Electrochemical biosensor based on palindromic structure nucleic acid and CRISPR / Cas12a and detection method thereof

Abstract

The invention relates to the technical field of biological detection medicines. The invention provides an electrochemical biosensor based on palindromic structure nucleic acid and CRISPR / Cas12a, the electrochemical biosensor comprises a gold electrode and a CRISPR / Cas12a system, the surface of the gold electrode is modified with a palindromic structure DNA probe, and the sequence of the palindromic structure DNA probe comprises a spacer region and a palindromic sequence region. The invention also provides a detection method using the electrochemical biosensor, and the detection method has the advantages of high sensitivity, strong specificity, fast detection speed, low cost and strong versatility, and can detect a plurality of pathogens.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to an electrochemical biosensor based on palindromic nucleic acid and CRISPR / Cas12a and a detection method thereof. BACKGROUND

[0002] At present, nucleic acid detection technologies include PCR, fluorescent probe method, etc., but have defects such as complex operation, high cost, and dependence on professional equipment. The electrochemical biosensor has become a research hotspot due to its portability and low cost, but the traditional method (such as enzyme cutting amplification, nanoparticle labeling) still has problems such as poor signal stability, insufficient specificity, limited signal amplification efficiency, high detection limit (usually nM level), weak single-base mismatch recognition ability, easy interference of non-specific hybridization, long detection time (> 2 hours) caused by step operation, etc. Therefore, it is necessary to obtain a nucleic acid detection method with higher detection sensitivity, faster detection speed and lower detection cost. SUMMARY

[0003] The present application aims to provide an electrochemical biosensor based on palindromic nucleic acid and CRISPR / Cas12a and a detection method thereof, which has high detection sensitivity, strong specificity, fast speed and low cost by using the gold electrode with surface modification of palindromic DNA probe and the CRISPR / Cas12a system.

[0004] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions: The present application provides an electrochemical biosensor based on palindromic nucleic acid and CRISPR / Cas12a, which comprises a gold electrode with surface modification of palindromic DNA probe and a CRISPR / Cas12a system.

[0005] As a preferred, the sequence of the palindromic DNA probe comprises a spacer region and a palindromic sequence region.

[0006] As a preferred, the sequence of the spacer region is shown as SEQ ID NO. 1.

[0007] As a preferred, the palindromic sequence region comprises a DNA sequence shown as SEQ ID NO. 2 and an RNA sequence shown as SEQ ID NO. 3.

[0008] As a preferred, the CRISPR / Cas12a system comprises a Cas12a protein and a gRNA.

[0009] As a preferred, the palindromic DNA probe is modified by thiol at the 5' end.

[0010] The application further provides a detection method using the electrochemical biosensor, comprising the following steps: (1) incubating the polished and cleaned gold electrode with the palindromic structure DNA probe, and then performing blocking to obtain a palindromic structure DNA probe modified electrode; (2) mixing a complex of Cas12a protein and gRNA with a sample solution containing DNA to be detected to obtain mixed solution 1; (3) adding the mixed solution 1 to the modified electrode in step (1), and then performing incubation, staining and recording of the SWV signal; if the sample solution contains the target DNA, the current difference is higher than that of the negative group; if the sample solution does not contain the target DNA, the current difference is lower than that of the negative group.

[0011] Preferably, the palindromic structure DNA probe in step (1) is added in an amount of 15-25 μL.

[0012] Preferably, the incubation in step (1) is performed at a temperature of 36-38℃ for 11-13 h.

[0013] Preferably, the blocking in step (1) is performed using methylcyclohexenone at a temperature of 36-38℃ for 0.8-1.2 h.

[0014] Preferably, the volume ratio of the complex of Cas12a protein and gRNA to the sample solution containing DNA to be detected in step (2) is (2-4):1.

[0015] Preferably, the mixed solution 1 in step (3) is added in an amount of 15-25 μL.

[0016] Preferably, the incubation in step (3) is performed at a temperature of 36-38℃ for 15-25 min.

[0017] Preferably, the staining in step (3) is performed for 3-8 min.

[0018] By adopting the above technical solution, the application has the following beneficial effects: The application discloses an electrochemical biosensor comprising a gold electrode modified with a palindromic DNA probe and a CRISPR / Cas12a system. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A schematic diagram of MB adsorption and current change mechanism in the presence or absence of a target DNA; Figure 2 A schematic diagram of the construction and detection process of the electrochemical biosensor; Figure 3 Detection limit verification results of Example 2 of the application; Figure 4 Specificity test results of Example 2 of the application. DETAILED DESCRIPTION

[0020] The application provides an electrochemical biosensor based on palindromic nucleic acid and CRISPR / Cas12a, comprising a gold electrode modified with a palindromic DNA probe and a CRISPR / Cas12a system.

[0021] In the application, the sequence of the palindromic DNA probe comprises a spacer region and a palindromic sequence region, the sequence of the spacer region is shown as SEQ ID NO. 1, and the specific sequence is TTTTTT.

[0022] In the application, the palindromic sequence region comprises a DNA sequence and an RNA sequence, the DNA sequence is shown as SEQ ID NO. 2, and the specific sequence is 5'-CAGATCTGTG-3'; the RNA sequence is shown as SEQ ID NO. 3, and the specific sequence is 5'-ACACAGAUCUG-3'. The palindromic sequence can self-hybridize to form a hairpin structure.

[0023] In the application, the sequence of the palindromic sequence region is specifically as follows: 5'-CAGATCTGTG-AATCAGGCTCTTGAACCTCA-ACACAGAUCUG-3'.

[0024] In the application, the 5' end of the palindromic DNA probe is modified by thiolation.

[0025] In the present application, the sequence of the palindromic structure DNA probe is specifically as follows: 5'-SH-(CH2)6-TTTTTT-CAGATCTGTG-AATCAGGCTCTTGAACCTCA-ACACAGAUCUG-3'.

[0026] In the present application, the CRISPR / Cas12a system comprises a Cas12a protein and a gRNA.

[0027] The present application also provides a detection method using the electrochemical biosensor, comprising the following steps: (1) incubating the polished and washed gold electrode with the palindromic structure DNA probe, and then blocking to obtain a palindromic structure DNA probe modified electrode; (2) mixing the complex of Cas12a protein and gRNA with a sample solution containing the DNA to be detected to obtain mixed solution 1; (3) adding mixed solution 1 to the modified electrode of step (1), incubating, then staining, and recording the SWV signal.

[0028] In the present application, the gold electrode is polished, then washed with Piranha solution, and then washed with PBS three times and blown dry with an ear ball. Then incubated with the palindromic structure DNA probe, the incubation temperature is preferably 36-38℃, further preferably 37℃; the incubation time is preferably 11-13h, further preferably 12h. The addition amount of the palindromic structure DNA probe is preferably 15-25μL, further preferably 18-22μL, and more preferably 20μL.

[0029] In the present application, methylcyclohexenone (MCH) is used for blocking, blocking non-specific sites, the blocking temperature is preferably 36-38℃, further preferably 37℃; the blocking time is preferably 0.8-1.2h, further preferably 1h. Then washed with PBS three times and blown dry with an ear ball to obtain a palindromic structure DNA probe modified electrode, which can be stored in a 4℃ refrigerator for standby.

[0030] In the present application, the Cas12a protein and gRNA in the CRISPR / Cas12a system are mixed to obtain a Cas12a protein / gRNA complex, and then the Cas12a protein / gRNA complex is mixed with a sample solution containing the DNA to be tested to obtain mixed solution 1. The sequence of the gRNA described in the present application is shown in SEQ ID NO. 4, and the specific sequence is 5'-CCAACAACUUCAGCAUCGAU-3'. The volume ratio of the Cas12a protein to the gRNA described in the present application is preferably 1:(4-7), further preferably 1:(5-6.5), and more preferably 1:5. The volume ratio of the Cas12a protein / gRNA complex described in the present application to the sample solution containing the DNA to be tested is preferably (2-4):1, further preferably (2.5-3.5):1, and more preferably 3:1.

[0031] In the present application, the mixed solution 1 is added dropwise to the modified electrode described above, and the dropwise addition amount of the mixed solution 1 is preferably 15-25 μL, further preferably 18-22 μL, and more preferably 20 μL. Then, incubation is performed, and the temperature of the incubation is preferably 36-38°C, further preferably 37°C; and the time of the incubation is preferably 15-25 min, further preferably 18-22 min, and more preferably 20 min.

[0032] In the present application, after the incubation is completed, methylene blue (MB) staining is performed, and then the SWV signal is recorded after washing. The time of the staining described in the present application is preferably 3-8 min, further preferably 4-6 min, and more preferably 5 min.

[0033] In the present application, if the sample solution contains the target DNA of interest, the current difference is higher than that of the negative group; if the sample solution does not contain the target DNA of interest, the current difference is lower than that of the negative group.

[0034] In the present application, when the target DNA of interest is contained, the target DNA of interest binds to the Cas12a protein / gRNA complex, activates the transcleavage activity, cuts the spacer, destroys the hairpin structure, releases MB, realizes the conversion of the electrochemical signal, and the current returns to the baseline (ΔI≤2 μA).

[0035] In the present application, when the target DNA of interest is not contained, no binding occurs, the palindromic probe self-hybridizes to form a hairpin structure, a large amount of MB is adsorbed, and in the presence of an electronic medium, the current is significantly increased (ΔI≥10 μA) as shown in Figure 1

[0036] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.​

[0037] Example 1

[0038] Construction of electrochemical biosensor based on palindromic structure nucleic acid and CRISPR / Cas12a: (I) Design of palindromic DNA probe The sequence of the palindromic DNA probe includes a spacer region and a palindromic sequence region, and is thiol-modified at the 5' end. The palindromic sequence can self-hybridize to form a hairpin structure.

[0039] The sequence of the spacer region is shown in SEQ ID NO. 1, and the specific sequence is TTTTTT.

[0040] The palindromic sequence region includes a DNA sequence and an RNA sequence, the DNA sequence is shown in SEQ ID NO. 2, and the specific sequence is 5'-CAGATCTGTG-3'; the RNA sequence is shown in SEQ ID NO. 3, and the specific sequence is 5'-ACACAGAUCUG-3'.

[0041] The sequence of the palindromic DNA probe is 5'-SH-(CH2)6-TTTTTT-CAGATCTGTG-AATCAGGCTCTTGAACCTCA-ACACAGAUCUG-3'.

[0042] (II) Modification of palindromic DNA probe electrode

[0043] The gold electrode is polished, then cleaned with Piranha solution, and then cleaned with PBS three times, and blown dry with an ear bulb. Take 20 μL of the above palindromic DNA probe and add it to the chip with the gold electrode fixed (purchased from Shenzhen Yixin Intelligent Technology Co., Ltd.), incubate at 37℃ in the dark for 12 hours, then block the non-specific site with MCH at 37℃ for 1 hour, then clean with PBS three times, blow dry with an ear bulb, and store in a 4℃ refrigerator for standby.

[0044] Example 2

[0045] The detection method of the electrochemical biosensor described in Example 1 is used: the Cas12a protein (10 uM) and gRNA (1 uM, sequence shown in SEQ ID NO. 4, 5'-CCAACAACUUCAGCAUCGAU-3') in the CRISPR / Cas12a system are mixed according to a volume ratio of 1:6 to obtain a Cas12a protein / gRNA complex, then 5 μL of a sample solution containing the DNA to be detected is added to the Cas12a protein / gRNA complex to obtain mixed solution 1. 20 μL of mixed solution 1 is added dropwise to the modified electrode prepared in Example 1, incubated at 37°C for 20 minutes, and the chip after the reaction is dyed with 50 μM MB solution for 5 minutes. After washing, the SWV signal is recorded (as shown in Figure 2 ). (I)

[0047] 1 μM, 10 nM, 100 pM, 1 pM, 10 fM, and 1 fM of Mycoplasma hominis DNA (provided by Nanjing Kebai Biological Technology Co., Ltd., GenBank: NC_013511.1) are set respectively, and a negative group (ddH2O) is set as a control. Each group is detected according to the above detection method to verify the detection limit.

[0048] Table 1 shows the peak difference of different groups

[0049] The results show (as shown in Figure 3 ): the peak difference of the negative group is 11.08 uA, the peak difference of the 1 μM group is 0.27 uA, and the peak difference of the 1 fM group is 5.75 uA; the detection limit reaches 1 fM, and the linear range spans 9 orders of magnitude. (II)

[0051] 1 μM of completely matched Mycoplasma hominis DNA, 1 μM of single-base mismatched DNA, 1 μM of four-base mismatched DNA, and a negative group (ddH2O) are set as test samples, and specific tests are performed according to the above detection method.

[0052] Mycoplasma hominis DNA: GenBank number NC_013511.1.

[0053] Single-base mismatched DNA: according to the normal GenBank: NC_013511.1 Mycoplasma hominis DNA sequence, the 871th T base is mismatched to G.

[0054] Four-base mismatched DNA: according to the normal GenBank: NC_013511.1 Mycoplasma hominis DNA sequence, the 871th-874th TGAA four bases are mismatched to CCGC.

[0055] Table 2 Wave peak difference and current change of different groups

[0056] The results show (as shown in Figure 4 ): the current of the completely matched sample decreases by 87%, the current of the single-base mismatched sample decreases by only 12%, and the current of the four-base mismatched sample decreases by 9%.

[0057] In summary, the detection method using the electrochemical biosensor has high sensitivity, strong specificity, fast detection speed, low cost, and strong universality, and can detect various pathogens.

[0058] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. An electrochemical biosensor based on palindromic nucleic acid and CRISPR / Cas12a, characterized in that, It includes a gold electrode with a surface-modified palindromic DNA probe and a CRISPR / Cas12a system; The palindromic DNA probe sequence includes a spacer region and a palindromic sequence region; The sequence of the interval region is shown in SEQ ID NO.1; The palindromic sequence region includes a DNA sequence as shown in SEQ ID NO.2 and an RNA sequence as shown in SEQ ID NO.3; The CRISPR / Cas12a system includes the Cas12a protein and gRNA.

2. The electrochemical biosensor according to claim 1, characterized in that, Thiolation modification was performed at the 5' end of the palindromic DNA probe.

3. A detection method using the electrochemical biosensor described in claim 1 or 2, characterized in that, Includes the following steps: (1) The polished and cleaned gold electrode was incubated with the palindromic DNA probe and then blocked to obtain the palindromic DNA probe modified electrode. (2) Mix the complex of Cas12a protein and gRNA with the sample solution containing the DNA to be tested to obtain Mixture 1; (3) Add the mixture 1 droplet to the modified electrode described in step (1), incubate, stain, and record the SWV signal; If the sample solution contains the target DNA, the current difference is higher than that of the negative group; if the sample solution does not contain the target DNA, the current difference is lower than that of the negative group.

4. The detection method according to claim 3, characterized in that, The amount of palindromic DNA probe added in step (1) is 15-25 μL; The incubation temperature in step (1) is 36-38℃, and the incubation time is 11-13h.

5. The detection method according to claim 3, characterized in that, Step (1) uses methylcyclohexenone for sealing at a temperature of 36-38°C for a time of 0.8-1.2 h.

6. The detection method according to claim 3, characterized in that, In step (2), the volume ratio of the Cas12a protein and gRNA complex to the sample solution containing the DNA to be tested is (2-4):

1.

7. The detection method according to claim 3, characterized in that, The amount of the mixed solution 1 added in step (3) is 15-25 μL.

8. The detection method according to claim 3, characterized in that, The incubation temperature in step (3) is 36-38℃, and the incubation time is 15-25 min.

9. The detection method according to claim 3, characterized in that, The staining time in step (3) is 3-8 minutes.

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