Metal-organic framework mediated steady-state electrochemiluminescence sensor and application thereof in escherichia coli detection

Abstract

The application discloses a metal covalent organic framework material, a mediated steady-state electrochemiluminescence sensor and application of the sensor in the field of Escherichia coli detection, wherein the preparation method of the metal covalent organic framework material is as follows: tris(4,4'-dicarboxylic acid-2,2'-bipyridyl) dichlororuthenium, 1,2,4,5-benzene tetraamine tetrahydrochloride and polyphosphoric acid are added into a polytetrafluoroethylene autoclave, heated at 150 DEG C for 24 hours, and then heated at 180 DEG C for another 24 hours to obtain a mixture; after the pH of the obtained mixture is adjusted to 8.5 by using a saturated NaHCO3 solution, black powder is precipitated, the black powder is collected by centrifugation, and the black powder is washed with water, methanol and acetone in sequence by using a Soxhlet extractor for 12 hours; the washed black powder is vacuum dried at 100 DEG C for 6 hours to obtain a ruthenium metal covalent organic framework material; the ruthenium metal covalent organic framework material is used as a signal source of an electrochemical sensor, and a PEG-CS modified interface is constructed on an electrode to enhance the antifouling performance of the sensor in a complex matrix; a molecular amplification method (polymerase chain reaction PCR and HCR) is adopted to fix a large amount of Ru-MCOFs materials on the surface of the electrode, so that a stable electrochemical sensor is developed and assembled, and the stable electrochemical sensor can be used for detecting common foodborne pathogenic bacteria.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of electrochemical sensors, in particular to a metal covalent organic framework material, a mediated steady-state electrochemiluminescence sensor and application thereof in the field of Escherichia coli detection. BACKGROUND

[0002] Electrochemiluminescence (ECL) is a chemical luminescence reaction triggered by electrochemistry at the electrode interface, representing a perfect combination of electrochemistry and chemiluminescence processes. In recent years, electrochemiluminescence biosensors have attracted widespread attention in the detection of foodborne pathogens due to their high sensitivity, wide linear range, strong controllability and good selectivity. Since nucleic acids are the material basis of biological heredity, electrochemiluminescence nucleic acid sensors targeting specific nucleic acid sequences of foodborne pathogens have comparable sensitivity and selectivity to standard methods. However, most current electrochemiluminescence nucleic acid sensing strategies mainly focus on improving sensitivity, often ignoring the stability of the sensor itself. Therefore, ensuring the stability of electrochemiluminescence sensors is crucial to meet the needs of practical applications.

[0003] Ru(bpy)3 2+ Among many electrochemiluminescence reagents, it has excellent electrochemiluminescence performance, but it has the defects of free diffusion and low luminescence intensity. While metal organic frameworks (MOFs) are widely used due to their large surface area, structural diversity and adjustable porosity, which can immobilize Ru(bpy)3 2+ inside their pores and enhance luminescence intensity. However, due to relatively weak coordination bonds, MOF-based electrochemiluminescence materials usually have poor chemical stability, leading to reagent leakage during testing and compromising the stability of electrochemiluminescence signals. In addition, non-specific adsorption on the electrode surface is a common problem during sensing of complex biological samples, which can adversely affect stability.

[0004] Based on these considerations, how to design a metal covalent organic framework material and its mediated steady-state electrochemiluminescence sensor, and apply it to the detection of O157:H7 Escherichia coli, becomes a technical problem that needs to be solved by the present application. SUMMARY

[0005] The present application aims to provide a metal covalent organic framework material, a mediated steady-state electrochemiluminescence sensor and application thereof in the field of Escherichia coli detection, to solve the problems raised in the background art.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0007] The metal covalent organic framework material is prepared by the following method:

[0008] Tris(4,4'-dicarboxylic acid-2,2'-bipyridyl) dichloride ruthenium ([Ru(dcbpy)3]Cl2), 1,2,4,5-benzene tetraamine tetrahydrochloride (BTA·4HCl) and polyphosphoric acid (PPA) were added into a polytetrafluoroethylene autoclave, heated at 150°C for 24 hours, and then heated at 180°C for another 24 hours to obtain a mixture;

[0009] After adjusting the pH of the obtained mixture to 8.5 using a saturated NaHCO3 solution, a black powder was precipitated, the black powder was collected by centrifugation, and was washed with water, methanol and acetone in sequence using a Soxhlet extractor for 12 hours, and the washed black powder was vacuum dried at 100°C for 6 hours to obtain a ruthenium metal covalent organic framework material (Ru-MCOFs).

[0010] A method for constructing a metal covalent organic framework material-mediated steady-state electrochemiluminescence sensor is as follows:

[0011] Based on the above-mentioned ruthenium metal covalent organic framework material, a streptavidin-modified ruthenium metal covalent organic framework material was prepared;

[0012] A mixed solution containing polyethylene glycol (PEG), chitosan (CS) and streptavidin (SAV) was dropped on the surface of a glassy carbon electrode and incubated for a period of time to form a PEG-CS modified interface on the glassy carbon electrode;

[0013] The non-specific active sites of the glassy carbon electrode were blocked using a BSA blocking solution;

[0014] Then, after being thoroughly rinsed with ultrapure water and dried with nitrogen, the O157:H7 E. coli PCR amplification product was dropped on the glassy carbon electrode, and the O157:H7 E. coli PCR amplification product was used to bind to the streptavidin (SAV) on the glassy carbon electrode;

[0015] The electrode was immersed in a gold nanoparticle (AuNPs) complex for a period of time, and the gold nanoparticle (AuNPs) complex was used to bind to the O157:H7 E. coli PCR amplification product, and the hybridization chain reaction system used to bind to the gold nanoparticle (AuNPs) complex was dropped on the surface of the electrode and incubated for a period of time;

[0016] Finally, the streptavidin-modified ruthenium metal covalent organic framework material (Ru-MCOFs-SAV) was dropped on the surface of the electrode and incubated for a period of time, and the ruthenium metal covalent organic framework material was used to bind to the hybridization product of the hybridization chain reaction system, and finally, the electrode was rinsed with deionized water to obtain an electrochemical sensor for detecting O157:H7 E. coli in a matrix.

[0017] As a further scheme of the present application, the method for preparing the ruthenium metal-covalent organic framework material modified with streptavidin is as follows:

[0018] 80 μL of N-hydroxysuccinimide (NHS) with a concentration of 50 mg / mL, 80 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) with a concentration of 50 mg / mL and 240 μL of MES buffer with a pH of 5.5 are added to activate the carboxyl group of 25 mg of ruthenium metal-covalent organic framework material (Ru-MCO Fs);

[0019] Then, 10 μL of streptavidin (SAV) with a concentration of 1 mg / mL is added, and the mixture is incubated at 37°C for 2 hours;

[0020] After centrifugation at 10000 rpm for 5 min, the supernatant is removed, and the ruthenium metal-covalent organic framework material modified with streptavidin is obtained, and the ruthenium metal-covalent organic framework material modified with streptavidin (Ru-MCOFs-SAV) is resuspended in PBS buffer (500 μL, pH 7.4) containing 1% (w / v) bovine serum albumin (BSA).

[0021] As a further scheme of the present application, the O157:H7 E. coli PCR amplification product is prepared by a PCR amplification reaction system, and the total volume of the PCR amplification reaction system is 25 μL, which includes 2 μL of O157:H7 E. coli DNA template, 12.5 μL of 2×PCR Master Mix, 1 μL of FITC-labeled forward primer (FP) with a concentration of 10 μM, 1 μL of biotin-labeled reverse primer (RP) with a concentration of 10 μM and 8.5 μL of ddH2O;

[0022] The hybridization chain reaction system includes 2 μL of biotin-labeled probe H1 with a concentration of 2 μM, 2 μL of biotin-labeled probe H2 with a concentration of 2 μM and 6 μL of TE buffer;

[0023] The sequence of the FITC-labeled forward primer (FP) is as follows: 5'-FITC-CGGACATCCATGTGATATGG-3';

[0024] The sequence of the biotin-labeled reverse primer (RP) is as follows: 5'-Biotin-TTGCCTATCTACAGCTAATCC-3';

[0025] The sequence of the biotin-labeled probe H1 is as follows:

[0026] The sequence of the biotin-labeled probe H2 is: 5'-AGTCTAGGATTCGGCGTGGGTTAACACGCCGAATCCTAGACTACTTTG-biotin-3'.

[0027] As a further scheme of the present application, the preparation method of the gold nanoparticle (AuNPs) complex is: adjusting the pH of the AuNPs solution to 6.8 with 100 mM K2CO3, mixing 1 mL of the AuNPs solution with 2.5 μL of anti-FITC antibody with a concentration of 2 mg / mL, and incubating at room temperature for 1 hour;

[0028] Then, the solution is centrifuged and concentrated ten times to 100 μL to form a 10×AuNPs solution;

[0029] Mixing 10 μL of thiol primer (SH Primer) with a concentration of 2 μM, 2 μL of TCEP with a concentration of 1 mM, and 2 μL of AB buffer with a concentration of 500 mM, and incubating for activation at room temperature for 1 hour;

[0030] After activation, the aforementioned 100 μL of 10×AuNPs solution is added, and the mixture is shaken for 1 hour to achieve coupling;

[0031] Subsequently, 10 μL of dATP solution with a concentration of 100 μM is added, and the mixture is shaken for 30 minutes to block unreacted sites;

[0032] Then, 20 μL of NaCl solution with a concentration of 0.1 M is added, and the mixture is aged at room temperature for 30 minutes;

[0033] Finally, centrifugation is performed at 7800 rpm for 10 minutes, and the obtained gold nanoparticle (AuNPs) complex is collected, resuspended in 1 mL of PBS buffer (pH 7.4, 10 mM PB, 1% BSA), and stored at 4°C until use;

[0034] The sequence of the thiol primer (SH Primer) is:

[0035] As a further scheme of the present application, the extraction method of the O157:H7 E. coli DNA template is:

[0036] 20 μL of anti-O157:H7 E. coli antibody with a concentration of 1 mg / mL is added to a solution containing 100 μL of protein G purification magnetic beads with a concentration of 50 mg beads / mL, and incubated at room temperature for 30 minutes;

[0037] Then, centrifuge at 1000 rpm for 5 minutes, discard the supernatant containing unbound antibodies;

[0038] The precipitate was resuspended in 100 μL of phosphate buffer (PBS, pH 7.4) containing 1% (w / v) bovine serum albumin (BSA) and 0.01% (v / v) Tween-20, i.e. the antibody functionalized magnetic beads were obtained, and stored in a brown bottle at 4°C;

[0039] 10 μL of antibody functionalized magnetic beads were placed in an EP tube with 1 mL of O157:H7 E. coli solution of unknown or known concentration, and mixed at 37°C for 30 minutes of incubation;

[0040] After magnetic separation, the supernatant was removed; resuspended in 100 μL of TE buffer, and treated with ultrasound for 5 minutes (100 W, 40 kHz) to release the O157:H7 E. coli DNA into the supernatant;

[0041] Finally, the supernatant containing the O157:H7 E. coli DNA template was collected for subsequent PCR amplification.

[0042] As a further aspect of the present application, the preparation method of the AuNPs solution is as follows: in a 150 mL flask, 50 mL of 0.01% HAuCl4 solution was added, after boiling with vigorous stirring, 1.0 mL of 1% trisodium citrate was quickly added, the color of the solution changed from deep red to purple, and finally to wine red, then continue to heat and stir for 10 minutes, after cooling to room temperature, the AuNPs solution was obtained, and stored at 4°C for subsequent use.

[0043] The application of the metal-organic framework material-mediated steady-state electrochemiluminescence sensor in the detection of E. coli, specifically O157:H7 E. coli, includes the following steps:

[0044] S1, culture of O157:H7 E. coli and positive control bacteria;

[0045] S2, preparation of various standard concentrations of O157:H7 E. coli solution;

[0046] S3, various standard concentrations of O157:H7 E. coli solution were treated with protein G purified magnetic beads, and PCR amplification was performed to obtain the corresponding O157:H7 E. coli PCR amplification product;

[0047] S4, the electrochemical sensor of claim 1 was assembled by O157:H7 E. coli PCR amplification product, electrochemiluminescence detection was performed, and a standard curve of electrochemiluminescence signal related to O157:H7 E. coli concentration was constructed;

[0048] S5, extract the contaminated milk sample containing unknown concentration of O157:H7 E. coli, and perform electrochemiluminescence detection by the same treatment method as steps S3 and S4, and the concentration of O157:H7 E. coli in the contaminated milk sample can be obtained by substituting the detected electrochemiluminescence signal into the standard curve.

[0049] Compared with the prior art, the beneficial effects of the present application are:

[0050] 1. The Ru-MCOFs material with excellent stability is prepared, which is used as a signal source of an electrochemical sensor, and a PEG-CS modified interface is constructed on the electrode to enhance the anti-fouling performance of the sensor in a complex matrix, and a stable electrochemical sensor is developed by using a molecular amplification method (polymerase chain reaction PCR and HCR) to fix a large amount of Ru-MCOFs material on the electrode surface, so that the common foodborne pathogenic bacteria (such as O157:H7 E. coli) can be detected.

[0051] 2. The Ru(dcbpy)3 2+ is used as a building unit to synthesize and prepare the Ru-MCOFs material, wherein the Ru-MCOFs material inherits the advantages of MOF and COF, has high porosity, high specific surface area and adjustable structure and function, and also provides excellent chemical stability and uniformly distributed metal active sites, which can significantly improve the stability of electrochemiluminescence performance;

[0052] 3. The PEG and CS are introduced into the electrode interface, and the synergistic effect of PEG and CS shows excellent hydrophilicity, which can effectively resist non-specific protein adsorption and further improve the stability of the sensor in a complex matrix, so that the performance of the sensor is maintained.

[0053] 4. The protein G purification magnetic beads are used to separate and capture the target (O157:H7 E. coli DNA) in a complex substrate, and PCR amplification is performed, so that the specificity and sensitivity of the determination result of the present application are ensured. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 It is a schematic diagram of the Ru-MCOFs material and the PEG-CS modified interface mediated electrochemiluminescence biosensor for detecting O157:H7 E. coli.

[0055] Figure 2 It is a characterization of the Ru-MCOFs material: (A) TEM image of Ru-MCOFs, (B) STEM image of Ru-MCOFs, (C-F) EDX element mapping of Ru-MOFs, (G) elemental analysis of Ru-MCOFs and (H) stability test of Ru-MCOF.

[0056] Figure 3 Characteristics of O157:H7 E. coli recognition process: (A and B) SEM images of G protein purified magnetic beads, (C and D) SEM images of O157:H7 E. coli, and (E and F) SEM images of G protein purified magnetic beads / O157:H7 E. coli antibody / O157:H7 E. coli.

[0057] Figure 4 Characteristics of O157:H7 E. coli recognition process: (A and B) SEM images of G protein purified magnetic beads, (C and D) SEM images of O157:H7 E. coli, and (E and F) SEM images of G protein purified magnetic beads / O157:H7 E. coli antibody / O157:H7 E. coli.

[0058] Figure 5 Performance of the electrochemical sensor: (A) ECL measurements of the electrochemical sensor for O157:H7 E. coli (from a to g) were 0 CFU / mL, 0.5 CFU / mL, 10 CFU / mL, 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, and 5 x 10 4 CFU / mL; (B) plot of ECL intensity versus target O157:H7 E. coli concentration, inset: relationship between ECL intensity and the logarithm of target O157:H7 E. coli concentration, error bars represent standard deviations of three parallel tests.

[0059] Figure 6 Specificity and actual sample detection results: (A) ECL signal enhancement of different bacteria, (B) column chart of O157:H7 E. coli recovery rate in milk samples at different dilution multiples. DETAILED DESCRIPTION

[0060] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0061] 1. Reagents and instruments:

[0062] The bacterial strains used in the experiments were from the Anhui Institute for Product Quality Supervision and Inspection. Tris(4,4'-dicarboxylic acid-2,2'-bipyridyl) ruthenium(II) dichloride, with the chemical formula [Ru(dcbpy)3]Cl2, was purchased from SunaTecch Inc. (Suzhou, China). 1,2,4,5-benzene tetraamine tetrahydrochloride (BTA-4HC1) and polyphosphoric acid (PPA) were purchased from Adamas (Shanghai, China). E. coli O157:H7 antibody was from Thermo Fisher Scientific. Chloroauric acid (HAuCl4) was from J&K Scientific (Shanghai, China). Protein G purified magnetic beads (0.5 pm), streptavidin (SAV), bovine serum albumin (BSA), and HPLC purified probes (Table S1) were purchased from Shanghai Generay Biotech Co., Ltd. (Shanghai, China). Fluorescein isothiocyanate (FITC) antibody was provided by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0063] Table S1: Sequences used and their names and purification grades

[0064]

[0065] Electrochemiluminescence (ECL) and electrochemical measurements were performed using an MPI-EII ECL analyzer (Xi'an Ruimai Electronic Technology Co., Ltd., Xi'an, China) and a CHI 660E electrochemical workstation (Shanghai Chenhua Instrument, Shanghai, China), respectively. All experiments were performed using a conventional three-electrode system with a glassy carbon electrode as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode. Dynamic light scattering (DLS) and zeta potential analysis were performed on a Microtrac Nanotrac Wave II (USA). Morphology and elemental mapping were characterized by scanning electron microscopy (EVO18, ZEISS, Germany), high-resolution transmission electron microscopy (JEM 2100F, JEOL, Japan), and scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray spectroscopy (EDS).

[0066] 2. Preparation of gold nanoparticles and their complexes

[0067] 2.1. Preparation of gold nanoparticle (AuNPs) solution:

[0068] In a 150 mL flask, 50 mL of 0.01% HAuCl4 solution was added, and after boiling with vigorous stirring, 1.0 mL of 1% trisodium citrate solution was quickly added. The color of the solution changed from dark red to purple and finally to wine red. After 10 minutes of continued heating and stirring, the AuNPs solution was obtained after cooling to room temperature and stored at 4°C for subsequent use.

[0069] 2.2, Preparation of gold nanoparticle (AuNPs) complex:

[0070] The pH of the AuNPs solution was adjusted to 6.8 with 100 mM K2CO3, 1 mL of AuNPs solution was mixed with 2.5 μL of anti-FITC antibody with a concentration of 2 mg / mL, and incubated at room temperature for 1 hour;

[0071] Then, the solution was centrifuged and concentrated ten times to 100 μL to form a 10× AuNPs solution;

[0072] 2 μL of 1 mM TCEP and 2 μL of 500 mM AB buffer were mixed and incubated at room temperature for 1 hour to activate;

[0073] After activation, the aforementioned 100 μL of 10× AuNPs solution was added, and the mixture was shaken for 1 hour to achieve coupling;

[0074] Subsequently, 10 μL of 100 μM dATP solution was added, and the mixture was shaken for 30 minutes to block unreacted sites;

[0075] Then, 20 μL of 0.1 M NaCl solution was added, and the mixture was aged at room temperature for 30 minutes;

[0076] Finally, centrifugation was performed at 7800 rpm for 10 minutes, and the resulting gold nanoparticle (AuNPs) complex was collected, resuspended in 1 mL of PBS buffer (pH 7.4, 10 mM PB, 1% BSA), and stored at 4°C until use.

[0077] 3, Pretreatment of glassy carbon electrode

[0078] First, a glassy carbon electrode (GCE, 3 mm in diameter) was polished with 0.05 pm alumina on a leather pad for 5 minutes to obtain a mirror surface; then, the electrode was thoroughly rinsed with ultrapure water and sonicated in ethanol, nitric acid (HNO3 / H2O, 1 : 1 by volume) and ultrapure water for 5 minutes to remove residual impurities; after that, the electrode was electrochemically cleaned in a 0.5 M H2SO4solution by potential scanning between -1 V and 1 V at a scan rate of 100 mV / s until a clear voltammetric peak was observed; finally, the electrode was rinsed with ultrapure water and dried with nitrogen (N2) gas.

[0079] 4. Bacterial culture

[0080] E. coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, Enterobacter sakazakii, Pseudomonas aeruginosa and Streptococcus enterica were placed in tryptic soy broth (TSB) medium and incubated at 37 °C in a shaking incubator for 24 hours. Their concentration was determined by the traditional plate count method. To prepare the E. coli O157:H7 at the specified concentration, 10 10 CFU / mL of the E. coli O157:H7 solution was centrifuged at 6500 rpm for 5 minutes to remove the culture medium; then, the obtained E. coli O157:H7 was serially diluted with 1 x PBS to obtain the desired concentration for testing.

[0081] 5. Extraction of the E. coli O157:H7 DNA template

[0082] 20 pL of anti-E. coli O157:H7 antibody at a concentration of 1 mg / mL was added to a solution containing 100 pL of protein G purification magnetic beads at a concentration of 50 mg beads / mL and incubated at room temperature for 30 minutes;

[0083] Then, it was centrifuged at 1000 rpm for 5 minutes and the supernatant containing the unbound antibody was discarded;

[0084] The pellet was resuspended in 100 pL of phosphate buffer (PBS, pH 7.4) containing 1% (w / v) bovine serum albumin (BSA) and 0.01% (v / v) Tween-20, obtaining antibody-functionalized magnetic beads, and stored in a brown bottle at 4 °C;

[0085] 10 pL of antibody-functionalized magnetic beads were placed in an EP tube with 1 mL of E. coli O157:H7 solution at an unknown or known concentration and mixed at 37 °C for 30 minutes of incubation;

[0086] After magnetic separation, the supernatant was removed; resuspended in 100 μL of TE buffer and treated with ultrasound for 5 minutes (100 W, 40 kHz) to release the E. coli O157:H7 DNA into the supernatant;

[0087] Finally, the supernatant containing the E. coli O157:H7 DNA template was collected for subsequent PCR amplification.

[0088] 6. PCR amplification procedure

[0089] The total volume of the PCR amplification reaction system was 25 μL, which included 2 μL of the E. coli O157:H7 DNA template, 12.5 μL of 2x PCR Master Mix, 1 μL of the FITC-labeled forward primer (FP) with a concentration of 10 μM, 1 μL of the biotin-labeled reverse primer (RP) with a concentration of 10 μM, and 8.5 μL of ddH2O; the amplification procedure was set as follows: 95°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 30 seconds, 72°C re-extension for 5 minutes, 35 cycles; after the amplification was completed, the E. coli O157:H7 PCR amplification product was stored at 12°C for standby.

[0090] 7. Preparation of Ru-MCOFs and Ru-MCOFs-SAV

[0091] 7.1. Preparation of ruthenium metal- covalent organic framework (Ru-MCOFs):

[0092] 27 mg of tris(4,4'-dicarboxylic acid-2,2'-bipyridyl) ruthenium dichloride ([Ru(dcbpy)3]Cl2) with a concentration of 30 μM, 26 mg of 1,2,4,5-benzene tetraamine tetrahydrochloride (BTA·4HCl) with a concentration of 90 μM, and 3 mL of polyphosphoric acid (PPA) were added to a polytetrafluoroethylene autoclave, heated at 150°C for 24 hours, and then heated at 180°C for another 24 hours to obtain a mixture;

[0093] After the pH of the obtained mixture was adjusted to 8.5 using a saturated NaHCO3 solution, a black powder was precipitated, the black powder was collected by centrifugation, and the black powder was washed with water, methanol, and acetone for 12 hours in sequence using a Soxhlet extractor, and the washed black powder was vacuum dried at 100°C for 6 hours to obtain the ruthenium metal-covalent organic framework (Ru-MCOFs).

[0094] 7.2. Preparation of streptavidin-modified ruthenium metal-covalent organic framework (Ru-MCOFs-SAV):

[0095] The carboxyl groups of 25 mg of ruthenium metal-covalent organic framework material (Ru-MCO Fs) were activated with 80 pL of N-hydroxysuccinimide (NHS) at a concentration of 50 mg / mL, 80 pL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) at a concentration of 50 mg / mL, and 240 pL of MES buffer at a pH of 5.5;

[0096] Then, 10 pL of streptavidin (SAV) at a concentration of 1 mg / mL was added, and incubated at 37 °C for 2 hours;

[0097] After centrifugation at 10,000 rpm for 5 min, the supernatant was removed, and the ruthenium metal-covalent organic framework material modified with streptavidin was obtained. The ruthenium metal-covalent organic framework material modified with streptavidin (Ru-MCO Fs-SAV) was resuspended in PBS buffer (500 pL, pH 7.4) containing 1% (w / v) bovine serum albumin (BSA).

[0098] 8. Preparation of an ECL electrochemical sensor

[0099] A 10 pL solution containing 4 pL of polyethylene glycol (PEG) at a concentration of 0.5% (w / w), 4 pL of chitosan (CS) at a concentration of 0.5% (w / w) in 1% (v / v) acetic acid, and 2 pL of streptavidin (SAV) at a concentration of 10 pg / mL was dropped onto the electrode surface and incubated at 37 °C for 1 hour to form a PEG-CS modified interface on the glassy carbon electrode.

[0100] Next, 5 pL of a 1% BSA blocking solution was added to the electrode surface and incubated for 1 hour to block unbound sites.

[0101] After being thoroughly rinsed with ultrapure water and dried with nitrogen, the O157:H7 E. coli PCR amplification product was dropped onto the glassy carbon electrode, which was used to bind to the streptavidin (SAV) on the glassy carbon electrode.

[0102] The electrode was immersed in 10 pL of AuNPs complex for 30 min, and then a hybridization chain reaction (HCR) system containing 2 pL of biotin-labeled probe H1 at a concentration of 2 pM, 2 pL of biotin-labeled probe H2 at a concentration of 2 pM, and 6 pL of TE buffer was dropped onto the electrode surface and incubated at 37 °C for 1 hour.

[0103] Subsequently, 5 μL of the pre-prepared Ru-MCOFs-SAV complex was dropped onto the electrode surface and incubated for 30 min, and was bound to the electrode through the SAV-biotin system; finally, the prepared ECL electrode was measured in a buffer containing 0.1 M PBS (pH 7.4) and 10 mM K2S2O8 using a photomultiplier tube at 800 V, scanning the potential from -1.6 to 0 V.

[0104] 9. Characterization of Ru-MCOFs, PEG-CS modified interface and AuNPs complex

[0105] Ru-MCOFs were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS), and the corresponding image results are shown in FIGS. 1A-1C, respectively. The TEM results Figure 2 Figure 2 ) and EDS mapping confirmed the successful synthesis of Ru-MCOFs and proved the presence of various elements, including carbon (C), nitrogen (N), oxygen (O) and ruthenium (Ru). Notably, the detection of ruthenium (Ru) confirmed the presence of Ru(bpy)3 2+ in the Ru-MCOFs complex. Importantly, Ru-MCOFs exhibited excellent chemical and ECL stability during repeated tests, which was attributed to the strong covalent bond between the Ru(bpy)3 2+ units and the benzobisimidazole units.

[0106] 10. Characterization of electrochemical behavior

[0107] To carefully characterize the modification process, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measurements were used to verify the step-by-step assembly of the electrochemiluminescence biosensor on the electrode surface. As shown in FIGS. 2A-2B, the EIS results Figure 4 ​EIS plots reflect the modifications made to the electrode at each immobilization stage. Initially, the EIS of the bare electrode exhibits a nearly straight line (curve a), which indicates excellent conductivity due to the absence of any significant electronic transfer barrier. Upon decorating the electrode with PEG / CS / SAV by drop casting, the charge transfer resistance (Ret) increases significantly (curve b), which is attributed to the attenuation of the electron transfer induced by the SAV layer. Upon the introduction of O157:H7 E. coli, the double-stranded amplification product is immobilized on the electrode surface, and the electrode surface is further blocked using BSA, which results in a continuous increase in Ret (curve c). Subsequently, upon the addition of the AuNPs complex and the induction of HCR assembly, a significant decrease in Ret is observed (curve d), thus enhancing the electron transfer process. Finally, the successive addition of Ru-MCOFs (curve e) results in a continuous increase in Ret, which can be attributed to their significant blocking effect on the electron transfer at the electrode interface. The observed changes in EIS at each modification step confirm the occurrence of specific chemical and physical changes on the electrode surface, verifying the successful fabrication of a stable electrochemiluminescent biosensor. The trends in CV measurements Figure 4 B) are closely related to the EIS data, further verifying that the electrochemical biosensor was effectively constructed through the stepwise modification process.

[0108] 11. Feasibility verification of O157:H7 E. coli

[0109] The feasibility of the electrochemiluminescent biosensor was further evaluated by analyzing its ECL performance. As shown in Figure 4 C, the bare electrode exhibits almost no ECL signal response (curve a). In the absence of E. coli O157:H7, only a very weak ECL background response is observed, mainly due to the nonspecific adsorption of a small amount of Ru-MCOFs on the electrode surface (curves b, c, and d). Notably, the ECL intensity of curve d is lower than that of curves b and c, which can be attributed to the excellent antifouling ability of the PEG / CS-modified interface. In sharp contrast, upon the introduction of the target E. coli O157:H7, a significant enhancement in the ECL signal intensity (curve e) is observed. This significant change is attributed to the large accumulation of Ru-MCOFs on the electrode surface through the classic SAV-biotin interaction. These results collectively verify the feasibility of the sensor from the perspective of electrochemiluminescent signal response.

[0110] 12. Investigation of the detection performance of O157:H7 E. coli

[0111] After the above investigations and characterizations, the analytical performance of the electrochemiluminescent biosensor was evaluated by analyzing the dynamic range and linear relationship between the ECL intensity and different concentrations of O157:H7 E. coli under the optimal conditions Figure 5). ECL intensity measurements of different concentrations of O157:H7 E. coli are shown in Figure 5 As expected, increasing the concentration of O157:H7 E. coli from 5 CFU / mL to 5 x 104CFU / mL resulted in a gradual increase in ECL intensity, demonstrating the effectiveness of the design approach of the present application. Based on 3o / slope (o = standard deviation of blank samples), the limit of detection was calculated to be 2.8 CFU / mL. In addition, the stability of the sensor was verified by repeating the ECL signal values of O157:H7 E. coli at the same concentration, which showed excellent relative standard deviation (RSD) values, as shown in Figure 5 These results confirm the advantages of the stability design and sensitivity enhancement strategies proposed in the present application study. The method of the present application has superior pathogen detection analysis capabilities, particularly in terms of sensitivity and linear range.

[0112] 13. Specificity and real sample detection

[0113] Specificity and real sample evaluation are essential for accurate bacterial identification. As shown in Figure 6 A, by defining the ECL signal enhancement of the target as 100%, the relative signals of other bacteria (Listeria monocytogenes, Staphylococcus aureus, Enterobacter sakazakii, Pseudomonas aeruginosa, and Streptococcus enterica) and blanks are almost negligible. This substantial difference can be attributed to the magnetic bead enrichment and PCR amplification techniques specifically for the target bacteria, demonstrating the high specificity of the method for bacterial detection and identification. To further verify the practicality of the method, different concentrations of O157:H7 E. coli were added to fresh milk at different dilution factors to prepare simulated real samples (it is important to note that all milk samples were pre-verified by traditional culture methods to ensure the absence of target bacteria). Figure 6 The recovery results shown in

[0114] Table S2: Recovery and RSD in real samples

[0115]

[0116] The working principle of the present application is that when there is target E. coli O157:H7, the target bacteria is first separated and enriched by protein G purified magnetic beads, then a specific sequence of E. coli O157:H7 is amplified by PCR to produce a large amount of biotin and FITC modified double-stranded product, one end of the amplification product is fixed on the PEG-CS and SAV modified electrode interface through SAV-biotin, and the other end is combined with the gold nanoparticle complex, then hybridization chain reaction (HCR) is triggered, a large amount of Ru-MCOFs is combined with the hybridization product, thereby accumulating on the electrode surface to produce a significant ECL signal, and as the concentration of E. coli O157:H7 increases, the ECL signal is also stronger, thereby realizing quantitative detection.

[0117] In summary, based on the Ru-MCOFs material and the PEG-CS modified interface, the present application prepares a simple and stable electrochemiluminescence biosensor for sensitive detection of E. coli O157:H7 in complex samples. The experimental scheme proposed in the present application includes three key components: (1) Ru(dcbpy)3 2+ As a building block, Ru-MCOFs with excellent electrochemiluminescence performance are synthesized, which can effectively improve the stability of the signal source; (2) the synergistic effect of PEG and CS improves the antifouling performance of the electrochemiluminescence sensing interface, and further improves the stability of the sensor; (3) the magnetic bead capture step and PCR amplification in complex substrates ensure the specificity and sensitivity of the method. These characteristics demonstrate that the electrochemical analysis platform can meet the needs of practical applications. Overall, this innovative stability enhancement strategy represents a promising approach to improving the detection performance of electrochemiluminescence biosensors, and has potential applications in diagnostics, environmental monitoring and biomedical research.

[0118] It is obvious to those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be regarded as exemplary and non-limiting, and the scope of the present application is defined by the appended claims rather than the above description, and therefore all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present application. Any reference signs in the claims should not be regarded as limiting the claims involved.

Claims

1. A metal-covalent organic framework material mediated steady-state electrochemiluminescence sensor characterized in that, The construction method is as follows: A ruthenium metal covalent organic framework material modified with streptavidin is prepared based on the ruthenium metal covalent organic framework material; A mixed solution containing polyethylene glycol, chitosan acetic acid solution and streptavidin is dropped on the surface of the glassy carbon electrode and incubated for a period of time to form a PEG-CS modified interface on the glassy carbon electrode; The non-specific active sites of the glassy carbon electrode are blocked by BSA blocking solution; Then, the glassy carbon electrode is thoroughly washed with ultrapure water and dried with nitrogen, and then the O157:H7 E. coli PCR amplification product is dropped on the glassy carbon electrode, which is used to bind to the streptavidin on the glassy carbon electrode; The electrode is immersed in a gold nanoparticle complex for a period of time, and the gold nanoparticle complex is used to bind to the O157:H7 E. coli PCR amplification product, and then the hybridization chain reaction system used to bind to the gold nanoparticle complex is dropped on the surface of the electrode and incubated for a period of time; The ruthenium metal covalent organic framework material modified with streptavidin is dropped on the surface of the electrode and incubated for a period of time, and the ruthenium metal covalent organic framework material is used to bind to the hybridization product of the hybridization chain reaction system, and finally, the electrode is washed with deionized water to obtain an electrochemical sensor for detecting O157:H7 E. coli in a matrix; The preparation method of the ruthenium metal covalent organic framework material is as follows: Tris(4,4'-dicarboxylic acid-2,2'-bipyridyl) ruthenium dichloride, 1,2,4,5-benzene tetramine tetrahydrochloride and polyphosphoric acid are added to a polytetrafluoroethylene autoclave, heated at 150 DEG C for 24 hours, and then heated at 180 DEG C for another 24 hours to obtain a mixture; After adjusting the pH of the obtained mixture to 8.5 using a saturated NaHCO3 solution, a black powder is precipitated, the black powder is collected by centrifugation, and the black powder is washed with water, methanol and acetone for 12 hours using a Soxhlet extractor, and the washed black powder is vacuum dried at 100 DEG C for 6 hours to obtain the metal covalent organic framework material.

2. The metal-coordinated covalent organic framework material-mediated steady-state electrochemiluminescence sensor of claim 1, wherein, The preparation method of the ruthenium metal covalent organic framework material modified with streptavidin is as follows: The carboxyl groups of 25 mg of the ruthenium metal covalent organic framework material are activated using 80 μL of N-hydroxysuccinimide with a concentration of 50 mg / mL, 80 μL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide with a concentration of 50 mg / mL and 240 μL of MES buffer with a pH of 5.5; Then, 10 μL of streptavidin with a concentration of 1 mg / mL is added, and incubated at 37 DEG C for 2 hours; After centrifugation at 10000 rpm for 5 min and removal of the supernatant, the ruthenium metal covalent organic framework material modified with streptavidin is obtained, and the ruthenium metal covalent organic framework material modified with streptavidin is resuspended in a PBS buffer containing 1% (w / v) bovine serum albumin, the volume of the PBS buffer is 500 μL, and the pH is 7.

4.

3. The metal-coordinated covalent organic framework material-mediated steady-state electrochemiluminescence sensor of claim 1, wherein, The O157:H7 E. coli PCR amplification product is prepared by PCR amplification reaction system, and the total volume of the PCR amplification reaction system is 25 μL, which includes 2 μL of O157:H7 E. coli DNA template, 12.5 μL of 2×PCR premix, 1 μL of FITC-labeled forward primer with a concentration of 10 μM, 1 μL of biotin-labeled reverse primer with a concentration of 10 μM, and 8.5 μL of ddH2O; The hybridization chain reaction system includes 2 μL of biotin-labeled probe H1 with a concentration of 2 μM, 2 μL of biotin-labeled probe H2 with a concentration of 2 μM, and 6 μL of TE buffer; The sequence of the FITC-labeled forward primer is 5'-FITC-CGGACATCCATGTG ATATGG-3'; The sequence of the biotin-labeled reverse primer is 5'-Biotin-TTGCCTATCTACA GCTAATCC-3'; The sequence of the biotin-labeled probe H1 is: The sequence of the biotin-labeled probe H2 is: 5'-AGTCTAGGATTCGGCGTGGGTTAACACGCCGAATCCTA GACTACTTTG-biotin-3'.

4. The MOF-mediated steady-state electrochemiluminescence sensor of claim 3, wherein, The preparation method of the gold nanoparticle complex is that 1 mL of AuNPs solution is mixed with 2.5 μL of anti-FITC antibody with a concentration of 2 mg / mL, and incubated at room temperature for 1 hour; Then, the solution is centrifuged and concentrated ten times to 100 μL to form a 10×AuNPs solution; 2 μL of TCEP with a concentration of 1 mM and 2 μL of AB buffer with a concentration of 500 mM are mixed, and activated at room temperature for 1 hour; After activation, the aforementioned 100 μL of 10×AuNPs solution is added, and the mixture is shaken for 1 hour to realize coupling; Subsequently, 10 μL of dATP solution with a concentration of 100 μM is added, and the mixture is shaken for 30 minutes to block unreacted sites; Then, 20 μL of NaCl solution with a concentration of 0.1 M is added, and the mixture is aged at room temperature for 30 minutes; Finally, centrifugation is performed at 7800 rpm for 10 minutes, and the obtained gold nanoparticle complex is collected, resuspended in 1 mL of PBS buffer, and stored at 4°C until use; The sequence of the thiol primer is:

5. The metal-coordinated covalent organic framework material-mediated steady-state electrochemiluminescence sensor of claim 3, wherein, The extraction method of the O157:H7 E. coli DNA template is: 20 μL of anti-O157:H7 E. coli antibody with a concentration of 1 mg / mL is added to a solution containing 100 μL of protein G purification magnetic beads with a concentration of 50 mg beads / mL, and incubated at room temperature for 30 minutes; Then, centrifugation is performed at 1000 rpm for 5 minutes, and the supernatant containing unbound antibody is discarded; The precipitate is resuspended in 100 μL of phosphate buffer containing 1% (w / v) bovine serum albumin and 0.01% (v / v) Tween-20 to obtain antibody functionalized magnetic beads, and stored in a brown bottle at 4°C; Put 10 μL antibody functionalized magnetic beads into an EP tube with 1 mL unknown or known concentration of O157:H7 E. coli solution, and mix at 37℃ for 30 minutes; After magnetic separation, remove the supernatant; resuspend in 100 μL TE buffer, and treat with ultrasonic wave for 5 minutes to release O157:H7 E. coli DNA into the supernatant; Finally, collect the supernatant containing O157:H7 E. coli DNA template for subsequent PCR amplification.

6. The metal-coordinated covalent organic framework material-mediated steady-state electrochemiluminescence sensor of claim 4, wherein, The preparation method of the AuNPs solution is as follows: In a 150 mL flask, add 50 mL of 0.01% HAuCl4 solution, and under vigorous stirring, boil, then quickly add 1.0 mL of 1% trisodium citrate, and the color of the solution changes from deep red to purple and finally to wine red; continue to heat and stir for 10 minutes, and then cool to room temperature to obtain the AuNPs solution, which is stored at 4℃ for subsequent use.

7. Application of metal-covalent organic framework material mediated steady-state electrochemiluminescence sensor in the field of Escherichia coli detection, characterized in that, The E. coli is specifically O157:H7 E. coli, and the detection comprises the following steps: S1, culture of O157:H7 E. coli and positive control bacteria; S2, preparation of various standard concentrations of O157:H7 E. coli solution; S3, various standard concentrations of O157:H7 E. coli solution are treated with protein G purified magnetic beads, and PCR amplification is performed to obtain the corresponding O157:H7 E. coli PCR amplification product; S4, assemble the electrochemiluminescence sensor of any one of claims 1-6 by using the O157:H7 E. coli PCR amplification product, perform electrochemiluminescence detection, and construct a standard curve of electrochemiluminescence signal related to O157:H7 E. coli concentration; S5, extract the contaminated milk sample containing O157:H7 E. coli with unknown concentration, and perform electrochemiluminescence detection by the same treatment method as steps S3 and S4, and substitute the detected electrochemiluminescence signal into the standard curve to obtain the concentration of O157:H7 E. coli in the contaminated milk sample.

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