Electrochemiluminescence sensor based on HE-MHOF enhanced PSA-HOF as well as preparation method and application of electrochemical luminescence sensor

Abstract

The invention discloses a pyrenyl HOF (PSA-HOF) / high-entropy metal hydroxide organic framework (HE-MHOF) electrochemical luminescence sensor for antibiotic resistance gene detection as well as a preparation method and application of the pyrenyl HOF / HE-MHOF electrochemical luminescence sensor, and belongs to the technical field of electrochemical luminescence analysis and detection. The method comprises the following steps: respectively modifying a glassy carbon electrode with PSA-HOF, HE-MHOF and a DNA hairpin H2 to obtain an H2-modified H2 / HE-MHOF / PSA-HOF / GCE electrode, co-incubating the modified electrode and an antibiotic resistance gene, and dispensing a DNA hairpin H1-Fc. And detecting the resistance gene ARGAMP in a PBS (phosphate buffer solution) containing potassium persulfate by adopting a three-electrode system. The prepared HE-MMOF / PSA-HOF system electrochemical luminescence sensor has the characteristics of simplicity, convenience and flexibility in operation, wide detection range, low detection limit, low detection cost and the like.

Description

Technical Field

[0001] This invention belongs to the field of electrochemiluminescence analysis and detection technology, and relates to an electrochemiluminescence sensor, its preparation method, and its application. In particular, it relates to an electrochemiluminescence sensor based on a high-entropy metal hydroxide organic framework (HE-MHOF) enhanced pyrene self-assembled hydrogen-bonded organic framework (PSA-HOF), its preparation method, and its application in detecting ampicillin antibiotic resistance genes (ARGs). AMP Applications in ). Background Technology

[0002] In modern human production and living systems, antibiotics, as important agents for disease prevention and control, are widely used in aquaculture and animal husbandry. Although most antibiotics have a limited half-life, long-term and high-frequency use leads to their continuous accumulation in the environment, making them a typical type of persistent organic pollutant. Antibiotics in the environment not only directly harm human health by interfering with metabolic homeostasis and disrupting the structure of the gut microbiota, but also induce the development of antibiotic resistance genes, promoting the spread of drug-resistant bacteria and having long-term and complex negative impacts on ecosystems.

[0003] Overuse of antibiotics or sustained low-dose exposure can exert strong selective pressure on microbial communities, inducing the emergence of antibiotic resistance genes (ARGs), a new type of environmental pollutant in the 21st century. As key genetic material for the generation and spread of bacterial resistance, ARGs have been proven to be widely present in various environmental media, including water bodies, soil, riverbed sediments, and wastewater treatment systems. These resistance genes can be horizontally transferred via mobile genetic elements such as plasmids and transposons. Once foodborne resistant bacteria carrying ARGs enter the food chain, they directly threaten human health and safety. Therefore, developing rapid, sensitive, and efficient detection technologies is of significant practical importance for effectively controlling the spread of ARGs and ensuring public health security.

[0004] Monitoring and regulating the proliferation and spread of antibiotic resistance genes has become a prominent and urgent research hotspot in environmental assessment and clinical diagnosis. Currently, while polymerase chain reaction (PCR) technology can provide accurate results in detecting antibiotic resistance genes, it still suffers from drawbacks such as being time-consuming, labor-intensive, requiring expensive equipment, and demanding specialized personnel. This severely hinders its application in resource-constrained environments. Genomic detection methods that do not involve any PCR amplification steps are extremely lacking. In recent years, in addition to PCR-based techniques, some researchers have attempted to overcome these limitations by using photoelectrochemical and electrochemical impedance spectroscopy to identify antibiotic resistance genes. Although these methods have achieved some success, further efforts are needed to develop new sensing strategies to detect resistance genes in complex environmental samples with lower detection limits.

[0005] In recent years, electrochemiluminescence (ECL) has demonstrated remarkable application potential in various fields such as environmental monitoring, food safety testing, and clinical diagnosis due to its advantages of high sensitivity, rapid response, and convenient operation, becoming a research hotspot in analytical chemistry. ECL technology is based on a light signal emission mechanism driven by electrochemical reactions, achieving light signal output through precise control of the electrode reaction process. To further improve the analytical performance of ECL sensing platforms, the development of high-performance luminescent reagents and the optimization of co-reaction system efficiency have become core research directions. Among these, the introduction of highly efficient co-reaction promoters plays a crucial role in enhancing the intensity and stability of ECL signals.

[0006] High-entropy metal hydroxide organic frameworks (HE-MHOFs), as an emerging class of nanomaterials, consist of five metal cations randomly or uniformly distributed in a single-phase structure. Their unique multi-metal active centers endow them with differentiated redox properties. This structural advantage gives HE-MHOFs both excellent chemical and structural stability, exhibiting outstanding electrocatalytic performance. Leveraging its multi-metal cation-constructed active sites, HE-MHOFs demonstrate potential catalytic applications in various chemical reactions, providing an innovative material basis for the development of novel ECL co-reaction promoters. Summary of the Invention

[0007] To address the problems existing in the aforementioned related technologies, this invention provides an electrochemiluminescence sensor based on a high-entropy metal hydroxide organic framework (HE-MHOF) enhanced pyrene self-assembled hydrogen-bonded organic framework (PSA-HOF), its preparation method, and its application in detecting ampicillin antibiotic resistance genes (ARGs). AMP Applications in ARG (Anaerobic Gamma-Rayet). The aim is to obtain an electrochemical sensor with high detection sensitivity, wide detection range, and low detection limit, and to apply it to ARG. AMP The testing process is inexpensive.

[0008] In a first aspect, the present invention provides an electrochemiluminescence sensor comprising a glassy carbon electrode H2 / HE-MHOF / PSA-HOF / GCE co-modified with an HE-MHOF / PSA-HOF complex and an aptamer having specific recognition function; wherein the HE-MHOF / PSA-HOF complex and the aptamer are connected by an amide bond formed by an amino group on chitosan and a carboxyl group on the aptamer.

[0009] In some optional embodiments, the aptamer with specific recognition function can specifically recognize ARG. In some optional embodiments, the ARG is an ampicillin antibiotic resistance gene, labeled ARG. AMP The aptamer consists of two DNA hairpins with the following nucleotide sequences: Hairpin DNA2 (H2): COOH-TTT TGAGGC GGA TAA AGT TGC AGG AAT CCG CCT CAT CCT GCA ACT TT and Hairpin DNA1 (H1-Fc): Fc-TTT TGC AGG ATG AGG CGG ATT CCT GCA ACT TTA TCC GCC TCA. When both hairpins specifically recognize ARG... AMP DNA hairpins can self-assemble and cycle with ARG. AMP The specific binding forms a double-stranded complex. The ferrocene (Fc) linked to H1 near the electrode surface produces a quenching effect, leading to a weakening of the ECL signal and a decrease in light intensity compared to ARG. AMP The concentration of ARG exhibits a linear relationship, which can then be used for quantitative detection and analysis. AMP content.

[0010] Secondly, the present invention also provides a method for preparing the electrochemiluminescence sensor, comprising: A certain amount of PSA-HOF and HE-MHOF were dispersed in chitosan solution to obtain PSA-HOF dispersion and HE-MHOF dispersion, respectively. The PSA-HOF and HE-MHOF dispersions were respectively drop-coated onto the surface of a clean glassy carbon electrode for drop-coating modification to obtain an HE-MHOF / PSA-HOF / GCE modified electrode. The DNA hairpin was incubated on the HE-MHOF / PSA-HOF / GCE modified electrode to obtain the electrochemiluminescence sensor H2 / HE-MHOF / PSA-HOF / GCE.

[0011] The modification mass ratio of PSA-HOF to HE-MHOF is 1:1 to 1.5. In some optional embodiments, the mass ratio of PSA-HOF to HE-MHOF is 4:4, 4:5, or 4:6; the mass-to-volume ratio of PSA-HOF or HE-MHOF to chitosan is 1:1 (g / L); and the mass fraction of the chitosan solution is 0.5%.

[0012] In some optional embodiments, the amount of HE-MHOF dispersion or PSA-HOF dispersion dropped onto the glassy carbon electrode surface is 2-6 μL.

[0013] In some optional embodiments, the concentration of the DNA hairpin is 0.5~2.5 μmol / L, preferably 1~2.5 μmol / L, and the drop volume is 3.0 μL.

[0014] Thirdly, the present invention also provides the electrochemiluminescence sensor or the electrochemiluminescence sensor obtained according to the above-described method for preparing an electrochemiluminescence sensor for detecting ARG. AMP Applications in [the context of] specific recognition of ARG. AMP DNA hairpins can self-assemble and cycle with ARG. AMP The specific binding forms a double-stranded complex. The ferrocene (Fc) linked to H1 near the electrode surface produces a quenching effect, leading to a weakening of the ECL signal and a decrease in light intensity compared to ARG. AMP The concentration of ARG exhibits a linear relationship, which can then be used for quantitative detection and analysis. AMP content.

[0015] In some alternative embodiments, the application includes the following steps: Prepare a series of ARGs with different concentrations AMP Standard solution; A three-electrode system was constructed using H2 / HE-MHOF / PSA-HOF / GCE as the working electrode, a platinum electrode as the counter electrode, and Ag / AgCl as the reference electrode. The three-electrode system was placed in a PBS phosphate buffer solution containing potassium persulfate. Within an electrochemical window of -2.0 to 0 V, a photomultiplier tube voltage of 300 to 600 V and a scan rate of 0.05 to 0.25 V / s were used to record the time-luminescence intensity curve. The series of ARG standard solutions of different concentrations were then incubated on the H2 / HE-MHOF / PSA-HOF / GCE. The electrode was then incubated with H1-Fc at 37°C to obtain H1-Fc / ARG / H2 / HE-MHOF / PSA-HOF / GCE. Cyclic voltammetry was then performed within the electrochemical window of -2.0 to 0 V, with a photomultiplier tube voltage of 300 to 600 V and a scan rate of 0.05 to 0.25 V / s. The time-luminescence intensity curves before and after ARG binding to the DNA hairpin were recorded. The ARG concentration in the sample solution was calculated based on the linear regression equation obtained from the linear relationship between the difference in luminescence intensity and the logarithm of the ARG concentration.

[0016] In some optional embodiments, the pH of the PBS phosphate buffer solution containing potassium persulfate is 5-9, and the concentration of PBS is 0.1 mol / L.

[0017] In some optional embodiments, the binding time of the ARG to the DNA hairpin in the electrochemiluminescence sensor H2 / HE-MHOF / PSA-HOF / GCE is 20-60 min.

[0018] This invention utilizes HE-MHOF nanomaterials as a co-reaction accelerator and DNA hairpins with specific recognition capabilities as recognition elements. HE-MHOF / PSA-HOF and the H2 group of the DNA hairpin are connected via chitosan (CS) (the amino groups on CS form amide bonds with the carboxyl groups on the DNA hairpin), and are co-modified onto a glassy carbon electrode (GCE). A pyrene-based self-assembled hydrogen-bonded organic framework (PSA-HOF) is introduced as a luminescent agent into a PBS phosphate buffer solution containing potassium persulfate. The H2 / HE-MHOF / PSA-HOF / GCE modified electrode is then used as the working electrode for electrochemiluminescence detection. The introduction of HE-MHOF significantly promotes the reaction of persulfate in the PBS phosphate buffer solution, thereby obtaining a strong and stable ECL signal.

[0019] The electrochemiluminescence sensor provided by this invention is an electrochemiluminescence sensor based on HE-MHOF nanomaterials enhanced PSA-HOF. Compared with ordinary electrochemiluminescence sensors, HE-MHOF nanomaterials can promote the reaction of persulfate, thereby improving the ECL signal.

[0020] This invention enables the quantitative detection of ARG by using HE-MHOF nanomaterials to enhance PSA-HOF electrochemiluminescence sensors. These sensors not only possess the advantages of high sensitivity, wide linear range, and simple operation in electrochemiluminescence analysis, but also hold significant practical importance for the specific detection of ARG in river water using a high-performance HE-MHOF / PSA-HOF ECL system. Attached Figure Description

[0021] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 This is a simplified flowchart illustrating the fabrication of the electrochemiluminescence sensor and the detection of ARG according to an exemplary embodiment of the present invention.

[0022] Figure 2 This is a linear curve showing the luminescence intensity of a three-electrode system after adding different concentrations of ARG in an exemplary embodiment of the present invention, where a ~ g: 1.0 × 10⁻⁶. -15 mol / L(a), 1.0 × 10 -14 mol / L(b), 1.0 × 10 -13mol / L(c), 1.0 × 10 -12 mol / L(d), 1.0×10 -11 mol / L(e), 1.0×10 -10 mol / L(f), 1.0×10 -9 mol / L(g).

[0023] Figure 3 This is a standard curve of luminescence intensity and the logarithm of ARG concentration after adding ARG to a three-electrode system according to an exemplary embodiment of the present invention.

[0024] Figure 4 This describes the effect of the mass ratio of PSA-HOF to HE-MHOF on the luminescence intensity of ECL in an exemplary embodiment of the present invention.

[0025] Figure 5 This is an exemplary embodiment of the present invention showing the effect of H2 concentration on luminescence intensity in DNA hairpins.

[0026] Figure 6 This is an exemplary embodiment of the present invention showing the effect of the binding time of ARG to DNA hairpin H2,H1-Fc on the luminescence intensity.

[0027] Figure 7 This relates to the effect of the pH of the PBS solution on the ECL luminescence intensity in the exemplary embodiment of the application. Detailed Implementation

[0028] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0029] The HE-MHOF nanomaterials and PSA-HOF in the specific embodiments of the present invention can be obtained using methods already available in the prior art. For example, they are prepared using the following method: (1) Preparation of PSA-HOF First, a 9-10 mM solution of 1,3,6,8-tetra(p-benzoic acid)pyrene (H4TBAPy)N,N-dimethylformamide (DMF) was prepared under ultrasonic conditions. Then, 3-4 times the volume of CH3OH was added to the solution and stirred for 5-10 minutes. Finally, the mixture was stored at room temperature for 10-12 hours, centrifuged, and washed three times with CH3OH and acetone, respectively, to remove impurities. The product was then placed in a vacuum drying oven and dried at 60 °C for 10-12 hours to obtain a yellow PSA-HOF powder.

[0030] (2) Preparation of HE-MHOF nanomaterials

[0031] Synthesis of Mmix(OH)₂ (Mmix = Mn, Ni, Co, Cu, Zn): All equimolar amounts of the metal chloride salts (0.5 mmol) were dissolved in 10 mL of water, and double the stoichiometric amount of NaOH was dissolved in 10 mL of water. The two solutions were then mixed and stirred at room temperature for 1 hour. After centrifugation, the product was collected, washed with water, and the pH of the filtrate was checked with pH paper until the pH of the filtrate reached neutral. Finally, the product was dried under vacuum to obtain the Mmix(OH)₂ precursor. The freshly prepared Mmix(OH)₂ precursor (0.3–0.5 mmol) and terephthalic acid (0.3–0.5 mmol) were added to a solution containing N,N-dimethylformamide (6 mL), water (4 mL), and ethanol (2 mL). The mixture was reacted in a 25 mL high-pressure vessel capped with polytetrafluoroethylene at 100–120 °C for 48 hours. After cooling to room temperature, the product was collected by centrifugation, washed with DMF and methanol, and further dried at room temperature.

[0032] The method for preparing the electrochemiluminescence sensor according to a specific embodiment of the present invention includes: A certain amount of PSA-HOF and HE-MHOF were dispersed in a 0.5% chitosan solution, wherein the mass-volume ratio of PSA-HOF or HE-MHOF to the chitosan solution was 1:1 (g / L); PSA-HOF dispersion and HE-MHOF dispersion were obtained respectively.

[0033] The PSA-HOF and HE-MHOF dispersions were dropped onto a clean glassy carbon electrode surface at a certain volume ratio to perform drop coating modification, thereby obtaining an HE-MHOF / PSA-HOF modified electrode.

[0034] The DNA hairpin H2 was drop-coated and incubated on the HE-MHOF / PSA-HOF / GCE modified electrode to obtain the electrochemiluminescence sensor H2 / HE-MHOF / PSA-HOF / GCE.

[0035] The specific detection method of this invention is as follows: Step 1: Prepare a series of ARG standard solutions with different concentrations, ranging from 1.0 × 10⁻⁶. -15 mol / L ~ 1.0 × 10 -9 mol / L; Step 2: Using the modified electrode H2 / HE-MHOF / PSA-HOF / GCE as the working electrode, the platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode, a three-electrode system was formed. This system was placed in a PBS phosphate buffer solution containing potassium persulfate. A series of ARG standard solutions with different concentrations prepared in Step 1 were added dropwise to the GCE. Cyclic voltammetry was performed within an electrochemical window of -2.0 to 0 V, using a photomultiplier tube with a high voltage of 300 V and a scan rate of 0.1 V. The time-luminescence intensity curves were recorded, and a linear relationship was established between the difference in luminescence intensity before and after the addition of ARG and the logarithm of the ARG concentration, yielding the corresponding linear regression equation. Step 3, Sample detection: Perform the test according to Step 2 above and obtain the luminescence intensity. The obtained luminescence intensity is calculated using the linear regression equation obtained in Step 2 to obtain the concentration of ARG in the sample.

[0036] The technical solution of the present invention will be further described below with reference to the embodiments, but the present invention is not limited to the following embodiments. Example 1

[0037] Fabrication of a high-sensitivity electrochemiluminescence sensor for ARG detection: (1) Preparation of PSA-HOF First, under ultrasonic conditions, 150 mg of 1,3,6,8-tetra(p-benzoic acid)pyrene (H4TBAPy) was dissolved in 22.5 mL of N,N-dimethylformamide (DMF). Then, 90 mL of CH3OH was added to the above solution and stirred for 5 minutes. Finally, the mixture was stored at room temperature for 12 hours, centrifuged, and washed three times with CH3OH and acetone, respectively, to remove impurities. The product was placed in a vacuum drying oven and dried at 60 °C for 12 hours to obtain a yellow PSA-HOF powder.

[0038] (2) Preparation of HE-MHOF nanomaterials

[0039] Synthesis of Mmix(OH)₂ (Mmix = Mn, Ni, Co, Cu, Zn): All equimolar amounts of the metal chloride salts (0.5 mmol) were dissolved in 10 mL of water, and 1.0 mmol of NaOH was dissolved in 10 mL of water. The two solutions were then mixed and stirred at room temperature for 1 hour. After centrifugation, the solution was washed, and the pH of the filtrate was checked with pH paper until the pH of the filtrate reached neutral. Finally, the product was dried under vacuum. Freshly prepared Mmix(OH)₂ precursor (0.5 mmol) and terephthalic acid (0.5 mmol) were added to a mixed solvent of N,N-dimethylformamide (6 mL), water (4 mL), and ethanol (2 mL), respectively, and reacted at 120 °C for 48 hours in a 25 mL PTFE-capped autoclave. After cooling to room temperature, the product was collected by centrifugation, washed with DMF and methanol, and further dried at room temperature.

[0040] (3) Preparation of PSA-HOF and HE-MHOF dispersions

[0041] Take 1 mg of the prepared PSA-HOF powder and 1 mg of the prepared HE-MHOF powder and dissolve them in 1 mL of chitosan, mix them evenly, and disperse them by ultrasonication to obtain two dispersions with a concentration of 1 mg / mL.

[0042] (4) Preparation of an electrochemiluminescence sensor for ARG detection

[0043] Clean the glassy carbon electrode using, but not limited to, the following methods: polish the glassy carbon electrode sequentially on chamois leather with Al2O3, sonicate it for 15 min each with anhydrous ethanol and ultrapure water to remove surface contaminants, and then air dry it for later use.

[0044] 4 μL of 1 mg / mL PSA-HOF and 4 μL of 1 mg / mL HE-MHOF were sequentially added dropwise onto a clean GCE surface to modify it, thus obtaining HE-MHOF / PSA-HOF / GCE. The modified electrode was then incubated with 3 μL of 1 μmol / mL H2 for 12 hours.

[0045] The aptamer sequence for the aforementioned sensor is shown below: (Ordered from Sangon Biotech (Shanghai) Co., Ltd.)

[0046] The nucleotide sequences of H2 and H1-Fc are as follows: Hairpin DNA2 (H2): COOH-TTT TGA GGC GGA TAAAGT TGC AGG AAT CCG CCT CAT CCT GCA ACT TT and Hairpin DNA1 (H1-Fc): Fc-TTT TGCAGG ATG AGG CGG ATT CCT GCA ACT TTA TCC GCC TCA.

[0047] (5) Drawing the standard curve

[0048] Prepare a series of ARG standard solutions with different concentrations, where the ARG concentration is 1.0 × 10⁻⁶. -15 mol / L, 1.0×10 -14 mol / L, 1.0×10 -13 mol / L, 1.0×10 -12 mol / L, 1.0×10 -11 mol / L, 1.0×10 -10 mol / L, 1.0×10 -9 mol / L; ARG is a segment derived from ARG AMP The gene fragment has the following nucleotide sequence: TCC TGC AAC TTTATC CGC CTC.

[0049] The modified electrode H2 / HE-MHOF / PSA-HOF / GCE was used as the working electrode, the platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode to form a three-electrode system. This system was placed in a PBS phosphate buffer solution containing potassium persulfate. Within an electrochemical window of -2.0 to 0 V, a photomultiplier tube voltage of 300 V and a scan rate of 0.1 V / s were used to record the time-emission intensity curve (T-ECL). (See [reference needed]). Figure 2 ; The modified electrode was then mixed with 3 μL of ARG at different concentrations. AMP The gene was incubated at 37°C for 30 min, and then the electrode was incubated with H1-Fc at 37°C for 20 min to obtain H1-Fc / ARG / H2 / HE-MHOF / PSA-HOF / GCE corresponding to a series of ARG standard solutions of different concentrations. The three-electrode system was formed by using H1-Fc / ARG / H2 / HE-MHOF / PSA-HOF / GCE as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode. The three-electrode system was placed in PBS phosphate buffer solution containing potassium persulfate. Within the electrochemical window range of -2.0 to 0 V, the photomultiplier tube was operated at a high voltage of 300 V and a scan rate of 0.1 V / s. The time-luminescence intensity curve (T-ECL) was recorded. To establish a linear relationship between the difference in luminescence intensity before and after the addition of ARG and the logarithm of ARG concentration, see [reference needed]. Figure 3 The corresponding linear regression equation is obtained as: Δ I ECL =1067.14282 LogC(mol / L) The value was 20176.07448, with a correlation coefficient (R) of 0.9974. The detection range of the linear regression equation was 1.0 × 10⁻⁶. -15 ~1.0×10 -9 mol / L, with a detection limit of 4.1 × 10⁻⁶. -16 mol / L.

[0050] (6) Sample testing

[0051] The three-electrode system was immersed in filtered and impurity-removed river water for 20 min, and electrochemiluminescence detection was performed according to the above standard solution detection method. The sample was detected in parallel 3 times, and the concentration of ARG in the sample was calculated according to the regression equation corresponding to the above standard curve. The results are listed in Table 1. Example 2

[0052] The effect of different mass ratios of PSA-HOF and HE-MHOF on the ECL luminescence performance of the HE-MHOF / PSA-HOF electrochemiluminescence sensor: (1) Preparation of PSA-HOF nanomaterials: Same as in Example 1; (2) Preparation of HE-MHOF material: Same as in Example 1; (3) Preparation of PSA-HOF and HE-MHOF dispersions: Same as in Example 1; (4) Fabrication of HE-MHOF / PSA-HOF electrochemiluminescence sensor: 4 μL of 1 mg / mL PSA-HOF and different amounts (2, 3, 4, 5, 6 μL) of 1 mg / mL HE-MHOF were added dropwise onto a clean GCE surface to modify it, so as to obtain HE-MHOF / PSA-HOF / GCE.

[0053] (5) Electrochemiluminescence detection: A three-electrode system was formed by using HE-MHOF / PSA-HOF / GCE with different mass ratios as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode. The three-electrode system was placed in a PBS phosphate buffer solution containing potassium persulfate. Within the electrochemical window of -2.0 to 0 V, the photomultiplier tube was operated at a high voltage of 300 V and a scan rate of 0.1 V / s to record the luminescence intensity.

[0054] like Figure 4 As shown, when the mass ratio of PSA-HOF to HE-MHOF decreases from 4:2 to 4:4, the ECL response gradually increases. However, as the mass ratio of PSA-HOF to HE-MHOF decreases from 4:4 to 4:6, the ECL intensity decreases instead. Therefore, a mass ratio of PSA-HOF to HE-MHOF of 4:4 is preferred. Example 3

[0055] Effect of different H2 concentrations on the ECL luminescence performance of the HE-MHOF / PSA-HOF electrochemiluminescence sensor: (1) Preparation of PSA-HOF nanomaterials: Same as in Example 1 (2) Preparation of HE-MHOF material: Same as in Example 1 (3) Preparation of PSA-HOF and HE-MHOF dispersions: Same as in Example 1 (4) Preparation of ARG detection electrochemical sensor: The glassy carbon electrode was polished and then sonicated sequentially with nitric acid, anhydrous ethanol, and deionized water. After air-drying, 6.0 μL of 0.01 M PBS buffer (pH 7.4) containing 0.005 M NHS and 0.01 M EDC was dropped onto a clean, bare glassy carbon electrode for 1 h to activate it. Subsequently, 4.0 μL of PSA-HOF and HE-MHOF dispersions were added layer by layer onto the GCE to obtain the HE-MHOF / PSA-HOF / GCE modified electrode. Then, 3 μL of ARG aptamer tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) solutions at concentrations of 0.5 μmol / L, 1 μmol / L, 1.5 μmol / L, 2 μmol / L, and 2.5 μmol / L were added to the electrode to obtain the H2 / HE-MHOF / PSA-HOF / GCE modified electrode, denoted as H2. 0.5 / HE-MHOF / PSA-HOF / GCE,H21 / HE-MHOF / PSA-HOF / GCE,H2 1.5 / HE-MHOF / PSA-HOF / GCE,H22 / HE-MHOF / PSA-HOF / GCE,H2 2.5 / HE-MHOF / PSA-HOF / GCE.

[0056] In the above sensors, the aptamer sequence is the same as in Example 1.

[0057] (5) Electrochemiluminescence detection: Within the electrochemical window range of -2.0~0 V, the photomultiplier tube is charged at 300 V and the scan rate is 0.1 V / s, and the luminescence intensity is recorded.

[0058] like Figure 5 As shown, the change in ECL response gradually increases with increasing H2 concentration. When the H2 concentration increases from 0.5 μmol / L to above 1 μmol / L, the change in ECL intensity gradually stabilizes. Therefore, an H2 concentration of 1 μmol / L is preferred. Example 4

[0059] The effect of the difference in ARG binding time to the aptamer on the luminescence performance of the HE-MHOF nanomaterial-enhanced PSA-HOF electrochemiluminescence sensor ECL: (1) Preparation of PSA-HOF nanomaterials: Same as in Example 1 (2) Preparation of HE-MHOF material: Same as in Example 1 (3) Preparation of PSA-HOF and HE-MHOF dispersions: Same as in Example 1 (4) Preparation of ARG detection HE-MHOF / PSA-HOF electrochemical sensor: The glassy carbon electrode was polished, then sonicated sequentially with nitric acid, anhydrous ethanol, and deionized water, and air-dried. At room temperature, 6.0 μL of 0.01 M PBS buffer (pH 7.4) containing 0.005 M NHS and 0.01 M EDC was dropped onto a clean, bare glassy carbon electrode for 1 h to activate the electrode. Subsequently, 4.0 μL of PSA-HOF and HE-MHOF solutions were added layer by layer onto the GCE to obtain an HE-MHOF / PSA-HOF / GCE modified electrode. Then, 3 μL of 1 μmol / L Tris-HCl solution was added to each electrode to obtain an H2 / HE-MHOF / PSA-HOF / GCE modified electrode. A concentration of 1.0 × 10⁻⁶ was prepared. -10 A mol / L ARG standard solution, with 1.0 × 10⁻⁶ mol / L -10 A mol / L ARG standard solution was added dropwise to the H2 / HE-MHOF / PSA-HOF / GCE modified electrode at 37°C. Then, H1-Fc was modified onto the surface of the ARG / H2 / HE-MHOF / PSA-HOF / GCE and incubated for 20, 30, 40, 50, and 60 min. The sequences of H2 and H1-Fc were the same as in Example 1.

[0060] (5) Electrochemiluminescence detection: Within the electrochemical window range of -2.0~0 V, the photomultiplier tube is charged at 300 V and the scan rate is 0.1 V / s, and the luminescence intensity is recorded.

[0061] like Figure 6 As shown, as the binding time of H2 and H1-Fc to ARG increased from 20 min to 60 min, the change in ECL response value did not show a significant upward trend, and tended to stabilize after 20 min, indicating that the binding of ARG to the two DNA hairpins had reached a stable state at 20 min. Therefore, we selected 20 min as the optimal binding time. Example 5

[0062] The effect of PBS phosphate buffer solutions with different pH values ​​on the ECL luminescence performance of HE-MHOF nanomaterial-enhanced PSA-HOF electrochemiluminescence sensor:

[0063] (1) Preparation of PSA-HOF nanomaterials: Same as in Example 1

[0064] (2) Preparation of HE-MHOF material: Same as in Example 1

[0065] (3) Preparation of PSA-HOF and HE-MHOF dispersions: Same as in Example 1

[0066] (4) Preparation of PBS phosphate buffer solution containing persulfate: 8.4758 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) and 0.2028 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) were dissolved in 250 mL of ultrapure water containing potassium persulfate. The pH was adjusted to 5, 6, 7, 8 and 9 to obtain PBS phosphate buffer solutions with different pH values ​​containing potassium persulfate. Finally, the solutions were stored for later use. (5) Preparation of ARG detection HE-MHOF / PSA-HOF electrochemical sensor: The glassy carbon electrode was polished, then sonicated sequentially with nitric acid, anhydrous ethanol, and deionized water, and air-dried. At room temperature, 6.0 μL of 0.01 M PBS buffer (pH 7.4) containing 0.005 M NHS and 0.01 M EDC was dropped onto a clean, bare glassy carbon electrode for 1 h to activate the electrode. Subsequently, 4.0 μL of PSA-HOF and HE-MHOF solutions were added layer by layer onto the GCE to obtain an HE-MHOF / PSA-HOF / GCE modified electrode. Then, 3 μL of 1 μmol / L Tris-HCl solution was added to each electrode to obtain an H2 / HE-MHOF / PSA-HOF / GCE modified electrode. A concentration of 1.0 × 10⁻⁶ was prepared.-10 A mol / L ARG standard solution, with 1.0 × 10⁻⁶ mol / L -10 A mol / L ARG standard solution was added dropwise to the H2 / HE-MHOF / PSA-HOF / GCE modified electrode at 37°C. Then, H1-Fc was modified onto the surface of ARG / H2 / HE-MHOF / PSA-HOF / GCE and incubated for 20 min to obtain H1-Fc / ARG / H2 / HE-MHOF / PSA-HOF / GCE. The DNA hairpin H2 and H1-Fc sequences were the same as in Example 1.

[0067] (6) Electrochemiluminescence detection: The modified electrode H1-Fc / ARG / H2 / HE-MHOF / PSA-HOF / GCE was used as the working electrode, the platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode to form a three-electrode system. The three-electrode system was placed in PBS phosphate buffer solution containing potassium persulfate with pH values ​​of 5, 6, 7, 8 and 9, respectively. Within the electrochemical window range of -2.0~0V, the photomultiplier tube was operated at a high voltage of 300V and a scan rate of 0.1V / s, and the luminescence intensity was recorded.

[0068] like Figure 7 As shown, the ECL response gradually increases with increasing pH value, but the ECL intensity decreases as the pH increases from 8 to 9. Therefore, a pH value of 7-8 is preferred. Comparative Example 1

[0069] Comparative Example 1 differs from Example 1 in that it uses an H1-Fc / ARG / H2 / PSA-HOF / GCE modified electrode as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode to form a three-electrode system. This three-electrode system is placed in a PBS phosphate buffer solution containing 0.1 M potassium persulfate, and the detection method is the same as in Example 1. The detection results show that the sensor prepared in Comparative Example 1 exhibits lower light intensity, lower recovery rate, and a higher RSD value for ARG detection when used for ARG detection. The results are listed in Table 1. Comparative Example 2

[0070] Comparative Example 2 differs from Example 1 in that it uses an H1-Fc / ARG / H2 / HE-MHOF / GCE modified electrode as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode to form a three-electrode system. This three-electrode system is placed in a PBS phosphate buffer solution containing 0.1 M potassium persulfate, and the detection method is the same as in Example 1. The detection results show that the sensor prepared in Comparative Example 2 has a low light intensity when used for ARG detection, making it difficult to detect ARG. Comparative Example 3

[0071] Comparative Example 3 differs from Example 1 in that it uses a three-electrode system: an H1-Fc / ARG / H2 / HE-MHOF / PSA-HOF / GCE modified electrode as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode. This three-electrode system is placed in a PBS phosphate buffer solution without potassium persulfate, and the detection method is the same as in Example 1. The detection results show that the light intensity of the detection system is very low, making it difficult to detect ARG.

[0072] Table 1. Results of ARG measurement in river water

[0073] Remark: a This is the average of three measurements.

[0074] As shown in Table 1, the samples were analyzed in parallel three times, with a relative standard deviation of less than 5% and a spiked recovery rate ranging from 96.8% to 105%. These results indicate that ARG cannot be detected using the H1-Fc / ARG / H2 / HE-MHOF / PSA-HOF / GCE modified solution placed in PBS phosphate buffer without potassium persulfate. The H1-Fc / ARG / H2 / HE-MHOF / PSA-HOF / GCE electrochemiluminescence sensor provided in this invention is effective and feasible for detecting ARG, and it is simple and flexible to operate, has a wide detection range, and a low detection limit.

[0075] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An electrochemiluminescence sensor, characterized in that, The modified electrode comprises a glassy carbon electrode surface constructed by sequentially modifying the surface of the electrode with PSA-HOF, HE-MHOF and a nucleic acid aptamer with specific recognition function; wherein M in HE-MHOF is an equimolar amount of Mn, Ni, Co, Cu and Zn, and the mass ratio of PSA-HOF to HE-MHOF is 1:1 to 1.5; wherein the nucleic acid aptamer with specific recognition function can specifically recognize antibiotic resistance genes.

2. The electrochemiluminescence sensor according to claim 1, characterized in that, The nucleic acid aptamer with specific recognition function can specifically recognize the ampicillin resistance gene. The nucleic acid aptamer includes DNA hairpin H2 and DNA hairpin H1-Fe. The nucleotide sequence of DNA hairpin H2 is COOH-TTT TGA GGC GGA TAA AGT TGC AGG AATCCG CCT CAT CCT GCA ACT TT. The nucleotide sequence of DNA hairpin H1-Fe is Fc-TTT TGC AGG ATGAGG CGG ATT CCT GCA ACT TTA TCC GCC TCA.

3. A method for preparing an electrochemiluminescence sensor as described in claim 1 or 2, characterized in that, Includes the following steps: PSA-HOF and HE-MHOF were dispersed in chitosan solution, respectively, to obtain PSA-HOF dispersion and HE-MHOF dispersion; The PSA-HOF dispersion and HE-MHOF dispersion were drop-coated layer by layer onto a clean glassy carbon electrode surface to obtain an HE-MHOF / PSA-HOF / GCE modified electrode. The DNA hairpin H2 was drop-coated and incubated on the HE-MHOF / PSA-HOF / GCE modified electrode to obtain H2 / HE-MHOF / PSA-HOF / GCE.

4. The method for preparing the electrochemiluminescence sensor according to claim 3, characterized in that, The mass-to-volume ratio of the PSA-HOF or HE-MHOF to the chitosan solution is 1 g: 1 L; the mass fraction of the chitosan solution is 0.5%.

5. The method for preparing the electrochemiluminescence sensor according to claim 3, characterized in that, The drop volume of the PSA-HOF dispersion or the HE-MHOF dispersion is 2~6 μL.

6. The method for preparing the electrochemiluminescence sensor according to claim 3, characterized in that, The concentration of H2 in the DNA hairpin is 1~2.5 μmol / L, and the drop volume is 3.0 μL.

7. The application of an electrochemiluminescence sensor as described in claim 1 or 2, or an electrochemiluminescence sensor obtained by the preparation method according to any one of claims 3 to 6, in the detection of antibiotic resistance genes.

8. The application according to claim 7, characterized in that, Includes the following steps: A series of ARG standard solutions of different concentrations were prepared using antibiotic resistance gene fragments as standards. A three-electrode system was used, with the modified electrodes before and after incubation with standard solution as working electrodes, and PBS phosphate buffer solution containing potassium persulfate as electrolyte. Cyclic voltammetry was used to record the time-luminescence intensity curves before and after incubation, and a linear relationship was established between the difference in luminescence intensity before and after the addition of ARG and the logarithm of ARG concentration, and the corresponding linear regression equation was obtained. Using the modified electrodes before and after incubation of the sample solution as working electrodes, cyclic voltammetry scans were performed to record the difference in luminescence intensity before and after incubation. The concentration of antibiotic resistance genes in the sample solution was calculated based on the linear regression equation.

9. The application according to claim 8, characterized in that, The cyclic voltammetric scan is performed within an electrochemical window of -2.0 to 0 V, with a photomultiplier tube voltage of 300 to 600 V and a scan rate of 0.05 to 0.25 V / s.

10. The application according to claim 8, characterized in that, The pH of the PBS buffer solution containing potassium persulfate is 5-9, and the concentration of PBS is 0.1 mol / L.

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