CN114460159B - ALP activity detection kit based on photo-ATRP signal amplification strategy and application method thereof - Google Patents
ALP activity detection kit based on photo-ATRP signal amplification strategy and application method thereof Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
Abstract
The invention discloses an ALP activity detection kit based on a photo-ATRP signal amplification strategy and a use method thereof, wherein the kit comprises the following raw materials: electrode, MPA, EDC, NHS, O-phosphoethanolamine, BIBB, DMSO, EY, me 6 TREN、FMMA、LiClO 4 . The invention uses the photo-ATRP reaction as a polymerization reaction signal amplification strategy to obviously improve the sensitivity of ALP activity detection, and simultaneously avoids the use of transition metal catalysts, and has the advantages of high efficiency, convenient operation and environmental friendliness. Under optimal experimental conditions, the linear range of the method for ALP activity detection is10-150 mU/mL, and the detection Limit (LOD) is 2.12mU/mL, which shows that the method has good sensitivity. The method has satisfactory selectivity, anti-interference performance, reproducibility and stability, and good experimental results are obtained in clinical serum samples and inhibition rate experiments.
Description
Technical Field
The invention relates to an alkaline phosphatase (ALP) activity detection kit based on a photo-mediated atom transfer radical polymerization (photo-ATRP) signal amplification strategy and a use method thereof, belonging to the technical field of biological analysis.
Background
Alkaline phosphatase (ALP) is a homodimeric metalloprotease widely occurring in prokaryotes and eukaryotes, where zinc and magnesium atoms in the structure play a critical role in ALP dephosphorylation. ALP catalyzes the hydrolysis of phosphomonoester structures in proteins, nucleic acids, and small molecules under alkaline conditions. ALP therefore plays an important role in physiological functions such as cell division, osteogenesis, mineralization, detoxification, etc. Studies have shown that abnormal ALP activity is closely associated with a variety of diseases, for example elevated ALP activity in serum is often associated with biliary tract obstruction, osteoblastic bone tumors, osteomalacia, leukemia-like response or lymphomas, etc.; in contrast, some metabolic diseases, such as wilson's disease, chronic granulocytic leukemia, etc., cause pathological inhibition of ALP activity, which is below normal. Therefore, ALP activity detection is of great importance for diagnosis and treatment of related diseases. Currently, the detection methods of ALP mainly comprise chromatography, surface enhanced Raman scattering, chemiluminescence, fluorescence, colorimetry, electrochemiluminescence and the like. With further understanding of the role of ALP in different body fluids or cellular locations, and with the ever-expanding types of samples, higher demands are placed on the precision and sensitivity of ALP detection. Therefore, developing a sensitive, simple, and efficient ALP detection method is a current urgent problem to be solved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an ALP activity detection kit based on a photo-ATRP signal amplification strategy and a use method thereof, which not only avoid the use of a transition metal catalyst, but also remarkably increase the output of electrochemical signals, and have the advantages of high sensitivity, good selectivity, simple operation, environmental friendliness and the like.
In order to achieve the above object, the technical scheme of the present invention is as follows:
ALP activity detection kit based on photo-ATRP signal amplification strategy, including the following raw materials: electrode, MPA, EDC, NHS, O-phosphoethanolamine, BIBB, DMSO, EY, me 6 TREN、FMMA、LiClO 4 。
When in use, part of raw materials are prepared into a solution, wherein the concentration of MPA solution is 10mM, the concentration of EDC in EDC/NHS mixed solution is 20mM, the concentration of NHS is 5mM, the concentration of O-phosphoethanolamine solution is 10mM, the concentration of BIBB solution is 10mM, the concentration of EY solution is 5mM, and Me 6 TREN solution concentration of 10mM, FMMA solution concentration of 10mM, liClO 4 The concentration of the solution was 1M.
A method of using an ALP activity assay kit comprising the steps of:
(1) MPA modification
Immersing the gold electrode in MPA solution, and incubating for 2-8 h at 25-37 ℃;
(2) Activation of MPA carboxyl group and modified phosphoethanolamine
Soaking the electrode obtained in the step (1) in EDC/NHS mixed solution, incubating for 0.5-1 h at 37 ℃, then soaking the electrode in O-phosphoethanolamine solution, and incubating for 1-2 h at 37 ℃;
(3) ALP dephosphorylation and BIBB modification
Dropping the solution to be detected on the surface of the electrode obtained in the step (2), incubating for 1-2 h at 37 ℃, then soaking the electrode in BIBB solution, and incubating for 0.5-2 h at 37 ℃;
(4) Light mediated ATRP reaction
Immersing the electrode obtained in the step (3) in a photo-ATRP reaction liquid, irradiating with blue light, and reacting for 3-6 h at 25-37 ℃;
(5) SWV detection
Immersing the electrode obtained in the step (4) into LiClO 4 In solution, and Square Wave Voltammetry (SWV) electrochemical measurements were performed.
Further, the gold electrode is pretreated firstly, and the pretreatment method comprises the following steps:
(1) respectively ultrasonically cleaning a gold electrode by using absolute ethyl alcohol and water, polishing the electrode by using alumina polishing powder with the particle size of 0.3 mu m and alumina polishing powder with the particle size of 0.05 mu m, and respectively ultrasonically cleaning the electrode by using absolute ethyl alcohol and ultrapure water;
(2) after washing, the gold electrode is put into a water tiger fish acid solution to be soaked, and then ultrasonic washing is respectively carried out by absolute ethyl alcohol and ultrapure water;
(3) the electrode is soaked in 0.5M sulfuric acid solution, the potential is set to be-0.3-1.5V, the scanning speed is 0.1V/s, the scanning section number is 40, until a repeatable cyclic voltammogram is obtained, ultra-pure water is ultrasonically cleaned, and nitrogen is blown dry for later use.
Further, the photo-ATRP reaction solution was prepared from 5mL of ultrapure water, 3990. Mu.L of DMSO, 5. Mu.L of EY solution, and 5. Mu.L of Me 6 TREN solution and 1mL FMMA solution.
Further, square Wave Voltammetry (SWV) electrochemical measurements range from 0 to 0.6V potential.
An application of the kit in detecting ALP activity.
An application of the kit in screening ALP inhibitors.
The detection principle of the invention is schematically shown in figure 1.
The invention has the beneficial effects that:
1. the invention uses the photo-ATRP reaction as a polymerization reaction signal amplification strategy to obviously improve the sensitivity of ALP activity detection.
2. The invention uses the photo-ATRP reaction as a signal amplifying strategy, avoids the use of transition metal catalysts, and has the advantages of high efficiency, convenient operation and environmental protection.
3. According to the ALP activity detection method based on the photo-ATRP signal amplification strategy, firstly, 3-mercaptopropionic acid (MPA) is self-assembled and fixed on the surface of a gold electrode through gold-sulfur bonds, so that an ALP substrate O-phosphoethanolamine can be connected to the surface of the electrode through amide bonds; in the presence of ALP, the phosphoric monoester structure in O-phosphoethanolamine is hydrolyzed into hydroxyl, and can be connected with an ATRP initiator BIBB; finally, under 470nm blue light irradiation, EY is used as a photocatalyst to carry out polymerization reaction, so that Ferrocenyl Methyl Methacrylate (FMMA) monomer is polymerized on the surface of the electrode. The polymer grafting containing a large amount of ferrocene signal labels obviously enhances the electrochemical signal output. Under the optimal experimental conditions, the linear range of the method for ALP activity detection is 10-150 mU/mL, and the detection Limit (LOD) is 2.12mU/mL, which shows that the method has good sensitivity. The method has satisfactory selectivity, anti-interference performance, reproducibility and stability, and good experimental results are obtained in clinical serum samples and inhibition rate experiments. Therefore, the photo-ATRP signal amplification strategy has good application potential in the aspect of clinical ALP related disease diagnosis and inhibitor screening.
Drawings
FIG. 1 is a schematic diagram of the detection principle of the present invention.
FIG. 2 is a graph showing the feasibility of the method of the invention for ALP activity detection.
FIG. 3 is an Electrochemical Impedance Spectroscopy (EIS) diagram of a step-wise modified electrode.
FIG. 4 is a CV plot of modified electrodes at different scan rates (inset: showing the linear relationship between reduction and oxidation current peaks and different scan rates).
FIG. 5 is an Atomic Force Microscope (AFM) image of a modified electrode before and after polymerization, A is before the photo-ATRP reaction, and B is after the photo-ATRP reaction.
FIG. 6 is a graph of Water Contact Angle (WCA) for a step-wise modified electrode.
FIG. 7 is an optimization of BIBB reaction time.
FIG. 8 EY and Me 6 The concentration ratio of TREN is optimized.
FIG. 9 is a graph showing the linear relationship between ALP activity and electrical signal intensity.
FIG. 10 is a selective study of ALP detection.
FIG. 11 is a study of the interference resistance of the electrochemical test of the present invention.
FIG. 12 is a study of the inhibition of ALP activity by sodium vanadate.
Detailed Description
The following describes the embodiments of the present invention in further detail with reference to examples.
Example 1: kit for detecting a substance in a sample
ALP activity detection kit based on photo-ATRP signal amplification strategy, including the following raw materials: electrode, 3-mercaptopropionic acid (MPA), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), O-phosphoethanolamine, 2-bromoisobutyryl bromide (BIBB), dimethyl sulfoxide (DMSO), eosin Y (EY), tris- (N, N-dimethylaminoethyl) amine (Me) 6 TREN), ferrocenylmethacrylate (FMMA), liClO 4 。
When in use, part of raw materials are prepared into a solution, wherein the concentration of MPA solution is 10mM, the concentration of EDC in EDC/NHS mixed solution is 20mM, the concentration of NHS is 5mM, the concentration of O-phosphoethanolamine solution is 10mM, the concentration of BIBB solution is 10mM, the concentration of EY solution is 5mM, and Me 6 TREN solution concentration of 10mM, FMMA solution concentration of 10mM, liClO 4 The concentration of the solution was 1M.
Example 2: ALP activity detection method
(1) MPA modification
The pretreated gold electrode was immersed in 400. Mu.L of 10mM MPA solution and incubated at 37℃for 2h;
(2) Activation of MPA carboxyl group and modified phosphoethanolamine
Immersing the electrode obtained in the step (1) in 400 mu L of EDC/NHS mixed solution (EDC concentration is 20mM, NHS concentration is 5 mM), incubating at 37 ℃ for 0.5h, and then immersing the electrode in 400 mu L of 10mM O-phosphoethanolamine solution, incubating at 37 ℃ for 1h;
(3) ALP dephosphorylation and BIBB modification
Dropping 10 mu L of solution to be detected (containing ALP) on the surface of the electrode obtained in the step (2), incubating for 1h at 37 ℃, and then immersing the electrode in 400 mu L of 10mM BIBB solution, and incubating for 1h at 37 ℃;
(4) Light mediated ATRP reaction
Immersing the electrode obtained in the step (3) in a photo-ATRP reaction solution (the reaction solution is prepared by sequentially adding 3990. Mu.L of DMSO, 5. Mu.L of 5mM EY solution, 5. Mu.L of 10mM Me to 5mL of ultra-pure water) 6 TREN solution, 1mL of 10mM FMMA solution), using 470nm blue light irradiation, and reacting for 3h at room temperature;
(5) SWV detection
Immersing the electrode obtained in the step (4) into 1M LiClO 4 Square Wave Voltammetry (SWV) electrochemical measurements were performed in solution and at a scan potential ranging from 0 to 0.6V.
The pretreatment method of the gold electrode comprises the following steps:
(1) ultrasonically cleaning a gold electrode by using absolute ethyl alcohol and water for 1min respectively, polishing the electrode by using alumina polishing powder with the particle size of 0.3 mu m and alumina polishing powder with the particle size of 0.05 mu m for 3-5 min respectively, and ultrasonically cleaning the electrode by using absolute ethyl alcohol and ultrapure water for 1min respectively;
(2) after washing, the gold electrode is put into a water tiger fish acid solution to be soaked for 15-20 min, and then the gold electrode is respectively washed by absolute ethyl alcohol and ultrapure water in an ultrasonic manner for 1min; the preparation method of the water tiger fish acid solution comprises the following steps: 98% H 2 SO 4 And H 2 O 2 Mixing in a volume ratio of 3:1;
(3) the electrode is soaked in 0.5M sulfuric acid solution, the potential is set to be-0.3-1.5V, the scanning speed is 0.1V/s, the scanning section number is 40, until a repeatable cyclic voltammogram is obtained, ultra-pure water is ultrasonically cleaned, and nitrogen is blown dry for later use.
Example 3: feasibility study
This study investigated the feasibility of the method of the invention to detect alkaline phosphatase (ALP) activity by a series of blank control experiments. The SWV curves for the various modified electrodes can be seen in fig. 2. In the absence of MPA (curve b), O-phosphoethanolamine (curve c), ALP (curve d), BIBB (curve e), FMMA (curve f),EY (curve g), me 6 In TREN (curve h) and no light (curve i), there is little electrochemical signal response, except for a weak background signal. When the electrode is modified stepwise according to the method of the present invention, a distinct oxidation current signal (curve a) is observed, with a peak potential of about 0.28V, with ferrocene electroactive molecules at LiClO 4 The potential ranges in the solutions are consistent. The generation of the oxidation current can be attributed to the electrochemical oxidation of the ferrocene electroactive molecule, demonstrating that monomeric FMMA was successfully attached to the gold electrode by the photo-ATRP reaction. Thus, the fully modified electrode is feasible for ALP activity detection.
Example 4: electrochemical characterization and morphological characterization
1. Electrochemical characterization
Electrochemical Impedance Spectroscopy (EIS) can sensitively detect solid-liquid interface transitions, which are used to characterize the gradual modification of the electrode surface. In the Nyquist diagram, the diameter of the semicircle in the high frequency region is equal to the charge transfer resistance (R ct ). The EIS diagram of the electrode is shown in figure 3, and the R of the bare gold electrode is due to the good conductivity of the gold electrode ct Minimum, about 0.29kΩ (a). MPA self-assembly hinders electron transfer at the electrode surface, resulting in R ct Up to 0.44kΩ (b). After EDC, NHS activates the carboxyl group of MPA, the negatively charged carboxyl group is replaced by succinimidyl ester, R ct Down to 0.23kΩ (c). Modification of O-phosphoethanolamine to R ct To 0.63kΩ (d). After ALP incubation, R ct Slightly drop (e). After modification of initiator BIBB, R ct Further to 1.02kΩ (f). Finally, because the polymer chain is grafted on the surface of the electrode, R ct Significantly increased to 1.99kΩ (g). These results demonstrate that the construction of the fully modified electrode was successful and successful. Successful grafting of electroactive polymers onto gold electrodes by the photo-ATRP strategy was demonstrated.
To demonstrate the success of the ATRP reaction, cyclic Voltammetry (CV) scan was used for the different modified electrodes. In LiClO 4 And scanning different modified electrodes CV in the solution, wherein the potential range is 0-0.6V. As can be seen from FIG. 4, the redox increases as the scan rate increases from 0.01V/s to 1.0V/sThe peak current varies linearly. This demonstrates that the redox process of ferrocene molecules is not by electrostatic adsorption, but rather by covalent bonding to the gold electrode surface.
2. Morphological characterization
The morphology of the modified electrode was observed with an Atomic Force Microscope (AFM), and by comparing the change in the surface height of the gold electrode before and after polymerization, it was confirmed that the monomer successfully formed a polymer chain by the target ATRP reaction (fig. 5). As can be seen from FIG. 5, after modification of initiator BIBB, the gold electrode height was 8.9nm (A), and the surface height was increased to 29.7nm (B) after the photo-ATRP reaction. The increase in the height of the gold electrode surface demonstrates the successful formation of the monomer into a polymer by the photo-ATRP reaction.
The stepwise modified electrode was characterized by Water Contact Angle (WCA) depending on the hydrophilicity of the modified electrode surface groups. As a result, as shown in FIG. 6, since the gold electrode has a very strong hydrophobicity, the WCA of the bare gold electrode is 90.6. After MPA modification of the gold electrode, the introduction of carboxyl groups reduced WCA (84.7 °). Subsequently, the WCA increased (88.2 °) after modification with the hydrophobic O-phosphoethanolamine of the carbon chain. After ALP catalyzes the hydrolysis of O-phosphoethanolamine, hydroxyl formation reduces WCA to 86.6. Modification of initiator BIBB, in turn, resulted in an increase in WCA (90.2). Finally, the WCA increased significantly to 93.6 ° due to the high hydrophobicity of the polymer chains. The change in WCA further indicates successful modification of the electrode.
Example 5: detection condition optimization
In order to achieve the best performance of the detection method, the invention investigated the reaction (incubation) time of BIBB and EY and ME 6 TERN molar ratio.
(1) Optimization of BIBB reaction time
In this study, signal responses were recorded with SWV over 15-90 min for BIBB modification time. The results are shown in FIG. 7, where the SWV response gradually increases with increasing reaction time. Subsequently, there was no significant change in current intensity after 60 min. Thus, the optimum reaction time for BIBB in this method is 60min and is used in the subsequent studies.
(2) EY and Me 6 Concentration ratio optimization of TREN
The study is based on the redox reaction of ferrocene molecules, EY and ME 6 The molar ratio of TERN is critical and must be optimized. Control EY and ME 6 TERN's are 5. Mu.L in volume and ME 6 The concentration of TERN was 10mM, and the EY concentration was changed. As a result, as shown in FIG. 8, the current signal increases with decreasing EY concentration, at EY and ME 6 The molar ratio of TERN was 0.5: maximum value is reached at 1. Along with EY and ME 6 The molar ratio of TERN is gradually less than 0.5:1, and the current signal drops rapidly with decreasing EY concentration. Thus EY and ME 6 The optimal molar ratio of TERN is 0.5:1.
In summary, BIBB has an optimal reaction time of 60min, EY and ME 6 The optimal molar concentration ratio of TERN was 0.5:1.
Example 6: analytical performance
The assay performance of the method of the invention was studied using a series of ALPs of different activities under the best experimental conditions obtained in example 5. As a result, as shown in FIG. 9, in the range of 10 to 150mU/mL of ALP activity, the current intensity increased with increasing ALP activity and in good linear relationship, the linear equation was I (μA) = 0.01933C ALP (mU/mL)+0.19188，R 2 =0.998, the lowest limit of detection LOD was calculated to be 2.12mU/mL. Compared with the reported ALP activity detection method, the strategy has relatively high sensitivity. Therefore, the proposed method has good application potential in detecting ALP activity clinically.
Example 7: selectivity, interference resistance, stability and reproducibility
To verify the selectivity of the strategy of the invention for ALP activity detection, the oxidation current intensity of Pepsin (Pepsin), bovine Serum Albumin (BSA), glucose oxidase (GOx) and ALP were compared under the same conditions under the optimal experimental conditions obtained in example 5. The concentrations of ALP, pepsin, GOx and BSA were 50mU/mL, 50mU/mL and 50. Mu.M, respectively. As a result, as shown in fig. 10, a significant current response was observed only in the ALP group. The results show that the strategy has higher selectivity for ALP activity detection.
To evaluate the anti-interference ability of the constructed fully modified electrode, the electrochemical signals of different active ALPs in Tris-HCl buffer and 10% human serum were compared under the optimal experimental conditions obtained in example 5. As a result, as shown in FIG. 11, the current signal of 10% human serum was 97.5% (80 mU/mL), 100.4% (100 mU/mL) and 97.4% (120 mU/mL) of Tris-HCl buffer. The results show that the method has good anti-interference capability in serum matrix.
To verify the stability of the fully modified electrode, the modified electrode was tested after storage for 3 weeks in a 4 ℃ moisture saturated environment and the current intensity was found to be 92.30% of the signal intensity of the freshly prepared electrode. This indicates that the fully modified electrode has good stability. Reproducibility of the fully modified electrodes was evaluated by intra-and inter-group experiments (n=5), with relative standard deviations of 3.54% and 4.13%, respectively, between groups. The results show that the completely modified electrode has good reproducibility.
Example 8: detection Performance in actual samples
The reliability of the method of the present invention was verified using clinical human serum samples, 5 clinical human serum samples were obtained from a third affiliated hospital at Henan traditional Chinese medicine university, ALP activity in serum was detected using the method of the present invention and compared with the provided clinical data (AMP buffer method detection). The results are shown in Table 1, and the relative error between the results obtained by the method and clinical data is less than 5%, which indicates that the detection method is reliable.
Table 1: comparison of AMP buffer method with results of the method of the invention
Example 9: inhibition test
To investigate the effectiveness of the method of the invention in the screening of ALP activity inhibitors, sodium vanadate Na 3 VO 4 Inhibition experiments were performed for the model. Pre-activating Na at different concentrations 3 VO 4 The solution was mixed with ALP sample and Na was investigated by the change of current intensity 3 VO 4 Inhibition of ALP activity. The results are shown in FIG. 12, as Na 3 VO 4 The concentration increases, and the inhibition efficiency increases. Description of Na 3 VO 4 Inhibition of ALP activity is concentration dependent. Thus, the fully modified electrode of the invention has proven to be suitable for screening alkaline phosphatase inhibitors.
Claims (6)
1. ALP activity detection kit based on photo-ATRP signal amplification strategy, characterized by comprising the following raw materials: electrode, MPA, EDC, NHS, O-phosphoethanolamine, BIBB, DMSO, EY, me 6 TREN、FMMA、LiClO 4 The method comprises the steps of carrying out a first treatment on the surface of the The application method comprises the following steps:
(1) MPA modification of 3-mercaptopropionic acid
Immersing a gold electrode in an MPA solution, and incubating at 25-37 ℃ for 2-8 h;
(2) Activation of MPA carboxyl group and modified phosphoethanolamine
Immersing the electrode obtained in the step (1) in EDC/NHS mixed solution, incubating at 37 ℃ for 0.5-1 h, immersing the electrode in O-phosphoethanolamine solution, and incubating at 37 ℃ for 1-2 h;
(3) ALP dephosphorylation and BIBB modification
Dropping the solution to be detected on the surface of the electrode obtained in the step (2), incubating 1-2 h at 37 ℃, then soaking the electrode in BIBB solution, and incubating 0.5-2 h at 37 ℃;
(4) Light mediated ATRP reaction
Immersing the electrode obtained in the step (3) in a photo-ATRP reaction liquid, irradiating with blue light, and reacting at 25-37 ℃ for 3-6 h;
(5) SWV detection
Immersing the electrode obtained in the step (4) into LiClO 4 In the solution, performing square wave voltammetry SWV electrochemical measurement;
the photo-ATRP reaction solution was prepared from 5mL ultrapure water, 3990. Mu.L DMSO, 5. Mu.L EY solution, and 5. Mu.L Me 6 The TREN solution and 1mL of FMMA solution were mixed.
2. The ALP activity detection kit according to claim 1, whereinWhen in use, part of raw materials are prepared into a solution, wherein the concentration of MPA solution is 10mM, the concentration of EDC in EDC/NHS mixed solution is 20mM, the concentration of NHS is 5mM, the concentration of O-phosphoethanolamine solution is 10mM, the concentration of BIBB solution is 10mM, the concentration of EY solution is 5mM, and Me 6 TREN solution concentration of 10mM, FMMA solution concentration of 10mM, liClO 4 The solution concentration was 1M.
3. The ALP activity detection kit of claim 1, wherein the gold electrode is pretreated by:
(1) respectively ultrasonically cleaning a gold electrode by using absolute ethyl alcohol and water, polishing the electrode by using alumina polishing powder with the particle size of 0.3 mu m and alumina polishing powder with the particle size of 0.05 mu m, and respectively ultrasonically cleaning the electrode by using absolute ethyl alcohol and ultrapure water;
(2) after washing, the gold electrode is put into a water tiger fish acid solution to be soaked, and then ultrasonic washing is respectively carried out by absolute ethyl alcohol and ultrapure water;
(3) the electrode is soaked in 0.5M sulfuric acid solution, the potential is set to be-0.3-1.5V, the scanning speed is 0.1V/s, the scanning section number is 40, until a repeatable cyclic voltammogram is obtained, ultra-pure water is used for ultrasonic cleaning, and nitrogen is used for drying for standby.
4. The ALP activity detection kit of claim 1, wherein the square wave voltammetry SWV electrochemical measurement is scanned at a potential ranging from 0 to 0.6V.
5. Use of a kit according to claim 1 or 2 for detecting ALP activity.
6. Use of a kit according to claim 1 or 2 for screening an ALP inhibitor.
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