A choline acetyltransferase dual mode assay based on antigen-antibody interaction

Abstract

The application patent creates a dual mode detection method, which relates to fluorescence technology and electrochemical technology. First, in a certain distance, the in-situ grown gold cluster covalent framework nanomaterial (Au@COF) can quench the fluorescence intensity of scGFP at 506 nm, and ChAT can become a bridge to shorten the distance between them and promote the quenching efficiency of the fluorescence intensity, so as to discuss the content of ChAT through the change of fluorescence intensity before and after; secondly, due to the electrostatic attraction, scGFP and GO can be well combined together, and the above material modified with ChAT antibody can be well modified to the electrode surface, when a certain amount of ChAT is added, the Au@COF modified with ChAT antibody can be further fixed to the electrode surface, and the electrode has a good electrochemical response to H2O2, and the dual mode method provides a basic theory and test method for further research on the analysis and monitoring of low concentration ChAT in cells. So far, there is no report on the choline acetyltransferase dual mode analysis method and application based on the antigen-antibody effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the construction of a dual-mode analysis method and its application in choline acetyltransferase activity analysis, in particular to an analysis sensing method based on antigen-antibody interaction and the application of supercharged fluorescent protein and covalent framework material, belonging to the field of nanobiomaterials and chemical biosensing technology. BACKGROUND

[0002] Cholinergic neurons, as an important functional neuron, exist widely in the central nervous system, have different morphological characteristics and perform various functions, such as participating in human physiological activities such as movement, learning and memory, and are also the target cells of various central nervous system degenerative diseases in clinic. Therefore, the occurrence and mechanism of cholinergic neurons have always been a hot spot in neuroscience research, and are also the basis for understanding the function of the central nervous system and exploring the treatment of its diseases. In recent years, there has been great progress in the development of the telencephalon at both cellular and molecular levels, but little is known about the embryonic development of cholinergic neurons in the telencephalon. Among them, acetylcholine transferase (ChAT) is a rate-limiting enzyme for the synthesis of neurotransmitter acetylcholine, and as a marker protein of cholinergic neurons, its expression region can be shown by immunohistochemical staining, so as to track the occurrence, migration and final distribution of cholinergic neurons. However, what interval does the ChAT expressed by radial glia belong to? Due to its extremely low abundance, there is an urgent need for some simple and sensitive analysis sensing methods to realize the analysis and monitoring of low-concentration ChAT in cells.

[0003] Green fluorescent protein (GFP) is a biologically self-encoded, non-toxic, high quantum yield, low-cost fluorescent marker molecule, which has been widely used in biology, medicine and other fields. Green fluorescent protein can emit strong green light under ultraviolet lamp irradiation, and has strong resistance to photobleaching, high salt solution, heat or alkali. In 2007, Professor Liu's research group mutated the non-charged amino acids on the surface of GFP to positively charged amino acids, and obtained a super positive charged green fluorescent protein scGFP with 36 positive charges. This protein has good biocompatibility, good stability, single fluorescence spectrum, strong fluorescence intensity, strong photobleaching resistance and other advantages. At present, the research and application of super positive charged green fluorescent protein are still very few. It is mainly used for drug delivery, imaging research and other fields due to its good biocompatibility. By using its 36 positive charges and fluorescence properties, related biosensors can be designed. To our knowledge, there is no report on the use of scGFP for low-concentration ChAT analysis and monitoring in cells.

[0004] The application selects ChAT with low abundance in radial glial cells as the main research target, creates a dual-mode detection method involving fluorescence technology and electrochemical technology. First, in a certain distance, the in-situ grown gold cluster@covalent framework nanomaterial (Au@COF) can quench the fluorescence intensity of scGFP at 506 nm, and ChAT can become a bridge to shorten the distance between them and promote the quenching efficiency of the fluorescence intensity, so as to discuss the content of ChAT through the change of the fluorescence intensity before and after; second, due to the electrostatic attraction, scGFP and GO can be well combined together, and the material modified with the ChAT antibody can be well modified to the electrode surface, and when a certain amount of ChAT is added, the Au@COF modified with the ChAT antibody can be further fixed to the electrode surface, and the electrode has a good electrochemical response to H2O2, so as to discuss the concentration of ChAT through the change of the electrochemical signal before and after. In the dual-mode detection method, one presents signal attenuation, and the other presents signal increase, and through mutual support, the generation of false positive signals can be avoided, so that the analysis result is more accurate and reliable. The dual-mode method provides a basic theory and test method for further research on the analysis and monitoring of ChAT with low concentration in cells. So far, there is no related report on the choline acetyltransferase dual-mode analysis method and application based on antigen-antibody interaction. SUMMARY

[0005] The technical problem to be solved by the application is to provide a choline acetyltransferase dual-mode analysis method based on antigen-antibody interaction, which has good specificity, high sensitivity, fast detection speed, accurate and reliable results, and low cost.

[0006] The technical solution adopted by the application to solve the above technical problem is as follows: A choline acetyltransferase dual-mode analysis method based on antigen-antibody interaction, the specific steps are as follows:

[0007] (1) Au@COF synthesis

[0008] A. 2,4,6-tris(4-formylphenyl) triazine (0-16 mg, 0.01-0.063 mmol), tris(4- aminophenyl) amine (5-17 mg, 0.03-0.07 mmol), dichlorobenzene / n-butanol (V / V = 2-6 mL / 1-2 mL) are mixed in a round-bottom flask, 0.1-0.2 mL of acetic acid (1-6 M) is added after stirring for 2-10 min, and stirring is carried out at room temperature under nitrogen atmosphere for 12-72 h. After the reaction is completed, the precipitate is filtered out, and Soxhlet extraction is carried out with tetrahydrofuran (12-24 h), methanol (12-24 h) and acetone (12-24 h) respectively. After drying at 80-120℃ under vacuum overnight, yellow COF powder is obtained.

[0009] B. The above COF powder (0.010-0.020 g) was suspended in 100-250 μM (5-15 mL) of HAuCl4 aqueous solution and stirred vigorously in an ice bath, followed by dropwise addition of 0.1-1 mL of 0.005-0.01 M NaBH4 aqueous solution, and continued stirring for 5-10 min. The final product was obtained by centrifugation, water and ethanol washing, and vacuum drying. In 1-2 mg of the nanomaterial, 0.1-1 mL of dimethyl sulfoxide was added, mixed well, and then 0.1-1 mL of double-distilled water was added, dispersed into a 0.2-1 mg / mL solution, and ready for use.

[0010] C. 200-500 μL of 20-100 μg / mL choline acetyltransferase antibody (Ab1) and 500-1500 μL of 0.2-1 mg / mL Au@COF were mixed and incubated at 30-37 °C for 20-180 min, labeled as Ab1-Au@COF.

[0011] (2) Choline acetyltransferase analysis sensing method and application

[0012] A. 200-500 μL of 50-100 μg / mL choline acetyltransferase antibody (Ab2) and 500-1500 μL of 100-500 nM scGFP were mixed and incubated at 30-37 °C for 50-180 min, labeled as Ab2-scGFP.

[0013] B1. Fluorescence method - 2-10 μL of Ab1-Au@COF solution in step (1C) and 2-10 μL of Ab2-scGFP in step (2C), 10-50 μL of 0.01-0.05 M PBS (pH 7.2), 2-20 μL of ultrapure water, and 5-10 μL of 200-1000 mU / mL ChAT were added, and incubated at 32-37 °C for 5-10 min. The fluorescence intensity in the wavelength range of 430-650 nm (slit width of 10 nm) was measured in a fluorescence instrument.

[0014] B2. Electrochemical method - clean the bare glassy carbon electrode (GCE), then mix 1-2 μL of Ab2-scGFP in step (2C), 1-2 μL of 0.2-1 mg / mL graphene (GO) and 2-6 μL of 0.1-0.2 wt% chitosan, incubate at 30-37 °C for 2-10 min, then drop on the surface of GCE and incubate at 30-37 °C for 2-10 min, then clean the electrode slowly with 0.05 M PBS (pH 7.2) (electrode 2). Then take 1-5 μL of 200-1000 mU / mL ChAT and drop on the surface of the above electrode, stand at 30-37 °C for 5-10 min (electrode 3), then add 1-5 μL of Ab1-Au@COF solution in step (1C) without cleaning, incubate at 30-37 °C for an additional 10-20 min (electrode 4). Then place it in 0.05 M PBS (pH 7.2) containing 5 mM H2O2 for electrochemical detection, the potential range is -1.2-0 V (scanning speed is 50 mV / s). In addition, mix 1-2 μL of 0.1-1 mg / mL graphene (GO), 2-6 μL of 0.2 wt% chitosan and 1-2 μL of ultrapure water, incubate at 30-37 °C for 5-10 min, then drop on the surface of GCE and incubate at 25-37 °C for 5-10 min, clean the electrode slowly with 0.05 M PBS (pH 7.2) to prepare electrode 1 as a control.

[0015] Based on the above steps (1)-(2), for several different enzymes (such as protein kinase, etc.), by changing the type of enzyme in step (2), and keeping other steps unchanged, selective analysis of choline acetyltransferase can be achieved.

[0016] Based on the above steps (1)-(2), by changing the concentration of ChAT (fluorescence: final concentration 0-150 mU / mL; electrochemistry: final concentration 0-100 mU / mL), and keeping other steps unchanged, analysis of choline acetyltransferase with different concentrations can be achieved.

[0017] The fluorescence method in the enzyme label instrument is used, the wavelength range is set as 430nm-650nm, the slit width is 10nm, the prepared Au@COF is used to quench the fluorescence of scGFP, the change process is controlled through ChAT, the fluorescence analysis detection of ChAT is realized, a series of fluorescence intensity corresponding to ChAT with different concentrations is obtained by changing the concentration of ChAT, the linear relationship between the fluorescence intensity response and the concentration of ChAT is established, the concentration of ChAT in the actual cell sample is determined according to the linear relationship between the two. The cyclic voltammetry in the electrochemical workstation is used, the potential range is set as-1.2V-0V, the scanning speed is 50mV / s, the electrochemical catalysis of Au@COF on H2O2 is used, the change process is controlled through ChAT, the electrochemical analysis detection of ChAT is realized, a series of electrochemical current corresponding to ChAT with different concentrations is obtained by changing the concentration of ChAT, the linear relationship between the current response and the concentration of ChAT is established, and the concentration of ChAT in the actual cell sample is determined according to the linear relationship between the two.

[0018] Invention principle: the application is a ChAT dual-mode analysis method based on antigen-antibody interaction, scGFP has good fluorescence at 506nm, and based on the quenching mechanism of Au@COF on scGFP fluorescence, when the distance between them is large, there is no quenching phenomenon, and ChAT can shorten the distance between them through antigen-antibody interaction and promote the quenching effect; for the electrochemical mode, Au@COF can better catalyze the electrochemical reduction of H2O2, and scGFP@GO can be better fixed on the electrode surface, and ChAT can fix Au@COF on the electrode surface through antigen-antibody interaction and promote the electrochemical catalysis of H2O2. The patent uses the above analysis and detection principle, and based on the antigen-antibody interaction, not only realizes the analysis and detection of ChAT with different concentrations, but also provides a new idea for the high-sensitivity detection of ChAT in actual cell samples.

[0019] Compared with the prior art, the application has the following advantages: the application is a ChAT dual-mode analysis method based on antigen-antibody interaction, obviously, Au@COF can quench the fluorescence of scGFP, within a certain concentration range, the greater the concentration of ChAT, the more the fluorescence quenching; similarly, Au@COF can catalyze the electrochemical reduction of H2O2, within a certain concentration range, the greater the concentration of ChAT, the more obvious the electrochemical signal. Experimental results show that the size of the fluorescence (current) intensity and the concentration of ChAT present a linear relationship within a certain range, and the analysis and detection of ChAT are successfully realized. The advantages are:

[0020] (1) Novel design. The present patent is the first choline acetyltransferase dual-mode analysis method based on antigen-antibody interaction, which overcomes the false positive signal caused by single signal and realizes accurate detection of choline acetyltransferase through opposite changes of two signals.

[0021] (2) High sensitivity. The present application is based on Au@COF which can quench scGFP fluorescence, and a linear equation is obtained: the linear correlation equation of fluorescence response to ChAT concentration is y=-1097.8x+3938.2, R 2 =0.9827, the linear range is 0.01-150mU / mL, and the lowest concentration detected is 0.01mU / mL; Similarly, based on the electrochemical reduction of Au@COF to H2O2, a linear equation is obtained: the linear correlation equation of current response to ChAT concentration is y=7.3316x+25.7063, R 2 =0.9907, the linear range is 0.005-100mU / mL, and the lowest concentration detected is 0.005mU / mL; It shows that the sensor can realize high sensitivity detection of ChAT.

[0022] (3) High specificity. The present patent uses ChAT and its antibody as the main force, so it has strong specificity and can realize selective detection of ChAT.

[0023] (4) Accurate results. The recovery rate is between 93% and 104%.

[0024] (5) The preparation and detection method requires less amount of reagents and has low cost. The present application can realize high sensitivity detection of ChAT by consuming a small amount of materials and reagents.

[0025] In summary, the present application is a choline acetyltransferase dual-mode analysis method based on antigen-antibody interaction and application, which has high sensitivity, good selectivity, simple operation, rapid analysis, easy operation and other advantages, and can realize detection of low concentration ChAT, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is the fluorescence intensity-concentration calibration curve of the sensor of the present application for different concentrations of ChAT;

[0027] Figure 2 is the electrochemical intensity-concentration calibration curve of the sensor of the present application for different concentrations of ChAT;

[0028] Figure 3 is the dual-mode specificity experiment graph of the sensor of the present application for ChAT;

[0029] Figure 4Figure 1 shows the detection of ChAT in complex cells by the sensor of the present application. DETAILED DESCRIPTION

[0030] The present application is further described in connection with the following examples and figures.

[0031] Example 1 Construction of the dual-mode analysis detection method and detection of ChAT at different concentrations

[0032] (1) Au@COF synthesis

[0033] A. 2,4,6-tris(4-formylphenyl)triazine (16 mg, 0.063 mmol), tris(4- aminophenyl)amine (17 mg, 0.07 mmol), dichlorobenzene / n-butanol (V / V = 6 mL / 2 mL) were mixed in a round-bottom flask, and 0.2 mL of acetic acid (6 M) was added after stirring for 10 min. The mixture was stirred at room temperature for 72 h under a nitrogen atmosphere. After the reaction was completed, the precipitate was filtered out and Soxhlet extracted with tetrahydrofuran (24 h), methanol (24 h), and acetone (24 h), respectively. After drying overnight under vacuum at 120 °C, a yellow COF powder was obtained.

[0034] B. The above COF powder (0.020 g) was suspended in 250 μM (15 mL) of an aqueous HAuCl4 solution and stirred vigorously in an ice bath, followed by the dropwise addition of 1 mL of a 0.01 M aqueous NaBH4 solution. The stirring was continued for 10 min, and the final product was obtained by centrifugation, water and ethanol washing, and vacuum drying. In 2 mg of the nanomaterial, 1 mL of dimethyl sulfoxide was added, mixed well, and then 1 mL of double-distilled water was added to disperse it into a 1 mg / mL solution, which was ready for use.

[0035] C. 500 μL of 100 μg / mL choline acetyltransferase antibody (Ab1) and 1500 μL of 1 mg / mL Au@COF were mixed and incubated at 37 °C for 180 min, labeled as Ab1-Au@COF.

[0036] (2) Choline acetyltransferase analysis sensor method and application

[0037] A. 500 μL of 100 μg / mL choline acetyltransferase antibody (Ab2) and 1500 μL of 500 nM scGFP were mixed and incubated at 37 °C for 180 min, labeled as Ab2-scGFP.

[0038] B1. Fluorescence method - Take 10 μL Ab1-Au@COF solution in step (1C) and 10 μL Ab2-scGFP in step (2C), 50 μL 0.05M PBS (pH 7.2), 20 μL ultrapure water, and then add 10 μL 1000 mU / mL ChAT, incubate at 37°C for 10 min. Place in a fluorescence instrument to measure the fluorescence intensity in the wavelength range of 430-650 nm (slit width is 10 nm).

[0039] B2. Electrochemical method - clean the bare glassy carbon electrode (GCE), then mix 2 μL Ab2-scGFP in step (2C), 2 μL 1 mg / mL graphene (GO), and 6 μL 0.2 wt% chitosan, incubate at 37°C for 10 min, then drop on the surface of GCE and incubate at 37°C for 10 min, gently wash the electrode with 0.05M PBS (pH 7.2) (electrode 2). Take 5 μL 1000 mU / mL ChAT and drop on the surface of the above electrode, stand at 37°C for 10 min (electrode 3), without washing, add 5 μL Ab1-Au@COF solution in step (1C), incubate at 37°C for an additional 20 min (electrode 4). Then place in 0.05M PBS (pH 7.2) containing 5 mM H2O2 for electrochemical detection, the potential range is -1.2-0 V (scanning speed is 50 mV / s). In addition, mix 2 μL 1 mg / mL graphene (GO), 6 μL 0.2 wt% chitosan, and 2 μL ultrapure water, incubate at 37°C for 10 min, then drop on the surface of GCE and incubate at 37°C for 10 min, gently wash the electrode with 0.05M PBS (pH 7.2) to prepare electrode 1 as a control.

[0040] As shown in Figure 1 A, without ChAT, the fluorescence intensity at 506 nm is large, and when there is ChAT, the fluorescence intensity at 506 nm is small, because when the interaction between ChAT and its antibody occurs, the distance between Au@COF and scGFP becomes smaller, and Au@COF has a quenching effect on scGFP fluorescence; as shown in Figure 1 B, as the concentration of ChAT (final concentration: 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 20, 50, 100, 150 mU / mL) increases, the fluorescence intensity decreases, and the fluorescence response is linearly related to the concentration of ChAT, the linear equation is y = -1097.8x + 3938.2, R 2 = 0.9827, the linear range is 0.01-150 mU / mL, and the lowest concentration detected is 0.01 mU / mL, indicating that this sensing method can realize fluorescence detection of ChAT with different concentrations.

[0041] As shown inFigure 2 As shown in A, when ChAT exists, there is a significant electrochemical response at -0.9V, and when ChAT does not exist, there is a smaller electrochemical response at -0.9V, because after the interaction between ChAT and its antibody, Au@COF is solidified to the electrode surface, and the material can electrocatalytically reduce H2O2 to produce a better electrochemical signal; as shown in Figure 2 B, as the concentration of ChAT (final concentration: 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 20, 50, 100 mU / mL) increases, the current intensity increases, and the current response is linearly related to the ChAT concentration equation y = 7.3316x + 25.7063, R 2 = 0.9907, the linear range is 0.005-100 mU / mL, and the lowest concentration detected is 0.005 mU / mL; it shows that the sensing method can realize electrochemical detection of ChAT with different concentrations.

[0042] Example 2 Selective analysis

[0043] As shown in Figure 3 Example 1, ChAT was selected as the research object, and the enzymes used in Example 2 were other enzymes such as protein kinase (PKA), thrombin, urease, glucose oxidase (GOx) and lysozyme, and the sensor only had a good response to ChAT in the two modes, and had good selectivity.

[0044] Example 3 ChAT recovery rate test in cell samples

[0045] After the myocardial tissue cells were pulverized, they were placed in a 0.15M sodium chloride solution for 1h, and then 10μL of the lysate was used instead of ChAT in Example 1 to complete the experiment. Because of the greater interference in the complex system, no ChAT originally present was found, so 5 different concentrations (final concentration: 0.05, 0.1, 0.5, 1, 10 mU / mL) of cell samples were added, and then the experiment was completed by replacing ChAT in Example 1, as shown in Figure 4 The recovery rates were 98.9%-102.3% in fluorescence mode and 97.2%-102.5% in electrochemical mode, respectively, indicating that the sensor can be well applied to ChAT analysis and detection in actual cells.

[0046] Of course, the above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by those of ordinary skill in the art within the scope of the present application should also be within the scope of the present application.

Claims

1. A choline acetyltransferase dual mode assay method based on antigen-antibody interaction for non-disease diagnosis and treatment purposes, characterized by, The specific steps are as follows: (1) Au@COF synthesis A. 2, 4, 6-tris (4-formylphenyl) triazine, tris (4-aminophenyl) amine, dichlorobenzene and n-butanol were mixed in a round-bottom flask, stirred for 2-10 min, then 0.1-0.2 mL of acetic acid was added, and stirred at room temperature under nitrogen atmosphere for 12-72 h; after the reaction was completed, the precipitate was filtered out, and was extracted with tetrahydrofuran, methanol and acetone respectively; after vacuum drying at 80-120℃ overnight, yellow COF powder was obtained; B. The above COF powder was suspended in 100-250 μM HAuCl4 aqueous solution and stirred vigorously in an ice bath, then 0.1-1 mL of 0.005-0.01 M NaBH4 aqueous solution was added dropwise, and stirring was continued for 5-10 min. The final product Au@COF nanomaterial was obtained by centrifugation, water and ethanol washing, and vacuum drying; 0.1-1 mL of dimethyl sulfoxide was added to 1-2 mg of the nanomaterial, mixed well, then 0.1-1 mL of double-distilled water was added, and a 0.2-1 mg / mL solution was prepared for use; C. 200-500 μL of 20-100 μg / mL choline acetyltransferase antibody Ab1 and 500-1500 μL of 0.2-1 mg / mL Au@COF were mixed and incubated at 30-37℃ for 20-180 min, labeled as Ab1-Au@COF; (2) Choline acetyltransferase analysis sensing method and application A. 200-500 μL of 50-100 μg / mL choline acetyltransferase antibody Ab2 and 500-1500 μL of 100-500 nM scGFP were mixed and incubated at 30-37℃ for 50-180 min, labeled as Ab2-scGFP; B1. Fluorescence method - 2-10 μL of Ab1-Au@COF solution in step (1) C and 2-10 μL of Ab2-scGFP in step (2) A, 10-50 μL of 0.01-0.05 M PBS, 2-20 μL of ultrapure water, and 5-10 μL of 200-1000 mU / mL ChAT were added, and incubated at 32-37℃ for 5-10 min; the fluorescence intensity in the wavelength range of 430-650 nm was measured in a fluorescence instrument; B2. Electrochemical method - clean the bare glassy carbon electrode GCE, then mix 1-2 μL of Ab2-scGFP in step (2) A, 1-2 μL of 0.2-1 mg / mL graphene GO and 2-6 μL of 0.1-0.2 wt% chitosan, incubate at 30-37 °C for 2-10 min, then drop on the surface of GCE and incubate at 30-37 °C for 2-10 min, and then wash the electrode with 0.05 M PBS; then take 1-5 μL of 200-1000 mU / mL ChAT and drop on the surface of the above electrode, and stand at 30-37 °C for 5-10 min, then add 1-5 μL of Ab1-Au@COF solution in step (1) C without washing, and incubate at 30-37 °C for an additional 10-20 min; then place it in 0.05 M PBS containing 5 mM H2O2 for electrochemical detection, with a potential range of -1.2-0 V; in addition, mix 1-2 μL of 0.1-1 mg / mL graphene GO, 2-6 μL of 0.2 wt% chitosan and 1-2 μL of ultrapure water, incubate at 30-37 °C for 5-10 min, then drop on the surface of GCE and incubate at 25-37 °C for 5-10 min, and then wash the electrode with 0.05 M PBS to prepare electrode 1 as a control.

2. The dual mode analysis method of claim 1, wherein, Based on steps (1)-(2), by changing the concentration of ChAT, and keeping other steps unchanged, the analysis of choline acetyltransferase of different concentrations can be realized.

3. The dual mode analysis method of claim 1, wherein, In the fluorescence method, the prepared Au@COF is used to quench the fluorescence of scGFP, and ChAT is used to control the change process, realizing the fluorescence analysis and detection of ChAT; by changing the concentration of ChAT, a series of fluorescence intensity corresponding to ChAT of different concentrations is obtained, and the linear relationship between fluorescence intensity response and ChAT concentration is established, and according to the linear relationship between them, the concentration of ChAT in the actual cell sample is determined.

4. The dual mode analysis method of claim 1, wherein, In the electrochemical method, the electrochemical catalytic effect of Au@COF on H2O2 is used, and ChAT is used to control the change process, realizing the electrochemical analysis and detection of ChAT; by changing the concentration of ChAT, a series of electrochemical current corresponding to ChAT of different concentrations is obtained, and the linear relationship between current response and ChAT concentration is established, and according to the linear relationship between them, the concentration of ChAT in the actual cell sample is determined.

5. The dual mode analysis method of claim 3, wherein, The linear equation of the fluorescence response to ChAT concentration was y = -1097.8x + 3938.2, R = 0.9827, the linear range was 0.01-150 mU / mL, and the lowest concentration detected was 0.01 mU / mL. 2 The linear equation of the fluorescence response to ChAT concentration was y = -1097.8x + 3938.2, R = 0.9827, the linear range was 0.01-150 mU / mL, and the lowest concentration detected was 0.01 mU / mL.

6. The dual mode analysis method of claim 4, wherein, The equation of linear correlation between current response and ChAT concentration was y = 7.3316x + 25.7063, R = 0.9907, the linear range was 0.005-100 mU / mL, and the lowest concentration detected was 0.005 mU / mL. 2 The equation of linear correlation between current response and ChAT concentration was y = 7.3316x + 25.7063, R = 0.9907, the linear range was 0.005-100 mU / mL, and the lowest concentration detected was 0.005 mU / mL.

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