mn2+. Mn2+ can be determined directly by ESR spectroscopy, and no further sensing probe is needed. ESR spectroscopy based on directly detecting Mn2+ is much simpler than those using other probes besides MnO2. The ESR signal of Mn2+ is proportional to the catalytic activity of BChE. OPs which inhibit the activity of BChE can also be detected by probing the ESR signal of Mn2+. Since there is no ESR signal of MnO2 nanosheets, the background signal in the absence of BChE was close to zero. The limit of detection (LOD) of BChE was as low as 0.042 U L-1. The standard curve for determining the OP paraoxon was established by measuring the inhibition of BChE by paraoxon, and the LOD of paraoxon was found to be 0.076 ng mL-1. The spiked Chinese cabbage extract samples were analyzed, and the experimental results indicated that the recoveries were from 96.5 to 102.8%. The planted Chinese cabbage was sprayed with the paraoxon solution, and the residue amount of paraoxon in the extract was estimated by the method. The result obtained by the present method was consistent with that obtained by HPLC, which proved the practicability of this new method.

Co, N co-doped porous carbon-based nanozyme as an oxidase mimic for fluorescence and colorimetric biosensing of butyrylcholinesterase activity.

A Co, N co-doped porous carbon-based nanozyme (Co-N-C nanozyme) has been fabricated. Taking advantages of the excellent oxidase catalytic activity and significant stability of Co-N-C nanozyme, we propose a fluorescence and colorimetric system based on Co-N-C nanozyme and red-emitting carbon quantum dots (RCDs) for butyrylcholinesterase (BChE) sensing. As the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) was catalyzed and oxidized by Co-N-C nanozyme, the generated oxTMB had a new absorption peak at 652 nm, which resulted in the significant quenching of the fluorescence of the carbon quantum dots at 610 nm. Under the catalysis of BChE, thiocholine was generated from the hydrolysis of S-butyrylthiocholine iodide (BTCh), and the as-generated thiocholine effectively inhibited the oxidation of TMB catalyzed by Co-N-C nanozyme, leading to a decrease of the absorption of oxTMB at 652 nm and effective fluorescence recovery of RCDs. By measuring the absorbance of produced oxTMB at 652 nm and the fluorescence of RCDs at 610 nm, the fluorescence and colorimetric system both exhibited an outstanding linear response to the activity of BChE in the range 0.5 to 40 U L-1, with a detection limit of 0.16 U L-1 and 0.21 U L-1, respectively. Furthermore, this established dual-channel biosensing strategy has been successfully applied to the determination of BChE in human serum samples. The present work has effectively expanded the development and application of nanozyme in biosensing.

An enhanced staining method K-B-2R staining for three-dimensional nerve reconstruction.

BACKGROUND: Three-dimensional (3D) reconstruction of human peripheral nerves, as a useful tool to understand the nerve internal information and functional basis, has become an important area of research in the peripheral nerve field. METHODS: In this study, we proposed a two-dimensional (2D) Karnovsky-Roots toluidine blue ponceau 2R (K-B-2R) staining method based upon conventional Karnovsky-Roots staining. It significantly improved the ability to display nerve fascicles, motor and sensory nerve fiber textures. In this method, Karnovsky-Roots staining was carried out, followed by toluidine blue counterstain and ponceau 2R counterstain. RESULTS: Comparisons were conducted between the three methods in staining of median nerve sections, which showed similar distribution characters in acetylcholinesterase-positive sites. The additional counterstaining did not change the basis of Karnovsky-Roots staining. However, the resulting images from this new method significantly facilitated the subsequent 3D nerve reconstruction and 3D printing. CONCLUSIONS: These results show that the new staining method significantly enhanced the display qualities of nerve fascicle edges and fiber textures of motor and sensory nerves and facilitated 3D nerve reconstruction.

A sensitive fluorescence assay of organophosphorus pesticides using acetylcholinesterase and copper-catalyzed click chemistry.

Organophosphorus pesticides (OPs) are widely used in agricultural fields, but exhibit high toxicity to human beings. A sensitive fluorescence assay for organophosphorus pesticides was developed using the inhibition of acetylcholinesterase (AChE) activity and the copper-catalyzed click chemical reaction. In the click reaction, two hybridized DNA probes can be ligated with copper ions, inducing a fluorescence quenching during the strand displacement reaction. AChE can hydrolyze acetylthiocholine (ATCh) to form thiocholine (TCh) which contains a thiol group. TCh will react with copper ions, blocking the click reaction and a high fluorescence signal is observed. But in the presence of OPs, the activity of AChE is inhibited, releasing a high concentration of copper ions that catalyze the click chemical reaction and resulting in decreased fluorescence signals. Taking advantage of the copper-mediated signal amplification effect, the sensitivity was improved. This assay has also been applied to detect OPs in river water samples with satisfactory results, which demonstrates that the method has great potential for practical applications in environmental protection and food safety fields.

Rapid colorimetric determination of the pesticides carbofuran and dichlorvos by exploiting their inhibitory effect on the aggregation of peroxidase-mimicking platinum nanoparticles.

A novel and highly sensitive enzyme inhibition assay was developed for the rapid detection of the organophosphate pesticide dichlorvos and the carbamate pesticide carbofuran. It achieves signal amplification by the secondary catalysis of platinum nanoparticles. Acetylcholinesterase (AChE) is capable of catalyzing the hydrolysis of acetylthiocholine to form thiocholine. Thiocholine causes the aggregation of citrate-capped platinum nanoparticles which then lose their peroxidase-mimicking properties. After addition of pesticides, the activity of AChE is inhibited, less thiocholine is produced, less aggregation occurs, and the peroxidase-mimetic properties are increasingly retained. In the presence of tetramethylbenzidine and H2O2, a deep blue coloration with an absorption maximum at 650 nm will be formed. The assay was applied to the determination of dichlorvos and carbofuran, and detection limits of 2.3 µg·L-1 and 1.4 µg·L-1 were obtained, respectively. Recovery experiments with spiked tap water and pears gave satisfactory relative standard deviations. Graphical abstract The blue product formed by platinum nanoparticle-catalyzed oxidation of 3,3'5,5'-tetramethylbenzidine (TMB) by H2O2 is reduced if acetylthiocholine (ATCh) is hydrolyzed by acetylcholinesterase (AChE) to form thiocholine. However, if AChE is inhibited by pesticides, color formation will recover.

A colorimetric assay for acetylcholinesterase activity and inhibitor screening based on the thiocholine-induced inhibition of the oxidative power of MnO2 nanosheets on 3,3',5,5'-tetramethylbenzidine.

The authors describe a colorimetric method for the determination of the activity of acetylcholinesterase (AChE). Manganese dioxide (MnO2) nanosheets directly reacts with 3,3',5,5'-tetramethylbenzidine (TMB) in the absence of hydrogen peroxide (H2O2). This leads to the formation of a blue product (oxTMB) with an absorption peak at 652 nm. If AChE hydrolyzes its substrate acetylthiocholine chloride, thiocholine is formed which blocks the oxidative power of the MnO2 nanosheets. Hence, oxTMB will not be formed. The decreased absorbance is directly related to the AChE activity in the 0.01-1.0 mU·mL-1 range. The detection limit is 0.01 mU·mL-1 and the relative standard deviation is 1.2% (for n = 11 at 0.5 mU·mL-1). The method was also applied to screen for inhibitors of AChE. Graphical abstract Based on the oxidizing properties of manganese dioxide nanosheets (MnO2 nanosheets), we report a colorimetric method for determining acetylcholinesterase activity with the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB).

Design and Development of Acetylthiocholine Electrochemical Biosensor Based on Zinc Oxide-Cerium Oxide Nanohybrid Modified Platinum Electrode.

Acetylcholinesterase (AChE) enzyme has been predominantly used for the detection of pesticides and metal ions. But, these sensors respond to pesticides as well as metal ions at certain concentration, which results in poor selectivity. Hence in this work, the amount of thiocholine produced during AChE inhibition has been estimated to detect the residual activity of AChE enzyme in-turn to enhance the efficiency of the biosensor. In this context, Pt/ZnO-CeO2/AChE/Chitosan based biosensor has been developed for sensitive voltammetric quantification of thiocholine in AChE. The sensor exhibited enhanced electron transfer rate, good conductivity and biocompatibility. Both the intrinsic and extrinsic parameters were simultaneously optimized using second order polynomial regression to get the best conditions for ATCh determination. Under optimized experimental conditions, the redox peak current was linear over the concentration range of 0.1-1.5 mM with detection and quantification limit of 0.05 and 0.15 µM respectively and the sensitivity of 1.47 µA mM-1.

An investigation into the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine using heavy atom kinetic isotope effects.

Heavy atom kinetic isotope effects (KIEs) were determined for the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine (FTC). The leaving-S, carbonyl-C, and carbonyl-O KIEs are (34)k=0.994±0.004, (13)k=1.0148±0.0007, and (18)k=0.999±0.002, respectively. The observed KIEs support a mechanism for both acylation and deacylation where the steps up to and including the formation of the tetrahedral intermediate are at least partially rate determining. These results, in contrast to previous studies with acetylthiocholine, suggest that the decomposition of a tetrahedral intermediate is not rate-determining for FTC hydrolysis. Structural differences between the two substrates are likely responsible for the observed mechanism change with FTC.

A mechanistic study of thioester hydrolysis with heavy atom kinetic isotope effects.

The carbonyl-C, carbonyl-O, and leaving-S kinetic isotope effects (KIEs) were determined for the hydrolysis of formylthiocholine. Under acidic conditions, (13)k(obs) = 1.0312, (18)k(obs) = 0.997, and (34)k(obs) = 0.995; for neutral conditions, (13)k(obs) = 1.022, (18)k(obs) = 1.010, and (34)k(obs) = 0.996; and for alkaline conditions, (13)k(obs) = 1.0263, (18)k(obs) = 0.992, and (34)k(obs) = 1.000. The observed KIEs provided helpful insights into a qualitative description of the bond orders in the transition state structure.

Optimal detection of cholinesterase activity in biological samples: modifications to the standard Ellman's assay.

Ellman's assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activity in biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman's assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence of DTNB. Then, the reaction is stopped by a cholinesterase inhibitor and the produced thiocholine is revealed by DTNB and quantified at 412 nm. Indeed, this modification of Ellman's method increases butyrylcholinesterase activity by 20 to 25%. Moreover, high stability of thiocholine enables separation of the two reactions of the Ellman's method into two successive steps that may be convenient for some applications.

Thiocholine mediated stabilization of in situ produced CdS quantum dots: application for the detection of acetylcholinesterase activity and inhibitors.

The use of acetylcholinesterase (AChE) inhibitors as chemical warfare agents or pesticides represents a strong hazard against human health. The high toxicity of these compounds arises from their ability to inhibit acetylcholinesterase from degrading acetylcholine (ACh), which could affect the physiology of the nervous system with serious or fatal consequences. Here we report a simple and fluorimetric system for a highly sensitive detection of AChE activity and inhibitors. The principle of this approach is based on the hydrolysis of acetylthiocholine (ATCh) by AChE, which yields the thiol-bearing compound thiocholine (TCh) that at trace concentrations stabilized the in situ generated CdS quantum dots (QDs). The system shows a linear relationship between the fluorescence intensity and AChE activity from 1 to 10 mU mL(-1) in buffer solution. The accuracy of the proposed system was further demonstrated through the determination of AChE activity in human serum (HS) by the standard addition method. Furthermore, this novel and highly sensitive sensing system allows the detection of 80 pM of the AChE inhibitor paraoxon and 100 nM of galanthamine. The reported methodology shows potential applications for the development of a simple and inexpensive assay for the routine quantification of AChE activity and inhibitors.

Toxicological and biochemical characterizations of AChE in phosalone-susceptible and resistant populations of the common pistachio psyllid, Agonoscena pistaciae.

The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in nine populations of the common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae), were investigated in Kerman Province, Iran. Nine A. pistaciae populations were collected from pistachio orchards, Pistacia vera L. (Sapindales: Anacardiaceae), located in Rafsanjan, Anar, Bam, Kerman, Shahrbabak, Herat, Sirjan, Pariz, and Paghaleh regions of Kerman province. The previous bioassay results showed these populations were susceptible or resistant to phosalone, and the Rafsanjan population was most resistant, with a resistance ratio of 11.3. The specific activity of AChE in the Rafsanjan population was significantly higher than in the susceptible population (Bam). The affinity (K(M)) and hydrolyzing efficiency (Vmax) of AChE on acetylthiocholine iodide, butyrylthiocholine iodide, and propionylthiocholine odide as artificial substrates were clearly lower in the Bam population than that in the Rafsanjan population. These results indicated that the AChE of the Rafsanjan population had lower affinity to these substrates than that of the susceptible population. The higher Vmax value in the Rafsanjan population compared to the susceptible population suggests a possible over expression of AChE in the Rafsanjan population. The in vitro inhibitory effect of several organophosphates and carbamates on AChE of the Rafsanjan and Bam populations was determined. Based on I50, the results showed that the ratios of AChE insensitivity of the resistant to susceptible populations were 23 and 21.7-fold to monocrotophos and phosphamidon, respectively. Whereas, the insensitivity ratios for Rafsanjan population were 0.86, 0.8, 0.78, 0.46, and 0.43 for carbaryl, eserine, propoxur, m-tolyl methyl carbamate, and carbofuran, respectively, suggesting negatively correlated sensitivity to organophosphate-insensitive AChE. Therefore, AChE from the Rafsanjan population showed negatively correlated sensitivity, being insensitive to phosphamidon and monocrotophos and sensitive to N-methyl carbamates.

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.

Smartphone-based colorimetric sensor array using gold nanoparticles for rapid distinguishment of multiple pesticides in real samples.

A simple, sensitive method for pesticide distinguishment based on a colorimetric sensor array using diverse gold nanoparticles (AuNPs) at room temperature is presented in this study. Acetylcholinesterase (AChE) hydrolysis ability was influenced by different pesticides and produced different concentrations of thiocholine by hydrolyzing acetylthiocholine iodide (ATCh). Thiocholine could be easily linked to the AuNPs through an Au - S covalent bond, and AuNPs underwent aggregation, resulting in a visible color change due to alteration of surface plasmon resonance properties. Based on these results, we successfully distinguished eight pesticides (glyphosate, thiram, imidacloprid, tribenuron methyl, nicosulfuron, thifensulfuron methyl, dichlorprop, and fenoprop) utilizing five different AuNPs by colorimetric assay. The limit of detection (LOD) of this visual method for all pesticides was less than 1.5 × 10-7 M, which was more sensitive than the U.S. Environmental Protection Agency regulations specify (1.18 âˆ¼ 3.91 × 10-6 M). This method was further improved by combining a portable smartphone device with a color picking application using (color name AR) and RGB (red, green, blue) values. The method was successfully applied to pesticide residue distinguishment in real samples by linear discriminant analysis (LDA).

Ultrasensitive Acetylcholinesterase detection based on a surface-enhanced Raman scattering lever strategy for identifying nerve fibers.

Accurate discriminating nerve fibers is a prerequisite for right suturing nerves in nerve transfer operation. Various methods have been developed for identification of motor and sensory fibers, but no simple method meets the requirements in clinic. In this study, a surface-enhanced Raman scattering (SERS) lever strategy is designed and developed to detect Acetylcholinesterase (AchE) ultrasensitively, in which using produced thiocholine with weak intrinsic Raman activity (four ounces) to adjust absorbance of Rhodamine B with strong intrinsic Raman activity (thousand catties) on SERS-active substrates is to increase the sensitivity. Employing a miniaturized SERS substrate, SERS-active microneedles, is to decrease the volume of enzymolysis systems. Adopting an internal reference is to increase the repeatability of collected signal. The ultrasensitive AchE detection method discriminate samples with four times of difference in enzyme activity between 1-1 × 10-4 U/mL in about 10 min of enzymolysis time. AchE amounts in 2-mm-long segments of ventral and dorsal roots were about 0.00025-0.001 U and 0.01-0.02 U, respectively. The developed method would be a reliable method met the requirements of identifying motor and sensory fibers in clinic.

Encapsulating gold nanoclusters into metal-organic frameworks to boost luminescence for sensitive detection of copper ions and organophosphorus pesticides.

Gold nanoclusters (Au NCs) with luminescence property are emerging as promising candidates in fluorescent methods for monitoring contaminants, but low luminescence efficiency hampers their extensive applications. Herein, GSH-Au NCs@ZIF-8 was designed by encapsulating GSH-Au NCs with AIE effect into metal-organic frameworks, achieving high luminescence efficiency and good stability through the confinement effect of ZIF-8. Accordingly, a fluorescent sensing platform was constructed for the sensitive detection of copper ions (Cu2+) and organophosphorus pesticides (OPs). Firstly, the as-prepared GSH-Au NCs@ZIF-8 could strongly accumulate Cu2+ due to the adsorption property of MOFs, accompanied by a significant fluorescence quenching effect with a low detection limit of 0.016 µM for Cu2+. Besides, thiocholine (Tch), the hydrolysis product of acetylthiocholine (ATch) by acetylcholinesterase (AchE), could coordinate with Cu2+ by sulfhydryl groups (-SH), leading to a significant fluorescence recovery, which was further used for the quantification of OPs owing to its inhibition to AChE activity. Furthermore, a hydrogel sensor was explored to accomplish equipment-free, visual, and quantitative monitoring of Cu2+ and OPs by a smartphone sensing platform. Overall, this work provides an effective and universal strategy for enhancing the luminescence efficiency and stability of Au NCs, which would greatly promote their applications in contaminants monitoring.

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.

Bivalve esterases as biomarker: identification and characterization in European cockles (Cerastoderma edule).

This study characterized esterase activity in Cerastoderma edule tissues using different substrates and specific inhibitors and identified the tissue distribution of esterases in this species. Synthetic thiocholines and thioacetate esters and specific inhibitors (eserine, BW284C51 and iso-OMPA) were used to identify and quantify cholines and carboxyl esterases. The results demonstrated the presence of a non-specific propionyl thiocholine (PrSCh)-cleaving cholinesterase (ChE) and a large amount of carboxylesterases (CaE). For further studies using C. edule esterases as biomarkers, our results suggest that the adductor muscle, with PrSCh (5 mM) as substrate should be used to analyze ChE, and for CaE analyses, phenyl thioacetate should be used in digestive gland extracts (PSA, 5 mM).

[Cholinesterase activity in tissues of some crab species from the Japan Sea].

The comparative study of the cholinesterase activity in some crab species was carried out for the first time with use of a set of thiocholine substrates. The substrate specificity was studied in stellar nerve, heart, and hemolymph of three crab species. The crab hemolymph was shown to be characterized by the highest enzyme activity. The enzyme from various crab organs has different structure o substrate specificity. Properties of crab enzymes was compared with acetylcholinesterase (AChE) of human blood erythrocytes, butyrylcholinesterase (BuChE) of horse blood serum, enzyme o squids and bivalve molluscs. The obtained data allow the conclusion to be made on differences in properties of enzymes both at the interspecies and at the tissue levels.