A CRISPR-Cas12a-based ATP visual detection system and detection method

Abstract

The present invention discloses an ATP visual detection system and detection method based on CRISPR-Cas12a, including an identification probe AP, hairpin DNAs H1 and H2, LbCas12a protein, crRNA, transducer DNA, and SSG4. This method uses probe AP to recognize ATP, trigger an enzyme-free isothermal amplification reaction, and then activate the nuclease activity of LbCas12a-crRNA to cleave transducer DNA. Since the peroxidase activity of the G-quadruplex nucleic acid SSG4 in the color reaction is regulated by transducer DNA, the amplification induced by ATP and the transducer DNA cleavage reaction can maintain the peroxidase activity of SSG4, and then catalyze the substrate oxidation reaction to make the solution show a significant blue-green color. This method avoids the nuclease resistance problem when G-quadruplex is directly used as the cleavage substrate of the LbCas12a protein-crRNA complex. When using this method to detect ATP, the method is simple, the nucleic acid does not require chemical modification, the detection sensitivity is high, and on-site instant detection based on color change can be achieved, which has broad application prospects in medical detection and biological research.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to an ATP visualization detection system and a detection method based on CRISPR-Cas12a. Background Art

[0002] Adenosine-Triphosphate (ATP), in addition to being an unstable high-energy compound, can directly provide energy for organisms. The ATP content can be used as an indicator of cell viability. Some studies have shown that the concentration fluctuations of ATP in organisms are closely related to diseases such as cardiovascular diseases, Parkinson's disease, and even cancer. Therefore, ATP can be used as a biomarker for non-invasive detection of various diseases.

[0003] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Cas proteins are widely present in bacteria and archaea, and are a unique adaptive immune defense system in prokaryotes. This system has been developed into a gene editing tool and is widely used in related fields of molecular biology. With the discovery of the trans-cleavage activities of some members of the Cas protein family (such as Cas12a, Cas13a, and Cas14), it has made gene detection using CRISPR possible. On the other hand, an aptamer is a structured oligonucleotide sequence obtained by an in vitro screening technique (Systematic evolution of ligands by exponential enrichment, SELEX), which has strict recognition ability and high affinity for the corresponding target. The targets of aptamers include ions, small molecules, polypeptides, and even cells, and are widely used in the construction of target recognition elements in biosensors. The binding of a non-nucleic acid target to the aptamer sequence releases a trigger strand, which in turn activates the trans-cleavage activity of the Cas12a protein to cleave the reporter probe and output a signal. Based on the above design concept, multiple non-nucleic acid detection biosensors based on the CRISPR system have been reported. Most of the CRISPR-based biosensors use single-stranded nucleic acids labeled with fluorescent groups as reporter probes, and the fluorescence released after their cleavage is used as signal output. However, the capture of fluorescence signals requires specific instruments, which greatly limits the application of this type of biosensor.

[0004] After G-quadruplex binds to hemin, a complex with peroxidase activity is formed, which can catalyze the oxidation-reduction reaction of the substrate and produce a color change distinguishable by the naked eye. Therefore, G-quadruplex is an ideal signal output element for visualizable biosensors. Due to the strong stability and nuclease tolerance of typical G-quadruplexes, they cannot be directly used as cleavage reporter probes for the CRISPR system, otherwise it will affect the sensitivity and accuracy of detection. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section, as well as in the abstract and title of the present application, to avoid obscuring the purpose of this section, the abstract, and the title. However, such simplifications or omissions should not be used to limit the scope of the present invention.

[0006] In view of the above problems and / or problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide an ATP visual detection system based on CRISPR-Cas12a.

[0008] To solve the above technical problems, the present invention provides the following technical solution: An ATP visual detection system based on CRISPR-Cas12a, characterized in that it includes: an identification probe AP, a hairpin DNA H1, a hairpin DNA H2, an LbCas12a protein, a crRNA, a transducer DNA, and a G-quadruplex nucleic acid SSG4;

[0009] Among them, the identification probe AP is obtained by annealing the nucleic acid sequence Aptamer and the nucleic acid sequence LC-8; the hairpin DNAs H1 and H2 are strand displacement isothermal amplification reaction elements; the transducer DNA is a single-stranded DNA and is a trans-cleavage substrate of the LbCas12a protein.

[0010] As a preferred embodiment of the ATP visual detection system of the present invention, wherein: the Aptamer sequence is as shown in SEQ ID No.1; the LC-8 sequence is as shown in SEQ ID No.2; the H1 sequence is as shown in SEQ ID No.3; the H2 sequence is as shown in SEQ ID No.4; the crRNA sequence is as shown in SEQ ID No.5; the transducer DNA sequence is as shown in SEQ ID No.6; the SSG4 sequence is as shown in SEQ ID No.7.

[0011] As a preferred embodiment of the ATP visualization detection system of the present invention, wherein: the recognition probe AP can specifically recognize ATP and induce subsequent amplification reactions; the hairpin DNAs H1 and H2 contain the recognition sequences and PAM sites of the CRISPR-Cas12a system.

[0012] As a preferred embodiment of the ATP visualization detection system of the present invention, wherein: the G-quadruplex nucleic acid SSG4 generates peroxidase activity after binding to hemin, and can catalyze the substrate ABTS to be oxidized into a blue-green product, thereby realizing the output of colorimetric signals.

[0013] As a preferred embodiment of the ATP visualization detection system of the present invention, wherein: the transducer DNA can regulate the peroxidase activity of SSG4 by binding to and changing the structure of the G-quadruplex nucleic acid SSG4.

[0014] Another object of the present invention is to overcome the deficiencies in the prior art and provide a detection method for an ATP visualization detection system based on CRISPR-Cas12a.

[0015] To solve the above technical problems, the present invention provides the following technical solution: a detection method for an ATP visualization detection system based on CRISPR-Cas12a, characterized in that the detection method is as follows:

[0016] Dilute Aptamer and LC-8 to 20 μM each in buffer A, mix them in a ratio of 1:1 so that the concentrations of Aptamer and LC-8 in the mixed solution are both 10 μM, then heat the mixed solution at 95 °C for 5 minutes, and then slowly cool it to room temperature for standby;

[0017] Dilute H1 and H2 to 10 μM respectively in buffer A, heat them at 95 °C for 5 minutes, and then slowly cool them to room temperature for standby;

[0018] Dilute SSG4 to 5 μM in buffer B, and heat it in a metal bath at 95 °C for 5 minutes, and then slowly cool it to room temperature for standby;

[0019] Take 1.0 μL of the annealed Aptamer and LC-8 solution prepared, add 1.6 μL of ATP standard solution or test solution with different concentrations, 0.8 μL of the annealed H1 prepared, 0.8 μL of the annealed H2 prepared, use buffer A to make up to the final volume of 20 μl, and incubate at room temperature for 2 hours to obtain reaction solution 1;

[0020] Take 1.0 μL of LbCas12a protein with a concentration of 200 nM, 2.0 μL of crRNA with a concentration of 50 nM, 2.0 μL of transducer DNA with a concentration of 60 μM, 2 μL of 10× buffer C, and 12 μL of water. After mixing, let it stand at room temperature for incubation for 5 minutes, then add 1 μL of the prepared reaction solution 1, react at 37 °C for 3 hours, and inactivate at 65 °C for 10 minutes to obtain reaction solution 2;

[0021] Take 10 μL of annealed SSG4 prepared, 10 μL of reaction solution 2 prepared in the above step, and 16 μL of hemin with a concentration of 25 μM and mix them in buffer B to a final volume of 190 μL, and react in the dark at room temperature for 5 minutes. Then add 5 μL of ABTS with a concentration of 40 mM and 5 μL of H2O2 with a concentration of 200 mM to a final volume of 200 μL, let it stand at room temperature for reaction for 10 minutes, observe the change in the color of the solution, and measure the absorbance value using a microplate reader or a UV-visible spectrophotometer.

[0022] As a preferred embodiment of the detection method of the present invention, wherein: the formulation of buffer A is: 25 mM Tris-HCl, 100 mM NaCl, 30 mM MgCl2, pH 7.4.

[0023] As a preferred embodiment of the detection method of the present invention, wherein: the formulation of buffer B is: 25 mM HEPES, 200 mM NaCl, 20 mM KCl, 150 mM NH4Cl, 0.025% Triton X-100, pH 5.3.

[0024] As a preferred embodiment of the detection method of the present invention, wherein: the formulation of 1× buffer C is: 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 100 μg / mL BSA, pH 7.9.

[0025] As a preferred embodiment of the detection method of the present invention, wherein: when measuring the absorbance value using a microplate reader or a UV-visible spectrophotometer, set the maximum absorption wavelength to 415 nm.

[0026] Advantages of the present invention:

[0027] The present invention provides an ATP visualization detection system based on CRISPR-Cas12a. The recognition probe in this detection system can specifically bind to ATP and change its structure, triggering the HCR amplification reaction to produce a product that can be recognized by the CRISPR-Cas12a system, activating the LbCas12a protein-crRNA complex to cleave the transducer DNA. Finally, the G-quadruplex DNA binds heme to generate peroxidase activity, which then catalyzes the substrate ABTS to be oxidized into a blue-green product. Through the analysis of the absorption light intensity, the visualization and quantitative detection of ATP can be realized. This method uses transducer DNA to regulate the G-quadruplex structure and peroxidase activity, avoiding the nuclease resistance problem when G-quadruplex is directly used as the cleavage substrate of the LbCas12a protein-crRNA complex. This method has high detection sensitivity, with a minimum detection limit of 1 nM, and can detect ATP in serum samples, showing broad application prospects in medical detection and biological research. Description of the Drawings

[0028] Figure 1 is the detection principle diagram of the present invention;

[0029] Figure 2 is the result of verifying the detection feasibility of ATP in Example 2 of the present invention;

[0030] Figure 3 is the schematic diagram of the structural optimization of the recognition probe AP in Example 3 of the present invention;

[0031] Figure 4 is the result of the structural optimization of the recognition probe AP in Example 3 of the present invention;

[0032] Figure 5 is the result of the concentration optimization of the recognition sequence AP in Example 4 of the present invention;

[0033] Figure 6 is the result of the optimization of the HCR reaction time in Example 5 of the present invention;

[0034] Figure 7 is the result of the optimization of the LbCas12a-crRNA cleavage substrate reaction time in Example 6 of the present invention;

[0035] Figure 8 is the analysis of the solution color and absorbance increase in response to different concentrations of ATP and the drawing of the standard curve in Example 7 of the present invention;

[0036] Figure 9 is the specificity test result in Example 8 of the present invention;

[0037] Figure 10This is the comparison of the detection of ATP in serum samples in Example 9 of the present invention with the detection results of a commercially available chemiluminescent ATP detection kit. Detailed implementation manners

[0038] To make the above objects, features and advantages of the present invention more obvious and understandable, the following describes the detailed implementation manners of the present invention in conjunction with the embodiments of the specification.

[0039] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0040] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure or characteristic that may be included in at least one implementation manner of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that excludes other embodiments.

[0041] Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available, as shown in Table 1.

[0042] Table 1

[0043]

[0044] The LbCas12a protein was obtained by prokaryotic induction expression and purification. For the specific method, see: Analytica Chimica Acta, 2023, 1283: 341950

[0045] Example 1

[0046] The detection principle diagram of the ATP detection system of the present invention is shown in Figure 1。The present invention includes three detection reaction modules, namely ATP recognition and signal amplification, cleavage of the substrate by the LbCas12a protein-crRNA complex, and regulation of the G-quadruplex nucleic acid SSG4 structure and colorimetric reaction signal output; in the ATP recognition and signal amplification module, the recognition probe AP is obtained by annealing Aptamer and LC-8, and the Aptamer sequence contains the aptamer sequence of ATP; when there is no ATP in the test solution, the recognition probe exists in the form of a hybrid double strand; when there is ATP in the test solution, the ATP aptamer sequence in the recognition probe specifically binds to ATP, causing the double-stranded structure of the recognition probe to be destroyed, and the Aptamer sequence to be released in a single-stranded form, thereby triggering the subsequent amplification reaction; the amplification reaction is a hybridization chain reaction (HCR), which is an enzyme-free isothermal amplification reaction, using two hairpin DNAs H1 and H2 as amplification units to produce a long double-stranded DNA product containing the recognition sequence and PAM site of the CRISPR-Cas12a system; in the LbCas12a protein-crRNA complex cleavage substrate module, single-stranded transducer DNA is used as the cleavage substrate, avoiding the nuclease resistance problem when G-quadruplex is directly used as the substrate; when the amplification reaction is not triggered, that is, when there is no ATP in the test solution, the LbCas12a protein-crRNA complex remains stable and has no cleavage activity, and the single-stranded transducer DNA structure in the cleavage solution is intact; when the amplification reaction is triggered, that is, when there is ATP in the test solution, the LbCas12a protein-crRNA complex will recognize the recognition sequence and PAM site in the amplification product, and at the same time activate the non-specific single-stranded DNA cleavage activity of the complex to cleave the single-stranded transducer DNA in the solution; in the G-quadruplex nucleic acid SSG4 structure regulation and colorimetric reaction signal output module, the SSG4 structure is regulated by transducer DNA; when there is no ATP in the test solution, the amplification reaction is not triggered, the LbCas12a protein-crRNA complex has no cleavage activity, and the single-stranded transducer DNA structure is intact, which can bind to and destroy the G-quadruplex structure of SSG4, making it unable to bind hemin, that is, it has no peroxidase activity; when there is ATP in the test solution, the amplification reaction is triggered, the LbCas12a protein-crRNA complex has cleavage activity, the single-stranded transducer DNA is degraded, the G-quadruplex structure of SSG4 remains intact, which can bind hemin and produce peroxidase activity, thereby catalyzing the substrate ABTS to be oxidized into a blue-green product, observing the color change of the solution, and measuring the absorbance value using an enzyme-labeled instrument or an ultraviolet-visible spectrophotometer.

[0047] The oligonucleotide sequences involved in the present invention are shown in Table 2.

[0048] Table 2

[0049]

[0050]

[0051] Note: The underlined part is the recognition region of the CRISPR-Cas12a system, and the italic part is the aptamer region.

[0052] The method for detecting ATP using the above detection system includes the following steps:

[0053] (1) Dilute Aptamer and LC-8 to 20 μM each in buffer A (25 mM Tris-HCl, 100 mM NaCl, 30 mM MgCl2, pH 7.4), mix them in a 1:1 ratio so that the concentrations of Aptamer and LC-8 in the mixed solution are both 10 μM, then heat the mixed solution at 95 °C for 5 minutes, and then slowly cool it to room temperature for standby;

[0054] (2) Dilute H1 and H2 to 10 μM respectively in buffer A (25 mM Tris-HCl, 100 mM NaCl, 30 mM MgCl2, pH 7.4), heat the above solutions at 95 °C for 5 minutes, and then slowly cool it to room temperature for standby;

[0055] (3) Dilute SSG4 to 5 μM in buffer B (25 mM HEPES, 200 mM NaCl, 20 mM KCl, 150 mM NH4Cl, 0.025% Triton X-100, pH 5.3), and heat it in a 95 °C metal bath for 5 minutes, and then slowly cool it to room temperature for standby;

[0056] (4) Take 1.0 μL of the annealed Aptamer and LC-8 solution prepared in step (1), add 1.6 μL of ATP standard solutions or test solutions with different concentrations, 0.8 μL of the annealed H1 prepared in step (2), 0.8 μL of the annealed H2 prepared in step (2), make up to a final volume of 20 μl with buffer A (25 mM Tris-HCl, 100 mM NaCl, 30 mM MgCl2, pH 7.4), and incubate at room temperature for 2 hours to obtain a reaction solution;

[0057] (5) Take 1.0 μL of LbCas12a protein (200 nM), 2.0 μL of crRNA (50 nM), 2.0 μL of transducer DNA (60 μM), 2 μL of 10× buffer C (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 100 μg / mL BSA, pH 7.9), and 12 μL of water. After mixing, let it stand at room temperature for incubation for 5 minutes, then add 1 μL of the reaction solution prepared in step (4), react at 37 °C for 3 hours, and inactivate at 65 °C for 10 minutes to obtain a reaction solution;

[0058] (6) Take 10 μL of the annealed SSG4 prepared in step (3), 10 μL of the reaction solution prepared in step (5), and 16 μL of hemin (25 μM). Mix them in buffer B (25 mM HEPES, 200 mM NaCl, 20 mM KCl, 150 mM NH4Cl, 0.025% Triton X-100, pH 5.3) to a final volume of 190 μL, and react in the dark at room temperature for 5 minutes. Then add 5 μL of ABTS (40 mM) and 5 μL of H2O2 (200 mM) to a final volume of 200 μL, and let it stand at room temperature for reaction for 10 minutes. Observe the change in the color of the solution. When measuring the absorbance value using a microplate reader or an ultraviolet-visible spectrophotometer, set the maximum absorption wavelength to 415 nm, and measure the absorbance value using a microplate reader or an ultraviolet-visible spectrophotometer.

[0059] Example 2

[0060] Figure 2It is the feasibility verification result of the ATP visualization detection method in Example 1: in the figure, the red line represents that the detection solution contains ATP, recognition probe (AP), hairpin DNA (H1), hairpin DNA (H2), Cas12a-crRNA, transducer DNA and SSG4; the blue line represents that the detection solution contains recognition probe (AP), hairpin DNA (H1), hairpin DNA (H2), Cas12a-crRNA, transducer DNA and SSG4; the green line represents that the detection solution contains ATP, hairpin DNA (H1), hairpin DNA (H2), Cas12a-crRNA, transducer DNA and SSG4; the yellow line represents that the detection solution contains ATP, recognition probe (AP), Cas12a-crRNA, transducer DNA and SSG4; the black line represents that the detection solution contains hairpin DNA (H1), Cas12a-crRNA and transducer DNA; the purple line represents that the detection solution contains hairpin DNA (H2), Cas12a-crRNA and transducer DNA. The experimental results show that only when ATP, Cas12a-crRNA and all DNA sequences are present at the same time, a strong absorption light signal can be detected, and the solution color presents a dark blue-green color, which is consistent with the detection principle described in Example 1.

[0061] Example 3

[0062] Figure 3 This is a schematic diagram of the structural optimization of the recognition probe AP of the present invention: the probe AP is obtained by annealing the nucleic acid sequence Aptamer and the nucleic acid sequence LC-n (n=4-9), and the single arrow represents the direction of the nucleic acid sequence (5'-3'). The effect of the subsequent HCR enzyme-free isothermal amplification is closely related to the structure of AP. The number of complementary bases between the 3' end of LC-n and the Aptamer will affect the detection background value, amplification effect and detection sensitivity. During the optimization process, the Aptamer sequence remains unchanged, the complementary bases (6bp) of the ATP aptamer sequence (green) in the LC-n sequence and the Aptamer sequence are fixed, and 4-9 nucleotides are added to the 3' end of LC-n to form a complementary pairing with the Aptamer.

[0063] Figure 4The structural optimization result of the recognition probe AP of the present invention: Since this optimization only involves the recognition and amplification module, a fluorescent probe in a traditional CRISPR sensor is used for signal output. As the 3'-end of LC-n is continuously lengthened, the background signal (F0) value continuously decreases, and the positive signal (F) also decreases. The background signal is the lowest at LC-8, and the signal-to-noise ratio (F / F0) value is the highest. Therefore, in the present invention, after annealing the Aptamer sequence and LC-8, the optimal AP recognition probe is produced.

[0064] Example 4

[0065] Figure 5 The concentration optimization result of the recognition probe AP in the present invention: To study the influence of the concentration of the recognition probe AP on the detection result, the ATP concentration was fixed at 100 nM, and different amounts of the recognition probe AP were added, namely 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, to explore the optimal concentration of the recognition probe AP. The results are as Figure 5 shown. As the concentration of the probe AP increases, the background signal (F0) value increases slowly, and the positive signal (F) increases significantly. When the concentration of the recognition probe AP gradually increases to 600 nM, the signal-to-noise ratio (F / F0) value is the highest. This indicates that when the concentration of the recognition probe AP is 600 nM, the reaction efficiency reaches the maximum. Therefore, 600 nM of AP is used for analysis in the present invention to ensure a high detection efficiency.

[0066] Example 5

[0067] Figure 6 The optimization result of the HCR reaction time of the present invention: To study the influence of the HCR reaction time of the enzyme-free strand displacement amplification reaction on the detection result, the ATP concentration was fixed at 100 nM, and different reaction times were set, namely 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, to explore the optimal HCR reaction time. The results are as Figure 6 shown. As the HCR reaction time increases, the background signal (F0) value increases slowly, and the positive signal (F) increases significantly. When the HCR reaction time increases to 2.0 h, the signal-to-noise ratio (F / F0) value is the highest. This indicates that when the HCR reaction time is 2.0 h, the reaction efficiency reaches the maximum. Therefore, the HCR reaction time of 2.0 h is used for analysis in the present invention to ensure a high detection efficiency.

[0068] Example 6

[0069] Figure 7Optimization results of the reaction time for the LbCas12a-crRNA cleavage substrate in the present invention: To study the effect of the reaction time of LbCas12a-crRNA cleavage substrate on the detection results, the ATP concentration was fixed at 100 nM, and different reaction times were set, namely 1 h, 2 h, 3 h, 4 h, and 5 h, to explore the optimal reaction time for the LbCas12a-crRNA cleavage substrate. Since the reaction cleavage substrate is involved in the subsequent color reaction, the color reaction system was optimized. The results are as Figure 7 shown. As the reaction time of the LbCas12a-crRNA cleavage substrate increases, both the background signal (A0) value and the positive signal (A) increase significantly. When the reaction time of the LbCas12a-crRNA cleavage substrate increases to 3 h, the signal-to-noise ratio (A / A0) value is the highest. This indicates that when the reaction time of the LbCas12a-crRNA cleavage substrate is 3 h, the reaction efficiency reaches the maximum. Therefore, in the present invention, the reaction time of the LbCas12a-crRNA cleavage substrate is 3 h for analysis to ensure a high detection efficiency.

[0070] Example 7

[0071] Different concentrations of ATP (concentrations are 0 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 70 nM, 100 nM respectively) were added to the biosensor designed in the present invention, the color change of the solution was observed, and according to the microplate reader, the relationship between the increase in absorbance value and the ATP concentration at the absorption wavelength of 415 nm was determined. The gradient response of the solution color change and absorbance intensity to different ATP concentration samples in the present ATP visualization detection invention was determined, and the same group of samples was measured three times repeatedly. The relationship between the absorbance increase rate and the ATP concentration is shown in Figure 8 A. It can be seen that in the range of 1 nM - 100 nM, as the ATP concentration increases, the absorbance value at 415 nm in the ultraviolet-visible spectrum also increases. The color of the solution gradually deepens visibly to the naked eye. Compared with the blank sample (Blank), the absorbance increase rates of all samples (this value is the increase in absorbance value of the sample within 10 min of the color reaction / the increase in absorbance value of the blank) have significant differences (C ATP At 1 nM, the p-value is 0.05; C ATP At 5 - 100 nM, the p-value is 0.001).

[0072] Select Figure 8 The data of ATP concentrations of 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, and 50 nM in A, with the APT concentration as the abscissa and the absorbance increase rate as the ordinate, to plot the standard curve, as shown in Figure 8A. The results showed that the enhancement rate of the absorbance value of the solution and the ATP concentration were linearly related in the range of 1 to 50 nM. The linear equation was y = 0.06912×C ATP + 1.181. The correlation coefficient (R 2 ) of the linear regression equation was 0.9957. The calculation formula for the detection limit of the present invention was based on the calculation method of the detection limit (LOD) of the Vashist team: the absorbance value corresponding to the lowest detection limit = the average blank absorbance value + 3σ (standard deviation) of the blank value, also known as the 3σ principle. According to this principle, the detection limit (LOD) of ATP in the present invention was estimated to be 1.0 pM. Based on the above results, the ATP visualization detection system designed in the present invention had good detection sensitivity.

[0073] Example 8

[0074] To evaluate the specificity of the ATP detection method designed in the present invention, under the optimal detection conditions, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP) and their mixture (NTP) were selected, and the detection method of Example 1 was applied for simultaneous detection, and the specificity of the detection method was verified by comparing the detection results. In the experiment, the concentration of ATP was 100 nM, and the concentrations of other nucleoside triphosphate samples were 1000 nM respectively. The absorption light intensity was measured at a wavelength of 415 nm by an ultraviolet-visible spectrophotometer. And the absorbance values between each nucleic acid triphosphate after detection were compared to evaluate the specificity of the detection method designed in the present invention.

[0075] The results of the specificity test are shown in Figure 9 . It can be seen that even when the concentration of non-target nucleoside triphosphate (1000 nM) was 10 times that of the target concentration, there was no obvious color change and no significant difference in absorbance value between non-target nucleoside triphosphates (GTP, CTP, UTP) and the blank sample (Blank). Only when ATP was present, the solution color became significantly darker compared with the blank sample (Blank), and the absorbance value increased significantly (p value was 0.001). Based on the above results, the constructed visualization ATP detection method had excellent specificity.

[0076] Comparative Example 1

[0077] A common commercially available chemiluminescent ATP detection kit (Name: Enhanced ATP Detection Kit; Manufacturer: Beyotime; Catalog Number: S0027).

[0078] Example 9

[0079] To evaluate the detection performance of the ATP detection method designed in the present invention in clinical or biological samples, ATP at different concentrations was added to a solution containing 5% calf serum to prepare the samples to be tested. Under the optimal detection conditions, the detection method of Example 1 was applied to detect these samples. The same group of samples was measured three times repeatedly. The detection results were compared with the added amounts, and the recovery rate and relative standard deviation were calculated. The results are shown in Table 3. When detecting ATP in serum samples, the average recovery rate calculated from the detection results of the present invention was between 100.34 - 109.31%, and the relative standard deviation of detection was 2.26 - 4.06%.

[0080] Table 3

[0081]

[0082] From Figure 10 the results, it can be seen that under the addition amounts of three concentrations (15 nM, 25 nM, 35 nM), the detection results of the present invention are basically consistent with those of Comparative Example 1, having reliability. However, the signal of the commercial ATP detection kit in Comparative Example 1 is chemiluminescence, and a chemiluminescence instrument is required to obtain the signal, while the invention of our side is a color signal, which can be directly distinguished by the naked eye, facilitating on-site instant detection, being convenient, fast and sensitive.

Claims

1. A CRISPR-Cas12a-based ATP visual detection system, characterized in that, Comprising: An identification probe AP, hairpin DNA H1, hairpin DNA H2, LbCas12a protein, crRNA, transducer DNA, and G-quadruplex nucleic acid SSG4; Among them, the identification probe AP is obtained by annealing the nucleic acid sequence Aptamer and the nucleic acid sequence LC-8; hairpin DNA H1 and hairpin DNA H2 are strand displacement isothermal amplification reaction elements; transducer DNA is single-stranded DNA and is the trans-cleavage substrate of LbCas12a protein; The G-quadruplex nucleic acid SSG4 generates peroxidase activity after binding to heme, and can catalyze the substrate ABTS to be oxidized into a blue-green product, thereby realizing colorimetric signal output; The transducer DNA can regulate the peroxidase activity of the G-quadruplex nucleic acid SSG4 by binding to and changing the structure of the G-quadruplex nucleic acid SSG4; The Aptamer sequence is as shown in SEQ ID No.1; the LC-8 sequence is as shown in SEQ ID No.2; the hairpin DNA H1 sequence is as shown in SEQ ID No.3; the hairpin DNA H2 sequence is as shown in SEQ ID No.4; the crRNA sequence is as shown in SEQ ID No.5; the transducer DNA sequence is as shown in SEQ ID No.6; the G-quadruplex nucleic acid SSG4 sequence is as shown in SEQ ID No.7; The identification probe AP can specifically recognize ATP and induce subsequent amplification reactions; hairpin DNA H1 and hairpin DNA H2 contain the recognition sequence and PAM site of the CRISPR-Cas12a system.

2. A detection method of a CRISPR-Cas12a-based ATP visualization detection system for non-diagnostic purposes, characterized in that, The detection method is as follows: Dilute Aptamer and the nucleic acid sequence LC-8 to 20 μM each in buffer A, mix them in a 1:1 ratio, so that the concentrations of Aptamer and the nucleic acid sequence LC-8 in the mixed solution are both 10 μM, then heat the mixed solution at 95°C for 5 minutes, and then slowly cool it to room temperature for standby; Dilute hairpin DNA H1 and hairpin DNA H2 to 10 μM respectively in buffer A, heat at 95°C for 5 minutes, and then slowly cool it to room temperature for standby; Dilute the G-quadruplex nucleic acid SSG4 to 5 μM in buffer B, and heat it in a 95°C metal bath for 5 minutes, and then slowly cool it to room temperature for standby; Take 1.0 μL of the annealed Aptamer and nucleic acid sequence LC-8 solution, add 1.6 μL of different concentrations of ATP standard solution or test solution, 0.8 μL of the annealed hairpin DNA H1, 0.8 μL of the annealed hairpin DNA H2, use buffer A to make up to a final volume of 20 μl, and incubate at room temperature for 2 hours to obtain reaction solution 1; Take 1.0 μL of LbCas12a protein with a concentration of 200 nM, 2.0 μL of crRNA with a concentration of 50 nM, 2.0 μL of transducer DNA with a concentration of 60 μM, 2 μL of 10× buffer C, and 12 μL of water. Mix them and incubate at room temperature for 5 minutes. Then add 1 μL of the prepared reaction solution 1, react at 37 °C for 3 hours, and inactivate at 65 °C for 10 minutes to obtain reaction solution 2; Take 10 μL of annealed G-quadruplex nucleic acid SSG4, 10 μL of reaction solution 2 prepared in the above step, and 16 μL of heme with a concentration of 25 μM. Mix them in buffer B to a final volume of 190 μL, and react in the dark at room temperature for 5 minutes. Then add 5 μL of ABTS with a concentration of 40 mM and 5 μL of H2O2 with a concentration of 200 mM to a final volume of 200 μL, and let it stand and react at room temperature for 10 minutes. Observe the color change of the solution and measure the absorbance using a microplate reader or ultraviolet-visible spectrophotometer; The Aptamer sequence is as shown in SEQ ID No.1; the LC-8 sequence is as shown in SEQ ID No.2; the hairpin DNA H1 sequence is as shown in SEQ ID No.3; the hairpin DNA H2 sequence is as shown in SEQ ID No.4; the crRNA sequence is as shown in SEQ ID No.5; the transducer DNA sequence is as shown in SEQ ID No.6; the G-quadruplex nucleic acid SSG4 sequence is as shown in SEQ ID No.7; The formula of buffer A is: 25 mM Tris-HCl, 100 mM NaCl, 30 mM MgCl2, pH 7.4; The formula of buffer B is: 25 mM HEPES, 200 mM NaCl, 20 mM KCl, 150 mM NH4Cl, 0.025% Triton X-100, pH 5.3; The formula of the 10× buffer C is: 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 100 μg / mL BSA, pH 7.

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3. The detection method according to claim 2, characterized in that: When measuring the absorbance using a microplate reader or ultraviolet-visible spectrophotometer, set the maximum absorption wavelength to 415 nm.

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