Inverted TDN modified electrode, CRISPR / Cas12a mediated DNA tetrahedral reporter probe interface cleavage ratio type electrochemical aptamer sensor and application

Abstract

The invention discloses an inverted TDN modified electrode, a CRISPR / Cas12a mediated DNA tetrahedron reporter probe interface cleavage ratio type electrochemical aptamer sensor and application of the ratio type electrochemical aptamer sensor and the CRISPR / Cas12a mediated DNA tetrahedron reporter probe interface cleavage ratio type electrochemical aptamer sensor. A sulfydryl-modified substrate chain is assembled on three edges of a DNA tetrahedron, and then is fixed on the surface of an electrode through a gold-sulfur bond, so that inverted fixation of the DNA tetrahedron is realized. The steric hindrance effect of a DNA tetrahedron hinders electron transfer of potassium ferricyanide on an electrode interface, and meanwhile, an electroactive molecule methylene blue is embedded into a double-chain skeleton, so that double-signal response is realized. When the trans-cleavage activity of CRISPR / Cas12a is activated, the co-block nucleic acid substrate chain is split, so that the DNA tetrahedral structure is dissociated from the surface of the electrode, and an MB signal is weakened. Meanwhile, the potassium ferricyanide signal is enhanced due to the recovery of the interface electron transmission efficiency, and a ratio signal is formed and output. According to the invention, the signal gain rate and the detection accuracy are effectively improved.

Description

Technical Field

[0001] The present invention relates to an inverted TDN-modified electrode, a ratiometric electrochemical aptasensor for CRISPR / Cas12a-mediated interfacial cleavage of DNA tetrahedron reporter probes, and applications thereof, belonging to the technical field of biosensing detection. Background Art

[0002] Combining CRISPR technology with electrochemical sensing (E-CRISPR) is expected to achieve breakthrough progress in point-of-care testing applications. However, inappropriate immobilization of reporter probes on the electrode surface results in a steric hindrance effect, leading to poor cleavage efficiency of CRISPR / Cas and thus affecting detection sensitivity. Although some studies have used upright DNA tetrahedron frameworks to improve the nucleic acid distribution on the electrode interface to enhance the cleavage efficiency. However, electrochemical reporter probes often need to be labeled with electroactive molecules such as methylene blue, which additionally increases the detection cost and probe purification steps of CRISPR electrochemical sensors. In addition, compared with the dual-labeled fluorophores in CRISPR-based fluorescence detection methods, the single response signal on the electrode interface further restricts the sensitivity and reliability of electrochemical sensors. Therefore, developing a label-free ratiometric signal output reporter probe is of great significance for constructing CRISPR-based electrochemical sensing. Summary of the Invention

[0003] The object of the present invention is: aiming at the deficiencies of the prior art, the present invention provides an inverted TDN-modified electrode, a ratiometric electrochemical aptasensor for CRISPR / Cas12a-mediated interfacial cleavage of DNA tetrahedron reporter probes, and applications thereof. By reasonably constructing the reporter probe for CRISPR / Cas trans-cleavage on the electrode interface, the trans-cleavage efficiency is improved, and label-free ratiometric electrochemical signal output is achieved.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] In the first aspect of the present invention, a method for preparing an inverted DNA tetrahedron probe-modified electrode is provided, comprising the following steps:

[0006] Step 1): Activate the glassy carbon electrode by CV scanning in a potassium ferricyanide electrolyte solution, wash and dry for standby. Then, drop the MXene aqueous solution on the surface of the glassy carbon electrode, dry it in an oven, and finally electrochemically deposit gold nanoparticles in a gold electrolyte solution to obtain the electrode Au-MXene / GCE;

[0007] Step 2): Incubate the DNA tetrahedron with a thiol-modified substrate strand probe SH-TE; the substrate strand probe contains a cleavage region that can be trans-cleaved by CRISPR-Cas12a and a thymine-rich anchoring region that can be assembled onto three edges of the DNA tetrahedron through Hoogsteen hydrogen bonds from the 5'-end to the 3'-end;

[0008] Step 3): Co-incubate the DNA tetrahedron obtained in Step 2) with the electrode Au-MXene / GCE obtained in Step 1), and then co-incubate with MCH to obtain an inverted DNA tetrahedron probe modified electrode SH-TE / TDN / Au-MXene / GCE.

[0009] In the above technical solution, a DNA tetrahedron is constructed by annealing, and a thiol-modified co-block nucleic acid substrate strand is assembled onto three edges of the DNA tetrahedron through Hoogsteen hydrogen bonds, and then fixed on the surface of the gold electrode through Au-S to achieve the inverted fixation of the DNA tetrahedron.

[0010] Preferably, the glassy carbon electrode in Step 1) is a glassy carbon electrode that has been polished and ultrasonically cleaned;

[0011] The potassium ferricyanide electrolyte solution is a potassium ferricyanide-potassium chloride solution, which contains 0.5 - 2 mM [Fe(CN)6] 3- / 4- and 0.05 - 0.2 M KCl;

[0012] The gold electrolyte solution is an aqueous solution of HAuCl4, and the concentration of the aqueous solution of HAuCl4 is 0.5 - 2 wt%.

[0013] Preferably, the conditions for electrochemically depositing gold nanoparticles are: depositing gold nanoparticles for 1 - 3 min at a potential of -0.5 to -0.1 V in an HAuCl4 solution with a mass fraction of 0.5 - 2%.

[0014] Preferably, the DNA tetrahedron in Step 2) is self-assembled from four single strands shown in SEQ ID NO: 1 - 4 through an annealing program;

[0015] and / or, the substrate strand probe in Step 2) is as shown in SEQ ID NO: 5.

[0016] In the second aspect of the present invention, a ratio-type electrochemical aptasensor for CRISPR / Cas12a-mediated DNA tetrahedron reporter probe interface cleavage is provided, which includes an inverted DNA tetrahedron probe modified electrode SH-TE / TDN / Au-MXene / GCE prepared by the preparation method described in the first aspect of the present invention, Cas12a / crRNA, and a recognition probe.

[0017] Preferably, the recognition probe is obtained by hybridizing an aptamer probe and an activation strand.

[0018] In the third aspect of the present invention, there is provided an inverted DNA tetrahedron probe modified electrode prepared by the preparation method described in the first aspect, and the application of the ratio-type electrochemical aptasensor described in the second aspect in detecting kanamycin. The detection is carried out with the inverted DNA tetrahedron probe modified electrode as the sensing electrode, and the probe obtained by the hybridization reaction of the aptamer probes kana-1 and kana-2 and the activation strand as the recognition probe, and is carried out under the mediation of Cas12a / crRNA and in the presence of the electroactive molecule methylene blue and the indicator potassium ferricyanide; wherein:

[0019] The sequence of the aptamer probe kana-1 is shown as SEQ ID NO: 7, the sequence of the aptamer probe kana-2 is shown as SEQ ID NO: 8, the sequence of the activation strand is shown as SEQ ID NO: 6, and the sequence of the crRNA is shown as SEQ ID NO: 9;

[0020] The principle of the detection is as follows: If the sample to be tested contains kanamycin, then kanamycin will bind to the nucleic acid aptamer in the recognition probe, release the activation strand to activate the trans-cleavage activity of Cas12a / crRNA, shear the substrate strand on the electrode, so that the inverted DNA tetrahedron dissociates from the electrode surface, restore the electron transfer ability of the electrode interface, increase the signal of potassium ferricyanide, and decrease the signal of MB embedded in the TDN framework, realizing ratio signal output.

[0021] Preferably, the preparation method of the recognition probe includes: first hybridize two 2 μM aptamer probes kana-1 and kana-2 with 1 μM activation strand (activator) in Tris-NaCl buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.5) to obtain the recognition probe; then dilute the recognition probe to 100 nM for standby.

[0022] Preferably, the temperature of the hybridization reaction is 95 °C, the time is 5 minutes, and it is slowly cooled to room temperature.

[0023] In the fourth aspect of the present invention, there is provided a method for detecting kanamycin, which includes: co-incubating the recognition probe (final concentration 5 nM) with the sample to be tested, Cas12a / crRNA (final concentration 15 nM) and the inverted DNA tetrahedron probe modified electrode, rinsing the electrode, adding the indicator potassium ferricyanide, and then co-incubating with the MB solution (10 μM), and qualitatively or quantitatively detecting kanamycin in the sample to be tested by electrochemically measuring the current intensities of potassium ferricyanide and MB.

[0024] Preferably, the quantitative detection further includes the step of establishing a standard curve: Mix Cas12a / crRNA (final concentration 15 nM) with the recognition probe (final concentration 5 nM) and kanamycin standard products at different concentrations. After rinsing the electrode, add the indicator potassium ferricyanide, and then mix with the MB solution (10 μM) and incubate (5 min). Use square wave voltammetry to record the current intensities of potassium ferricyanide and MB respectively in the range of 0.6 to -0.6 V, establish the linear relationship between the changes in the ratio of current intensities of potassium ferricyanide / methylene blue for different concentrations of antibiotics, and obtain the standard curve.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. In the present invention, the substrate strand probe for cleavage is assembled on three edges of the DNA tetrahedron through Hoogsteen hydrogen bonds to construct an inverted tetrahedron reporter probe, realizing the uniform distribution of the cleaved substrate strand on the electrode interface. At the same time, the inverted fixation of the DNA tetrahedron framework improves the distribution of the substrate strand on the electrode interface and enhances the cleavage efficiency.

[0027] 2. The steric hindrance effect of the DNA tetrahedron increases the signal change rate and amplifies the detection signal.

[0028] 3. By utilizing the signals of potassium ferricyanide and the MB (Methylene blue) embedded in the DNA tetrahedron, the ratio-type electrochemical signal output of MB and potassium ferricyanide before and after trans-cleavage can be realized, improving the detection sensitivity and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the principle of the present invention.

[0030] Figure 2 It is the square wave voltammetry images of using activated and unactivated Cas12a / crRNA to cleave (A) SH-TE and (B) SH-TE / TDN modified electrodes.

[0031] Figure 3 It is the cyclic voltammetry scanning diagrams of bare glassy carbon electrode (a), MXene / GCE (b), Au-MXene / GCE (c), SH-TE / TDN / Au-MXene / GCE (d), and Cas12a RNP / SH-TE / TDN / Au-MXene / GCE (e).

[0032] Figure 4 It is the square wave voltammograms of using the inverted DNA tetrahedron probe modified electrode with and without kanamycin.

[0033] Figure 5The linear relationship between the signal differences detected by the electrode modified with the inverted DNA tetrahedron probe and different concentrations of kanamycin.

[0034] Figure 6 The selectivity of the electrode modified with the inverted DNA tetrahedron probe for detecting kanamycin. Detailed implementation manners

[0035] To make the present invention more obvious and understandable, preferred embodiments are hereby described in detail in conjunction with the accompanying drawings as follows.

[0036] For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications; the materials, reagents, etc. used, unless otherwise specified, are all commercially available conventional products.

[0037] In the present invention, the four single strands used for assembling the DNA tetrahedron and the probe sequences involved are shown in Table 1:

[0038] Table 1 Sequence list

[0039]

[0040]

[0041] Example 1

[0042] A preparation method of an electrode based on an inverted DNA tetrahedron modification:

[0043] 1) Polishing of the glassy carbon electrode and preparation of the composite material modified electrode

[0044] The glassy carbon electrode was polished with 0.3 μm and 0.05 μm alumina powders respectively. Next, the glassy carbon electrode was sonicated in ethanol, ultrapure water, 50% nitric acid aqueous solution and ultrapure water respectively. Then, it was scanned by CV in a 1 mM potassium ferricyanide solution (1 mM [Fe(CN)6] 3- / 4- and 0.1 M KCl). Finally, the surface of the glassy carbon electrode was rinsed with Milli-Q water and dried with nitrogen for standby. Subsequently, an aqueous solution of Ti3C2Tx MXene (CAS No.: 12316-56-2, purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.) was dropped on the electrode surface, dried at 37 °C for 10 min, and then nanogold particles were deposited at a potential of -0.2 V in a 1 wt% HAuCl4 solution for 1 min to obtain the electrode Au-MXene / GCE.

[0045] 2) Preparation of the DNA tetrahedron probe

[0046] Mix four single-stranded Probes TA (SEQ ID NO: 1), TB (SEQ ID NO: 2), TC (SEQ ID NO: 3), and TD (SEQ ID NO: 4) in a molar ratio of 1:1:1:1 in TM buffer (pH 8.0), then add 30 mM of TCEP (Tris(2-carboxyethyl)phosphine) to make the final concentration of DNA (each nucleic acid strand) 5 μM. Then perform slow annealing using a PCR instrument and store at 4 °C for later use. At the same time, dilute the substrate strand (SH-TE, SEQ ID NO: 5) to 5 μM with TM buffer (the solution contains 3 mM of TCEP), then perform slow annealing using a PCR instrument and store at 4 °C for later use. Mix and dilute the prepared DNA tetrahedron and the annealed thiol-modified nucleic acid substrate strand in a molar ratio of 1:1.5 in TM buffer (pH 7.0) and incubate for 4 hours.

[0047] 3) Preparation of an inverted DNA tetrahedron probe-modified electrode

[0048] Subsequently, incubate the DNA tetrahedron probe prepared in step 2) with the prepared electrode Au-MXene / GCE overnight, and then co-incubate with 2 mM of MCH (6-mercaptohexanol) for 1 h to obtain an inverted TDN-modified electrode (SH-TE / TDN / Au-MXene / GCE).

[0049] Example 2

[0050] Method for measuring kanamycin:

[0051] 1) Preparation of the recognition probe

[0052] First, hybridize two 2 μM aptamer probes kana-1 (SEQ ID NO: 7) and kana-2 (SEQ ID NO: 8) with 1 μM of the activator strand (SEQ ID NO: 6) in Tris-NaCl buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.5). After the hybridization reaction at 95 °C for 5 minutes, slowly cool to room temperature, and then dilute the solution to 100 nM to obtain the recognition probe solution for later use.

[0053] 2) Detection of kanamycin

[0054] Mix Cas12a / crRNA (final concentration 15 nM, SEQ ID NO: 9) with the recognition probe (final concentration 5 nM) and different concentrations of kanamycin, and incubate with the inverted TDN-modified electrode prepared in Example 1 for 45 min. After rinsing the electrode, add the indicator potassium ferricyanide, mix and incubate with the MB solution (Methylene Blue solution, 10 μM) for 5 min, and finally perform electrochemical measurements.

[0055] Figure 1 This is the schematic diagram of the preparation and detection of the electrode of the present invention. The principle of the present invention is as follows: The thiol-modified substrate strand is assembled onto three edges of the DNA tetrahedron through Hoogsteen hydrogen bonds, and then fixed on the surface of the Au-MXene / GCE electrode through gold-sulfur bonds to achieve the inverted fixation of the DNA tetrahedron. The framework structure of the DNA tetrahedron hinders the electron transfer of the electrochemical indicator potassium ferricyanide at the electrode interface through steric hindrance. At the same time, the electroactive molecule methylene blue (MB) can also be embedded in the double-stranded backbone, thus realizing dual-signal response. When the trans-cleavage activity of CRISPR / Cas12a is activated, the co-block nucleic acid substrate strand is cleaved, causing the DNA tetrahedron structure to dissociate from the electrode surface, resulting in a decrease in the MB signal. At the same time, the potassium ferricyanide signal increases due to the restoration of the interfacial electron transfer efficiency, forming a ratio signal output.

[0056] Figure 2 Square wave voltammetry images of the (A) SH-TE and (B) SH-TE / TDN modified electrodes cut with activated and unactivated Cas12a / crRNA; as shown in the figure, the substrate strand probe with the inverted tetrahedron produced a large signal gain before and after cleavage, indicating the signal advantage of the inverted tetrahedron-assisted fixation of the substrate strand.

[0057] Figure 3Cyclic voltammetry scanning diagrams of bare glassy carbon electrode (a), MXene / GCE, Au-MXene / GCE (c), SH-TE / TDN / Au-MXene / GCE (d), and Cas12a RNP / SH-TE / TDN / Au-MXene / GCE (e). As shown in the figure, the current intensities of the bare glassy carbon electrode, MXene-modified glassy carbon electrode (MXene / GCE), Au-MXene-modified glassy carbon electrode, and SH-TE / TDN / Au-MXene / GCE decrease with DNA modification, indicating to a certain extent the success of the composite material-modified electrode and the successful assembly of SH-TE / TDN on the electrode surface; while the current intensity of Cas12a / SH-TE / Au-MXene / GCE increases, indicating that after Cas12a is activated, the substrate strand on the electrode surface is cleaved, resulting in the release of TDN and the restoration of the ability of electron transfer, verifying the feasibility of this immobilization method for cleavage.

[0058] Figure 4 Square wave voltammograms of the modified electrode using the inverted DNA tetrahedron probe with and without kanamycin. As shown in the figure, the difference in the electrochemical signals detected using this strategy with and without kanamycin indicates that this strategy can be applied to the detection of kanamycin.

[0059] Figure 5 Linear relationship between the signal difference detected by the electrode modified with the inverted DNA tetrahedron probe and different concentrations of kanamycin. As shown in the figure, there is a good linear relationship between the ratio of the current response signal for detecting kanamycin using this strategy and the concentration of kanamycin, indicating that the proposed strategy can be applied to the quantitative detection of kanamycin.

[0060] Figure 6 Selectivity of the electrode modified with the inverted DNA tetrahedron probe for detecting kanamycin. As shown in the figure, the electrochemical signals for detecting kanamycin and other interfering antibiotics using this strategy indicate that this strategy has good selectivity.

[0061] As described above, only the preferred embodiments of the present invention are provided, and there is no limitation in any form and substance to the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of an inverted DNA tetrahedron probe modified electrode, characterized in that, It includes the following steps: Step 1): Activate the glassy carbon electrode by CV scanning in a potassium ferricyanide electrolyte solution, wash and dry it for standby. Then, drop the MXene aqueous solution on the surface of the glassy carbon electrode, dry it in an oven. Finally, electrochemically deposit gold nanoparticles in the gold electrolyte solution to obtain the electrode Au-MXene / GCE; Step 2): Incubate the DNA tetrahedron with the thiol-modified substrate strand probe SH-TE; the substrate strand probe contains a cleavage region that can be trans-cleaved by CRISPR-Cas12a and a thymine-rich anchoring region that can be assembled onto three edges of the DNA tetrahedron through Hoogsteen hydrogen bonds from the 5'-end to the 3'-end; Step 3): Co-incubate the DNA tetrahedron obtained in Step 2) with the electrode Au-MXene / GCE obtained in Step 1), and then co-incubate with MCH to obtain the inverted DNA tetrahedron probe-modified electrode SH-TE / TDN / Au-MXene / GCE.

2. The preparation method according to claim 1, wherein The glassy carbon electrode in Step 1) is a glassy carbon electrode that has been polished and ultrasonically cleaned; The potassium ferricyanide electrolyte solution is a potassium ferricyanide - potassium chloride solution, which contains 0.5 - 2 mM [Fe(CN)6] 3- / 4- and 0.05 - 0.2 M KCl; The gold electrolyte solution is an aqueous solution of HAuCl4.

3. The preparation method according to claim 1, characterized in that, The DNA tetrahedron in Step 2) is self-assembled from four single strands with sequences shown in SEQ ID NO: 1-4 through an annealing procedure; And / or, the substrate strand probe in Step 2) is as shown in SEQ ID NO:

5.

4. A ratiometric electrochemical aptasensor based on CRISPR / Cas12a-mediated interfacial cleavage of DNA tetrahedron reporter probe, characterized in that, It includes the inverted DNA tetrahedron probe-modified electrode SH-TE / TDN / Au-MXene / GCE, Cas12a / crRNA, and recognition probe prepared by the preparation method described in any one of claims 1-3.

5. The ratiometric electrochemical aptasensor according to claim 4, characterized in that, The recognition probe is obtained by hybridization of an aptamer probe and an activation strand.

6. The inverted DNA tetrahedron probe modified electrode prepared by the preparation method according to any one of claims 1 to 3, and the application of the ratio-type electrochemical aptasensor according to claim 4 in detecting kanamycin, characterized in that, The detection uses the inverted DNA tetrahedron probe-modified electrode as the sensing electrode, and the probe obtained by hybridization of the aptamer probes kana-1, kana-2 and the activation strand as the recognition probe, and is carried out under the mediation of Cas12a / crRNA and the participation of the electroactive molecule methylene blue and the indicator potassium ferricyanide; where: The sequence of the aptamer probe kana-1 is as shown in SEQ ID NO: 7, the sequence of the aptamer probe kana-2 is as shown in SEQ ID NO: 8, the sequence of the activation strand is as shown in SEQ ID NO: 6, and the sequence of the crRNA is as shown in SEQ ID NO: 9; The principle of the detection is: If the test sample contains kanamycin, then kanamycin will bind to the nucleic acid aptamer in the recognition probe, release the activation strand to activate the trans-cleavage activity of Cas12a / crRNA, shear the substrate strand on the electrode, cause the inverted DNA tetrahedron to dissociate from the electrode surface, restore the electron transfer ability at the electrode interface, increase the signal of potassium ferricyanide, and decrease the signal of MB embedded in the TDN framework, realizing ratio signal output.

7. The application according to claim 6, wherein The preparation method of the recognition probe includes: First, mix two aptamer probes kana-1 and kana-2 with the activation strand in Tris-NaCl buffer, react at 95 °C for 5 minutes, and then slowly cool down to room temperature to obtain the recognition probe.

8. A method for detecting kanamycin, characterized in that, Including: Co-incubate the recognition probe with the sample to be tested, Cas12a / crRNA and the inverted DNA tetrahedron probe modified electrode. After rinsing the electrode, add the indicator potassium ferricyanide, and then co-incubate with methylene blue solution. Qualitative or quantitative detection of kanamycin in the sample to be tested is achieved by electrochemically measuring the current intensities of potassium ferricyanide and methylene blue.

9. The method according to claim 8, wherein The quantitative detection also includes the step of establishing a standard curve: Mix Cas12a / crRNA with the recognition probe and kanamycin standard products with different concentrations. After rinsing the electrode, add the indicator potassium ferricyanide, and then mix and incubate with methylene blue solution. Square wave voltammetry is used to record the current intensities of potassium ferricyanide and methylene blue in the range of 0.6~-0.6V respectively, establish the linear relationship between the change in the ratio of different concentrations of antibiotics to the current intensity ratio of potassium ferricyanide / methylene blue, and obtain the standard curve.

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