A method for detecting alkaline phosphatase activity based on CRISPR / Cas12a and DNAzyme

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a method for detecting alkaline phosphatase activity based on CRISPR / Cas12a and DNAzyme. The present invention utilizes alkaline phosphatase to specifically catalyze the hydrolysis reaction of PPi (pyrophosphate) to improve the detection specificity of the method for alkaline phosphatase. Specifically: after alkaline phosphatase catalyzes the hydrolysis of PPi, Cu 2+ that is not complexed by PPi is removed. 2+ It can assist DNAzyme in catalyzing the cutting of substrate chains. The cut substrate chains cannot further bind to the binary complex of Cas12a / crRNA, the trans-cutting activity of CRISPR / Cas12a is silenced, the single-stranded DNA reporter probe cannot be cleaved, the fluorescence is quenched, and the alkaline phosphatase can be specifically analyzed by the fluorescence intensity of the DNA reporter probe. The method of the present invention is simple to operate, highly sensitive, and has good selectivity, and can be used to detect alkaline phosphatase activity in a variety of bioactive substances.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for detecting alkaline phosphatase activity based on CRISPR / Cas12a and DNAzyme. Background Art

[0002] Alkaline phosphatase (ALP) is a phosphomonoester hydrolase that specifically acts on a variety of substrates such as proteins, nucleic acids and small molecules, and regulates dephosphorylation reactions in the human intestine, bones, liver and kidneys. Abnormal alkaline phosphatase activity affects the cell regulation process in the body. Studies have shown that abnormal alkaline phosphatase levels are closely related to a variety of diseases such as breast cancer, prostate cancer, diabetes, and liver dysfunction. Therefore, the development of an efficient and sensitive alkaline phosphatase activity analysis method is of great significance for clinical disease diagnosis and biomedical research. In recent years, various analytical methods have been used for alkaline phosphatase activity detection, such as colorimetry, fluorescence, electrochemistry, photoelectrochemistry, etc. Among them, the fluorescence method has received widespread attention due to its advantages such as high sensitivity, fast response, and convenient design.

[0003] Deoxyribozyme (DNAzyme) is a nucleic acid obtained through in vitro screening, with specific metal ions as coenzyme factors and catalytic cleavage activity on certain substrates. Compared with proteases, it has the advantages of good stability, low cost, and easy synthesis. It is widely used in heavy metal detection, enzyme activity analysis, antibiotic residue detection, etc. Metal ion-dependent DNAzyme catalyzes the cleavage of substrate chains, and the free enzyme chain and metal ions continue to cut the next substrate chain, triggering the next cycle. After multiple cycles of cleavage, a large number of broken substrate chains are generated, realizing signal cyclic amplification. Single amplification may no longer be able to meet clinical detection needs. Consider adding a new amplification technology to achieve dual signal amplification and further improve the sensitivity of detection.

[0004] The CRISPR / Cas12a system consists of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas proteins), which have cis- and trans-cutting activity. The target DNA specifically hybridizes with CRISPR RNA (crRNA), allosterically activates the Cas12a protein, and cis-cuts the target DNA. Fluorescence and quenching groups are respectively labeled at both ends of non-specific single-stranded DNA (ssDNA) as a signal giving method. After cutting the target DNA, the protein activity remains, and the non-target ssDNA near the Rucvc domain is cut in trans, with high cutting efficiency and fast speed. The CRISPR / Cas12a system is used to construct a biosensor to achieve signal amplification and significantly improve the sensitivity of the biosensor.

[0005] In the prior art, a kit is often used to detect the activity of alkaline phosphatase. For example, patent application CN111721756A discloses a kit for detecting the activity of alkaline phosphatase and a rapid detection method, which includes a colorimetric reagent and a standard colorimetric card. During the detection process, the colorimetric result is mainly compared with the standard colorimetric card to quickly determine the concentration and activity range of ALP in the sample and whether it has infiltrated into raw milk, thereby determining the degree of inactivation of pathogenic microorganisms or the infiltration and contamination of raw milk during processing; however, this method can only detect an approximate concentration range of ALP and cannot accurately measure its activity.

[0006] Similarly, Chinese patent application CN106866727A discloses the use of a 2-phosphate benzyl formate compound as a fluorescent probe in the detection of alkaline phosphatase. This compound specifically recognizes alkaline phosphatase and does not react with other phosphatases. However, this compound is prone to false positive problems when used as a fluorescent probe, thereby reducing the detection sensitivity. Summary of the invention

[0007] In view of the common defects of the prior art, the present invention provides a method for detecting alkaline phosphatase activity based on CRISPR / Cas12a and DNAzyme. The detection method provided by the present invention is simple to operate, highly sensitive, and has good selectivity, and can be used to detect alkaline phosphatase activity in human serum, mouse serum, human cervical cancer cells, etc.

[0008] In order to achieve the above technical effects, the present invention adopts the following technical solutions:

[0009] A method for detecting alkaline phosphatase activity based on CRISPR / Cas12a and DNAzyme, comprising the following steps:

[0010] S1. PPi hydrolysis: Alkaline phosphatase standards with different activity concentrations were mixed with PPi and Cu 2+ and buffer solution and then incubated to hydrolyze PPi so that PPi cannot react with Cu 2+ Combine;

[0011] S2, DNAzyme cleavage: Cu 2+ The specific DNAzyme is mixed with the solution obtained in step S1, and then ascorbic acid is added to mix well and then incubated to fully cut the substrate chain;

[0012] S3, CRISPR / Cas12a trans-cleavage: Cas12a / crRNA complex is mixed with the solution obtained in step S2, and then cutting buffer solution, dithiothreitol and single-stranded DNA reporter probe are added and mixed, and incubation is continued to allow the complete substrate chain to activate the trans-cleavage activity of Cas12a protein, resulting in the breakage of the single-stranded DNA reporter probe;

[0013] S4. Fluorescence spectrophotometer detection: The CRISPR / Cas12a trans-cleavage product obtains a signal through a fluorescence spectrophotometer, and a standard curve is constructed based on the relationship between the signal value and the activity concentration;

[0014] S5. Using the sample to be tested instead of the standard substance to obtain a fluorescent signal, and calculating the alkaline phosphatase activity in the sample to be tested based on the fluorescent signal value of the sample to be tested and the standard curve.

[0015] Preferably, the concentration range of the different concentrations of alkaline phosphatase in step S1 is 0.05-10 U / L; the concentration of PPi is 50-250 μM; the concentration of Cu 2+ The concentration is 30-80 nM; the incubation condition after mixing is 25-35° C. and incubation for 20-40 min.

[0016] Preferably, the Cu in step S2 2+ The formation conditions of specific DNAzyme are incubation at 25-35°C for 20-40 minutes, including substrate chain and enzyme chain.

[0017] Preferably, the concentration of the substrate chain is 2.5-20 nM, and the nucleotide sequence is as shown in SEQ ID NO.1; the concentration of the enzyme chain is 10-30 nM, and the nucleotide sequence is as shown in SEQ ID NO.2.

[0018] 5`-AGCTTCTTTCTAATACGGCTTACCTGC-3`(SEQ ID NO.1);

[0019] 5`-GGTAAGCCTGGGCCTCTTTCTTTTTAAGAAAGAAC-3` (SEQ ID NO. 2).

[0020] Preferably, the ascorbic acid concentration in step S2 is 30-80 μM; the incubation condition is 25-35° C., and the incubation time is 70-100 min.

[0021] Preferably, the formation conditions of the Cas12a / crRNA complex in step S3 are incubated at 25-35°C for 20-40 min; the Cas12a protein concentration is 10-20 nM; the crRNA concentration is 10-20 nM; the dithiothreitol concentration is 70-80 nM; and the single-stranded DNA reporter probe concentration is 220-260 nM.

[0022] Preferably, the nucleotide sequence of crRNA in step S3 is as shown in SEQ ID NO.3; the nucleotide sequence of the DNA reporter probe is as shown in SEQ ID NO.4; the cutting buffer solution is 10×NE buffer, with a formula of 500mM NaCl, 100mM Tris-HCl, 100mM MgCl2, 1mg / ml recombinant albumin, pH 7.9; the incubation conditions are at 20-45°C for 20-40min.

[0023] 5`-UAAUUUCUACUCUUGUAGAUGCAGGUAAGCCGUAUUAGAA-3`

[0024] (SEQ ID NO.3);

[0025] 5`-FAM-TTA TT-BHQ1-3` (SEQ ID NO. 4).

[0026] The present invention also provides an application of the detection method in detecting alkaline phosphatase activity in biological samples.

[0027] The present invention also provides a use of the CRISPR / Cas12a and DNAzyme in the preparation of an alkaline phosphatase activity detection reagent or kit.

[0028] The present invention also provides an alkaline phosphatase activity detection reagent, the detection reagent comprising the Cu 2+ Specific DNAzyme, Cas12a / crRNA complex, single-stranded DNA reporter probe (DNA Reporter).

[0029] When the fluorescence spectrophotometer is used for detection, the excitation wavelength is 480-490 nm, the emission wavelength is 500-600 nm, and the slit width is 5-10 nm.

[0030] The detection method provided by the present invention has the following detection principle: Figure 1 As shown: In the absence of alkaline phosphatase or inactivation of alkaline phosphatase, PPi molecules complex Cu 2+ , the DNAzyme catalytic cleavage activity is silenced, the substrate chain remains intact, the Cas12a / crRNA binary complex specifically recognizes the substrate chain, the CRISPR / Cas12a trans-cleavage activity is activated, the single-stranded DNAReporter is cleaved, and a fluorescent signal is generated. When alkaline phosphatase has a high activity, PPi (pyrophosphate) can be hydrolyzed. Because there are not enough PPi molecules to complex Cu 2+ , free Cu 2+ It can assist DNAzyme in catalyzing the cutting of substrate strand DNA. The cut substrate strand DNA cannot further bind to the binary complex of Cas12a / crRNA, and the trans-cutting activity of CRISPR / Cas12a is silenced, and the single-stranded DNA reporter cannot be cleaved, and the fluorescence is quenched. Therefore, sensitive detection of alkaline phosphatase activity can be achieved by analyzing the changes in the fluorescence intensity of the system.

[0031] Compared with the prior art, the detection method provided by the present invention has the following advantages:

[0032] (1) The detection method provided by the present invention uses alkaline phosphatase to specifically catalyze the hydrolysis reaction of PPi to improve the specificity of the method for alkaline phosphatase;

[0033] (2) The alkaline phosphatase activity detection method of the present invention utilizes Cu 2+ Specific DNAzyme and CRISPR / Cas12a dual amplification technology greatly improves the sensitivity of detecting alkaline phosphatase activity;

[0034] (3) The method of the present invention has the advantages of simple operation, high sensitivity, good selectivity, etc., and has a wide range of applications, and is suitable for detecting alkaline phosphatase activity in human serum, mouse serum and human cervical cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the detection principle of the alkaline phosphatase activity detection method of the present invention;

[0036] Figure 2 Fluorescence spectra of different samples analyzed for the present invention;

[0037] Figure 3The standard curve for detecting different concentrations of alkaline phosphatase by the method of the present invention;

[0038] Figure 4 This is a diagram showing the specificity verification result of the method of the present invention on alkaline phosphatase. DETAILED DESCRIPTION

[0039] The present invention is further explained below in conjunction with specific examples, but it should be noted that the following examples are only used to explain the present invention, and cannot be used to limit the present invention, and all technical solutions that are the same or similar to the present invention are within the scope of protection of the present invention. If no specific technology or conditions are specified in this embodiment, the operation is carried out according to the conventional technical methods and instrument instructions in the art; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0040] Example 1 A method for detecting alkaline phosphatase activity based on CRISPR / Cas12a and DNAzyme

[0041] The alkaline phosphatase activity detection method comprises the following process:

[0042] S1. PPi hydrolysis: 10U / L alkaline phosphatase standard solution (or HEPES buffer solution), 0.2mM PPi, 50nM Cu 2+ Mix well and incubate at 37°C for 30 min to hydrolyze PPi so that PPi cannot react with Cu 2+ Combine.

[0043] S2, DNAzyme cleavage: 20nM enzyme chain and 10nM substrate chain were incubated at 37°C for 30min to form Cu 2+ Specific DNAzyme. The solution was mixed with the solution obtained in step S1, and then 50 μM ascorbic acid was added to mix well, and the mixture was incubated for 80 min to fully cleave the substrate chain.

[0044] S3, CRISPR / Cas12a trans-cleavage: 15nM crRNA and 15nM Cas12a were mixed, 75nM dithiothreitol and 5μL 10×NE buffer were added, and the mixture was incubated for 30min to form a Cas12a / crRNA complex. The complex was mixed with the solution obtained in step S2, and 240nM single-stranded DNA reporter probe was added, and the incubation was continued for 30min to activate the trans-cleavage activity of Cas12a protein by the complete substrate chain, and the single-stranded DNA reporter probe was broken.

[0045] S4. Fluorescence spectrophotometer detection: The fluorescence intensity of the product after trans-cleavage of Cas12a protein was measured using a fluorescence spectrophotometer to analyze the activity of alkaline phosphatase. The excitation wavelength was 485 nm, the fluorescence spectrum between 500-600 nm was scanned, and the fluorescence intensity at 520 nm was used for data analysis to obtain a standard curve.

[0046] Results: Figure 2 , when alkaline phosphatase is absent or inactivated, the system has a high fluorescence intensity at 520 nm (curve a); when alkaline phosphatase is added, the fluorescence intensity decreases significantly (curve b). The results show that the method based on CRISPR / Cas12a and DNAzyme proposed in the present invention can be used for alkaline phosphatase activity detection.

[0047] Example 2 Alkaline phosphatase sensitivity detection

[0048] Under the optimal experimental conditions, different concentrations (0.05, 0.1, 1, 2, 4, 5, 8, 10 U / L) of alkaline phosphatase standard solutions were used as test samples to analyze the sensitivity of the method of the present invention in detecting alkaline phosphatase activity. The specific operation is as follows:

[0049] The alkaline phosphatase standard solution was mixed with 0.2 mM PPi, 50 nM Cu 2+ Mix well and incubate at 37°C for 30 min to hydrolyze PPi so that PPi cannot react with Cu 2+ Then, 20 nM enzyme chain and 10 nM substrate chain were incubated at 37°C for 30 min to form Cu 2+ Specific DNAzyme. The above solutions were mixed, 50 μM ascorbic acid was added and mixed, and the mixture was incubated for 80 min to fully cut the substrate chain. Next, 15 nM crRNA was mixed with 15 nM Cas12a, 75 nM dithiothreitol and 5 μL 10 × NE buffer were added, and the mixture was incubated for 30 min to form a Cas12a / crRNA complex. The above solution was incubated with 240 nM single-stranded DNA reporter probe for 30 min to activate the trans-cleavage activity of Cas12a protein by the complete substrate chain, and the DNA reporter probe was broken. Finally, the fluorescence intensity of the product after trans-cleavage of Cas12a protein was measured using a fluorescence spectrophotometer.

[0050] Results: Figure 3 In the range of 0.05U / L to 10U / L, the change in fluorescence intensity (ΔF=F0-F, F0 and F are the fluorescence intensity of the system at 520nm when there is no alkaline phosphatase and when there is alkaline phosphatase, respectively) shows a good linear relationship with the concentration of alkaline phosphatase. The linear equation is ΔF=54.98C ALP +8.018, the fitting degree of the relevant equation is R2 =0.991, and the detection limit is as low as 0.04U / L (S / N=3), indicating that the method of the present invention can achieve high-sensitivity detection of alkaline phosphatase.

[0051] Example 3 Specific detection of alkaline phosphatase

[0052] The samples to be tested in this embodiment are standard solutions of alkaline phosphatase (ALP), bovine serum albumin (BSA), glucose oxidase (GOx), glutathione reductase (GR), urease (Urease), reduced glutathione (GSH) and a mixed solution (Mixture) of the above substances. Among them, the concentration of alkaline phosphatase is 10U / L, and the concentrations of other substances are all 200U / L. The specific operation is as follows:

[0053] The sample to be tested was mixed with 0.2 mM PPi, 50 nM Cu 2+ Mix well and incubate at 37°C for 30 min to hydrolyze PPi so that PPi cannot react with Cu 2+ Then, 20 nM enzyme chain and 10 nM substrate chain were incubated at 37°C for 30 min to form Cu 2+ Specific DNAzyme. The above solutions were mixed, 50 μM ascorbic acid was added and mixed, and the mixture was incubated for 80 min to fully cut the substrate chain. Next, 15 nM crRNA was mixed with 15 nM Cas12a, 75 nM dithiothreitol and 5 μL 10 × NE buffer were added, and the mixture was incubated for 30 min to form a Cas12a / crRNA complex. The above solution was incubated with 240 nM single-stranded DNA reporter probe for 30 min to activate the trans-cleavage activity of Cas12a protein by the complete substrate chain, and the DNA reporter probe was broken. Finally, the fluorescence intensity of the product after trans-cleavage of Cas12a protein was measured using a fluorescence spectrophotometer.

[0054] Results: Taking the maximum fluorescence intensity as 1, other fluorescence intensities were normalized and calculated, such as Figure 4 When 10U / L alkaline phosphatase was added, the measured fluorescence intensity was low. After adding other interfering substances, the fluorescence intensity of the system was equivalent to that of the group without alkaline phosphatase (Blank), indicating that this method has good specificity for the detection of alkaline phosphatase.

[0055] Example 4 Detection of alkaline phosphatase activity in actual samples

[0056] In this example, the method of the present invention is used to detect the activity of alkaline phosphatase in various actual samples in order to investigate the practicality of the method of the present invention. The specific operation is as follows:

[0057] 1) Detection of alkaline phosphatase activity in human serum:

[0058] In this embodiment, the sample to be tested is a human serum sample containing alkaline phosphatase standard solution of different concentrations (0, 2, 20, 200 U / L), and the specific operation is as follows:

[0059] The human serum samples to be tested were mixed with 0.2mM PPi, 50nM Cu 2+ Mix well and incubate at 37°C for 30 min to hydrolyze PPi so that PPi cannot react with Cu 2+ Then, 20 nM enzyme chain and 10 nM substrate chain were incubated at 37°C for 30 min to form Cu 2+ Specific DNAzyme. The above solutions were mixed, 50 μM ascorbic acid was added and mixed, and the mixture was incubated for 80 min to fully cut the substrate chain. Next, 15 nM crRNA was mixed with 15 nM Cas12a, 75 nM dithiothreitol and 5 μL 10 × NE buffer were added, and the mixture was incubated for 30 min to form a Cas12a / crRNA complex. The above solution was incubated with 240 nM single-stranded DNA reporter probe for 30 min to activate the trans-cleavage activity of Cas12a protein by the complete substrate chain, and the DNA reporter probe was broken. Finally, the fluorescence intensity of the product after trans-cleavage of Cas12a protein was measured using a fluorescence spectrophotometer.

[0060] Table 1 Detection of alkaline phosphatase activity in human serum samples

[0061]

[0062] a Standard deviation of three measurements.

[0063] Results: As shown in Table 1, according to the linear equation in Example 2, the alkaline phosphatase activity in the human serum sample was calculated to be 337 U / L, and the recovery rate was between 94.6% and 103.9%, with a good overall recovery rate. This indicates that the overall anti-interference ability of this method in detecting alkaline phosphatase activity in human serum samples is good.

[0064] 2) Detection of alkaline phosphatase activity in mouse serum:

[0065] In this embodiment, the sample to be tested is a mouse serum sample containing different concentrations (0, 4, 8, 40 U / L) of alkaline phosphatase standard solution, and the specific operation is as follows:

[0066] The mouse serum samples to be tested were mixed with 0.2mM PPi, 50nM Cu 2+ Mix well and incubate at 37°C for 30 min to hydrolyze PPi so that PPi cannot react with Cu 2+ Then, 20 nM enzyme chain and 10 nM substrate chain were incubated at 37°C for 30 min to form Cu 2+Specific DNAzyme. The above solutions were mixed, 50 μM ascorbic acid was added and mixed, and the mixture was incubated for 80 min to fully cut the substrate chain. Next, 15 nM crRNA was mixed with 15 nM Cas12a, 75 nM dithiothreitol and 5 μL 10 × NE buffer were added, and the mixture was incubated for 30 min to form a Cas12a / crRNA complex. The above solution was incubated with 240 nM single-stranded DNA reporter probe for 30 min to activate the trans-cleavage activity of Cas12a protein by the complete substrate chain, and the DNA reporter probe was broken. Finally, the fluorescence intensity of the product after trans-cleavage of Cas12a protein was measured using a fluorescence spectrophotometer.

[0067] Table 2 Detection of alkaline phosphatase activity in mouse serum samples

[0068]

[0069] a Standard deviation of three measurements.

[0070] Results: As shown in Table 2, according to the standard curve obtained in the above example, the alkaline phosphatase activity in the mouse serum sample was calculated to be 189U / L, the recovery rate was between 89.6% and 111.9%, and the RSD value was less than 9.3%. The experimental results show that this method is practical in detecting the alkaline phosphatase activity in mouse serum samples and has good overall anti-interference ability.

[0071] 3) Detection of alkaline phosphatase activity in human cervical cancer cells (HeLa):

[0072] In this embodiment, the sample to be tested is HeLa cell lysate, and the specific operation is as follows:

[0073] The HeLa cell lysate to be tested was mixed with 0.2 mM PPi, 50 nM Cu 2+ Mix well and incubate at 37°C for 30 min to hydrolyze PPi so that PPi cannot react with Cu 2+ Then, 20 nM enzyme chain and 10 nM substrate chain were incubated at 37°C for 30 min to form Cu 2+Specific DNAzyme. Mix the above solutions, add 50μM ascorbic acid to mix, and continue incubation for 80min to fully cut the substrate chain. Next, mix 15nM crRNA with 15nM Cas12a, add 75nM dithiothreitol and 5μL 10×NE buffer, mix and continue incubation for 30min to form a Cas12a / crRNA complex. The above solution was incubated with 240nM single-stranded DNA reporter probe for another 30min to activate the trans-cleavage activity of Cas12a protein by the complete substrate chain, and the DNA reporter probe was broken. Finally, the fluorescence intensity of the product after trans-cleavage of Cas12a protein was measured using a fluorescence spectrophotometer.

[0074] Results: A significant decrease in fluorescence intensity was detected in the HeLa cell lysate to be tested, while the fluorescence intensity in the products of normal cells and the blank control group did not change significantly. This is because the alkaline phosphatase activity of HeLa cells is overexpressed, indicating that the CRISPR / Cas12a and DNAzyme-based method proposed in the present invention can realize the detection of HeLa cell alkaline phosphatase activity.

[0075] Finally, it should be noted that the above embodiments are only illustrative of the principles, performance and efficacy of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention. SEQUENCE LISTING <110> Guangdong Pharmaceutical University <120> A method for detecting alkaline phosphatase activity based on CRISPR / Cas12a and DNAzyme <130> 2022.6.1 <160> 4 <170> PatentIn version 3.3 <210> 1 <211> 27 <212> DNA <213> Substrate Strand Nucleotide Sequence <400> 1 agcttctttc taatacggct tacctgc 27 <210> 2 <211> 35 <212> DNA <213> Enzyme chain nucleotide sequence <400> 2 ggtaagcctg ggcctctttc tttttaagaa agaac 35 <210> 3 <211> 40 <212> RNA <213> Nucleotide sequence of crRNA <400> 3 uaauuucuac ucuuguagau gcagguaagc cguauuagaa 40 <210> 4 <211> 5 <212> DNA <213> Nucleotide sequences of DNA reporter probes <400> 4 ttatt 5

Claims

1. A method for detecting alkaline phosphatase activity based on CRISPR / Cas12a and DNAzyme for non-diagnostic purposes, characterized in that: The following steps are involved: S1. PPi hydrolysis: Alkaline phosphatase standards with different activity concentrations were mixed with PPi and Cu 2+ and buffer solution and then incubated to hydrolyze PPi so that PPi cannot react with Cu 2+ Combine; S2, DNAzyme cleavage: Cu 2+ The specific DNAzyme is mixed with the solution obtained in step S1, and then ascorbic acid is added to mix and then incubated to fully cut the substrate chain, wherein the DNAzyme includes a substrate chain and an enzyme chain; S3, CRISPR / Cas12a trans-cleavage: Cas12a / crRNA complex is mixed with the solution obtained in step S2, and then cutting buffer solution, dithiothreitol and single-stranded DNA reporter probe are added and mixed, and incubation is continued to allow the complete substrate chain to activate the trans-cleavage activity of Cas12a protein, resulting in the breakage of the single-stranded DNA reporter probe; S4. Fluorescence spectrophotometer detection: The CRISPR / Cas12a trans-cleavage product obtains a signal through a fluorescence spectrophotometer, and a standard curve is constructed based on the relationship between the signal value and the activity concentration; S5. Using the sample to be tested instead of the standard substance to obtain a fluorescent signal, and calculating the alkaline phosphatase activity in the sample to be tested based on the fluorescent signal value of the sample to be tested and the standard curve.

2. The method for detecting alkaline phosphatase activity according to claim 1, wherein: The concentration range of different concentrations of alkaline phosphatase in step S1 is 0.05-10 U / L; the concentration of PPi is 50-250 μM; the concentration of Cu 2+ The concentration is 30-80 nM; the incubation condition after mixing is 25-35° C. and incubation for 20-40 min.

3. The method for detecting the activity of alkaline phosphatase according to claim 1, characterized in that: Cu in step S2 2+ The conditions for the formation of specific DNAzyme are incubation at 25-35°C for 20-40 minutes.

4. The method for detecting the activity of alkaline phosphatase according to claim 3, characterized in that: The concentration of the substrate chain is 2.5-20 nM, and the nucleotide sequence is shown in SEQ ID NO.1; the concentration of the enzyme chain is 10-30 nM, and the nucleotide sequence is shown in SEQ ID NO.

2.

5. The method for detecting the activity of alkaline phosphatase according to claim 1, wherein: The ascorbic acid concentration in step S2 is 30-80 μM; the incubation condition is 25-35° C. and the incubation time is 70-100 min.

6. The method for detecting the activity of alkaline phosphatase according to claim 1, wherein: The formation conditions of the Cas12a / crRNA complex in step S3 are incubation at 25-35°C for 20-40 min; the Cas12a protein concentration is 10-20 nM; the crRNA concentration is 10-20 nM; the dithiothreitol concentration is 70-80 nM; and the single-stranded DNA reporter probe concentration is 220-260 nM.

7. The method for detecting the activity of alkaline phosphatase according to claim 1, wherein: The nucleotide sequence of crRNA in step S3 is shown in SEQ ID NO.3; the nucleotide sequence of the DNA reporter probe is shown in SEQ ID NO.4; the cutting buffer solution is 10 × NE buffer, the formula is 500mM NaCl, 100mM Tris-HCl, 100mM MgCl2, 1mg / ml recombinant albumin, pH 7.9; the incubation conditions are 20-45°C for 20-40min.

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