Electrochemical gene sensor based on Ti3C2@AuNP and preparation and application thereof

Abstract

This invention discloses an electrochemical gene sensor based on Ti3C2@AuNP, its preparation, and its application, belonging to the field of biosensing technology. The Ti3C2@AuNP-based electrochemical DNA sensor of this invention is prepared by drop-coating Ti3C2@AuNP onto the surface of a working electrode, followed by self-assembly of a methylene blue-labeled and thiol-modified hairpin HP-Sul2 probe onto the electrode surface, thus creating a ratiometric electrochemical DNA sensor capable of detecting the Sul2 antibiotic resistance gene. This sensor has advantages such as simple preparation process, low cost, high sensitivity, good reproducibility, and storage stability, providing a powerful tool for the sensitive and specific detection of antibiotic resistance genes in aquatic environments.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosensing, and particularly relates to a DNA electrochemical sensor, a preparation method and application thereof in detection of antibiotic resistance genes in a water environment. BACKGROUND

[0002] Global dissemination of environmental biological pollutants, such as antibiotic-resistant bacteria and their antibiotic resistance genes (ARGs), is one of the most concerned public health issues in recent years. ARGs in bacteria are ancient genes that have existed for a long time before the use of antibiotics. Bacteria can obtain ARGs through horizontal gene transfer by conjugation (intercellular contact), transduction (phage infection) and transformation (direct uptake of extracellular DNA), ultimately triggering resistant bacterial infections that are difficult to cure. Nowadays, with the overuse of antibiotics in the fields of aquaculture and medical health, antibiotic resistance genes have been detected in various environments such as water, air and soil, among which municipal wastewater treatment plants are an important reservoir of ARGs, and hundreds of resistance genes have been detected. As a key node of water circulation, sewage from wastewater treatment plants is removed by ultraviolet irradiation and other methods, resulting in a large amount of cell-free DNA (including ARGs) flowing into natural water bodies, and forming new antibiotic-resistant bacteria again through horizontal gene transfer. Due to the constraints of detection methods, most current studies only target traditional intracellular resistance genes (iARGs), and extracellular resistance genes (eARGs) have rarely been studied. eARGs are produced by active secretion by cells or released after cell death, can survive for a long time without being degraded, and can re-enter the intracellular expression of drug resistance under suitable conditions. More and more studies have shown that eARGs also exist widely in water environments, and have equally serious or even more hidden health risks. Therefore, it is necessary to study reliable analysis techniques to quickly and accurately quantify eARGs in water environments.

[0003] At present, the analysis of eARGs in water environments mainly relies on molecular biology techniques such as PCR, qPCR and metagenomic sequencing. Although these methods can provide accurate and complete information, they are time-consuming and costly. In contrast, electrochemical biosensing technology has the advantages of fast response, high sensitivity, low cost, and real-time data collection and multi-channel analysis in a single device, which can make up for the above shortcomings. It can be used as an effective supplement to traditional detection methods and can meet the demand for on-site rapid detection.

[0004] Isothermal strand displacement polymerization reaction (ISDPR) has been used as signal amplification in biosensors, combined with the designed single-labeled double-signal ratio sensing strategy, to further improve the sensitivity and detection range, because its requirements for repeated thermal cycling, specific targets, strict temperature regulation and complex sequence modeling are dispensable.

[0005] MXene, as a two-dimensional material, has been widely used in the field of biosensing due to its good electrical conductivity, rich surface functional groups and excellent hydrophilic ability, etc. Ti3C2@AuNP synthesized by seed growth method can significantly improve its conductivity, and provide more active sites for the fixation of DNA probes than Ti3C2@AuNP composite material synthesized by self-reduction method. At present, there is no related report on the application of Ti3C2@AuNP synthesized by seed growth method in electrochemical DNA sensor at home and abroad. SUMMARY

[0006] In view of this, the purpose of the present application is to provide a simple, rapid and efficient electrochemical DNA sensor for detecting antibiotic resistance genes in water environment and its preparation method and application.

[0007] The purpose of the present application is achieved by the following means:

[0008] The present application provides a ratio-type electrochemical DNA sensor based on two-dimensional monolayer Ti3C2@AuNP modification and its preparation and application. The two-dimensional monolayer Ti3C2@AuNP is synthesized by seed growth method, has high specific surface area and good electrical conductivity, not only provides more active sites for the fixation of hairpin probes, but also promotes the electron transfer between electrochemical indicators and electrode surface. By using ISDPR signal amplification strategy, KF exo - Catalytic primer extension, release of target Sul2 gene, trigger the next polymerization cycle, enhance the change of response signal of electrochemical indicator labeled on the probe; the second indicator is used to form a complex with the groove surface of DNA, and a second signal is introduced to construct a cheap double-signal ratio sensing platform; the electrochemical DNA sensor designed by the present application takes the widely distributed sulfonamide antibiotic resistance gene Sul2 in water environment as the eARGs model, realizes the quantitative analysis of antibiotic resistance genes with high sensitivity and wide detection range.

[0009] The present application provides a preparation method of a ratio-type electrochemical DNA sensor based on two-dimensional Ti3C2@AuNP, mainly comprising the following steps:

[0010] (1) 0.1-1 mg / mL Ti3C2 nanosheet solution is added dropwise into 1-3 times volume of 1-5 mg / mL polyallylamine hydrochloride solution, ultrasonic treatment for 10-60 min, then stirring for 1-5 h, centrifugation, water washing, adding water to disperse the Ti3C2-PAH solution obtained in an equal volume of nanosheet solution;

[0011] (2) The Ti3C2-PAH solution prepared in step (1) is added dropwise into 2-10 times volume of gold seed solution, ultrasonic treatment for 10-40 min, then stirring for 1-5 h, centrifugation, water washing, adding water to disperse the Ti3C2-Au seed solution obtained in an equal volume of Ti3C2-PAH solution;

[0012] (3) The Ti3C2-Au seed solution prepared in step (2) is added to 10-40 times volume of gold growth solution, stirring for 1-3 h, then adding dropwise a reducing agent solution, stirring for 0.5-3 h, centrifugation, water washing, adding water to disperse the Ti3C2@AuNP solution obtained;

[0013] (4) The Ti3C2@AuNP solution prepared in step (3) is added dropwise to the surface of the cleaned electrode, and dried to obtain a Ti3C2@AuNP covered electrode;

[0014] (5) The electrochemical indicator label and thiol-modified single-stranded DNA probe are modified to the surface of the Ti3C2@AuNP modified electrode prepared in step (4) to obtain a single-stranded DNA probe modified Ti3C2@AuNP electrode;

[0015] (6) The single-stranded DNA probe modified Ti3C2@AuNP electrode prepared in step (5) is immersed in an aqueous solution containing a blocking agent for 0.5-2 hours to obtain an electrochemical DNA sensor.

[0016] Based on the above technical solution, further, the gold seed solution in step (2) is: 5-20 μL of 80% tetrahydroxymethyl phosphonium hydroxide (THPC) and 0.1-1 mL of 1-3 M sodium hydroxide are added to 30-50 mL of ultrapure water, stirred uniformly, then 1-3 mL of 0.5-5% HAuCl4 is quickly added, and stirred overnight at room temperature in the dark to obtain.

[0017] Based on the above technical solution, further, the gold growth solution in step (3) is: 10-50 mg of potassium carbonate is added to 100 mL of ultrapure water containing 0.5-5 mL of 0.5-5% HAuCl4, and aged for 1 day to obtain; the reducing agent solution is a 10-30% formaldehyde solution.

[0018] Based on the above technical scheme, further, the electrode in step (4) includes a glassy carbon electrode, a gold electrode, a paper electrode, and a polymer electrode, the concentration of the Ti3C2@AuNP solution is 0.1-2 mg / mL, and the dropwise adding volume is 1-10 μL.

[0019] Based on the above technical scheme, further, the structure of the electrochemical indicator-labeled and thiol-modified single-stranded DNA probe in step (5) is as follows: 5'-electrochemical indicator-CAAGCGCCAGCAATCCCGACGCCGCGCCTGTTTCCTGGCGCTTG-(CH2)6-SH; the electrochemical indicator includes but is not limited to methylene blue and ferrocene; the concentration of the dilute solution of the electrochemical indicator-labeled and thiol-modified single-stranded DNA probe is 0.1-10 μM, and the dropwise adding volume is 2-10 μL.

[0020] Based on the above technical scheme, further, the blocking agent in step (6) includes mercaptohexanol and mercaptoethanol, and the concentration of the blocking agent is 1-10 mM.

[0021] Another aspect of the present application provides a two-dimensional Ti3C2@AuNP-based ratio-type electrochemical DNA sensor prepared by the preparation method.

[0022] Based on the above technical scheme, further, the two-dimensional Ti3C2@AuNP-based ratio-type electrochemical DNA sensor is applied to detection of antibiotic resistance genes in a water environment.

[0023] Based on the above technical scheme, further, the antibiotic resistance genes include sulfonamide, fluoroquinolone, and tetracycline antibiotic resistance genes.

[0024] The present application also provides a detection method of the two-dimensional Ti3C2@AuNP-based ratio-type electrochemical DNA sensor for detecting antibiotic resistance genes in a water environment.

[0025] (a) adding a polymerase buffer solution containing target antibiotic resistance genes, primers, KF exo - , and dNTPs to the surface of the electrode of the electrochemical DNA sensor, and incubating at 35-39 ℃ for 1-4 h to obtain a sensor after reaction and amplification with the antibiotic resistance genes;

[0026] (b) immersing the electrochemical DNA sensor obtained in step (a) into a buffer solution containing another electrochemical indicator, and recording the current intensity values of the two electrochemical indicators by differential pulse voltammetry scanning; wherein, when the electrochemical indicator labeled on the single-stranded DNA probe is methylene blue, the other electrochemical indicator is ferrocene; when the electrochemical indicator labeled on the single-stranded DNA probe is ferrocene, the other electrochemical indicator is methylene blue;

[0027] (c) establishing a standard curve of the ratio of the current value of the electrochemical indicator in the buffer solution to the current value of the electrochemical indicator labeled on the single-stranded DNA probe to the concentration of the target antibiotic resistance gene;

[0028] (d) detecting the sample to be tested according to steps (a)-(c), and substituting the detection result into the standard curve to determine the content of the target antibiotic resistance gene in the sample to be tested.

[0029] The present application has the following beneficial effects relative to the prior art:

[0030] 1、The Ti3C2@AuNP prepared by the seed growth method of the present application has a very high specific surface area, which is conducive to improving the loading capacity of the DNA probe, and the good conductivity of the Ti3C2@AuNP can enhance the current response, thereby effectively improving the sensitivity of the sensor.

[0031] 2、The DNA electrochemical sensor prepared by the present application has reliable and accurate detection results, and the DNA sensor has better stability than protein (enzyme) sensors, and the ex vivo DNA has better thermal stability than most protein (enzyme) molecules, so the prepared sensor can be stored for a long time.

[0032] 3、The DNA electrochemical sensor prepared by the present application has good specificity, and the double-stranded DNA molecules have very high specific recognition through specific base pairing, and the change of a single base can cause obvious changes in the electric signal.

[0033] 4、The DNA electrochemical sensor prepared by the present application is low in price, simple in operation and fast in reaction.

[0034] 5、The DNA electrochemical sensor prepared by the present application has the advantages of high sensitivity, fast detection speed and low detection limit in the detection of antibiotic resistance genes in the water environment. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present application, the drawings involved in the embodiments will be briefly introduced below.

[0036] Figure 1 The transmission electron microscope (TEM) image of the Ti3C2@AuNP prepared for Example 1 of the present application;

[0037] Figure 2 Preparation of DNA electrochemical sensor for Example 2 of the present application;

[0038] Figure 3 Linear correlation plot of the logarithm of Sul2 resistance gene concentration and peak current I of the DNA electrochemical sensor in Example 3 of the present application; Fc / I MB Linear correlation plot of the logarithm of Sul2 resistance gene concentration and peak current I of the DNA electrochemical sensor in Example 3 of the present application;

[0039] Figure 4 Specificity of the electrochemical DNA sensor for detecting Sul2 resistance gene in Example 4 of the present application;

[0040] Figure 5 Reproducibility of the electrochemical DNA sensor for detecting Sul2 resistance gene in Example 4 of the present application;

[0041] Figure 6 Stability of the electrochemical DNA sensor for detecting Sul2 resistance gene in Example 4 of the present application. DETAILED DESCRIPTION

[0042] The present application will be described in detail below with reference to the examples, but the embodiments of the present application are not limited thereto. It is obvious that the examples described below are only some of the embodiments of the present application, and other similar embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0043] The following are the main instruments and equipment used in the examples of the present application, and other experimental conditions not specifically mentioned are carried out according to the conventional or recommended conditions of the instrument manufacturers.

[0044] The instrument used for electrochemical detection is a portable electrochemical workstation PalmSens4, and the three-electrode system; the DNA sequences (including Sul2 gene fragments) are purchased from Shenguo Bioengineering (Shanghai) Co., Ltd. The DNA sequences in the examples are shown in Table 1.

[0045] Table 1. Base sequence of oligonucleotide

[0046]

[0047] Preparation of Ti3C2@AuNP in Example 1

[0048] Ti3C2 nanosheets (1 mg / mL) were added dropwise into 4 mL of polyallylamine hydrochloride (PAH) solution (Mw = 15,000, 2 mg / mL), and after ultrasonic treatment for 30 min, stirring for 4 h, centrifugation (14800 rpm, 5 min), washing with water for 3 times, and dispersion with 2 mL of water, a Ti3C2-PAH solution was obtained. Then, the above Ti3C2-PAH solution was added dropwise into 10 mL of gold seed solution, and after ultrasonic treatment for 30 min, stirring for 4 h, centrifugation purification for 2 times, and dispersion with 2 mL of water, a Ti3C2-Au seed solution was obtained. Finally, 1 mL of Ti3C2-Au seed solution (1 mg / mL Ti3C2) was added into 20 mL of gold growth solution, stirring for 2 h, followed by dropwise addition of a reducing agent formaldehyde (HCHO, 29%), stirring for 1 h, and repeated washing with water, to obtain a Ti3C2@AuNP composite material.

[0049] Preparation of gold seed solution: 12 μL of 80% tetrahydroxymethyl phosphonium hydroxide (THPC) and 0.25 mL of 2M sodium hydroxide solution were added into 45 mL of ultrapure water, and after stirring for 5 min, 2 mL of 1% HAuCl4 was quickly added, and the solution was stirred overnight at 25°C in the dark.

[0050] Preparation of gold growth solution: 25 mg of potassium carbonate was added into 100 mL of ultrapure water containing 1.5 mL of 1% HAuCl4, and the solution was stored for one day before use.

[0051] Preparation of an electrochemical DNA sensor

[0052] The preparation method of the DNA electrochemical biosensor of the present application mainly comprises the following steps:

[0053] (1) The surface area of the gold electrode was 0.02 cm 2 The gold electrode was cleaned in piranha solution (H2O2:H2SO4 = 1:3, v / v) for 15 min, and then repeatedly ultrasonically cleaned in anhydrous ethanol and deionized water for 3 times, each for 3 min; polished with alumina powder with particle sizes of 1 μm, 0.3 μm, and 0.05 μm in turn, and then repeatedly ultrasonically cleaned in anhydrous ethanol and deionized water for 3 times, each for 3 min; and then placed in a 0.5M sulfuric acid solution and cyclically scanned between 0-1.7V for activation, until the scanning curve completely overlapped, the electrode was taken out, washed with deionized water, and dried with nitrogen;

[0054] (2) The Ti3C2@AuNP prepared in Example 1 was prepared into a solution with a concentration of 1 mg / mL, ultrasonically treated for 30 min, and 4 μL was added dropwise on the surface of the above gold electrode and naturally dried;

[0055] (3) The methylene blue-labeled and thiol-modified single-stranded DNA probe HP-Sul2 solution (a hairpin probe HP-Sul2 containing a disulfide bond between the strands was obtained after reacting with a reducing agent tris(2-carboxyethyl)phosphine according to a molar ratio of 1:100) was diluted to 1 μM, 8 μL of which was added dropwise to the electrode surface obtained in step (2), and the reaction was carried out at 4°C for 16 hours. The electrode was repeatedly washed with deionized water and 10 mM PBS buffer solution (pH 7.0) and dried with nitrogen to obtain the HP-Sul2 / Ti3C2@AuNP / Au electrode.

[0056] (3) The HP-Sul2 / Ti3C2@AuNP / Au electrode prepared in step (2) was immersed in a 1 mM aqueous solution of mercaptohexanol (blocking agent) and reacted at room temperature for 1 hour to obtain the electrochemical DNA sensor.

[0057] Example 3 Detection of Sul2 antibiotic resistance gene by electrochemical DNA sensor

[0058] A 50 μL polymerase buffer solution containing different concentrations of target DNA, primers (5 μL, 500 nM), KF exo - (5U) and dNTPs (2.5 μL) was added dropwise to the surface of the HP-Sul2 / Ti3C2@AuNP / Au electrode, and then incubated at 37°C for 2.5 h. After washing the electrode with a large amount of ultrapure water for 3 times, the electrode was placed in a 10 mM PBS buffer solution containing 0.2 μg / mL ferrocene, and the differential pulse voltammogram was scanned between -0.5 and 0.5 V.I Fc / I MB The relationship between the ratio of the peak current of the target sul2 gene and the peak current of the non-target gene and the logarithm of the concentration of the sul2 target gene is shown in Figure 3 .

[0059] Example 4 Evaluation of specificity, reproducibility and storage stability of electrochemical DNA sensor

[0060] The specificity of the electrochemical DNA sensor of the present application was studied by differential pulse. The prepared electrochemical DNA sensor was exposed to PBS buffer solution (a), completely non-complementary gene (b), three-base mismatched gene TBM-Sul2 (c), single-base mismatched gene SBM-Sul2 (d) and completely complementary target gene sul2 (e), respectively. The results are shown in Figure 4 , and the significant change in the response signal of the sul2 gene indicates that the sensor has good specificity.

[0061] In order to evaluate the reproducibility of the electrochemical DNA sensor of the present application between different electrodes of the same batch, 10 sensors were independently prepared under the same conditions, and each 5 sensors were used to detect Sul2 resistance genes with concentrations of 10 pM and 100 fM, respectively. The results are shown in Figure 5As shown, the results show that the RSDs are 1.83% and 2.24% in turn, indicating that the electrodes prepared in different batches have good reproducibility.

[0062] In order to evaluate the storage stability of the DNA electrochemical biosensor of the present application, the same electrodes as those used for detecting 10 pM Sul2 resistance gene were stored at 4°C for 7 days, and then were used to detect Sul2 resistance gene, and the results are shown in Table 3. Figure 6 As shown, the I Fc / I MB values (RSD = 1.87%) only changed by 1.97% compared with the results one week ago, proving that the DNA sensor developed in the present application has good stability within the error range.

[0063] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a two-dimensional Ti3C2@AuNP-based ratio-type electrochemical DNA sensor, characterized by, The method mainly comprises the following steps: (1) adding 0.1-1 mg / mL Ti3C2 nanosheet solution into 1-3 times volume of 1-5 mg / mL polyallylamine hydrochloride solution, ultrasonic treatment for 10-60 min, then stirring for 1-5 h, centrifuging, water washing, adding water to disperse the Ti3C2-PAH solution to obtain Ti3C2-PAH solution; (2) adding the Ti3C2-PAH solution prepared in step (1) into 2-10 times volume of gold seed solution, ultrasonic treatment for 10-40 min, then stirring for 1-5 h, centrifuging, water washing, adding water to disperse the Ti3C2-Auseed solution to obtain Ti3C2-Auseed solution; (3) adding the Ti3C2-Au seed solution prepared in step (2) into 10-40 times volume of gold growth solution, stirring for 1-3 h, then adding reducing agent solution dropwise, stirring for 0.5-3 h, centrifuging, water washing, adding water to disperse the Ti3C2@AuNP solution to obtain Ti3C2@AuNP solution; (4) adding the Ti3C2@AuNP solution prepared in step (3) to the surface of the cleaned electrode, drying to obtain Ti3C2@AuNP covered electrode; (5) modifying electrochemical indicator labeled and thiol modified single-stranded DNA probe to the surface of the Ti3C2@AuNP modified electrode prepared in step (4) to obtain single-stranded DNA probe modified Ti3C2@AuNP electrode; (6) immersing the single-stranded DNA probe modified Ti3C2@AuNP electrode prepared in step (5) into water solution containing blocking agent, reacting for 0.5-2 h to obtain electrochemical DNA sensor.

2. The production method according to claim 1, characterized by, The gold seed solution in step (2) is prepared by adding 5-20 μL 80% tetrahydroxymethyl phosphonium hydroxide (THPC) and 0.1-1 mL 1-3 M sodium hydroxide into 30-50 mL ultrapure water, stirring uniformly, then quickly adding 1-3 mL 0.5-5% HAuCl4, stirring overnight at room temperature in dark.

3. The preparation method according to claim 1, characterized in that, The gold growth solution in step (3) is prepared by adding 10-50 mg potassium carbonate into 100 mL ultrapure water containing 0.5-5 mL 0.5-5% HAuCl4, aging for 1 day to obtain; the reducing agent solution is 10-30% formaldehyde solution.

4. The method of claim 1, wherein, The electrode in step (4) includes glassy carbon electrode, gold electrode, paper electrode, polymer electrode, and the concentration of Ti3C2@AuNP solution is 0.1-2 mg / mL, and the dropwise adding volume is 1-10 μL.

5. The preparation method according to claim 1, characterized in that, The structure of the electrochemical indicator labeled and thiol modified single-stranded DNA probe in step (5) is shown as follows: 5'-electrochemical indicator-CAAGCGCCAGC AATCCCGACGCCGCGCCTGTTTCCTGGCGCTTG-(CH2)6-SH; the electrochemical indicator includes but is not limited to methylene blue and ferrocene; the concentration of the dilute solution of the electrochemical indicator labeled and thiol modified single-stranded DNA probe is 0.1-10 μM, and the dropwise adding volume is 2-10 μL.

6. The method of claim 1, wherein, The blocking agent described in step (6) includes mercaptohexanol and mercaptoethanol, and the concentration of the blocking agent is 1-10 mM.

7. The two-dimensional Ti3C2@AuNP-based ratiometric electrochemical DNA sensor prepared by the method of any one of claims 1-6.

8. The use of the two-dimensional Ti3C2@AuNP-based ratiometric electrochemical DNA sensor of claim 7 in the detection of antibiotic resistance genes in water environment.

9. Use according to claim 8, characterized in that, The antibiotic resistance genes include, but are not limited to, sulfonamide, fluoroquinolone and tetracycline antibiotic resistance genes. 10.The method for detecting antibiotic resistance genes in water environment based on the two-dimensional Ti 3C 2 @AuNP ratio type electrochemical DNA sensor of claim 7, characterized in that, The method mainly comprises the following steps: (a) adding a polymerase buffer containing a target antibiotic resistance gene, primers, KF exo - , dNTPs to the surface of the electrochemical DNA sensor electrode, and incubating at 35-39°C for 1-4 h to obtain a sensor after reaction and amplification with the antibiotic resistance gene; (b) immersing the electrochemical DNA sensor obtained in step (a) into a buffer solution containing another electrochemical indicator, and recording the current intensity values of the two electrochemical indicators by differential pulse voltammetry scanning; wherein, when the electrochemical indicator labeled on the single-stranded DNA probe is methylene blue, the other electrochemical indicator is ferrocene; when the electrochemical indicator labeled on the single-stranded DNA probe is ferrocene, the other electrochemical indicator is methylene blue; (c) establishing a standard curve of the ratio of the current value of the electrochemical indicator in the buffer solution to the current value of the electrochemical indicator labeled on the single-stranded DNA probe to the concentration of the target antibiotic resistance gene; (d) detecting the sample to be tested according to steps (a)-(c), and substituting the detection result into the standard curve to determine the content of the target antibiotic resistance gene in the sample to be tested.

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