An RCA-Cas12a-DNA tetrahedral sensor for detecting vancomycin in serum and its preparation method.

Abstract

This invention discloses a method for preparing an RCA-Cas12a-DNA tetrahedral sensor for detecting vancomycin in serum. This invention utilizes the specific binding of aptamers to the target analyte, transforming non-nucleic acid targets into nucleic acid detection, thus expanding the application range of RCA and Cas12a. The binding of RCA and Cas12a amplifies the signal twice, resulting in a lower detection limit and higher sensitivity. Compared with traditional detection methods, this invention eliminates the need for expensive instruments, complex and time-consuming sample pretreatment, and skilled operators, making it more convenient and economical.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology, specifically relating to an RCA-Cas12a-DNA tetrahedral sensor for detecting vancomycin in serum and its preparation method. Background Technology

[0002] RCA technology is an isothermal nucleic acid amplification method with many significant advantages such as speed, efficiency, ease of operation, high sensitivity, and high specificity, which has led to its widespread application in biological detection, medical research, and industrial applications.

[0003] Cas12a, a nuclease in the CRISPR system, can specifically recognize and cleave specific DNA sequences, and after binding to a target, it can indiscriminately cleave nearby single-stranded DNA (ssDNA). This characteristic makes it widely used in molecular biology research and diagnosis.

[0004] Vancomycin is a glycopeptide antibiotic primarily used to treat serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive bacteria. Its mechanism of action involves inhibiting bacterial cell wall synthesis, altering bacterial cell membrane permeability, and selectively inhibiting bacterial RNA synthesis, thereby exhibiting potent antibacterial activity. However, vancomycin has a narrow effective therapeutic concentration range, necessitating monitoring of its therapeutic efficacy. Summary of the Invention

[0005] To address the aforementioned problems, this invention proposes an RCA-Cas12a-DNA tetrahedral sensor for detecting vancomycin in serum and its preparation method.

[0006] The technical solution adopted in this invention is as follows:

[0007] A first aspect of the present invention provides an RCA-Cas12a-DNA tetrahedral sensor for detecting vancomycin in serum, the sensor comprising: vancomycin as the target analyte; DNA strands TSP1, TSP2, and TSP3 for preparing DNA tetrahedra and the vancomycin aptamer; DNA strands T, S, and T4 DNA ligases, exonuclease I and exonuclease III for preparing circular DNA; DNA strand P and phi29 DNA polymerase for performing rolling circle amplification; and LbCas12a nuclease, crRNA, and ssDNA-FQ probes for preparing a reporter. TSP1: 5'-ATTGGCAGTCTGACTTCCGACTAGGAAGCTACCGAACAATAAGTA CT-3', TSP2: 5'-AGTCAGACTGCCAATTTTGCGGTACCCTCGGCCTACGTACAGCTAAC-3', TSP3: 5'-TAGCTTCCTAGTCGGTGTTAGCTGTACGTAGCCCCGACCCACAATAG-3', Aptamer: 5'-CCGAG GGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGG-3', T: 5'-P-TAGCGTAGAGTACTTATTGTTCGTTTCCAGTATCAGTCTATCTATCTATTGTGGGTCGGGGTACCATCGTGCAATTTCGGTTC-3',

[0008] S: 5'-AAGTACTCTACGCTAGAACCGAAATTGCAC-3',

[0009] P: 5'-GTACCATCGTGCAAT-3',

[0010] crRNA: 5'-UAAUUUCUACUAAGUGUAGAUCAGUAUCAGUCUAUCUAU-3',

[0011] ssDNA-FQ: 5'-Cy3-TTAATT-BHQ2-3'.

[0012] A second aspect of the present invention provides a method for preparing the aforementioned RCA-Cas12a-DNA tetrahedral sensor, comprising the following steps:

[0013] S1: Preparation of TSPA solution:

[0014] 10 -5 μmol of DNA strand TSP1, 10 -5μmol of DNA strand TSP2, 10 -5 μmol of DNA strand TSP3 and 10 -5 μmol of vancomycin aptamer is mixed thoroughly in 10 μL of 1×TM buffer and reacted to obtain a solution of DNA tetrahedra, also known as TSPA solution.

[0015] S2: Preparation of TSP solution:

[0016] The target substance, vancomycin, was dissolved in ultrapure water to obtain a target substance solution; 10 μL of the target substance solution was added to the TSPA solution in step S1, and the reaction was carried out to obtain a TSP solution.

[0017] S3: Preparation of TS solution:

[0018] S3-1: 2×10 -5 μmol of DNA strand T and 2×10 -5 μmol of DNA strand S was mixed thoroughly in 10 μL of 1×TM buffer and reacted to obtain reaction solution 1.

[0019] S3-2: Add 1 μL of 5 U / μL T4 DNA ligase, 3 μL of 10×T4 DNA ligase buffer and 16 μL of ultrapure water to reaction solution 1 in step S3-1, and carry out the reaction to obtain reaction solution 2.

[0020] S3-3: Add 4 μL of 20 U / μL exonuclease I, 0.4 μL of 200 U / μL exonuclease III, 5 μL of 10×ExoI Buffer, 5 μL of 10×ExoIII Buffer and 5.6 μL of ultrapure water to the reaction solution 2 in step S3-2, and carry out the reaction to obtain a solution of circular DNA, also known as TS solution;

[0021] S4: Rolling circle amplification reaction:

[0022] Add 1 μL of 10 U / μL Phi29 DNA polymerase, 1 μL of 10 μM DNA strand P, 1 μL of 0.4 μM circular DNA, 3 μL of 10× Phi29 DNA polymerase buffer, 1.2 μL of 5 mg / mL BSA standard solution, 1 μL of 10 mM dNTP™ spectrophotometer, and 1.8 μL of ultrapure water to the TSP solution in step S2 to obtain the rolling circle amplification reaction system. Perform the reaction to obtain the rolling circle amplification reaction product.

[0023] S5: Preparation of the reporter solution:

[0024] Mix 10 μL of 500 nM LbCas12a nuclease and 10 μL of 500 nM crRNA thoroughly and react. Then add 50 μL of 2 μM ssDNA-FQ probe and mix thoroughly to obtain the reporter solution.

[0025] S6: Sensor Assembly

[0026] The reporter solution from step S5 and the rolling circle amplification reaction product from step S4 are mixed and reacted to obtain a test solution containing the target analyte. The fluorescence intensity in the range of 540–650 nm is measured and recorded under an excitation wavelength of 525 nm.

[0027] In step S1 above, the reaction conditions are: 95℃ for 10 min, 4℃ for 15 min.

[0028] In step S2 above, the reaction conditions are: 37℃ for 2 hours.

[0029] In step S3-1 above, the reaction conditions are: 95℃ for 2 min, 65℃ for 2 min, 60℃ for 6 min, 20℃ for 30 s, and 4℃ for 10 min; in step S3-2 above, the reaction conditions are: 16℃ for 8 h and 65℃ for 10 min; in step S3-3 above, the reaction conditions are: 37℃ for 12 h and 80℃ for 15 min.

[0030] In step S4 above, the reaction conditions are: 30℃ for 90 min.

[0031] In step S5 above, the reaction conditions are: 25℃ for 30 min.

[0032] In step S6 above, the reaction conditions are: 37℃ for 90 min.

[0033] A third aspect of the present invention provides the application of the aforementioned RCA-Cas12a-DNA tetrahedral sensor in the detection of vancomycin, wherein the application is for non-disease diagnosis and treatment purposes.

[0034] Figure 1This is a schematic diagram of the detection principle of the RCA-Cas12a-DNA tetrahedron sensor of the present invention. When the target is absent, the DNA tetrahedron formed by DNA strand TSP1, DNA strand TSP2, DNA strand TSP3 and vancomycin aptamer maintains a stable structure with no 3' ends exposed, thus failing to induce rolling circle amplification and generating no signal. However, when the target is present, Aptamer binds to the target and detaches from the DNA tetrahedron, exposing two 3' ends. These two 3' ends act as primers to induce rolling circle amplification (RCA). LbCas12a and crRNA binders recognize specific DNA double-stranded fragments, LbCas12a is activated, and its trans-cleavage activity cleaves ssDNA-FQ. The fluorescent group moves away from the quenching group, and fluorescence is restored.

[0035] The significant advantages of this invention are:

[0036] (1) This invention utilizes the specific binding of aptamers to targets to convert non-nucleic acid targets into nucleic acid detection, thus expanding the application scope of RCA and Cas12a.

[0037] (2) This invention combines RCA with Cas12a, so that the signal is amplified twice, resulting in a lower detection limit and higher sensitivity;

[0038] (3) Compared with traditional detection methods, the present invention does not require expensive instruments and equipment, complex and time-consuming sample pretreatment and skilled operators, making it more convenient and economical. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the detection principle of the RCA-Cas12a-DNA tetrahedral sensor of the present invention.

[0040] Figure 2 These are fluorescence response diagrams and working curves after adding different concentrations of the target substance in Example 1.

[0041] Figure 3 This is a fluorescence response diagram after adding the target, non-target, and their mixture in Example 2. Detailed Implementation

[0042] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0043] The nucleotide chain sequences involved in the embodiments of this invention are as follows:

[0044] TSP1: 5'-ATTGGCAGTCTGACTTCCGACTAGGAAGCTACCGAACAATAAGTACT-3',

[0045] TSP2: 5'-AGTCAGACTGCCAATTTTGCGGTACCCTCGGCCTACGTACAGCTAAC-3',

[0046] TSP3: 5'-TAGCTTCCTAGTCGGTGTTAGCTGTACGTAGCCCCGACCCACAATAG-3',

[0047] Aptamer: 5'-CCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGG-3', T: 5'-P-TAGCGTAGAGTACTTATTGTTCGTTTCCAGTATCAGTCTATCTATCTATTGTGGGTCGGGGTACCATCGTGCAATTTCGGTTC-3',

[0048] S: 5'-AAGTACTCTACGCTAGAACCGAAATTGCAC-3',

[0049] P: 5'-GTACCATCGTGCAAT-3',

[0050] ssDNA-FQ: 5'-Cy3-TTAATT-BHQ2-3',

[0051] crRNA: 5'-UAAUUUCUACUAAGUGUAGAUCAGUAUCAGUCUAUCUAU-3'.

[0052] The formulation of the 1×TM buffer involved in this embodiment of the invention is: 10mM Tris-HCl, 5mM MgCl2; pH=8.0.

[0053] The LbCas12a nuclease involved in the embodiments of the present invention was purchased from New England Biolabs, catalog number: M0653T.

[0054] Example 1:

[0055] A method for preparing an RCA-Cas12a-DNA tetrahedral sensor for detecting vancomycin in serum includes the following steps:

[0056] S1: Preparation of TSPA solution:

[0057] 10 -5 μmol of DNA strand TSP1, 10 -5 μmol of DNA strand TSP2, 10 -5μmol of DNA strand TSP3 and 10 -5 μmol of vancomycin aptamer was mixed thoroughly in 10 μL of 1×TM buffer and reacted to obtain a DNA tetrahedral solution, also known as TSPA solution. The reaction conditions were: heating at 95°C for 10 min, then cooling to 4°C and holding for 15 min.

[0058] S2: Preparation of TSP solution:

[0059] Vancomycin was dissolved in ultrapure water to obtain target solutions of different concentrations; 10 μL of the target solution was added to the TSPA solution in step S1, and the reaction was carried out at 37°C for 2 h to obtain the TSP solution.

[0060] S3: Preparation of TS solution:

[0061] S3-1: 2×10 -5 μmol of DNA strand T and 2×10 -5 μmol of DNA strand S was mixed evenly in 10 μL of 1×TM buffer and reacted to obtain reaction solution 1. The reaction conditions were as follows: heating at 95℃ for 2 min, then cooling to 65℃ and holding for 2 min, then cooling to 60℃ and holding for 6 min, then cooling to 20℃ and holding for 30 s, and then cooling to 4℃ and holding for 10 min.

[0062] S3-2: Add 1 μL of 5 U / μL T4 DNA ligase, 3 μL of 10×T4 DNA ligase buffer and 16 μL of ultrapure water to reaction solution 1 in step S3-1, and carry out the reaction to obtain reaction solution 2; wherein, the reaction conditions are: react at 16℃ for 8 h, and then heat to 65℃ for 10 min.

[0063] S3-3: Add 4 μL of 20 U / μL exonuclease I, 0.4 μL of 200 U / μL exonuclease III, 5 μL of 10×ExoⅠ buffer, 5 μL of 10×ExoⅢ buffer, and 5.6 μL of ultrapure water to reaction solution 2 in step S3-2, and carry out the reaction to obtain a solution of circular DNA, also known as TS solution; wherein, the reaction conditions are: 37℃ for 12 h, and then the temperature is raised to 80℃ for 15 min.

[0064] S4: Rolling circle amplification reaction:

[0065] Add 1 μL of 10 U / μL Phi29 DNA polymerase, 1 μL of 10 μM DNA strand P, 1 μL of 0.4 μM circular DNA, 3 μL of 10× Phi29 DNA polymerase buffer, 1.2 μL of 5 mg / mL BSA standard solution, 1 μL of 10 mM dNTP™ spectrophotometer, and 1.8 μL of ultrapure water to the TSP solution in step S2 to obtain the rolling circle amplification reaction system. Perform the reaction to obtain the rolling circle amplification reaction product.

[0066] S5: Preparation of the reporter solution:

[0067] Mix 10 μL of 500 nM LbCas12a nuclease and 10 μL of 500 nM crRNA thoroughly, react at 25 °C for 30 min, then add 50 μL of 2 μM ssDNA-FQ probe and mix thoroughly to obtain the reporter solution.

[0068] S6: Sensor Assembly

[0069] The reporter solution from step S5 and the rolling circle amplification reaction product from step S4 were mixed and reacted at 37°C in the dark for 90 min to obtain test solutions containing different concentrations of the target analyte (0, 1 fM, 10 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM). The fluorescence intensity in the range of 540–650 nm was measured and recorded under an excitation wavelength of 525 nm.

[0070] The results are as follows Figure 2 As shown, Figure 2 The relationship between the fluorescence intensity of the RCA-Cas12a-DNA tetrahedral sensor and the concentration of the target analyte vancomycin is shown. As can be seen from the figure, the fluorescence intensity increases with increasing vancomycin concentration. Furthermore, the detection limit for vancomycin was calculated to be 1 fM using the blank plus 3σ (three times the standard deviation) method.

[0071] Example 2:

[0072] A method for preparing an RCA-Cas12a-DNA tetrahedral sensor for detecting vancomycin in serum includes the following steps:

[0073] S1: Same as Example 1.

[0074] S2: Prepare vancomycin solution, kanamycin solution, gentamicin solution, and vancomycin-kanamycin-gentamicin mixed solution using ultrapure water. Take 10 μL of each solution and add it to the TSPA solution in step S1. React at 37℃ for 2 h to obtain TSP solution.

[0075] S3 to S6: Same as Example 1.

[0076] The results are as follows Figure 3 As shown. From left to right: ultrapure water, vancomycin, kanamycin, gentamicin, and a mixture of vancomycin-kanamycin-gentamicin (each antibiotic's final concentration in the test solution is 1 nM). Compared with other interfering antibiotics, the target analyte vancomycin caused at least an 8-fold increase in relative fluorescence signal. Furthermore, a strong signal was also shown in the target analyte mixture containing the same concentration of interfering antibiotics, indicating that the RCA-Cas12a-DNA tetrahedral sensor developed in this invention has good specificity for vancomycin.

[0077] Example 3:

[0078] Human serum samples were diluted 10-fold with ultrapure water. Vancomycin of various concentrations was added to the diluted serum, and recovery experiments were performed using the standard spiking method as described in Example 1. The recovery rate and relative standard deviation (RSD) were calculated through three independent experiments.

[0079] The results are shown in Table 1. The recovery rate was 91.9%–124.0%, and the RSD was 7.21%–15.80%. This indicates that the RCA-Cas12a-DNA tetrahedral sensor of the present invention has good accuracy and precision and can be used for actual sample analysis.

[0080] Table 1

[0081]

[0082] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. An RCA-Cas12a-DNA tetrahedral sensor for detecting vancomycin in serum, characterized in that: The sensor includes: the target analyte vancomycin; DNA strands TSP1, TSP2, and TSP3 for preparing DNA tetrahedral DNA strands and the vancomycin aptamer; DNA strands T, S, and T4 for preparing circular DNA; DNA ligase, exonuclease I, and exonuclease III; DNA strand P and phi29 DNA polymerase for performing rolling circle amplification; and LbCas12a nuclease, crRNA, and ssDNA-FQ probe for preparing the reporter. TSP1: 5'-ATTGGCAGTCTGACTTCCGACTAGGAAGCTACCGAACAATAAGTACT-3', TSP2: 5'-AGTCAGACTGCCAATTTTGCGGTACCCTCGGCCTACGTACAGCTAAC-3', TSP3: 5'-TAGCTTCCTAGTCGGTGTTAGCTGTACGTAGCCCCGACCCACAATAG-3', Aptamer: 5'-CCGAGGGTACCGCAATAGTACTTATTGTTCGCCTATTGTGGGTCGGG-3', T: 5'-P-TAGCGTAGAGTACTTATTGTTCGTTTCCAGTATCAGTCTATCTATCTATTGTGGGTCGGGGTACCATCGTGCAATTTCGGTTC-3', S: 5'-AAGTACTCTACGCTAGAACCGAAATTGCAC-3', P: 5'-GTACCATCGTGCAAT-3', crRNA: 5'-UAAUUUCUACUAAGUGUAGAUCAGUAUCAGUCUAUCUAU-3', ssDNA-FQ: 5'-Cy3-TTAATT-BHQ2-3'.

2. The method for preparing the RCA-Cas12a-DNA tetrahedral sensor as described in claim 1, characterized in that: The steps are as follows: S1: Preparation of TSPA solution: 10 -5 μmol of DNA strand TSP1, 10 -5 μmol of DNA strand TSP2, 10 -5 μmol of DNA strand TSP3 and 10 -5 μmol of vancomycin aptamer is mixed thoroughly in 10 μL of 1×TM buffer and reacted to obtain a solution of DNA tetrahedra, also known as TSPA solution. S2: Preparation of TSP solution: The target substance, vancomycin, was dissolved in ultrapure water to obtain a target substance solution; 10 μL of the target substance solution was added to the TSPA solution in step S1, and the reaction was carried out to obtain a TSP solution. S3: Preparation of TS solution: S3-1: 2×10 -5 μmol of DNA strand T and 2×10 -5 μmol of DNA strand S was mixed thoroughly in 10 μL of 1×TM buffer and reacted to obtain reaction solution 1. S3-2: Add 1 μL of 5 U / μL T4 DNA ligase, 3 μL of 10×T4 DNA ligase buffer and 16 μL of ultrapure water to reaction solution 1 in step S3-1, and carry out the reaction to obtain reaction solution 2. S3-3: Add 4 μL of 20 U / μL exonuclease I, 0.4 μL of 200 U / μL exonuclease III, 5 μL of 10×Exo I Buffer, 5 μL of 10×Exo III Buffer and 5.6 μL of ultrapure water to the reaction solution 2 in step S3-2, and carry out the reaction to obtain a solution of circular DNA, also known as TS solution; S4: Rolling circle amplification reaction: Add 1 μL of 10 U / μL Phi29 DNA polymerase, 1 μL of 10 μM DNA strand P, 1 μL of 0.4 μM circular DNA, 3 μL of 10× Phi29 DNA polymerase buffer, 1.2 μL of 5 mg / mL BSA standard solution, 1 μL of 10 mM dNTP mixture, and 1.8 μL of ultrapure water to the TSP solution in step S2 to obtain the rolling circle amplification reaction system. Perform the reaction to obtain the rolling circle amplification reaction product. S5: Preparation of the reporter solution: Mix 10 μL of 500 nM LbCas12a nuclease and 10 μL of 500 nM crRNA thoroughly and react. Then add 50 μL of 2 μM ssDNA-FQ probe and mix thoroughly to obtain the reporter solution. S6: Sensor Assembly The reporter solution from step S5 and the rolling circle amplification reaction product from step S4 are mixed and reacted to obtain a test solution containing the target analyte. The fluorescence intensity in the range of 540-650 nm is measured and recorded under an excitation wavelength of 525 nm.

3. The preparation method according to claim 2, characterized in that: In step S1, the reaction conditions are: 95℃ for 10 min, 4℃ for 15 min.

4. The preparation method according to claim 2, characterized in that: In step S2, the reaction conditions are: 37℃ for 2 hours.

5. The preparation method according to claim 2, characterized in that: In step S3-1, the reaction conditions are: 95℃ for 2 min, 65℃ for 2 min, 60℃ for 6 min, 20℃ for 30 s, and 4℃ for 10 min; in step S3-2, the reaction conditions are: 16℃ for 8 h and 65℃ for 10 min; in step S3-3, the reaction conditions are: 37℃ for 12 h and 80℃ for 15 min.

6. The preparation method according to claim 2, characterized in that: In step S4, the reaction conditions are: 30℃ for 90 min.

7. The preparation method according to claim 2, characterized in that: In step S5, the reaction conditions are: 25℃ for 30 min.

8. The preparation method according to claim 2, characterized in that: In step S6, the reaction conditions are: 37℃ for 90 min.

9. The application of the RCA-Cas12a-DNA tetrahedral sensor as described in claim 1 in the detection of vancomycin, characterized in that: The application is for purposes other than disease diagnosis and treatment.

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