Au-at-Ti3C2MXene composite material-based aptamer regulation and control electrochemical sensor, preparation method and application of Au-at-Ti3C2MXene composite material-based aptamer regulation and control electrochemical sensor

Abstract

The invention discloses an aptamer regulation and control electrochemical sensor based on an Au (at) Ti3C2MXene composite material, and a preparation method and application of the aptamer regulation and control electrochemical sensor based on the Au (at) Ti3C2MXene composite material. A glassy carbon electrode is used as a working electrode, and the surface of the electrode is modified by an Au-coated Ti3C2MXene composite material prepared by an in-situ reduction method so as to enhance the electron transfer efficiency; a locking compound is formed by a specific aptamer of the lactobacillus acidophilus and an activation chain, and signal regulation and control are realized by combining the trans-cleavage activity of CRISPR-Cas12a. During detection, specific binding of the lactobacillus acidophilus and the aptamer causes conformational change, an activation chain is released to trigger the MB labeled DNA probe on the surface of the Cas12a cutting electrode, and quantification is realized through DPV detection signal change. The detection limit of the method is as low as 4 CFU / mL, the linear range is 10-106 CFU / mL, and the method is suitable for accurate detection of lactobacillus acidophilus in food and probiotic preparations.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, and in particular to an aptamer-regulated electrochemical sensor based on Au@Ti3C2MXene composite material, its preparation method, and its applications. Background Technology

[0002] Lactobacillus acidophilus, an important probiotic in the human gut, is also a core functional strain in yogurt, fermented dairy products, and probiotic preparations. Its content and activity directly affect food quality (such as the degree of fermentation in yogurt) and the efficacy of probiotic preparations. Therefore, establishing an accurate and reliable quantitative detection technology is of great significance for food quality control, efficacy verification of probiotic products, and clinical application evaluation.

[0003] Currently, mainstream detection methods all have significant limitations. Traditional culture methods can reflect the number of live bacteria, but they are time-consuming (usually requiring 3-7 days), have poor selectivity, and are difficult to distinguish between target bacteria and dormant bacteria in mixed bacterial communities. Molecular biological methods such as qPCR, although included in the new national standard and possessing high sensitivity, are expensive, complex to operate, and cannot distinguish between live and dead bacteria. They are also prone to false positives due to DNA residue, affecting the accuracy of the detection.

[0004] In recent years, electrochemical aptamer sensors have shown promising application prospects in the field of food safety analysis due to their advantages such as high sensitivity, high efficiency, portability, and low cost. However, achieving simultaneous detection of probiotics with high specificity and high sensitivity in complex food matrices still faces challenges. On the one hand, the design of the sensing interface directly affects electron transfer efficiency and signal output intensity; on the other hand, the identification of non-nucleic acid targets (such as intact bacteria) requires effective signal transduction mechanisms to activate the downstream signal amplification system. Summary of the Invention

[0005] To address the aforementioned issues, this invention proposes a novel detection platform based on the synergistic effect of electrochemical aptamer recognition and CRISPR-Cas12a signal amplification, specifically designed for rapid, specific, and sensitive quantitative analysis of Lactobacillus.

[0006] This invention constructs a high-performance electrochemical sensing interface: an Au@Ti3C2MXene heterocomposite material is synthesized using an in-situ reduction method, uniformly anchoring zero-dimensional gold nanoparticles (Au NPs) onto the surface of two-dimensional Ti3C2MXene ultrathin nanosheets. This structure not only effectively inhibits the stacking and aggregation of MXene sheets, significantly improving material dispersibility, but also greatly enhances the electron transfer rate and electroactive surface area of ​​the electrode through the strong electronic coupling between Au and MXene, providing an ideal substrate for highly sensitive electrochemical signal output.

[0007] This invention integrates aptamer recognition and CRISPR-Cas12a signal amplification: Since the CRISPR-Cas12a system can only be activated by specific single-stranded DNA (i.e., the activation strand) and trigger non-specific trans-cleavage activity, and *Lactobacillus* itself is a non-nucleic acid target, an intermediate transduction mechanism needs to be introduced. Therefore, this invention designs a "lock-and-release" strategy: the activation strand binds to the aptamer through a complementary sequence to form a thermodynamically stable composite structure (i.e., a "locked" state). When the target bacterium is present, the aptamer preferentially binds with high affinity to bacterial surface markers, leading to the release of the activation strand. The free activation strand then activates Cas12a, causing it to cleave the reporter probe (containing electrochemical signal molecules) modified on the electrode surface, thereby generating a detectable current change.

[0008] This cascaded design achieves an integrated detection process of "bacterial recognition → signal transduction → enzymatic amplification → electrochemical readout," significantly improving sensitivity while maintaining high specificity. Experiments show that this sensor performs well in the 10–10-10 range. 6 It exhibits a good linear response within the CFU / mL range, with a detection limit as low as 4 CFU / mL, fully meeting the quality control requirements for Lactobacillus in actual samples such as yogurt and probiotic preparations.

[0009] In summary, through the collaborative innovation of materials engineering and biometrics, this invention has successfully constructed an electrochemical sensing platform that combines high specificity, good sensitivity, ease of operation, and controllable cost, providing reliable technical support for the standardized detection of probiotic products and possessing significant application value and industrialization prospects.

[0010] To achieve the above objectives, the present invention provides an aptamer-regulated electrochemical sensor based on Au@Ti3C2MXene composite material, characterized in that it comprises a conventional three-electrode system, Au@Ti3C2MXene composite material, MB-labeled DNA probe, aptamer-activator chain-locking complex, and CRISPR-Cas12a / crRNA complex; the MB-labeled DNA probe is immobilized on the surface of the Au@Ti3C2MXene composite material via Au-S bonds;

[0011] The Au@Ti3C2MXene composite material was synthesized by in-situ reduction method. Au nanoparticles with a diameter of 2-4 nm were uniformly dispersed on the surface of Ti3C2MXene ultrathin layer and modified on GCE surface. The MB-labeled DNA probe contains a thiol group at its 5' end and an MB group at its 3' end; The aptamer-activator chain-locked complex is formed by annealing a lactobacillus-specific aptamer with an activation chain. In the CRISPR-Cas12a / crRNA complex, the crRNA is complementary to the activating strand; preferably, it is prepared by incubating buffer, LbCas12a and crRNA.

[0012] Furthermore, the Au@Ti3C2MXene composite material was prepared by the following method: 1) LiF was slowly dissolved in HCl solution to obtain HCl / LiF etching solution; Ti3AlC2 powder was then slowly added to the HCl / LiF etching solution and magnetically stirred at room temperature; after the reaction was completed, residual acid was removed by multiple centrifugations and the precipitate was collected by vacuum filtration; finally, a dark green monolayer Ti3C2MXene supernatant was obtained by centrifugation and ultrasonic dispersion; the obtained Ti3C2MXene supernatant was stored at 4℃ for later use. Preferably, the ratio of LiF:HCl:Ti3AlC2 powder is 1g:12mmol:0.1g; The magnetic stirring time is 12-36 hours, more preferably 24 hours; Centrifuge multiple times until the pH of the supernatant is 6 ± 0.2; 2) Place HAuCl4 solution and GSH solution in a reaction vessel and mix them evenly under continuous stirring; then add NaOH solution and Ti3C2MXene suspension in sequence, continue stirring until the mixture is homogeneous, and then let it stand at room temperature; centrifuge to obtain Au@Ti3C2MXene composite material; Preferably, the mass ratio of HAuCl4:GSH:NaOH:Ti3C2MXene is 1:1.5:45:50; The time for standing at room temperature is 4 ± 0.5 h.

[0013] Furthermore, the concentration of the MB-labeled DNA probe is 0.8–1.2 μM, and its sequence is shown in SEQ ID NO:1; Furthermore, the method for immobilizing the MB-labeled DNA probe on the surface of the Au@Ti3C2MXene composite material via Au-S bonds is as follows: S1. Polish the working electrode to a mirror finish using alumina powder, and then clean it with ultrasonic cleaning to remove residue. S2. Take a suspension of Au@Ti3C2MXene composite material and uniformly drop it onto the surface of the pretreated working electrode, and let it dry naturally at room temperature; S3. Add MB-labeled DNA probe to the working electrode surface and let stand. Covalent binding is achieved through Au-S bonds. S4. Add MCH solution and incubate at room temperature for 1 h to seal the remaining active sites on the electrode surface.

[0014] Furthermore, the Lactobacillus-specific aptamer sequence in the aptamer-activation chain-locked complex is shown in SEQ ID NO:2, and the activation chain sequence is shown in SEQ ID NO:3; Preferably, the preparation method of the aptamer-activator chain-locked complex is to mix the Lactobacillus-specific aptamer with the activation chain, heat to denature the DNA, then slowly cool to room temperature, and finally centrifuge to collect the supernatant.

[0015] More preferably, the molar ratio of the Lactobacillus-specific aptamer to the activating chain is 1.5:1.

[0016] This invention also provides a method for preparing an aptamer-controlled electrochemical sensor based on Au@Ti3C2MXene composite material, characterized in that the Au@Ti3C2MXene composite material in the aptamer-controlled electrochemical sensor is prepared by the following method: 1) LiF was slowly dissolved in HCl solution to obtain HCl / LiF etching solution; Ti3AlC2 powder was then slowly added to the HCl / LiF etching solution and magnetically stirred at room temperature; after the reaction was completed, residual acid was removed by multiple centrifugations and the precipitate was collected by vacuum filtration; finally, a dark green monolayer Ti3C2MXene supernatant was obtained by centrifugation and ultrasonic dispersion; the obtained Ti3C2MXene supernatant was stored at 4℃ for later use. Preferably, the ratio of LiF:HCl:Ti3AlC2 powder is 1g:12mmol:0.1g; The magnetic stirring time is 12-36 hours, more preferably 24 hours; Centrifuge multiple times until the pH of the supernatant is 6 ± 0.2; 2) Place HAuCl4 solution and GSH solution in a reaction vessel and mix them evenly under continuous stirring; then add NaOH solution and Ti3C2MXene suspension in sequence, continue stirring until the mixture is homogeneous, and then let it stand at room temperature; centrifuge to obtain Au@Ti3C2MXene composite material; Preferably, the mass ratio of HAuCl4:GSH:NaOH:Ti3C2MXene is 1:1.5:45:50; The standing time at room temperature is 4 ± 0.5 h; The preparation method of MB-labeled DNA probe is as follows: S1, polish the working electrode to a mirror finish using alumina powder, and remove residues by ultrasonic cleaning; S2. Take a suspension of Au@Ti3C2MXene composite material and uniformly drop it onto the surface of the pretreated working electrode, and let it dry naturally at room temperature; S3. Add MB-labeled DNA probe to the working electrode surface and let stand. Covalent binding is achieved through Au-S bonds. S4. Add MCH solution and incubate at room temperature for 1 h to seal the remaining active sites on the electrode surface. The preparation method of the aptamer-activator chain-locked complex is to mix the Lactobacillus-specific aptamer with the activation chain, heat to denature the DNA, then slowly cool to room temperature, and finally centrifuge to collect the supernatant. The CRISPR-Cas12a / crRNA complex can be prepared by incubating buffer, LbCas12a and crRNA; preferably, incubation at 37°C for 10 minutes.

[0017] The present invention also provides the application of the aptamer-regulated electrochemical sensor based on Au@Ti3C2 MXene composite material in the detection of Lactobacillus.

[0018] Furthermore, the detection method is as follows: (1) Take 5-15 μL of different concentrations of Lactobacillus acidophilus solution and mix it with 15-25 μL of aptamer-activator chain-locking complex. Incubate for 40-80 minutes to allow the aptamer to specifically bind to Lactobacillus acidophilus and release the activation chain. (2) The above reaction solution is mixed with the CRISPR-Cas12a / crRNA complex to form a Cas12a-crRNA-activation chain ternary complex, which activates the trans-cleavage activity of the Cas12a enzyme. (3) Take the activated Cas12a enzyme solution and drop it onto the treated working electrode to non-specifically cut the structure of the DNA probe on the electrode surface and switch the electrochemical signal to the "off" state. (4) The concentration of Lactobacillus was detected by differential pulse voltammetry in PBS buffer with a pH of 6.5-7.5 and calculated based on the standard curve.

[0019] The electrochemical sensor of this invention combines the signal enhancement effect of Au@Ti3C2MXene with the recognition amplification effect of CRISPR-Cas12a to achieve highly sensitive and specific detection of Lactobacillus.

[0020] Compared with the prior art, the present invention has the following advantages: (1) The present invention utilizes an in-situ reduction method to construct an Au@Ti3C2MXene composite material. Au nanoparticles (2-4 nm in diameter) are uniformly loaded onto the surface of an ultrathin Ti3C2MXene layer, suppressing van der Waals aggregation between MXene sheets through a nano-confinement effect. Simultaneously, Au@Ti3C2MXene provides a large electroactive region, which facilitates the accessibility of reactants, thereby significantly improving the electrochemical signal. Furthermore, this sensing substrate stably binds to methylene blue (MB)-labeled DNA probes via Au-S bonds, requiring no additional functional group modification.

[0021] (2) This invention combines the CRISPR-Cas12a system with aptamer recognition to form a cascade reaction system of "specific recognition - precise activation - efficient amplification". The activation chain is released only when the target Lactobacillus is present, avoiding background interference caused by non-specific activation; after the released activation chain binds complementary to the crRNA sequence, it precisely triggers the trans-cleavage activity of Cas12a, which is then used for efficient cleavage of MB-labeled DNA probes on the electrode surface to achieve signal amplification effect.

[0022] (3) Relying on the synergistic effect of signal enhancement from Au@Ti3C2MXene and amplification from CRISPR-Cas12a, the sensor of this invention has a detection limit as low as 4 CFU / mL and a linear detection range as wide as 10~10 6 CFU / mL; in addition, the sensor exhibits good stability and reproducibility.

[0023] The electrochemical sensor provided by this invention has the following specific mechanism for detecting Lactobacillus acidophilus: The *Lactobacillus*-specific aptamer and the activation strand form a stable double-stranded complex through base complementarity. At this point, the activation strand cannot bind to the CRISPR-Cas12a / crRNA complex, and Cas12a remains inactive. MB-labeled DNA probes immobilized on the electrode surface bring MBs close to the electrode, generating a strong electrochemical redox signal, and the sensor is in a "signal-on" state. When *Lactobacillus* is present in the system, the aptamer preferentially binds to *Lactobacillus* through its specific binding site, inducing a conformational transition from double-stranded to single-stranded folding of the aptamer, resulting in the release of the activation strand from the "aptamer-activation strand" complex. The free activation strand binds to crRNA through base complementarity, forming a "Cas12a / crRNA-activation strand" ternary complex, activating the trans-cleavage activity of Cas12a. Cas12a non-specifically cleaves the MB-labeled DNA probes on the electrode surface, causing MBs to detach from the electrode surface. The electrochemical signal decreases significantly with decreasing MB concentration. The magnitude of the signal decrease is positively correlated with the *Lactobacillus* concentration. Quantification of *Lactobacillus* is achieved by detecting peak current changes using DPV (Distributed Photovoltaic Detection).

[0024] This invention constructs an Au@Ti3C2MXene electrochemiluminescence sensor for detecting Lactobacillus, using a glassy carbon electrode as the working electrode. The electrode surface is modified with an Au@Ti3C2MXene heterostructure prepared via in-situ reduction to enhance electron transfer efficiency. A locking complex is formed between a Lactobacillus-specific aptamer and the activation strand, which, combined with the trans-cleavage activity of CRISPR-Cas12a, achieves signal modulation. During detection, the specific binding of Lactobacillus to the aptamer leads to a conformational change, releasing the activation strand to trigger Cas12a cleavage of a methylene blue (MB)-labeled DNA probe on the electrode surface. Quantification is achieved by detecting the signal change using differential pulse voltammetry (DPV). This invention has a detection limit as low as 4 CFU / mL and a linear range of 10⁻¹⁰–10⁻¹⁰. 6 The concentration of CFU / mL indicates a low detection limit, demonstrating promising application prospects in food safety testing. It is suitable for the accurate detection of Lactobacillus in food and probiotic preparations. Attached Figure Description

[0025] Figure 1 Transmission electron microscopy image of Ti3C2MXene nanosheets prepared in Example 1 of this invention.

[0026] Figure 2 This is a transmission electron microscope image of the Au@Ti3C2MXene composite material prepared in Example 1 of the present invention.

[0027] Figure 3 EIS images of GCE electrodes modified with Ti3C2MXene and Au@Ti3C2MXene.

[0028] Figure 4 Figure showing a feasibility study of using aptamer-regulated CRISPR-Cas12a for the detection of Lactobacillus acidophilus.

[0029] Figure 5 EIS curves for the construction process of an aptamer-regulated electrochemical sensor based on Au@Ti3C2MXene composite material.

[0030] Figure 6 This is a DPV response curve of the aptamer-regulated electrochemical sensor based on Au@Ti3C2MXene composite material in this invention for different concentrations of Lactobacillus acidophilus.

[0031] Figure 7 This is a linear curve showing the relationship between the peak current value of DPV and the logarithm of the concentration of Lactobacillus in this invention.

[0032] Figure 8 The response of six independently prepared aptamers based on Au@Ti3C2MXene composite materials to the same concentration of Lactobacillus is shown in the graph.

[0033] Figure 9 The figure shows the current signal stability results of the aptamer-controlled electrochemical sensor based on Au@Ti3C2MXene composite material provided by this invention after storage at 4°C for different times. Detailed Implementation

[0034] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0035] Example 1: Preparation of Au@Ti3C2MXene composite material A method for preparing Au@Ti3C2MXene composite material includes the following steps: S1. Preparation of monolayer Ti3C2MXene: The Ti3C2MXene was prepared by etching the Ti3C2MAX phase with LiF / HCl, with a thickness of 1~2 nm.

[0036] Ti3AlC2 was etched using LiF / HCl. Specifically, 1 g of LiF was slowly dissolved in 10 mL of HCl (12 M) solution to obtain an HCl / LiF etching solution. Then, 0.1 g of Ti3AlC2 powder was slowly added to this solution, and the mixture was magnetically stirred at room temperature for 24 h. After the reaction was completed, residual acid was removed by multiple centrifugations (until the pH of the supernatant was 6), and the precipitate was collected by vacuum filtration. Finally, a dark green monolayer Ti3C2MXene supernatant was obtained by centrifugation and ultrasonic dispersion, which is the Ti3C2MXene suspension. S2. Preparation of Au@Ti3C2MXene: 0.5 mL of 20 mM HAuCl4 solution and 0.15 mL of 0.1 M GSH solution were placed in a reaction vessel and mixed evenly under continuous stirring. Then, 4.5 mL of 0.1 M NaOH solution and 400 μL of 1.25 mg / mL Ti3C2MXene suspension were added sequentially, and stirring was continued until the mixture was homogeneous. The reaction mixture was then allowed to stand at room temperature for 4 h. Finally, the mixture was centrifuged to obtain Au@Ti3C2MXene composite material. The composite material was dissolved and sealed at 4 ℃ for later use. The particle size of Au NPs was 2~5 nm.

[0037] The monolayer Ti3C2MXene prepared by S1 and the Au@Ti3C2MXene composite material prepared by S2 were observed by transmission electron microscopy, and the results are as follows: Figure 1 and Figure 2 As shown.

[0038] Figure 1 The transmission electron microscope image of the single-layer Ti3C2MXene prepared in Example 1 of the present invention shows a typical ultrathin sheet structure with a smooth and defect-free surface.

[0039] Figure 2 This is a transmission electron microscope (TEM) image of the Au@Ti3C2MXene composite material prepared in Example 1 of this invention. As can be seen, high-density gold nanoparticles (3 nm) are distributed on the surface of the Ti3C2MXene layer, confirming the successful preparation of the Au@Ti3C2MXene composite material.

[0040] Electrochemical impedance spectroscopy (EIS) is used to study the conductivity of the prepared composite materials, such as... Figure 3 As shown, to investigate the electrochemical performance of Au@Ti3C2Mxene, this study used electrochemical impedance spectroscopy (EIS) for analysis. Charge transfer resistance (...) R ct It can reflect electron transport capability and electrochemical response performance, that is... R ct The smaller the value, the higher the electron transport efficiency and the better the electrochemical response performance. Working electrode, Ti3C2MXene and Au@Ti3C2MXene modified working electrode R ct The values ​​decreased respectively. Due to the high specific surface area of ​​Ti3C2MXene, the charge transfer resistance was significantly reduced. Notably, the Au@Ti3C2MXene-modified working electrode exhibited the smallest [value missing]. R ct The value confirms the synergistic effect of Au and Ti3C2MXene.

[0041] Example 2: Fabrication of an aptamer-controlled electrochemical sensor based on Au@Ti3C2MXene composite material Aptamer-regulated electrochemical sensors based on Au@Ti3C2MXene composite materials include: a conventional three-electrode system, Au@Ti3C2 MXene composite material, MB-labeled DNA probes, aptamer-activator chain-locking complexes, and CRISPR-Cas12a / crRNA complexes.

[0042] The conventional three-electrode system consists of a glassy carbon electrode (GCE) with a diameter of 4 mm, a platinum wire electrode as the counter electrode, and an Ag / AgCl electrode with saturated KCl solution as the reference electrode. Au@Ti3C2 MXene composite material: See Example 1; MB-labeled DNA probe: See step 1 below.

[0043] Aptamer-activator chain-locked complex: see step 2 below.

[0044] CRISPR-Cas12a / crRNA complex: see step 3 below.

[0045] Its preparation method includes the following steps: 1. MB-labeled DNA probes are immobilized on the Au@Ti3C2 MXene surface via Au-S bonds: S1. Polish the working electrode (GCE) sequentially to a mirror finish using alumina powder (0.3 μm and 0.05 μm), and then remove residues by ultrasonic cleaning; S2. Take 6 μL of 1.5 mg / mL Au@Ti3C2MXene suspension and uniformly drop it onto the GCE surface pretreated in S1 above, and let it air dry at room temperature. S3. Add 6 μL of MB-labeled DNA probe (1 μM) to the electrode surface and let stand for 1 h to achieve covalent binding through Au-S bonds; The concentration of the DNA probe was 0.8–1.2 μM, and its sequence is shown in SEQ ID NO:1. 5'-SH-GCTCCGAATTGGATGTTTTT-3'; S4. Add 3 μL of 1 mM MCH solution and incubate at room temperature for 1 h to seal the remaining active sites on the electrode surface.

[0046] 2. Aptamer-Activated Chain Locking Complex: 10 μL of Lactobacillus-specific aptamer (Apt, 1.5 μM) was mixed with 10 μL of activated chain (1.0 μM), heated at 95 °C for 5 min to denature the DNA, and then slowly cooled to room temperature (to promote specific hybridization). Finally, the mixture was centrifuged at 12,000 rpm for 5 min, and the supernatant was used for subsequent experiments.

[0047] The Lactobacillus-specific aptamer sequence is shown in SEQ ID NO:2: 5'-AGCAGCACAGAGGTCAGATGTAGCCCTTCAACATAGTAATATCTCTGCATTCTGTGTGCCTATGCGTGCTACCGTGAA-3'; the activation strand sequence is 5'-CACAAACCTTTTCACGGTAGCA-3' (SEQ ID NO:3).

[0048] 3. CRISPR-Cas12a / crRNA complex: LbCas12a was purchased from Tolobio (Shanghai), and the buffer solution was a matching reagent.

[0049] Add 4 μL of 10-fold buffer, 0.4 μL of LbCas12a (10 µM), 2 μL of crRNA (2 µM), and 29.6 μL of water (H2O) to a 40 μL reaction system, and incubate at 37°C for 10 minutes.

[0050] The sequence of the crRNA is 5'-UAAUUUCUACUAAGUGUAGAUUGCUACCGUGAAAACGUUUG-3' (SEQ ID NO:4).

[0051] The construction process of the electrochemical sensor prepared by the above method was verified as follows: Figure 4 The locking mechanism was verified using a single-stranded nucleotide (reporter probe) labeled with a FAM fluorescent group at the 5' end and a BHQ1 quencher group at the 3' end. As shown in the figure, the lowest fluorescence intensity was observed without the addition of the activating strand (i.e., Cas12a-CrRNA). In the system containing only the aptamer-activating strand mixture (i.e., Cas12a-crRNA+Apt-Activator), the fluorescence signal remained weak because the activating strand was locked. However, when the target, *Lactobacillus*, was introduced into the system (i.e., Cas12a-crRNA+Apt-Activator+target), the fluorescence signal was significantly enhanced. This is attributed to the specific binding of the aptamer to *Lactobacillus*, which induced the release of the activating strand, thereby activating Cas12a to cleave the reporter probe and triggering fluorescence recovery. The strongest fluorescence signal was produced when the activating strand was used alone (i.e., Cas12a-crRNA+Activator).

[0052] The modified electrode surface was tested using electrochemical impedance spectroscopy, such as... Figure 5As shown, (a) GCE, (b) Au@Ti3C2MXene / GCE, (c) DNA probe / Au@Ti3C2MXene / GCE, (d) MCH / DNA probe / Au@Ti3C2MXene / GCE, and (e) Cas12a / MCH / DNA probe / Au@Ti3C2MXene / GCE. After modifying the electrode surface with Au@Ti3C2MXene (curve b), a significant decrease in resistance was observed. This is attributed to the high conductivity of Au@Ti3C2MXene significantly enhancing the electron transport rate, thereby reducing the resistance. With progressive modification of the DNA probe (curve c) and MCH (curve d), the resistance increased. The biomolecules introduced at the electrode interface hindered the absorption of Fe(CN)6. 3- / 4- Electron transfer leads to a weakening of the electrochemical signal. After treatment with the Cas12a-crRNA system, due to the activated cleavage activity of Cas12a, most of the MB detaches from the Au@Ti3C2MXene-modified electrode surface, resulting in a decrease in electrode resistance. As expected, the charge transfer resistance decreases significantly (curve e), demonstrating the dynamic changes in the electrochemical signal during sensor construction. This series of ordered EIS responses validates the precise assembly and functionalization regulation mechanism of the aptamer-regulated CRISPR-Cas12a electrochemical sensing platform based on the Au@Ti3C2MXene heterostructure, providing a reliable signal conversion strategy for the detection of Lactobacillus.

[0053] Example 3 An aptamer-regulated CRISPR-Cas12a electrochemical sensor based on the Au@Ti3C2MXene heterostructure and its application in detecting Lactobacillus includes the following steps: S1. Mix 10 μL of Lactobacillus acidophilus solution with 20 μL of nucleic acid aptamer functional solution (i.e., aptamer-activator chain-locked complex) and incubate at 37 °C for 40 min to allow the aptamer to specifically bind to Lactobacillus acidophilus and release the activation chain. S2. The above reaction solution is mixed with the pre-prepared Cas12a-crRNA complex (i.e., CRISPR-Cas12a / crRNA complex), and the Cas12a / crRNA / activation chain ternary complex is formed by the activation chain, thereby activating the trans-cleavage activity of the Cas12a enzyme. S3. Take the Cas12a enzyme solution activated in S2 above and drop it onto the surface of the MCH / DNA probe / Au@Ti3C2MXene modified electrode (i.e. the electrode surface obtained in step 2 of Example 2). S4. Differential pulse voltammetry (DPV) was used to detect Lactobacillus in PBS buffer at pH 7, and the concentration was calculated based on the standard curve. The DPV detection parameters were: pulse width 0.05 s, sampling width 0.017 s, amplitude 0.05 V, and pulse period 0.5 s.

[0054] For 10~10 6 Quantitative detection of Lactobacillus was performed within a CFU / mL concentration gradient range. Results are as follows: Figure 6 and 7 As shown, Figure 6 This is the DPV response curve of the sensor in this invention to different concentrations of Lactobacillus acidophilus. Figure 7 This is a linear curve showing the relationship between the peak current value of DPV and the logarithm of the Lactobacillus concentration in this invention. The DPV current signal decreases as the Lactobacillus concentration increases. The logarithm of the Lactobacillus concentration has a linear relationship with the peak current, and the linear equation is I = -1.77 lgC + 13.67 (R 2 = 0.997), and the limit of detection (LOD) for Lactobacillus was 4 CFU / mL.

[0055] Example 4: Repeatability and stability of the aptamer-modulated electrochemical sensor based on Au@Ti3C2MXene composite material. To examine the reproducibility of the aptamer-regulated electrochemical sensor based on the Au@Ti3C2MXene composite material, radar graphs show the performance consistency of six identical sensors (prepared according to the method in Example 2) independently fabricated under the same conditions. Figure 8 As shown, Figure 8 The graphs show the response of six independently prepared sensors (i.e., aptamer-controlled electrochemical sensors based on Au@Ti3C2MXene composite material prepared according to the method of Example 2) to the same concentration of Lactobacillus. Each sensor exhibits similar response characteristics, and a relative standard deviation (RSD) of 1.1% indicates that the electrochemical sensor has good repeatability.

[0056] Next, the stability of the aptamer-controlled electrochemical sensor based on Au@Ti3C2MXene composite material prepared in Example 2 was tested for several weeks. Figure 9 The figure shows the current signal stability results of the aptamer-controlled electrochemical sensor based on Au@Ti3C2MXene composite material provided by this invention after storage at 4°C for different times. Figure 9The results showed that after being stored continuously at 4 °C for 6 weeks, the current value of the aptamer-regulated electrochemical sensor based on Au@Ti3C2MXene composite material remained at approximately 87.89% of its initial value. This indicates that the aptamer-regulated electrochemical sensor based on Au@Ti3C2MXene composite material has good stability and can meet the requirements for long-term application.

[0057] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. An aptamer-controlled electrochemical sensor based on Au@Ti3C2MXene composite material, characterized in that, The system includes a conventional three-electrode system, an Au@Ti3C2MXene composite material, an MB-labeled DNA probe, an aptamer-activator chain-locking complex, and a CRISPR-Cas12a / crRNA complex; the MB-labeled DNA probe is immobilized on the surface of the Au@Ti3C2MXene composite material via Au-S bonds; The Au@Ti3C2MXene composite material was synthesized by in-situ reduction method. Au nanoparticles with a diameter of 2-4 nm were uniformly dispersed on the surface of Ti3C2MXene ultrathin layer and modified on GCE surface. The MB-labeled DNA probe contains a thiol group at its 5' end and an MB group at its 3' end; The aptamer-activator chain-locked complex is formed by annealing a lactobacillus-specific aptamer with an activation chain. In the CRISPR-Cas12a / crRNA complex, the crRNA is complementary to the activating strand; preferably, it is prepared by incubating buffer, LbCas12a and crRNA.

2. The aptamer-controlled electrochemical sensor based on Au@Ti3C2MXene composite material as described in claim 1, characterized in that, The Au@Ti3C2MXene composite material was prepared by the following method: 1) LiF was slowly dissolved in HCl solution to obtain HCl / LiF etching solution; Ti3AlC2 powder was then slowly added to the HCl / LiF etching solution and magnetically stirred at room temperature; after the reaction was completed, residual acid was removed by multiple centrifugations and the precipitate was collected by vacuum filtration; finally, a dark green monolayer Ti3C2MXene supernatant was obtained by centrifugation and ultrasonic dispersion; the obtained Ti3C2MXene supernatant was stored at 4℃ for later use. Preferably, the ratio of LiF:HCl:Ti3AlC2 powder is 1g:12mmol:0.1g; The magnetic stirring time is 12-36 hours, more preferably 24 hours; Centrifuge multiple times until the pH of the supernatant is 6 ± 0.2; 2) Place HAuCl4 solution and GSH solution in a reaction vessel and mix them evenly under continuous stirring; then add NaOH solution and Ti3C2MXene suspension in sequence, continue stirring until the mixture is homogeneous, and then let it stand at room temperature; centrifuge to obtain Au@Ti3C2MXene composite material; Preferably, the mass ratio of HAuCl4:GSH:NaOH:Ti3C2MXene is 1:1.5:45:50; The time for standing at room temperature is 4 ± 0.5 h.

3. The aptamer-controlled electrochemical sensor based on Au@Ti3C2MXene composite material as described in claim 1, characterized in that, The concentration of the MB-labeled DNA probe is 0.8–1.2 μM, and its sequence is shown in SEQ ID NO:

1.

4. The aptamer-controlled electrochemical sensor based on Au@Ti3C2MXene composite material as described in claim 1, characterized in that, The method for immobilizing the MB-labeled DNA probe on the surface of the Au@Ti3C2MXene composite material via Au-S bonds is as follows: S1. Polish the working electrode to a mirror finish using alumina powder, and then clean it with ultrasonic cleaning to remove residue. S2. Take a suspension of Au@Ti3C2MXene composite material and uniformly drop it onto the surface of the pretreated working electrode, and let it dry naturally at room temperature; S3. Add MB-labeled DNA probe to the working electrode surface and let stand. Covalent binding is achieved through Au-S bonds. S4. Add MCH solution and incubate at room temperature for 1 h to seal the remaining active sites on the electrode surface.

5. The aptamer-controlled electrochemical sensor based on Au@Ti3C2MXene composite material as described in claim 1, characterized in that, The Lactobacillus-specific aptamer sequence in the aptamer-activation chain-locked complex is shown in SEQ ID NO:2, and the activation chain sequence is shown in SEQ ID NO:3; Preferably, the preparation method of the aptamer-activator chain-locked complex is to mix the Lactobacillus-specific aptamer with the activation chain, heat to denature the DNA, then slowly cool to room temperature, and finally centrifuge to collect the supernatant. More preferably, the molar ratio of the Lactobacillus-specific aptamer to the activating chain is 1.5:

1.

6. A method for preparing an aptamer-controlled electrochemical sensor based on Au@Ti3C2MXene composite material, characterized in that, The preparation method of the Au@Ti3C2MXene composite material in the aptamer-controlled electrochemical sensor based on the Au@Ti3C2MXene composite material is as follows: The Au@Ti3C2MXene composite material is prepared by the following method: 1) LiF was slowly dissolved in HCl solution to obtain HCl / LiF etching solution; Ti3AlC2 powder was then slowly added to the HCl / LiF etching solution and magnetically stirred at room temperature; after the reaction was completed, residual acid was removed by multiple centrifugations and the precipitate was collected by vacuum filtration; finally, a dark green monolayer Ti3C2MXene supernatant was obtained by centrifugation and ultrasonic dispersion; the obtained Ti3C2MXene supernatant was stored at 4℃ for later use. Preferably, the ratio of LiF:HCl:Ti3AlC2 powder is 1g:12mmol:0.1g; The magnetic stirring time is 12-36 hours, more preferably 24 hours; Centrifuge multiple times until the pH of the supernatant is 6 ± 0.2; 2) Place HAuCl4 solution and GSH solution in a reaction vessel and mix them evenly under continuous stirring; then add NaOH solution and Ti3C2MXene suspension in sequence, continue stirring until the mixture is homogeneous, and then let it stand at room temperature; centrifuge to obtain Au@Ti3C2MXene composite material; Preferably, the mass ratio of HAuCl4:GSH:NaOH:Ti3C2MXene is 1:1.5:45:50; The standing time at room temperature is 4 ± 0.5 h; The preparation method of MB-labeled DNA probe is as follows: S1, polish the working electrode to a mirror finish using alumina powder, and remove residues by ultrasonic cleaning; S2. Take a suspension of Au@Ti3C2MXene composite material and uniformly drop it onto the surface of the pretreated working electrode, and let it dry naturally at room temperature; S3. Add MB-labeled DNA probe to the working electrode surface and let stand. Covalent binding is achieved through Au-S bonds. S4. Add MCH solution and incubate at room temperature for 1 h to seal the remaining active sites on the electrode surface. The preparation method of the aptamer-activator chain-locked complex is to mix the Lactobacillus-specific aptamer with the activation chain, heat to denature the DNA, then slowly cool to room temperature, and finally centrifuge to collect the supernatant. The CRISPR-Cas12a / crRNA complex can be prepared by incubating buffer, LbCas12a and crRNA; preferably, incubation at 37°C for 10 minutes.

7. The application of the aptamer-regulated electrochemical sensor based on Au@Ti3C2 MXene composite material as described in any one of claims 1-5 in the detection of Lactobacillus.

8. The application as described in claim 7, characterized in that, The detection method is as follows: (1) Take 5-15 μL of different concentrations of Lactobacillus acidophilus solution and mix it with 15-25 μL of aptamer-activator chain-locking complex. Incubate for 40-80 minutes to allow the aptamer to specifically bind to Lactobacillus acidophilus and release the activation chain. (2) The above reaction solution is mixed with the CRISPR-Cas12a / crRNA complex to form a Cas12a-crRNA-activation chain ternary complex, which activates the trans-cleavage activity of the Cas12a enzyme. (3) Take the activated Cas12a enzyme solution and drop it onto the treated working electrode to non-specifically cut the structure of the DNA probe on the electrode surface and switch the electrochemical signal to the "off" state. (4) The concentration of Lactobacillus was detected by differential pulse voltammetry in PBS buffer with a pH of 6.5-7.5 and calculated based on the standard curve.

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