Cholinesterase Detection Systems: Reaction Principles, Method Types, and Experimental Design

Cholinesterase detection systems are mainly used to measure the activities of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and total cholinesterase (ChE). The differences among detection methods are mainly reflected in substrate type, signal generation mode, readout format, ability to distinguish enzyme species, and sample compatibility.

Keywords: cholinesterase; acetylcholinesterase; butyrylcholinesterase; AChE; BChE; ChE; Ellman method; DTNB; thiocholine substrates; enzyme activity detection; fluorescence method; electrochemical method; histochemical method

1 Basic Methodological Framework of Cholinesterase Detection

1.1 Detection Targets

(1) Acetylcholinesterase

Acetylcholinesterase mainly hydrolyzes acetylcholine and is the key enzyme responsible for terminating cholinergic neurotransmission. AChE detection is commonly performed in erythrocyte membranes, brain tissue, nerve cells, neuromuscular junctions, and insect nervous tissue. AChE assays are often used in neurotoxicology, Alzheimer’s disease-related research, pesticide exposure assessment, and inhibitor screening.

(2) Butyrylcholinesterase

Butyrylcholinesterase is mainly found in serum, plasma, liver, and some tissues, and has a broader substrate spectrum. Serum or plasma cholinesterase assays mostly reflect BChE activity and are commonly used to evaluate hepatic synthetic function, drug metabolism, organophosphate exposure, and cholinesterase inhibition status.

(3) Total cholinesterase

Total ChE detection reflects the combined activity of AChE, BChE, and other cholinesterase-like activities in a sample. Total activity is suitable for rapid screening, but it is not suitable for directly inferring the source of a specific enzyme species. If a study needs to distinguish AChE from BChE, substrate selection, specific inhibitors, or protein detection should be combined for interpretation.

1.2 Method Selection Logic

(1) Substrate determines enzyme preference

Acetylthiocholine is commonly used for AChE detection, butyrylthiocholine is commonly used for BChE detection, and propionylthiocholine can be used for some total ChE or substrate-spectrum analyses. Substrate selection can only provide an enzyme-species preference and cannot fully replace specificity validation.

(2) Signal determines readout mode

Cholinesterase detection can be achieved through colorimetric, fluorescent, luminescent, electrochemical, or in situ precipitated signals. Colorimetric methods are suitable for routine enzyme activity detection, fluorescence methods are suitable for low-activity samples and high-throughput screening, electrochemical methods are suitable for rapid sensor-based detection, and histochemical methods are suitable for spatial localization.

(3) Reaction rate determines enzyme activity calculation

Cholinesterase activity detection should preferably use kinetic readings and calculate the signal change per unit time during the initial linear phase of the reaction. Endpoint readings are suitable for certain screening or low-throughput experiments, but they are more easily affected by substrate depletion, product accumulation, and background reactions.

Table 1 Method Types and Application Positioning of Cholinesterase Detection

Method type	Signal source	Main advantages	Main limitations	Applicable scenarios
Ellman colorimetric method	Thiocholine reacts with DTNB to generate yellow TNB	Classical, stable, high-throughput, easy to standardize	Interference from thiol compounds, color, and turbidity	Routine activity detection of AChE, BChE, and total ChE
pH indicator method	pH change caused by substrate hydrolysis	Intuitive principle, simple system	Low sensitivity; affected by buffer system	Basic validation, teaching experiments
Fluorogenic substrate method	Hydrolysis releases or activates fluorescent signal	High sensitivity, suitable for micro-samples	Affected by autofluorescence and quenching	Inhibitor screening, low-activity samples
Luminescent coupling method	Enzymatic coupling generates luminescent signal	High sensitivity, low background	Complex system, relatively high cost	High-throughput screening, drug evaluation
Electrochemical method	Hydrolysis product or coupled product produces current change	Rapid, portable, suitable for field detection	Electrode stability and matrix interference are significant	Pesticide residue testing, environmental toxicology, biosensors
Histochemical method	In situ color development or precipitation	Displays spatial localization	Limited quantitative capability	Tissue sections, neuromuscular junctions, insect tissues
Zymography / in-gel activity assay	Substrate color development or fluorescence after electrophoresis	Distinguishes active bands	Highly condition-dependent, semi-quantitative	Analysis of active enzyme components

2 Ellman Colorimetric Method

2.1 Reaction Principle

(1) Substrate hydrolysis

The Ellman method usually uses acetylthiocholine, butyrylthiocholine, or propionylthiocholine as artificial substrates. Cholinesterase catalyzes the hydrolysis of thiocholine substrates, releasing the corresponding acidic product and thiocholine.

(2) DTNB color development

The released thiocholine reacts with DTNB to generate the yellow 5-thio-2-nitrobenzoate anion (TNB). TNB has a clear absorption peak near 412 nm, so cholinesterase activity can be calculated from the rate of increase in absorbance.

(3) Kinetic readout

The Ellman method is more suitable for continuous readout mode. In experiments, absorbance changes are usually recorded immediately after substrate addition, and the slope is calculated from the initial linear phase of the reaction. If only endpoint absorbance is read, the result is more easily affected by spontaneous substrate hydrolysis and differences in reaction time.

2.2 Composition of the Experimental System

(1) Buffer

Phosphate buffer or other pH-stable systems are commonly used. pH affects both enzyme activity and DTNB reaction efficiency; therefore, pH, ionic strength, and reaction temperature should be fixed within the same experimental batch. For AChE and BChE detection, the buffer system should not be changed arbitrarily, otherwise comparability across experiments will decrease.

(2) Substrate

Acetylthiocholine iodide or acetylthiocholine chloride is commonly used for AChE detection; butyrylthiocholine iodide is commonly used for BChE detection. Substrate concentration should be optimized through preliminary experiments. Too low a concentration limits the reaction rate, whereas too high a concentration may cause substrate inhibition or increase non-enzymatic hydrolysis background.

(3) Chromogenic reagent

DTNB is the core chromogenic reagent of the Ellman method. DTNB is sensitive to thiol compounds, so if samples contain glutathione, cysteine, DTT, β-mercaptoethanol, or other reducing or thiol-containing components, the background reading may be significantly affected.

(4) Sample

Serum, plasma, erythrocyte lysate, tissue homogenate, cell lysate, and insect tissue extracts can all be used in the Ellman method, but different samples require different dilution factors, blank settings, and normalization approaches.

2.3 Endpoint Method and Rate Method

(1) Endpoint method

The endpoint method reads absorbance after a fixed reaction time. It is simple to operate and suitable for preliminary screening experiments or samples with large activity differences. This method is sensitive to incubation time, sample addition order, and background subtraction, and is not suitable for precise kinetic analysis.

(2) Rate method

The rate method continuously records absorbance changes and calculates enzyme activity from the slope of absorbance change per unit time. This method is more suitable for comparing activity differences among treatment groups and is also better for identifying substrate depletion, excessively rapid reactions, or nonlinearity.

(3) Microplate method

The microplate Ellman method is suitable for batch samples and inhibitor screening. During the experiment, sample addition order, reading delay, edge-well evaporation, and bubbles between wells should be controlled. For high-activity samples, dilution should be used to keep absorbance changes within the linear range.

2.4 Method Compatibility and Limitations

(1) Suitable for routine enzyme activity detection

The Ellman method is the most commonly used system for AChE, BChE, and total ChE detection, and is suitable for blood samples, tissue homogenates, cell lysates, and insect samples.

(2) Affected by thiol background

DTNB can react with free thiols. Reducing-agent-containing lysates, glutathione-rich tissue samples, or plant/insect extracts may show high background; therefore, sample blanks must be included.

(3) Interference from color and turbidity

Hemoglobin, bilirubin, lipemia, tissue pigments, and particle turbidity can all affect absorbance readings. For complex samples, sample blanks, centrifugal clarification, dilution linearity validation, or confirmation by fluorescence/electrochemical methods can be used.

3 pH Indicator Method and Acid-Base Titration Method

3.1 pH Indicator Method

(1) Reaction basis

When cholinesterase hydrolyzes acetylcholine or related choline ester substrates, acidic products can be generated, causing a pH change in the reaction system. pH indicators can convert this pH change into a color change, thereby indirectly evaluating enzyme activity.

(2) Detection characteristics

This method has a simple system and does not rely on DTNB or fluorogenic substrates, but its sensitivity is low and it is highly sensitive to buffer capacity and sample background pH. If the buffer capacity is too strong, the pH change is not obvious; if the buffer capacity is too weak, the intrinsic pH differences among samples can cause large errors.

(3) Application positioning

The pH indicator method is suitable for teaching demonstrations, early method validation, or rough observation of enzyme activity changes. For complex samples such as serum, tissue homogenates, or cell lysates, it should not be used as the preferred quantitative method.

3.2 Acid-Base Titration Method

(1) Method principle

The acid-base titration method indirectly calculates cholinesterase activity by quantitatively measuring the acidic products generated from substrate hydrolysis. This method was used historically, but its procedure is relatively cumbersome.

(2) Technical limitations

The titration method requires a larger sample volume and a longer reaction time, and is difficult to adapt to high-throughput testing. Sample buffering capacity, volatile components, and exogenous acid-base contamination can all affect results.

(3) Scope of application

This method is more suitable for methodological comparison or historical method description. In modern cholinesterase detection, it is usually replaced by the Ellman method, fluorescence methods, and automated biochemical methods.

4 Fluorescence and Luminescence Methods

4.1 Fluorogenic Substrate Method

(1) Reaction principle

Fluorescence methods use fluorogenic substrates that can be hydrolyzed by cholinesterase. After hydrolysis, the substrate releases a fluorescent molecule or changes fluorescence intensity. Compared with colorimetric methods, fluorescence methods are more sensitive to low activity changes.

(2) Detection workflow

A typical workflow includes sample pre-dilution, substrate addition, constant-temperature incubation, and continuous or endpoint fluorescence reading. Kinetic fluorescence readings are more suitable for enzyme activity calculation, whereas endpoint fluorescence readings are more suitable for preliminary screening.

(3) Applicable scenarios

Fluorescence methods are suitable for low-enzyme-activity samples, small cell samples, micro tissue samples, and high-throughput cholinesterase inhibitor screening. In drug screening, concentration gradients can be set and inhibition rates or IC50 values calculated.

4.2 Fluorescence-Coupled Reactions

(1) Coupled detection

Some fluorescence systems do not directly detect cholinesterase substrate hydrolysis. Instead, they couple choline or thiocholine products to subsequent enzymatic or chemical reactions, ultimately generating a fluorescent signal. This design can improve sensitivity, but the reaction chain is more complex.

(2) Background control

Insufficiency in any substrate, enzyme, or cofactor in the coupled system will affect the final signal. During method development, it is necessary to verify separately whether both the cholinesterase reaction and the coupled reaction are within their linear ranges.

(3) Matrix effects

Tissue homogenates, blood samples, plant extracts, and insect samples may have autofluorescence or fluorescence quenching effects. Sample blanks should be included, and excitation/emission wavelengths should be selected to avoid sample background as much as possible.

4.3 Luminescence Method

(1) Reaction characteristics

Luminescence methods amplify cholinesterase hydrolysis signals through enzymatic coupling or chemiluminescence systems, offering low background and high sensitivity. This method is suitable for high-throughput screening and low-abundance enzyme activity detection.

(2) Experimental control

Luminescence reactions are sensitive to time and temperature, and the reading window after adding detection reagents must be strictly consistent. In batch plate experiments, large reaction-time differences among wells should be avoided.

(3) Application boundaries

Luminescence methods have high sensitivity, but they are more costly and reagent-dependent. If used for mechanistic studies, they should be cross-validated with the Ellman method or protein detection.

Table 2 Technical Characteristics of Different Signal Systems

Signal system	Typical readout	Sensitivity	Throughput	Main interference	Recommended use
Ellman colorimetric method	A412 kinetic slope	Medium	High	Thiol compounds, color, turbidity	Routine AChE/BChE activity detection
pH indicator method	Color or pH change	Low	Medium	Sample pH, buffer capacity	Basic validation
Fluorescence method	Change in fluorescence intensity	High	High	Autofluorescence, fluorescence quenching	Low-activity samples, inhibitor screening
Luminescence method	Luminescence intensity	High	High	Reading time, coupled reaction stability	High-throughput screening
Electrochemical method	Current or potential change	Medium to high	Medium	Electroactive substances, electrode fouling	Rapid testing, sensor development
Histochemical method	In situ color development or precipitation	Qualitative/semi-quantitative	Low	Section thickness, substrate diffusion	Spatial localization

5 Electrochemical Detection Methods

5.1 Direct Electrochemical Detection

(1) Thiocholine oxidation

After cholinesterase hydrolyzes thiocholine substrates, thiocholine is produced. Thiocholine can be oxidized on the electrode surface, generating a detectable current. This method converts enzyme activity into an electrochemical signal.

(2) Response characteristics

Direct electrochemical methods respond rapidly, require small sample volumes, and are suitable for miniaturized and portable detection. Sensor performance depends on electrode materials, modification layers, enzyme immobilization methods, and the electrochemical window.

(3) Interference sources

Ascorbic acid, uric acid, glutathione, polyphenols, and other electroactive components in samples may produce background currents. Before testing complex samples, matrix validation or selective membrane modification should be performed.

5.2 Choline Oxidase-Coupled Electrochemical Method

(1) Coupling principle

Some systems use choline generated from acetylcholine hydrolysis as an intermediate. Choline oxidase then catalyzes the generation of hydrogen peroxide, which subsequently produces a signal through electrochemical oxidation-reduction of hydrogen peroxide.

(2) System advantages

This method is closer to the natural substrate conversion logic and is suitable for constructing cholinesterase-choline oxidase coupled sensors. By immobilizing enzyme layers, rapid detection and repeated use can be achieved.

(3) System limitations

The coupled system is affected by the activities of both cholinesterase and choline oxidase. If choline oxidase is insufficient or hydrogen peroxide is consumed by reducing substances in the sample, cholinesterase activity will be underestimated.

5.3 Inhibition-Based Sensor Detection

(1) Detection logic

Organophosphates or carbamates can inhibit cholinesterase activity. After immobilizing cholinesterase on the electrode surface, the decrease in signal before and after inhibition can be used to evaluate cholinesterase-inhibiting contaminants in samples.

(2) Application scenarios

This method is suitable for rapid screening of pesticide residues, water pollution, food safety, and environmental toxicology. Its advantages include fast response, miniaturizable equipment, and suitability for on-site detection.

(3) Confirmation requirements

Inhibition-based sensors detect an “enzyme activity inhibition effect” and cannot directly identify the specific pollutant type or concentration. Positive samples usually require further confirmation by GC-MS, LC-MS/MS, or targeted chemical analysis.

6 Histochemical and In Situ Detection Methods

6.1 Koelle Method and Karnovsky-Roots Method

(1) Method basis

Histochemical methods usually rely on cholinesterase in tissue sections to hydrolyze thiocholine substrates. The generated thiocholine reacts with metal salts or chromogenic systems to form insoluble precipitates, thereby showing the spatial distribution of enzyme activity.

(2) Localization advantage

These methods can show AChE or ChE localization in neuromuscular junctions, brain regions, peripheral nerves, insect nervous systems, or tissue sections. Compared with homogenate enzyme activity assays, histochemical methods preserve spatial information.

(3) Fixation conditions

Over-fixation reduces enzyme activity, whereas insufficient fixation affects tissue structure and background control. Before detection, fixative type, fixation time, section thickness, and incubation conditions must be optimized.

6.2 Inhibitor-Assisted Localization

(1) AChE localization

Adding a BChE inhibitor to a histochemical system can reduce BChE background and highlight AChE-related signals. This strategy is suitable for AChE localization in complex tissues.

(2) BChE localization

Adding an AChE inhibitor and selecting a BChE-preferred substrate can help observe BChE distribution. Regions rich in blood vessels, liver tissue, glial cells, or plasma components should be interpreted cautiously.

(3) Control of nonspecific esterases

Tissue sections often contain nonspecific esterases. Without inhibitor groups or substrate-negative controls, nonspecific esterase precipitates may be misinterpreted as cholinesterase activity.

6.3 Image Analysis

(1) Semi-quantitative analysis

Histochemical staining can be semi-quantitatively analyzed using image grayscale, positive area, or integrated optical density. Section batch, color development time, and background correction must be standardized.

(2) Spatial colocalization

If AChE or BChE activity in specific cell types needs to be determined, immunofluorescence, cell markers, or serial section analysis can be combined. Histochemical staining alone usually cannot accurately identify the cellular source.

(3) Result boundaries

Histochemical methods show in situ enzyme activity distribution and are not equivalent to protein expression levels. If expression regulation needs to be explained, qPCR, Western blot, ELISA, or immunostaining should be combined.

7 Zymography and Molecular Typing Methods

7.1 Native PAGE Activity Staining

(1) Method principle

Native PAGE can separate different cholinesterase components under non-denaturing conditions, followed by activity staining using substrate and chromogenic systems. This method can reveal active bands with different migration rates.

(2) Applicable scenarios

This method is suitable for analyzing serum BChE multimers, different ChE active components in tissues, insect AChE variants, or active components in enzyme preparations.

(3) Limitations

Active bands are affected by electrophoresis conditions, protein conformation, substrate penetration, and staining time. This method is usually qualitative or semi-quantitative. If precise quantification is required, routine enzyme activity assays should be combined.

7.2 Protein Expression Detection

(1) Western blot

Western blot can be used to detect AChE or BChE protein levels and to help determine whether changes in enzyme activity arise from expression changes. Different splice forms, glycosylation, and polymerization states may cause band differences.

(2) ELISA

ELISA is suitable for batch detection of AChE or BChE protein content. Unlike enzyme activity assays, ELISA reflects protein amount and does not necessarily reflect enzyme activity status. Inhibitor binding, oxidative modification, or conformational changes may result in “unchanged protein level but decreased enzyme activity.”

(3) qPCR

qPCR can analyze ACHE or BCHE gene transcription levels and is suitable for studying expression regulation. mRNA level cannot directly replace enzyme activity and should be interpreted together with protein and functional readouts.

7.3 Mass Spectrometry and Structural Analysis

(1) Protein mass spectrometry

Mass spectrometry can be used to identify cholinesterase proteins, modification sites, and inhibitor adducts. In organophosphate exposure studies, mass spectrometry helps identify adduct modifications related to the enzyme active center.

(2) Substrate conversion analysis

LC-MS/MS can directly analyze changes in acetylcholine, choline, or related metabolites, providing a supplement to enzyme activity detection. This method has high specificity but requires expensive instruments and higher technical expertise.

(3) Mechanism validation

When enzyme activity decreases while expression level remains unchanged, mass spectrometry or targeted modification analysis can help determine whether inhibitor adducts, oxidative modifications, or post-translational modifications are present.

8 Sample Processing Methods

8.1 Serum and Plasma Samples

(1) Sampling requirements

Serum or plasma is commonly used for BChE activity detection. After collection, serum or plasma should be separated as soon as possible to avoid prolonged room-temperature exposure. Obvious hemolysis introduces erythrocyte AChE and hemoglobin interference.

(2) Anticoagulant selection

For plasma samples, anticoagulant compatibility should be considered. Different anticoagulants may affect enzyme activity or detection background, so the same anticoagulant type should be used throughout the experiment.

(3) Storage conditions

Cholinesterase activity is affected by storage temperature and freeze-thaw cycles. For long-term storage, samples should be aliquoted and frozen at low temperature to avoid repeated freeze-thaw cycles. Samples from different batches should use the same storage duration and freeze-thaw history as much as possible.

8.2 Erythrocyte Samples

(1) Sample preparation

Erythrocyte AChE detection usually requires erythrocyte separation, washing, and preparation of erythrocyte suspensions or membrane-related samples. Inadequate washing can leave residual plasma BChE and affect AChE interpretation.

(2) Hemolysis treatment

Erythrocyte AChE is mainly located on the cell membrane, and sample preparation affects enzyme accessibility. Hemolysis, membrane preparation, and dilution conditions should be standardized.

(3) Normalization approach

Erythrocyte AChE can be normalized to hemoglobin concentration, erythrocyte count, or membrane protein amount. Different normalization approaches should not be mixed, especially in samples with anemia, hemolysis, or abnormal hematocrit.

8.3 Tissue and Cell Samples

(1) Tissue homogenates

Brain tissue, muscle, liver, and insect tissue should be homogenized at low temperature before detection. Reducing agents such as DTT or β-mercaptoethanol, which interfere with DTNB color development, should be avoided in the buffer. If detergents must be used, their effects on enzyme activity and reading background should be validated.

(2) Cell lysates

Cell samples can be lysed under mild conditions to preserve enzyme activity. Strong denaturing lysis buffers are not suitable for enzyme activity assays and are only suitable for protein expression analysis. After lysis, debris should be removed by centrifugation, and protein concentration should be measured for normalization.

(3) Insect samples

Insect tissues often contain high nonspecific esterase activity. AChE detection should be validated using substrate selection and specific inhibitors. In resistance studies, AChE mutations, expression changes, and metabolic enzyme contributions should also be considered.

Table 3 Detection Method Compatibility for Different Samples

Sample type	Recommended method	Main detection target	Key control points
Serum	Butyrylthiocholine-Ellman method	Mainly BChE	Avoid hemolysis; standardize storage conditions
Plasma	Butyrylthiocholine-Ellman method or automated ChE detection	Mainly BChE	Use consistent anticoagulants; set sample blanks
Erythrocytes	Acetylthiocholine-Ellman method	Mainly AChE	Wash sufficiently; normalize to Hb or membrane protein
Brain tissue	Ellman method, histochemical method, fluorescence method	Mainly AChE	Homogenize at low temperature; avoid reducing-agent interference
Liver tissue	Ellman method, protein detection	BChE and total ChE	Pay attention to nonspecific esterase background
Cell lysate	Ellman method, fluorescence method, Western blot	AChE or BChE	Use mild lysis; normalize to protein amount
Insect tissue	Ellman method, inhibitor typing, zymography	AChE and esterase background	Combine with specific inhibitor validation
Tissue sections	Histochemical method	In situ ChE activity	Control fixation, section thickness, and incubation time
Environmental samples	Inhibition-based electrochemical method, enzyme inhibition screening	Inhibitory effect	Positive results require chemical confirmation

9 Enzyme Activity Calculation and Result Expression

9.1 Kinetic Slope Calculation

(1) Linear range selection

The slope should be calculated from the region where absorbance or fluorescence intensity increases linearly over time. The initial lag phase, late substrate-depletion phase, and signal-saturation region are not suitable for enzyme activity calculation.

(2) Blank subtraction

Before enzyme activity calculation, reagent blanks and sample blanks should be subtracted. Reagent blanks reflect spontaneous substrate hydrolysis and chromogenic reagent background; sample blanks reflect intrinsic sample color, turbidity, or endogenous reactions.

(3) Unit conversion

Colorimetric methods can calculate enzyme activity units based on molar extinction coefficient, optical path length, sample volume, and dilution factor. In microplate assays, the actual optical path is shorter than 1 cm. If absolute enzyme activity is calculated, pathlength correction or a standard curve should be used.

9.2 Result Expression

(1) Serum or plasma

Serum or plasma ChE/BChE activity is commonly expressed as U/L or U/mL. When comparing results from different platforms, detection temperature, substrate, unit definition, and calibration method must be considered.

(2) Tissue and cells

Tissue homogenates and cell lysates are commonly expressed as U/mg protein. If tissue blood content differs significantly, the influence of blood cholinesterase background should be considered.

(3) Erythrocytes

Erythrocyte AChE can be expressed as U/g Hb, U/mL packed RBC, U/10⁹ RBC, or U/mg membrane protein. In toxicological studies, the normalization method most consistent with the experimental design should be selected and kept consistent throughout.

9.3 Inhibition Rate Calculation

(1) Basic formula

Inhibition rate is usually calculated as the ratio of “control enzyme activity minus treated enzyme activity” to control enzyme activity. In inhibitor screening, comparison should be performed under the same reaction time, substrate concentration, and sample concentration.

(2) IC50 determination

In drug screening, inhibitor concentration gradients can be established and IC50 values fitted based on residual enzyme activity. For irreversible inhibitors, pre-incubation time and enzyme concentration should also be considered because inhibition is time-dependent.

(3) Reversibility analysis

Carbamate inhibition is relatively reversible, whereas organophosphate inhibition is usually more persistent. If inhibition recovery is being studied, elution, reactivator, or time-recovery experiments should be included.

10 Selection of Reagents and Materials for Cholinesterase Detection

Table 4 Reagents and Detection Tools Related to Cholinesterase Detection Systems

Cat. No.	Product Name	Grade/Specification	Corresponding detection step	Application positioning
A1506759	Acetylcholinesterase (AchE) activity detection kit (DNTB, microcalorimetry)	BioReagent	AChE activity detection	Used to measure AChE activity in micro-samples; suitable for Ellman-like detection systems in tissues, cells, or insect samples
A1515940	Acetylcholinesterase (AchE) Activity Assay Kit (DTNB, Colorimetric Method)	BioReagent	AChE activity detection	Used for colorimetric detection of AChE activity; suitable for routine enzyme activity assays and inhibitor evaluation
A486188	Acetylcholinesterase Activity Assay Kit	110T/50S	AChE activity detection	Used for quantitative AChE activity measurement and can serve as a routine AChE detection tool
EJ1513770	Human Acetylcholinesterase (AchE) ELISA Kit	BioReagent	AChE protein quantification	Used for detecting AChE protein levels in human samples; should be interpreted separately from enzyme activity results
EJ1512621	Mouse Acetylcholinesterase (AchE) ELISA Kit	BioReagent	AChE protein quantification	Used for AChE protein detection in mouse samples; suitable for neurotoxicology and cholinergic system research
EJ1513107	Mouse Cholinesterase(CHE) ELISA Kit	BioReagent	Total ChE/CHE detection	Used to detect cholinesterase-related protein in mouse samples; suitable for combined analysis with total ChE activity results
A1520586	Acetylcholinesterase	200u/g	AChE positive enzyme source	Used as a positive control enzyme source for AChE detection systems, substrate hydrolysis validation, and inhibitor screening
B1510310	Butyrylcholinesterase (BCHE)	Bioactive,ActiBioPure™,Native,High Performance,EnzymoPure™,from Equine Serum; ≥50 U/mg enzyme powder; ≥300 U/mg protein	BChE positive enzyme source	Used for establishing BChE activity systems, validating butyrylthiocholine substrates, and evaluating inhibitors
B1510978	Butyrylcholinesterase (BChE)	Bioactive,ActiBioPure™,Native,High Performance,EnzymoPure™,from Equine Serum; Protein content: See COA; ≥10 U/mg protein	BChE positive enzyme source	Used as a control for serum/plasma BChE detection systems and enzymatic method validation
np001121	Butyrylcholinesterase from Human Plasma	ActiBioPure™, Bioactive, High Performance, EnzymoPure™, ≥90%(SDS-PAGE), Pre-lyophilization Protein Concentration	Human BChE enzyme source	Used for human BChE activity studies, plasma cholinesterase assay development, and inhibitor response validation
A113536	Acetylthiocholine chloride	≥99%	AChE substrate	Common AChE thio-substrate in the Ellman method; hydrolysis releases thiocholine for DTNB color development
A100869	Acetylthiocholine iodide	≥98%	AChE substrate	Used for AChE activity detection, kinetic analysis, and inhibition-rate evaluation
B100871	S-Butyrylthiocholine iodide	≥98%	BChE substrate	Used for BChE activity detection; suitable for serum, plasma, and BChE enzyme source systems
P348764	Propionylthiocholine iodide	≥98%	ChE substrate / substrate-spectrum analysis	Used for total ChE activity detection or comparison of substrate preference among different cholinesterases
B152178	Benzoylthiocholine Iodide	≥98%	Cholinesterase substrate	Can be used for BChE-related substrate-spectrum analysis or evaluation of cholinesterase hydrolysis capacity
T304468	thiocholine iodide	≥95%	Chromogenic product / methodological control	Used for DTNB color reaction validation, thiocholine product response, and standardized controls
D755430	5,5′-Dithio-bis-(2-nitrobenzoic Acid)	Sulfhydryl reagent used to characterize reactive SH groups.	DTNB chromogenic reagent	Core chromogenic reagent of the Ellman method; reacts with thiocholine to generate TNB for 412 nm reading
D425581	5,5＇-Dithio bis-(2-nitrobenzoic acid)	10mM in DMSO	DTNB chromogenic reagent	Suitable for direct addition to Ellman chromogenic systems for microplate or colorimetric detection
D105559	5,5＇-Dithio bis-(2-nitrobenzoic acid)	≥98%	DTNB chromogenic reagent	Used in cholinesterase activity assays for thiocholine color development
A111014	Acetylcholine chloride（ACh）	≥99%	Natural substrate / standard	Used for acetylcholine hydrolysis, choline generation, and validation of coupled detection systems
A425009	Acetylcholine chloride（ACh）	10mM in DMSO	Natural substrate / standard solution	Used for natural substrate systems, cell treatment, or methodological controls
A111015	Acetylcholine chloride（ACh）	for cell culture, ≥99%	Natural substrate / cell experiment	Suitable for cholinergic cell experiments and AChE hydrolysis-system validation
A106023	Acetylcholine bromide	≥98%	Natural substrate salt form	Used for acetylcholine-related substrate validation and cholinesterase hydrolysis research
A422713	Acetylcholine iodide	10mM in DMSO	Natural substrate salt form	Used for ACh-related detection systems or cholinesterase substrate reaction validation
A111033	Acetylcholine iodide	≥98%	Natural substrate salt form	Used for acetylcholine substrate hydrolysis and methodological controls
A353807	Acetylcholine-d9 Chloride	≥98 atom%,≥98%	Isotope-labeled ACh	Used for LC-MS/MS acetylcholine detection, substrate conversion tracing, and method validation
A982545	Acetylcholine-d9 Bromide	≥98%	Isotope-labeled ACh	Used for tracing acetylcholine hydrolysis by mass spectrometry or establishing an internal-standard system
B770284	Butyrylcholine Iodide	≥99%	BChE natural/simulated substrate	Used for BChE substrate hydrolysis research and butyrylcholine-related method validation
B152286	Benzoylcholine Chloride	≥98%(T)	BChE-related substrate	Used for benzoylcholine hydrolysis experiments and BChE substrate-spectrum studies
B152285	Benzoylcholine Bromide	≥98%	BChE-related substrate	Can be used for BChE substrate specificity and hydrolytic activity analysis
B152287	Benzoylcholine Iodide	≥98%(T)	BChE-related substrate	Used for cholinesterase substrate-spectrum analysis and methodological comparison
C108897	Choline chloride	for cell culture, suitable for insect cell culture, ≥99%	Choline standard / product control	Used for choline generation, coupled detection, choline oxidase reaction, and standard curve validation
C108898	Choline chloride	≥99%	Choline standard	Used for validation of natural substrate hydrolysis products and calibration of coupled systems
C755647	Choline chloride	UltraBio™, ≥99%(AT)	High-purity choline standard	Suitable for choline oxidase-coupled detection requiring stronger background control
C425396	Choline chloride	10mM in DMSO	Choline standard solution	Suitable for direct use in standard curves, product recovery, and method validation
C651832	Choline-d4 Chloride	≥99%,≥98atom%D	Isotope-labeled choline	Used as an LC-MS/MS internal standard for choline quantification or tracing acetylcholine hydrolysis products
C472029	Choline chloride-(trimethyl-d₉)	≥98 atom% D,≥95%	Isotope-labeled choline	Used for mass spectrometric choline detection, coupled-system validation, and product quantification correction
C464602	Choline chloride-¹⁵N	≥98 atom% 15N,≥99%	Isotope-labeled choline	Used for mass spectrometric quantification of choline metabolites and tracing hydrolysis products
C474001	Choline bromide-(methyl-¹³C)	≥99 atom% 13C	Isotope-labeled choline	Used as an LC-MS/MS internal standard or for choline product validation
C471988	Choline-1,1,2,2-d₄bromide	≥98 atom% D,≥98%	Isotope-labeled choline	Used for choline quantification and analysis of acetylcholine hydrolysis products
C774081	Choline Oxidase (CODA)	Bioactive,Recombinant,ActiBioPure™,High Performance,EnzymoPure™,≥5 U/mg enzyme powder; ≥15 U/mg protein	Choline oxidase coupling enzyme	Used for oxidation detection of choline generated from acetylcholine hydrolysis; suitable for fluorescence, electrochemical, or HRP-coupled systems
C1492995	Choline Oxidase (CHO) from Alcaligenes sp.	Bioactive,ActiBioPure™,Native,High Performance,EnzymoPure™,≥10 U/mg enzyme powder	Choline oxidase coupling enzyme	Used for oxidizing choline to generate hydrogen peroxide; suitable for natural substrate coupled detection and sensor systems
P105528	Horseradish Peroxidase (HRP)	Bioactive,ActiBioPure™,Native,High Performance,EnzymoPure™,from Horseradish; ≥250 U/mg enzyme powder; RZ≥3	HRP-coupled colorimetric/fluorescent enzyme	Used for POD/HRP colorimetric or fluorescent signal amplification after H₂O₂ generation by choline oxidase
H1508159	Horseradish Peroxidase (HRP)	Bioactive,ActiBioPure™,Native,High Performance,EnzymoPure™,≥300U/mg enzyme powder, Rz≥3; from Horseradish	High-activity HRP	Suitable for high-sensitivity choline oxidase-HRP coupled systems
R1507818	Horseradish Peroxidase (HRP)	Bioactive,Recombinant,ActiBioPure™,High Performance,EnzymoPure™,≥250U/mg enzyme powder, Rz ≥3; expressed in Nicotiana benthamiana	Recombinant HRP	Used in recombinant-source HRP coupled systems; suitable for experiments requiring high formulation consistency and background control
P128534	Peroxidase from horseradish(EIA Grade,Purified)	EnzymoPure™, RZ 2.9,≥500 units/mg protein	High-purity HRP	Suitable for coupled detection systems requiring high background control, purity, and signal amplification efficiency
F274954	Fasciculin-II	≥99%	AChE inhibitor	Used as an AChE-specific inhibitory control, suitable for confirming AChE signal origin
P337839	Physostigmine salicylate	Moligand™, ≥98%	Cholinesterase inhibitor	Used in AChE/BChE inhibition experiments, positive inhibitory controls, and method validation
P1499516	Physostigmine salicylate	Moligand™, 10 mM in DMSO	Cholinesterase inhibitor solution	Suitable for direct use in inhibition-rate assays and inhibitor screening systems
N159835	Neostigmine Bromide	Moligand™, ≥99%(T)	Reversible cholinesterase inhibitor	Used for reversible cholinesterase inhibition models and positive controls
N420722	Neostigmine Bromide	10mM in Water	Reversible cholinesterase inhibitor solution	Used for microplate inhibition experiments, cell treatment, and method validation
C141096	Carbaryl	≥97%	Carbamate inhibitor	Used for reversible cholinesterase inhibition, pesticide toxicology, and insect resistance research
C109839	Carbaryl	analytical standard, ≥98%	Pesticide toxicology inhibitor standard	Used for cholinesterase inhibition-rate evaluation and positive method controls
C109840	Carbaryl	analytical standard, ≥99.5%(HPLC)	High-purity inhibitor standard	Suitable for quantitative inhibition experiments, standard curves, and toxicological method validation
C425206	Carbaryl	10mM in DMSO	Inhibitor stock solution	Used for cholinesterase inhibitor screening and dose-response experiments
C109842	Carbaryl solution	analytical standard, 10ug/ml in acetone	Inhibitor standard solution	Used for low-concentration inhibition experiments and pesticide residue-related method validation
C109841	Carbaryl solution	analytical standard, 100ug/ml in acetone	Inhibitor standard solution	Used for cholinesterase inhibition-rate assays and standardized controls
C141256	Carbaryl	1000ug/ml in Acetonitrile	Inhibitor standard solution	Used for high-concentration stocks, toxicological exposure gradients, and instrumental method comparison
BWY272968	Carbaryl in Methanol	100μg/mL in Methanol	Inhibitor reference material	Used for pesticide inhibitor standardization, method validation, and quality control
BWY272973	Carbaryl in Methanol	1000μg/mL in Methanol	Inhibitor reference material	Used for high-concentration stock preparation, toxicological gradients, and detection-system quality control

The core of cholinesterase detection systems is to accurately reflect AChE, BChE, or total ChE activity using appropriate substrates and signal systems. The Ellman method is suitable as the main system for routine enzyme activity detection, fluorescence and luminescence methods are suitable for high-sensitivity or high-throughput applications, electrochemical methods are suitable for rapid screening and sensor-based detection, and histochemical methods are used for in situ localization.

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[1] The Key Molecule in Neurotransmission—Acetylcholine