Inverted TDN-modified electrodes, ratiometric electrochemical aptamer sensors mediated by CRISPR / Cas12a-mediated DNA tetrahedral reporter probe interface cleavage, and their applications.
By modifying the electrode with an inverted DNA tetrahedral probe, the problem of electrode surface fixation in CRISPR/Cas electrochemical sensors was solved, achieving efficient ratiometric electrochemical signal output and improving detection sensitivity and accuracy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- UNIV OF SHANGHAI FOR SCI & TECH
- Filing Date
- 2025-04-09
- Publication Date
- 2026-06-30
AI Technical Summary
In existing CRISPR/Cas electrochemical sensors, unsuitable reporter probe fixation on the electrode surface leads to poor CRISPR/Cas cleavage efficiency, affecting detection sensitivity. Furthermore, electrochemical sensors require additional labeling of electroactive molecules, increasing cost and complexity. A single response signal limits sensitivity and reliability.
An electrode was modified with an inverted DNA tetrahedral probe. The substrate chain probe was assembled by Hoogsteen hydrogen bonds and fixed on the surface of a gold electrode to construct a ratiometric electrochemical signal output. The ratiometric electrochemical signal output was realized by embedding MB signals in potassium ferricyanide and DNA tetrahedrons.
This improved the efficiency of CRISPR/Cas trans-cutting, enabled label-free ratiometric electrochemical signal output, and enhanced detection sensitivity and accuracy.
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Figure CN120275470B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to an inverted TDN-modified electrode, a ratiometric electrochemical aptamer sensor with CRISPR / Cas12a-mediated DNA tetrahedral reporter probe interface cleavage, and their applications, belonging to the field of biosensing and detection technology. Background Technology
[0002] Combining CRISPR technology with electrochemical sensing (E-CRISPR) holds promise for breakthroughs in point-of-care testing applications. However, unsuitable reporter probe immobilization on the electrode surface leads to steric hindrance, resulting in poor CRISPR / Cas cleavage efficiency and consequently affecting detection sensitivity. Although some studies have employed upright DNA tetrahedral frameworks to improve nucleic acid distribution at the electrode interface and enhance cleavage efficiency, electrochemical reporter probes often require labeling with electroactive molecules such as methylene blue, adding to the detection cost and probe purification steps of CRISPR electrochemical sensors. Furthermore, compared to dual-labeled fluorophores in CRISPR-based fluorescence detection methods, the single response signal at the electrode interface further limits the sensitivity and reliability of electrochemical sensors. Therefore, developing a label-free ratiometric signal output reporter probe is crucial for constructing CRISPR-based electrochemical sensors. Summary of the Invention
[0003] The purpose of this invention is to address the shortcomings of existing technologies by providing an inverted TDN-modified electrode, a ratiometric electrochemical aptamer sensor for CRISPR / Cas12a-mediated DNA tetrahedral reporter probe interface cleavage, and its application. By rationally constructing the CRISPR / Cas trans-cleavage reporter probe at the electrode interface, the trans-cleavage efficiency is improved, and label-free ratiometric electrochemical signal output is achieved.
[0004] To achieve the above objectives, the present invention adopts the following technical solution:
[0005] A first aspect of the present invention provides a method for preparing an inverted DNA tetrahedral probe-modified electrode, comprising the following steps:
[0006] Step 1): The glassy carbon electrode was activated in potassium ferricyanide electrolyte solution by CV scanning, cleaned and dried for later use. Then, MXene aqueous solution was dropped onto the surface of the glassy carbon electrode and dried. Finally, gold nanoparticles were electrochemically deposited in gold electrolyte solution to obtain the electrode Au-MXene / GCE.
[0007] Step 2): Incubate the DNA tetrahedron with the thiol-modified substrate chain probe SH-TE; the substrate chain probe contains a cleavage region that can be trans-cleaved by CRISPR-Cas12a and a thymine-rich anchoring region that can be assembled to the three edges of the DNA tetrahedron via 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 it with MCH to obtain the 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 chain is assembled onto three edges of the DNA tetrahedron via Hoogsteen hydrogen bonds. Subsequently, it is fixed on the surface of a gold electrode by 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 containing 0.5-2 mM [Fe(CN)6]. 3- / 4- and 0.05-0.2M 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 electrochemical deposition of gold nanoparticles are as follows: gold nanoparticles are deposited in a 0.5-2% (w / w) HAuCl4 solution at a potential of -0.5 to -0.1 V for 1 to 3 minutes.
[0014] Preferably, the DNA tetrahedron in step 2) is formed by the self-assembly of four single strands with sequences as shown in SEQ ID NO: 1-4 through an annealing process;
[0015] And / or, the substrate chain probe in step 2) is as shown in SEQ ID NO: 5.
[0016] A second aspect of the present invention provides a ratiometric electrochemical aptamer sensor for CRISPR / Cas12a-mediated DNA tetrahedral reporter probe interface cleavage, comprising an inverted DNA tetrahedral probe-modified electrode SH-TE / TDN / Au-MXene / GCE, Cas12a / crRNA, and a recognition probe prepared by the preparation method described in the first aspect of the present invention.
[0017] Preferably, the recognition probe is obtained by hybridization of an aptamer probe and an activation strand.
[0018] A third aspect of the present invention provides an inverted DNA tetrahedral probe-modified electrode prepared by the method described in the first aspect, and the application of the ratiometric electrochemical aptamer sensor described in the second aspect in the detection of kanamycin. The detection uses the inverted DNA tetrahedral probe-modified electrode as the sensing electrode, and probes obtained by hybridization of aptamer probes kana-1 and kana-2 with the activating strand as recognition probes. Detection is performed under the mediation of Cas12a / crRNA and with the participation of the electroactive molecule methylene blue and the indicator potassium ferricyanide. Wherein:
[0019] The sequence of the aptamer probe kana-1 is shown in SEQ ID NO: 7, the sequence of the aptamer probe kana-2 is shown in SEQ ID NO: 8, the sequence of the activation strand is shown in SEQ ID NO: 6, and the sequence of the crRNA is shown in SEQ ID NO: 9.
[0020] The detection principle 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, cleave the substrate strand on the electrode, so that the inverted DNA tetrahedron dissociates from the electrode surface, thereby restoring the electron transfer ability of the electrode interface, increasing the potassium ferricyanide signal, and decreasing the MB signal embedded in the TDN framework, thus realizing the ratio signal output.
[0021] Preferably, the method for preparing the recognition probe includes: firstly, hybridizing two 2μM aptamer probes kana-1 and kana-2 with a 1μM activator in Tris-NaCl buffer (20mM Tris-HCl, 150mM NaCl, pH 7.5) to obtain the recognition probe; then diluting the recognition probe to 100nM for later use.
[0022] Preferably, the hybridization reaction is carried out at a temperature of 95°C for 5 minutes, followed by slow cooling to room temperature.
[0023] A fourth aspect of the present invention provides a method for detecting kanamycin, comprising: co-incubating a recognition probe (final concentration 5 nM) with a sample to be tested, Cas12a / crRNA (final concentration 15 nM) and an electrode modified with an inverted DNA tetrahedral probe; rinsing the electrode; adding potassium ferricyanide as an indicator; and then co-incubating with an MB solution (10 μM); and measuring the current intensity of potassium ferricyanide and MB electrochemically to achieve qualitative or quantitative detection of kanamycin in the sample to be tested.
[0024] Preferably, the quantitative detection further includes the step of establishing a standard curve: Cas12a / crRNA (final concentration 15 nM) is mixed with the recognition probe (final concentration 5 nM) and kanamycin standards of different concentrations. After rinsing the electrode, potassium ferricyanide indicator is added, followed by incubation with MB solution (10 μM) for 5 min. The current intensities of potassium ferricyanide and MB are recorded in the range of 0.6 to -0.6 V using square wave voltammetry to establish a linear relationship between the changes in the ratio of potassium ferricyanide / methylene blue current intensities for different concentrations of antibiotics, thus obtaining the standard curve.
[0025] Compared with the prior art, the present invention has the following beneficial effects:
[0026] 1. This invention assembles the substrate chain probe for cleavage onto three edges of a DNA tetrahedron via Hoogsteen hydrogen bonds, constructing an inverted tetrahedral reporter probe. This achieves a uniform distribution of the enzyme-cleaved substrate chain at the electrode interface. Simultaneously, the inverted DNA tetrahedral framework improves the substrate chain distribution at the electrode interface, thereby increasing cleavage efficiency.
[0027] 2. The steric hindrance effect of DNA tetrahedrons increases the rate of signal change and amplifies the detection signal.
[0028] 3. By utilizing the embedded MB (methylene blue) signal in potassium ferricyanide and DNA tetrahedrons, a ratio-type electrochemical signal output of MB to potassium ferricyanide before and after trans-cleavage can be achieved, thereby improving detection sensitivity and accuracy. Attached Figure Description
[0029] Figure 1 This is a schematic diagram illustrating the principle of the present invention.
[0030] Figure 2 Square wave voltammetry images of (A) SH-TE and (B) SH-TE / TDN modified electrodes cleaved using activated and inactivated Cas12a / crRNA.
[0031] Figure 3 Cyclic voltammetric scans of bare glassy carbon electrodes (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 Square wave voltammetry plots of electrodes modified with inverted DNA tetrahedral probes with and without kanamycin.
[0033] Figure 5The linear relationship between signal differences detected using electrodes modified with inverted DNA tetrahedral probes and different concentrations of kanamycin was established.
[0034] Figure 6 To detect the selectivity of kanamycin using an electrode modified with an inverted DNA tetrahedral probe. Detailed Implementation
[0035] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.
[0036] Unless otherwise specified, the experimental methods in the following examples are performed according to conventional methods and conditions or according to the product instructions; the materials and reagents used are all commercially available products unless otherwise specified.
[0037] In this invention, the four single strands used to assemble 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 method for preparing an electrode based on an inverted DNA tetrahedron modified electrode:
[0043] 1) Polishing of glassy carbon electrodes and preparation of composite material modified electrodes
[0044] The glassy carbon electrode was polished with 0.3 μm and 0.05 μm alumina powder, respectively. Next, the glassy carbon electrode was ultrasonically treated in ethanol, ultrapure water, 50% nitric acid aqueous solution, and ultrapure water, respectively. Then, it was immersed in 1 mM potassium ferricyanide solution (1 mM [Fe(CN)6]). 3- / 4- The glassy carbon electrode surface was then scanned by CV with 0.1 M KCl. Finally, the surface was rinsed with Milli-Q water and dried with nitrogen for later use. Subsequently, an aqueous solution of Ti3C2Tx MXene (CAS No.: 12316-56-2, purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.) was dropped onto the electrode surface and dried at 37 °C for 10 min. Then, gold nanoparticles were deposited in a 1 wt% HAuCl4 solution at a potential of -0.2 V for 1 min to obtain the Au-MXene / GCE electrode.
[0045] 2) Preparation of DNA tetrahedral probes
[0046] Four single-stranded Probe TA (SEQ ID NO: 1), TB (SEQ ID NO: 2), TC (SEQ ID NO: 3), and TD (SEQ ID NO: 4) were mixed in a molar ratio of 1:1:1:1 in TM buffer (pH 8.0). Then, 30 mM of TCEP (tris(2-carboxyethyl)phosphine) was added to bring the final concentration of DNA (per nucleic acid strand) to 5 μM. The mixture was then subjected to slow annealing using a PCR instrument and stored at 4°C for later use. Simultaneously, the substrate strand (SH-TE, SEQ ID NO: 5) was diluted to 5 μM (containing 3 mM TCEP) with TM buffer and subjected to slow annealing using a PCR instrument. The mixture was then stored at 4°C for later use. The prepared DNA tetrahedra were then mixed and diluted with the annealed thiol-modified nucleic acid substrate strand at a molar ratio of 1:1.5 in TM buffer (pH 7.0) and incubated for 4 hours.
[0047] 3) Fabrication of electrodes modified with inverted DNA tetrahedral probes
[0048] The DNA tetrahedral probe prepared in step 2) was then incubated overnight with the prepared electrode Au-MXene / GCE, and then co-incubated with 2 mM MCH (6-mercaptohexanol) for 1 h to obtain the inverted TDN modified electrode (SH-TE / TDN / Au-MXene / GCE).
[0049] Example 2
[0050] Methods for measuring kanamycin:
[0051] 1) Preparation of recognition probes
[0052] First, two 2 μM aptamer probes, kana-1 (SEQ ID NO: 7) and kana-2 (SEQ ID NO: 8), were hybridized with a 1 μM activator (SEQ ID NO: 6) in Tris-NaCl buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.5). The hybridization reaction was carried out at 95 °C for 5 minutes, and then slowly cooled to room temperature. The solution was then diluted to 100 nM to obtain the recognition probe solution for later use.
[0053] 2) Detection of kanamycin
[0054] Cas12a / crRNA (final concentration 15 nM, SEQ ID NO: 9) was mixed with recognition probe (final concentration 5 nM) and different concentrations of kanamycin, and incubated with the inverted TDN-modified electrode prepared in Example 1 for 45 min. After rinsing the electrode, potassium ferricyanide indicator was added, and the mixture was incubated with MB solution (Methylene Blue, 10 μM) for 5 min. Finally, electrochemical measurements were performed.
[0055] Figure 1 This diagram illustrates the electrode preparation and detection principle of the present invention. The principle is as follows: A thiol-modified substrate chain is assembled onto three edges of a DNA tetrahedron via Hoogsteen hydrogen bonds, and then fixed to the Au-MXene / GCE electrode surface via gold-sulfur bonds, achieving inverted fixation of the DNA tetrahedron. The framework structure of the DNA tetrahedron hinders electron transfer of the electrochemical indicator potassium ferricyanide at the electrode interface through steric hindrance. Simultaneously, the electroactive molecule methylene blue (MB) can also be embedded in the double-stranded backbone, thus achieving a dual-signal response. When the trans-cleavage activity of CRISPR / Cas12a is activated, the co-block nucleic acid substrate chain is cleaved, causing the DNA tetrahedron structure to dissociate from the electrode surface, resulting in a weakened MB signal. Simultaneously, the potassium ferricyanide signal is enhanced due to the restoration of interfacial electron transport efficiency, forming a ratio signal output.
[0056] Figure 2 Square wave voltammetry images of (A) SH-TE and (B) SH-TE / TDN modified electrodes cleaved with activated and inactivated Cas12a / crRNA; the figures show that the substrate strand probe with inverted tetrahedrons produced a large signal gain before and after cleavage, indicating the signal advantage of inverted tetrahedrons in assisted substrate strand immobilization.
[0057] Figure 3Cyclic voltammetry scans of the 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) are shown. The figures reveal that the current intensity of the bare glassy carbon electrode, the MXene-modified glassy carbon electrode (MXene / GCE), the Au-MXene-modified glassy carbon electrode, and the SH-TE / TDN / Au-MXene / GCE decreases with DNA modification, indicating to some extent the success of the composite material-modified electrode and the successful assembly of SH-TE / TDN onto the electrode surface. The current intensity of the Cas12a / SH-TE / Au-MXene / GCE recovers, indicating that Cas12a activation cleaves the substrate chain on the electrode surface, releasing TDN and restoring its electron transfer capability, thus verifying the feasibility of this immobilization method for cleavage.
[0058] Figure 4 Square wave voltammetry plots of the modified electrode using an inverted DNA tetrahedral probe with and without kanamycin are shown. The figure illustrates the difference in electrochemical signals detected using this strategy with and without kanamycin, demonstrating that this strategy can be applied to the detection of kanamycin.
[0059] Figure 5 The figure shows the linear relationship between the signal difference detected using an electrode modified with an inverted DNA tetrahedron probe and different concentrations of kanamycin. The figure demonstrates that the ratio of the kanamycin current response signal to the kanamycin concentration obtained using this strategy has a good linear relationship, indicating that the proposed strategy can be applied to the quantitative detection of kanamycin.
[0060] Figure 6 The selective detection of kanamycin was performed using an electrode modified with an inverted DNA tetrahedral probe. The figure shows the electrochemical signals of kanamycin and other interfering antibiotics detected using this strategy, demonstrating its good selectivity.
[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.
Claims
1. The application of an inverted DNA tetrahedral probe-modified electrode in the detection of kanamycin, characterized in that, The detection uses an inverted DNA tetrahedral probe-modified electrode as the sensing electrode, and probes obtained by hybridization of aptamer probes kana-1 and kana-2 with the activated strand as recognition probes. Detection is performed under the mediation of Cas12a / crRNA and with the participation of the electroactive molecule methylene blue and the indicator potassium ferricyanide; wherein: The preparation method of the inverted DNA tetrahedral probe modified electrode includes the following steps: Step 1): The glassy carbon electrode was activated in potassium ferricyanide electrolyte solution by CV scanning, cleaned and dried for later use. Then, MXene aqueous solution was dropped onto the surface of the glassy carbon electrode and dried. Finally, gold nanoparticles were electrochemically deposited in gold electrolyte solution to obtain the electrode Au-MXene / GCE. Step 2): Incubate the DNA tetrahedron with the thiol-modified substrate chain probe SH-TE; the substrate chain probe contains a cleavage region that can be trans-cleaved by CRISPR-Cas12a and a thymine-rich anchoring region that can be assembled to the three edges of the DNA tetrahedron via 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 it with MCH to obtain the inverted DNA tetrahedron probe modified electrode SH-TE / TDN / Au-MXene / GCE; The sequence of the aptamer probe kana-1 is shown in SEQ ID NO: 7, the sequence of the aptamer probe kana-2 is shown in SEQ ID NO: 8, the sequence of the activation strand is shown in SEQ ID NO: 6, and the sequence of the crRNA is shown in SEQ ID NO:
9. The detection principle 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, cleave the substrate strand on the electrode, causing the inverted DNA tetrahedron to dissociate from the electrode surface, thereby restoring the electron transfer ability of the electrode interface, increasing the potassium ferricyanide signal, and decreasing the MB signal embedded in the TDN framework, thus achieving ratio signal output.
2. The application according to claim 1, characterized in that, 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 containing 0.5-2 mM [Fe(CN)6]. 3- / 4- and 0.05-0.2M KCl; The gold electrolyte solution is an aqueous solution of HAuCl4.
3. The application according to claim 1, characterized in that, The DNA tetrahedron in step 2) is formed by the self-assembly of four single strands with sequences as shown in SEQ ID NO: 1~4 through an annealing process; And / or, the substrate chain probe in step 2) is as shown in SEQ ID NO:
5.
4. The application according to claim 1, characterized in that, The method for preparing the recognition probe includes: first, mixing two aptamer probes kana-1 and kana-2 with the activation chain in Tris-NaCl buffer, reacting at 95°C for 5 minutes, and then slowly cooling to room temperature to obtain the recognition probe.
5. A method for detecting kanamycin, characterized in that, include: The recognition probe was co-incubated with the test sample, Cas12a / crRNA, and an electrode modified with an inverted DNA tetrahedral probe. After rinsing the electrode, potassium ferricyanide indicator was added, followed by co-incubation with methylene blue solution. The qualitative or quantitative detection of kanamycin in the test sample was achieved by electrochemically measuring the current intensity of potassium ferricyanide and methylene blue. The recognition probe is obtained by hybridization reaction of aptamer probes kana-1 and kana-2 with the activation strand that can activate the trans-cleavage activity of Cas12a / crRNA. The preparation method of the inverted DNA tetrahedral probe modified electrode includes the following steps: Step 1): The glassy carbon electrode was activated in potassium ferricyanide electrolyte solution by CV scanning, cleaned and dried for later use. Then, MXene aqueous solution was dropped onto the surface of the glassy carbon electrode and dried. Finally, gold nanoparticles were electrochemically deposited in gold electrolyte solution to obtain the electrode Au-MXene / GCE. Step 2): Incubate the DNA tetrahedron with the thiol-modified substrate chain probe SH-TE; the substrate chain probe contains a cleavage region that can be trans-cleaved by CRISPR-Cas12a and a thymine-rich anchoring region that can be assembled to the three edges of the DNA tetrahedron via 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 it with MCH to obtain the inverted DNA tetrahedron probe modified electrode SH-TE / TDN / Au-MXene / GCE.
6. The method according to claim 5, characterized in that, The quantitative detection also includes the step of establishing a standard curve: Cas12a / crRNA is mixed with the recognition probe and kanamycin standards of different concentrations. After rinsing the electrode, potassium ferricyanide indicator is added, followed by incubation with methylene blue solution. The current intensities of potassium ferricyanide and methylene blue are recorded in the range of 0.6 to -0.6 V using square wave voltammetry to establish a linear relationship between the changes in the ratio of potassium ferricyanide / methylene blue current intensities for different concentrations of antibiotics, thus obtaining the standard curve.