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.

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.

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.

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.

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.

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).

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.

Carbon Dot-Anchored Cobalt Oxyhydroxide Composite-Based Hydrogel Sensor for On-Site Monitoring of Organophosphorus Pesticides.

The development of a portable, quantitative, and user-friendly sensor for on-site monitoring of organophosphorus pesticides (OPs) is significantly urgent to guarantee food safety. Herein, a carbon dot/cobalt oxyhydroxide composite (CD/CoOOH)-based fluorescent hydrogel sensor is constructed for precisely quantifying OPs using a homemade portable auxiliary device. As a fluorescence signal indicator, the orange-emissive CD/CoOOH composite is encapsulated into an agarose hydrogel kit for amplifying the detection signals, shielding background interference, and enhancing stability. Acetylcholinesterase (AChE) catalyzes the hydrolysis of the substrate to produce thiocholine, which induces the decomposition of CoOOH and makes the fluorescence enhancement of the hydrogel platform possible. OPs can specifically block the AChE activity to limit thiocholine production, resulting in a decrease in platform fluorescence. The image color of the fluorescent hydrogel kit is transformed into digital information using a homemade auxiliary device, achieving on-site quantitative detection of paraoxon (model target) with a detection limit of 10 ng mL-1. Harnessing CD/CoOOH composite signatures, hydrogel encapsulation, and portable optical devices, the proposed fluorescence hydrogel platform demonstrated high sensitivity and good anti-interference performance in agricultural sample analysis, indicating considerable potential in the on-site application.

A Novel Paper-Based Electrochemical Biosensor Based on N,O-Rich Covalent Organic Frameworks for Carbaryl Detection.

A new N,O-rich covalent organic framework (COFDHNDA-BTH) was synthesized by an amine-aldehyde condensation reaction between 2,6-dialdehyde-1,5-dihydroxynaphthalene (DHNDA) and 1,3,5-phenyltriformylhydrazine (BTH) for carbaryl detection. The free NH, OH, and C=O groups of COFDHNDA-BTH not only covalently couples with acetylcholinesterase (AChE) into the pores of COFDHNDA-BTH, but also greatly improves the catalytic activity of AChE in the constrained environment of COFDHNDA-BTH's pore. Under the catalysis of AChE, the acetylthiocholine (ATCl) was decomposed into positively charged thiocholine (TCl), which was captured on the COFDHNDA-BTH modified electrode. The positive charges of TCl can attract anionic probe [Fe(CN)6]3-/4- on the COFDHNDA-BTH-modified electrode to show a good oxidation peak at 0.25 V (versus a saturated calomel electrode). The carbaryl detection can inhibit the activity of AChE, resulting in the decrease in the oxidation peak. Therefore, a turn-off electrochemical carbaryl biosensor based on a flexible carbon paper electrode loaded with COFDHNDA-BTH and AChE was constructed using the oxidation peak of an anionic probe [Fe(CN)6]3-/4- as the detection signal. The detection limit was 0.16 µM (S/N = 3), and the linear range was 0.48~35.0 µM. The sensor has good selectivity, repeatability, and stability, and has a good application prospect in pesticide detection.

Dual-mode colorimetric-photothermal sensing platform of acetylcholinesterase activity based on the peroxidase-like activity of Fe-N-C nanozyme.

Sensors based on colorimetry, fluorescence, and electrochemistry have been widely employed to detect acetylcholinesterase and its inhibitors, however, there are only a minority of strategies for AChE detection based on photothermal method. This work reports a versatile dual-mode colorimetric and photothermal biosensing platform for acetylcholinesterase (AChE) detection and its inhibitor (paraoxon-ethyl, a model of AChE inhibitors) monitor based on Fe-N-C/H2O2/3,3',5,5'-tetramethylbenzidine (TMB) system. The Fe-N-C with abundant active Fe-Nx sites shows outstanding peroxidase-mimicking activity and can be used to promote the generation of •OH by H2O2 to oxidize TMB. However, the introduction of mercapto molecules tending to coordinate with metal atoms result in the block of action site in Fe-N-C, thereby decrease its peroxidase-mimetic activity. The designed biosensor principle is based on the block of active sites of Fe-N-C by thiocholine (TCh, one kind of mercapto molecules) that can be produced by acetylthiocholine (ATCh) in the presence of AChE. Under optimum conditions, the limit of detection (LOD) for AChE activity is 1.9 mU mL-1 (colorimetric) and 2.2 mU mL-1 (photothermal), while for paraoxon-ethyl is 0.012 µg mL-1 (colorimetric) and 0.013 µg mL-1 (photothermal), respectively. The assay we proposed not only can be designed to monitor AChE detection and its inhibitors, but also can be easily extended for the detection of other biomolecules relate to the generation or consumption of H2O2.

Flow-Through Acetylcholinesterase Sensor with Replaceable Enzyme Reactor.

Fast and reliable determination of enzyme inhibitors are of great importance in environmental monitoring and biomedicine because of the high biological activity and toxicity of such species and the necessity of their reliable assessment in many media. In this work, a flow-through biosensor has been developed and produced by 3D printing from poly(lactic acid). Acetylcholinesterase from an electric eel was immobilized on the inner walls of the reactor cell. The concentration of thiocholine formed in enzymatic hydrolysis of the substrate was monitored amperometrically with a screen-printed carbon electrode modified with carbon black particles, pillar[5]arene, electropolymerized Methylene blue and thionine. In the presence of thiocholine, the cathodic current at -0.25 V decreased because of an alternative chemical reaction of the macrocycle. The conditions of enzyme immobilization and signal measurements were optimized and the performance of the biosensor was assessed in the determination of reversible (donepezil, berberine) and irreversible (carbofuran) inhibitors. In the optimal conditions, the flow-through biosensor made it possible to determine 1.0 nM-1.0 µM donepezil, 1.0 µM-1.0 mM berberine and 10 nM to 0.1 µM carbofuran. The AChE biosensor was tested on spiked samples of artificial urine for drugs and peanuts for carbofuran. Possible interference of the sample components was eliminated by dilution of the samples with phosphate buffer. Easy mounting, low cost of replaceable parts of the cell and satisfactory analytical and metrological characteristics made the biosensor a promising future application as a point-of-care or point-of-demand device outside of a chemical laboratory.

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.

Branched poly(ethylenimine) carbon dots-MnO2 nanosheets based fluorescent sensory system for sensing of malachite green in fish samples.

Malachite green (MG) is an organic dye compound that is frequently used as a fungicide and antiseptic in aquaculture. However, human or animal exposure to MG causes carcinogenic, teratogenic and mutagenic effects. Herein, a novel fluorescent assay was designed for the detection of MG using manganese dioxide nanosheets (MnO2 NS) as an energy acceptor to quench the fluorescence of branched poly(ethylenimine) carbon dots (BPEI-CDs) via Förster resonance energy transfer. When butyrylcholinesterase is introduced to form thiocholine in the presence of S-butyrylthiocholine iodide, MnO2 NS can be recovered by thiocholine to Mn2+, resulting in restoration of the fluorescence of BPEI-CDs. Exploiting these changes in fluorescence intensity in the above system, a fluorescence probe was successfully developed for the quantitative detection of MG. Besides, this assay was applied to fish samples, verifying the high potential for practical application of the proposed sensor for the monitoring of MG in aquatic products.

A green photocatalytic-biosensor for colorimetric detection of pesticide (carbaryl) based on inhibition of acetylcholinesterase.

Carbaryl is a widely-used carbamate pesticide and the detection of its residues in environmental, food and clinical samples is of great importance. In this sturdy, we developed a green photocatalytic-biosensor based on double strand DNA-SYBR green I complex for sensitively colorimetric detection of carbaryl. This green photocatalytic-biosensor can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) into blue ox-TMB. Meanwhile thiocholine is catalytically produced by acetylcholinesterase (AChE) to directly reduce blue ox-TMB into colorless TMB. But the activity of AChE will be suppressed by carbaryl, thus generating less thiocholine and resulting in more ox-TMB for colorimetric analysis. After the careful optimization of sensing conditions (2 µM for DNA concentration, 50 × concentration for SYBR Green I, 10 min for illumination time), the lowest detectable concentration for carbaryl is 0.008 ng/mL with a linear response in the range of 0.01-0.25 ng/mL. In addition, this photocatalytic-biosensor has good selectivity over non-target chemicals (acetamiprid, atrazine, carbendazim, melamine, bisphenol A, estradiol). It also allows detection of pesticides in real samples verified by a standard HPLC method.

In-situ preparation of hollow CdCoS2 heterojunction with enhanced photocurrent response for highly photoelectrochemical sensing of organophosphorus pesticides.

In this study, a porous hollow CdCoS2(2) microsphere was synthesized based on the ZIF-67-S MOFs derived method of sulfurization reaction and calcination process. Under visible light irradiation, the resulting CdCoS2(2) composite showed a markedly enhanced photoelectrochemical (PEC) response. The photocurrent value of the CdCoS2(2) modified ITO electrode was 93-fold and 41-fold than that of CoS and CdS materials, respectively. Promoting the photo-absorption ability by internal multilight scattering/reflection was due to the porous and hollow nature of CdCoS2(2). Furthermore, obtained CdCoS2(2) heterostructure in-situ with a close contact interface could facilitate the separation/migration of photo-induced carriers. The CdCoS2(2) was also mixed with Ag nanoparticles (NPs) to further improve the PEC response. Acetylcholinesterase (AChE) as a bio-recognition molecule was immobilized on the glutaraldehyde-chitosan (GLD-CS) modified CdCoS2(2)@Ag electrode surface by cross-linking effect. AChE could hydrolyze the acetylcholine chloride (ATCl) to produce an electron donor of thiocholine which led to the elevated photocurrent output. When the bioactivity of AChE was inhibited by the organophosphate pesticides (chlorpyrifos as substrate), the reduced production of thiocholine resulted in a decline in photocurrent. Under optimal conditions, the structured AChE/GLD-CS/CdCoS2(2)@Ag/ITO sensing platform was successfully achieved for chlorpyrifos detection. The wide linear response range was from 0.001 to 270 µg mL-1 and with a low detection limit of 0.57 ng mL-1. The proposed PEC biosensor also exhibited excellent selectivity and good stability, demonstrating the designed porous hollow CdCoS2(2)@Ag heterostructured composite promised to be a great application in the PEC sensors.

Water-soluble non-conjugated polymer dots with strong green fluorescence for sensitive detection of organophosphate pesticides.

Water-soluble non-conjugated polymer dots (PDs) have been synthesized using hyperbranched polyethyleneimine (PEI) and dihydroxybenzaldehyde (DHB) for the first time via the Schiff base reaction at room temperature. The yielded non-conjugated PDs of PEI-DHB could display the well-defined spheric structure and good water solubility. In contrast to the common PDs otherwise showing blue emission, the PEI-DHB PDs could give out strong green fluorescence in aqueous media. Especially, the fluorescence of the PEI-DHB PDs could be specifically quenched by MnO2 nanosheets through the inner filter effects and further restored by the thiocholine that could reduce MnO2 nanosheets into Mn2+. Herein, thiocholine could be produced in hydrolysis reaction of acetylthiocholine catalyzed by the acetylcholinesterase (AChE), of which the catalytic activity could be irreversibly inhibitted by the introduction of organophosphates. A highly selective fluorimetric method was thereby been developed for the detection of organophosphorus pesticides using dimethyl-dichloro-vinyl phosphate as a model. The linear concentrations ranges from 0.050 to 2.5 µM. Importantly, the non-conjugated PDs probes with strong green fluorescence and high water solubility may promise the extensive applications in the environmental, food, and clinical analysis fields.

Interactions of human acetylcholinesterase with phenyl valerate and acetylthiocholine: Thiocholine as an enhancer of phenyl valerate esterase activity.

Phenyl valerate (PV) is a neutral substrate for measuring the PVase activity of neuropathy target esterase (NTE), a key molecular event of organophosphorus-induced delayed neuropathy. This substrate has been used to discriminate and identify other proteins with esterase activity and potential targets of organophosphorus (OP) binding. A protein with PVase activity in chicken (model for delayed neurotoxicity) was identified as butyrylcholinesterase (BChE). Further studies in human BChE suggest that other sites might be involved in PVase activity. From the theoretical docking analysis, other more favorable sites for binding PV related to the Asn289 residue located far from the catalytic site ("PVsite") were deduced.In this paper, we demonstrate that acetylcholinesterase is also able to hydrolyze PV. Robust kinetic studies of interactions between substrates PV and acetylthiocholine (AtCh) were performed. The kinetics did not fit the classic competition models among substrates. While PV interacts as a competitive inhibitor in AChE activity, AtCh at low concentrations enhances PVase activity and inhibits this activity at high concentrations. Kinetic behavior suggests that the potentiation effect is caused by thiocholine released at the active site, where AtCh could act as a Trojan Horse. We conclude that the products released at the active site could play an important role in the hydrolysis reactions of different substrates in biological systems.

Broad-spectrum pesticide screening by multiple cholinesterases and thiocholine sensors assembled high-throughput optical array system.

Accurate screening of organophosphorus and carbamates pesticides from the complex real sample is crucial for water quality analysis and food safety control. Herein, a simple, low-cost and accurate pesticides screening method based on a high-throughput optical array system assembled by multiple cholinesterases (ChE) and thiocholine (TCh) sensors is described. The detection mechanism is that the inhibition of ChE activity by pesticides reduces the TCh produced by the hydrolysis of butyryl/acetylthiocholine iodide, thus changing the fluorescence intensity of TCh sensor. The diverse response of ChEs to pesticides and different affinity of sensors to TCh ensure the high-throughput and distinguishable signal output, which allow the establishment of high discrete pesticide database with intra-cluster agglomeration and inter-cluster dispersion. By using the database, the screening of unknown real contaminated samples were successfully operated, and the screened pesticide species and concentrations were consistent with high-performance liquid chromatography. This screening strategy demonstrates the feasibility of replacing existing complex mass spectrometry-based screening strategy with simple optical analysis, providing a new idea for the development of simple accurate screening technologies for widespread organic pollutants including pesticides.

A tunable bifunctional hollow Co3O4/MO3 (M = Mo, W) mixed-metal oxide nanozyme for sensing H2O2 and screening acetylcholinesterase activity and its inhibitor.

A self-templated strategy was adopted to design hollow Co3O4/MO3 (M = Mo, W) mixed-metal oxides via the Mo or W doping of ZIF-67, and subsequent pyrolysis under an atmosphere of air at a low temperature of 450 °C. The hollow Co3O4/MO3 (M = Mo, W) mixed-metal oxides displayed tunable oxidase-like and peroxidase-like activities able to efficiently catalyze the oxidation of TMB to generate a deep blue color in the absence or presence of H2O2. Relative to that of the un-doped Co3O4, the oxidase mimic activity of the Mo-doped Co3O4 increased to 1.3 to 2.1-fold, while its peroxidase mimic activity increased to 7.1 to 19.9-fold, depending on different Mo doping amounts. The oxidase mimic activity of the W-doped Co3O4 increased to 2.1 to 2.3-fold, while its peroxidase mimic activity increased to 4.8 to 5.9-fold, depending on the different W doping amounts. The Mo- and W-doped Co3O4 nanohybrid exhibited both higher O2 and H2O2 activating capability, and their H2O2 activating capacity was superior to the O2 activating capability. Furthermore, the Mo- and W-doped Co3O4 nanohybrids exhibited similar O2 activating abilities, while the Mo-doped one displayed a higher H2O2 activating capability than the W-doped one. The discrepant peroxidase-like nature of Mo- and W-doped Co3O4 nanohybrids is likely attributed to their different catalytic mechanisms. The peroxidase-like activity of Mo-doped Co3O4 is highly related to the ˙OH free radical, while that of W-doped Co3O4 is likely ascribed to the electron transfer between TMB and H2O2. The Km values of Co3O4/MoO3 for TMB and H2O2 were 0.0352 mM and 0.134 mM, which were 3.2- and 1.9-fold lower than that of pure Co3O4, respectively. A Co3O4/MoO3-based colorimetric platform was developed for the determination of H2O2 in the 0.1-200 µM range, with a limit of detection of 0.08 µM (3σ). Based on the thiocholine (TCh) inhibition of the excellent peroxidase-like activity of Co3O4/MoO3 and the TCh generation via acetylcholinesterase (AChE) catalyzed hydrolysis of acetylthiocholine chloride (ATCh), the colorimetric platform was extended to screen AChE activity and its inhibitor.

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.