CN-121978180-A - Au@Ti-based alloy3C2Aptamer-controlled electrochemical sensor of MXene composite material, preparation method and application

CN121978180ACN 121978180 ACN121978180 ACN 121978180ACN-121978180-A

Abstract

The invention discloses an aptamer-controlled electrochemical sensor based on an Au@Ti 3 C 2 MXene composite material, a preparation method and application. The method takes a glassy carbon electrode as a working electrode, and utilizes an Au@Ti 3 C 2 MXene composite material prepared by an in-situ reduction method to modify the surface of the electrode so as to enhance the electron transfer efficiency, and utilizes a lactobacillus acidophilus specific aptamer and an activation chain to form a locking complex, and the signal regulation and control are realized by combining the trans-cutting activity of CRISPR-Cas12 a. During detection, the specific binding of lactobacillus acidophilus and the aptamer causes conformational change, an activation chain is released to trigger the Cas12a to cut the MB marked DNA probe on the surface of the electrode, and quantification is realized through DPV detection signal change. The detection limit of the invention is as low as 4 CFU/mL, the linear range is 10-10 6 CFU/mL, and the invention is suitable for the accurate detection of lactobacillus acidophilus in food and probiotic preparations.

Inventors

WANG WEI
ZHANG XIAOBO
XIAO JING
ZHENG QIUYUE
CAO JIJUAN

Assignees

国家食品安全风险评估中心
大连民族大学
内蒙古国家乳业技术创新中心有限责任公司

Dates

Publication Date: 20260505
Application Date: 20260206

Claims (8)

1. An aptamer-controlled electrochemical sensor based on an Au@Ti 3 C 2 MXene composite material is characterized by comprising a conventional three-electrode system, an Au@Ti 3 C 2 MXene composite material, an MB marked DNA probe, an aptamer-activated chain locking complex and a CRISPR-Cas12a/crRNA complex, wherein the MB marked DNA probe is fixed on the surface of the Au@Ti 3 C 2 MXene composite material through an Au-S bond; The Au@Ti 3 C 2 MXene composite material is synthesized by an in-situ reduction method, wherein the diameter of Au nano particles is 2-4 nm, and the Au nano particles are uniformly dispersed on the surface of the Ti 3 C 2 MXene ultrathin layer and modified on the surface of the GCE; the 5 'end of the MB marked DNA probe contains sulfhydryl and the 3' end contains MB; the aptamer-activation chain locking complex is formed by annealing a lactobacillus acidophilus specific aptamer to an activation chain; In the CRISPR-Cas12a/crRNA complex, crRNA is complementary to an activation strand, and preferably, the CRRNA is prepared by incubating a buffer solution, lbCas a and the crRNA.
2. The aptamer-controlled electrochemical sensor based on an Au@Ti 3 C 2 MXene composite material according to claim 1, wherein the Au@Ti 3 C 2 MXene composite material is prepared by the following preparation method: 1) Slowly dissolving LiF in an HCl solution to obtain an HCl/LiF etching solution, slowly adding Ti 3 AlC 2 powder into the HCl/LiF etching solution, magnetically stirring at room temperature, centrifuging for several times after the reaction is finished to remove residual acid liquor, vacuum filtering to collect precipitate, centrifuging and ultrasonically dispersing to obtain dark green single-layer Ti 3 C 2 MXene supernatant, and storing the obtained Ti 3 C 2 MXene supernatant at 4 ℃ for later use; Preferably, the ratio of LiF to HCl to Ti 3 AlC 2 powder is 1g to 12mmol to 0.1g; The magnetic stirring time is 12-36h, more preferably 24h; Centrifugation was repeated until supernatant ph=6±0.2; 2) Placing HAuCl 4 solution and GSH solution in a reaction vessel, uniformly mixing under the condition of continuous stirring, then sequentially adding NaOH solution and Ti 3 C 2 MXene suspension, continuously stirring until the mixed system is in a uniform state, standing at room temperature, and centrifuging to obtain Au@Ti 3 C 2 MXene composite material; preferably, the mass usage of HAuCl 4 ：GSH：NaOH：Ti 3 C 2 MXene is 1:1.5:45:50; the time of standing at room temperature was 4.+ -. 0.5 h.
3. The aptamer-controlled electrochemical sensor based on the Au@Ti 3 C 2 MXene composite material according to claim 1, wherein the concentration of the MB-marked DNA probe is 0.8-1.2 mu M, and the sequence is shown as SEQ ID NO. 1.
4. The aptamer-controlled electrochemical sensor based on an Au@Ti 3 C 2 MXene composite material according to claim 1, wherein the method for fixing the MB-marked DNA probe on the surface of the Au@Ti 3 C 2 MXene composite material through an Au-S bond is as follows: s1, polishing a working electrode to a mirror surface by using alumina powder, and removing residues by ultrasonic cleaning; S2, uniformly dripping a suspension of the Au@Ti 3 C 2 MXene composite material on the surface of the pretreated working electrode, and naturally drying at room temperature; S3, dropwise adding MB marked DNA probes to the surface of the working electrode, standing, and realizing covalent bonding through Au-S bonds; S4, dropwise adding MCH solution, incubating at room temperature for 1 h, and closing the residual active sites on the surface of the electrode.
5. The aptamer-controlled electrochemical sensor based on the Au@Ti 3 C 2 MXene composite material according to claim 1, wherein the sequence of the lactobacillus acidophilus specific aptamer in the aptamer-activation chain locking complex is shown as SEQ ID NO. 2, and the sequence of the activation chain is shown as SEQ ID NO. 3; Preferably, the aptamer-activating chain locking complex is prepared by mixing a lactobacillus acidophilus specific aptamer with an activating chain, heating to denature DNA, slowly cooling to room temperature, and centrifuging to obtain supernatant. More preferably, the molar amount of lactobacillus acidophilus specific aptamer to activating strand is 1.5:1.
6. The preparation method of the aptamer-controlled electrochemical sensor based on the Au@Ti 3 C 2 MXene composite material is characterized in that the preparation method of the Au@Ti3C2 MXene composite material in the Au@Ti 3 C 2 MXene composite material-based aptamer-controlled electrochemical sensor is characterized in that the Au@Ti 3 C 2 MXene composite material is prepared by the following preparation method: 1) Slowly dissolving LiF in an HCl solution to obtain an HCl/LiF etching solution, slowly adding Ti 3 AlC 2 powder into the HCl/LiF etching solution, magnetically stirring at room temperature, centrifuging for several times after the reaction is finished to remove residual acid liquor, vacuum filtering to collect precipitate, centrifuging and ultrasonically dispersing to obtain dark green single-layer Ti 3 C 2 MXene supernatant, and storing the obtained Ti 3 C 2 MXene supernatant at 4 ℃ for later use; Preferably, the ratio of LiF to HCl to Ti 3 AlC 2 powder is 1g to 12mmol to 0.1g; The magnetic stirring time is 12-36h, more preferably 24h; Centrifugation was repeated until supernatant ph=6±0.2; 2) Placing HAuCl 4 solution and GSH solution in a reaction vessel, uniformly mixing under the condition of continuous stirring, then sequentially adding NaOH solution and Ti 3 C 2 MXene suspension, continuously stirring until the mixed system is in a uniform state, standing at room temperature, and centrifuging to obtain Au@Ti 3 C 2 MXene composite material; preferably, the mass usage of HAuCl 4 ：GSH：NaOH：Ti 3 C 2 MXene is 1:1.5:45:50; The standing time at room temperature is 4+/-0.5 h; s1, polishing a working electrode to a mirror surface by using alumina powder, and removing residues by ultrasonic cleaning; S2, uniformly dripping a suspension of the Au@Ti 3 C 2 MXene composite material on the surface of the pretreated working electrode, and naturally drying at room temperature; S3, dropwise adding MB marked DNA probes to the surface of the working electrode, standing, and realizing covalent bonding through Au-S bonds; S4, dropwise adding MCH solution, incubating at room temperature for 1 h, and closing the residual active sites on the surface of the electrode; The aptamer-activated chain locking complex is prepared by mixing lactobacillus acidophilus specific aptamer with activated chain, heating to denature DNA, slowly cooling to room temperature, and centrifuging to obtain supernatant; The CRISPR-Cas12a/crRNA complex is prepared by incubating buffer, lbCas a and crRNA, preferably at 37℃for 10 min.
7. Use of an aptamer-controlled electrochemical sensor based on an au@ti3c2mxene composite material according to any one of claims 1-5 for lactobacillus acidophilus detection.
8. The use of claim 7, wherein the method of detecting is: (1) Mixing lactobacillus acidophilus solutions with different concentrations and volumes of 5-15 mu L with an aptamer-activation chain locking compound with a volume of 15-25 mu L, and incubating for 40-80 minutes to enable the aptamer to be specifically combined with lactobacillus acidophilus and release an activation chain; (2) Mixing the reaction solution with a CRISPR-Cas12a/crRNA complex, reacting to form a Cas12 a-crRNA-activation chain ternary complex, and activating trans-cleavage activity of Cas12a enzyme; (3) Dripping the activated Cas12a enzyme onto the treated working electrode to enable the structure of the DNA probe on the surface of the non-specific cutting electrode to switch an electrochemical signal to a 'closed' state; (4) And detecting in PBS buffer solution with pH value of 6.5-7.5 by adopting differential pulse voltammetry, and calculating the concentration of the lactobacillus acidophilus according to a standard curve.

Description

Aptamer-controlled electrochemical sensor based on Au@Ti 3C2 MXene composite material, preparation method and application Technical Field The invention relates to the technical field of microorganism detection, in particular to an aptamer-controlled electrochemical sensor based on an Au@Ti 3C2 MXene composite material, a preparation method and application. Background Lactobacillus acidophilus is used as an important probiotic for human intestinal tracts, and is also a core functional strain in yoghurt, fermented dairy products and probiotic preparations, and the content and activity of the lactobacillus acidophilus are directly related to the food quality (such as the fermentation degree of the yoghurt) and the efficacy of the probiotic preparations. Therefore, an accurate and reliable quantitative detection technology is established, and the method has important significance for food quality control, efficacy verification of probiotic products and clinical application evaluation. The existing mainstream detection method has obvious limitations, while the traditional culture method can reflect the number of living bacteria, the traditional culture method is long in time (usually 3-7 days are needed), the selectivity is poor, and the target bacteria in the mixed flora and the bacteria in a dormant state are difficult to distinguish, while the molecular biological method such as qPCR is brought into a new national standard, has higher sensitivity, but the equipment is expensive, the operation is complex, the living bacteria and the dead bacteria cannot be distinguished, and the false positive is easily caused by DNA residues, so that the detection accuracy is influenced. In recent years, the electrochemical aptamer sensor has good application prospect in the field of food safety analysis due to the advantages of high sensitivity, high efficiency, portability of equipment, low cost and the like. However, achieving high specificity and high sensitivity simultaneous detection of probiotics in complex food matrices remains a challenge. On the one hand, the design of the sensing interface directly affects the electron transfer efficiency and the signal output intensity, and on the other hand, the identification of non-nucleic acid targets (such as whole bacteria) needs to use an effective signal transduction mechanism to activate a downstream signal amplification system. Disclosure of Invention Aiming at the problems, the invention provides a novel detection platform based on the synergistic effect of electrochemical aptamer recognition and CRISPR-Cas12a signal amplification, which is special for rapid, specific and sensitive quantitative analysis of lactobacillus acidophilus. The invention constructs a high-performance electrochemical sensing interface by adopting an in-situ reduction method to synthesize an Au@Ti 3C2 MXene heterogeneous composite material and uniformly anchoring zero-dimensional gold nano particles (Au NPs) on the surface of a two-dimensional Ti 3C2 MXene ultrathin nano sheet. The structure not only effectively inhibits the stacking and agglomeration of MXene sheets and remarkably improves the dispersibility of materials, but also greatly enhances the electron transfer rate and the electroactive surface area of the electrode through the strong electron coupling effect between Au and MXene, thereby providing an ideal substrate for high-sensitivity electrochemical signal output. The invention combines proper ligand recognition with CRISPR-Cas12a signal amplification, namely, because the CRISPR-Cas12a system can only be activated by specific single-stranded DNA (namely an activation chain) and trigger nonspecific trans-cutting activity, lactobacillus acidophilus is a non-nucleic acid target and an intermediate transduction mechanism needs to be introduced. For this purpose, the invention designs a 'lock-release' strategy, wherein an activation strand is combined with an aptamer through a complementary sequence to form a thermodynamically stable composite structure (namely a 'locked' state), the aptamer preferentially binds with high affinity to a bacterial surface marker when a target bacterium exists, so that the activation strand is released, and a free activation strand immediately activates Cas12a to enable the Cas12a to cleave a reporter probe (containing an electrochemical signal molecule) modified on the surface of an electrode, so that a detectable current change is generated. The cascade design realizes the integrated detection flow of bacterial identification, signal transduction, enzymatic amplification and electrochemical reading, and obviously improves the sensitivity while ensuring high specificity. Experiments show that the sensor has good linear response within the range of 10-10 6 CFU/mL, the detection limit is as low as 4 CFU/mL, and the quality control requirements of lactobacillus acidophilus in practical samples such as yoghurt, probiotic preparations and the like are completel