Metal covalent organic framework material, mediated steady-state electrochemical luminescence sensor and application of metal covalent organic framework material and mediated steady-state electrochemical luminescence sensor in field of escherichia coli detection

Abstract

The invention discloses a metal covalent organic framework material, a mediated steady-state electrochemical luminescence sensor and application of the mediated steady-state electrochemical luminescence sensor in the field of escherichia coli detection.The preparation method of the metal covalent organic framework material comprises the steps that tris (4, 4 '-dicarboxylic acid-2, 2'-bipyridyl) ruthenium dichloride, 1, 2, 4, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, 5-trimethyl-1, 3, the preparation method comprises the following steps: adding 1, 3, 5-phenylenetetramine tetrahydrochloride and polyphosphoric acid into a polytetrafluoroethylene autoclave, heating at 150 DEG C for 24 hours, and then heating at 180 DEG C for 24 hours to obtain a mixture; and adjusting the pH value of the obtained mixture to 8.5 by using a saturated NaHCO3 solution, precipitating black powder, collecting the black powder by centrifugation, washing for 12 hours by using a Soxhlet extractor in sequence by using water, methanol and acetone, and carrying out vacuum drying on the washed black powder at 100 DEG C for 6 hours to obtain the ruthenium metal covalent organic framework material. According to the invention, the Ru-MCOFs material is used as a signal source of an electrochemical sensor, a PEG-CS modified interface is constructed on an electrode so as to enhance the antifouling property of the sensor in a complex matrix, and a large amount of Ru-MCOFs material is fixed on the surface of the electrode by adopting a molecular amplification method (polymerase chain reaction PCR and HCR), so that the stable electrochemical sensor is developed and assembled; the method can be used for detecting common food-borne pathogenic bacteria.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical sensors, in particular to metal covalent organic framework materials, mediated steady-state electrochemical luminescence sensors and applications thereof in the field of Escherichia coli detection. Background Art

[0002] Electrochemiluminescence (ECL) is a chemiluminescent reaction triggered by electrochemistry at the electrode interface, representing a perfect combination of electrochemical and chemiluminescent processes. In recent years, electrochemiluminescent 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 inheritance, electrochemiluminescent nucleic acid sensors targeting specific nucleic acid sequences of foodborne pathogens have comparable sensitivity and selectivity to standard methods. However, most current electrochemiluminescent nucleic acid sensing strategies focus on improving sensitivity, often ignoring the stability of the sensor itself. Therefore, ensuring the stability of electrochemiluminescent sensors is crucial to meet the needs of practical applications.

[0003] Ru(bpy)3 2+ Among many electrochemiluminescent reagents, it has excellent electrochemiluminescence performance, but it has the defects of free diffusion and low luminescence intensity. Metal organic frameworks (MOFs) are widely used due to their large surface area, structural diversity and adjustable porosity, which can convert Ru(bpy)3 2+ However, due to relatively weak coordination bonds, MOF-based electrochemiluminescent materials generally have poor chemical stability, resulting in reagent leakage during the test and compromising the stability of the electrochemiluminescent signal. In addition, nonspecific adsorption on the electrode surface is a common problem during the sensing of complex biological samples, which may adversely affect stability.

[0004] Based on these considerations, how to design metal covalent organic framework materials and steady-state electrochemiluminescence sensors mediated by them and apply them to the detection of O157:H7 Escherichia coli becomes a technical problem that needs to be solved in this application. Summary of the invention

[0005] The purpose of the present invention is to provide a metal covalent organic framework material, a mediated steady-state electrochemiluminescence sensor and its application in the field of Escherichia coli detection, so as to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] Metal covalent organic framework material, and its preparation method is:

[0008] Tris(4,4'-dicarboxylic acid-2,2'-bipyridyl)dichlororuthenium ([Ru(dcbpy)3]Cl2), 1,2,4,5-phenyltetraamine 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 the pH of the resulting mixture was adjusted to 8.5 using a saturated NaHCO3 solution, a black powder was precipitated. The black powder was collected by centrifugation and washed with water, methanol and acetone in sequence for 12 hours using a Soxhlet extractor. 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 metal covalent organic framework-mediated steady-state electrochemiluminescence sensor, the construction method of which is as follows:

[0011] Based on the above ruthenium metal covalent organic framework material, preparing a ruthenium metal covalent organic framework material modified with streptavidin;

[0012] A mixed solution containing polyethylene glycol (PEG), chitosan (CS) acetic acid solution and streptavidin (SAV) was dropped onto 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;

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

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

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

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

[0017] As a further embodiment of the present invention, the preparation method of the ruthenium metal covalent organic framework material modified with streptavidin is as follows:

[0018] The carboxyl groups of 25 mg of ruthenium metal covalent organic framework (Ru-MCO Fs) were activated with 80 μL of 50 mg / mL N-hydroxysuccinimide (NHS), 80 μL of 50 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and 240 μL of MES buffer at pH 5.5.

[0019] Then, 10 μL of 1 mg / mL streptavidin (SAV) was added and incubated at 37°C for 2 h;

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

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

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

[0023] The sequence of the FITC-labeled forward primer (FP) was: 5′-FITC-CGGACATCCATGTGATATGG-3′;

[0024] The sequence of the biotin-labeled reverse primer (RP) was: 5′-Biotin-TTGCCTATCTACAGCTAATCC-3′;

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

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

[0027] As a further embodiment of the present invention, the gold nanoparticle (AuNPs) complex is prepared by 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 an anti-FITC antibody having a concentration of 2 mg / mL, and incubating at room temperature for 1 hour;

[0028] Then, the solution was centrifuged and concentrated tenfold to 100 μL to form a 10× AuNPs solution;

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

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

[0031] Subsequently, 10 μL of 100 μM dATP solution was added, and the mixture was shaken for 30 min to block unreacted sites;

[0032] Then, 20 μL of 0.1 M NaCl solution was added, and the mixture was aged at room temperature for 30 min;

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

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

[0035] As a further solution of the present invention, the method for extracting the O157:H7 Escherichia coli DNA template is:

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

[0037] Then, the mixture was centrifuged at 1000 rpm for 5 min, and the supernatant containing unbound antibody was discarded;

[0038] The precipitate was resuspended in 100 μL phosphate buffered saline (PBS, pH 7.4) containing 1% (w / v) bovine serum albumin (BSA) and 0.01% (v / v) Tween-20 to obtain antibody-functionalized magnetic beads, and stored in a brown bottle at 4°C;

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

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

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

[0042] As a further scheme of the present invention, the preparation method of the AuNPs solution is as follows: 50mL of 0.01% HAuCl4 solution is added to a 150mL flask, and after boiling under vigorous stirring, 1.0mL of 1% trisodium citrate is quickly added, and the color of the solution changes from dark red to purple and finally to wine red. Then, heating and stirring are continued for 10 minutes. After cooling to room temperature, the obtained Au NPs solution is stored at 4°C for subsequent use.

[0043] The application of the metal covalent organic framework material-mediated steady-state electrochemiluminescence sensor in the field of Escherichia coli detection, wherein the Escherichia coli is specifically O157:H7 Escherichia coli, and the detection steps are as follows:

[0044] Culture of S1, O157:H7 Escherichia coli and positive control bacteria;

[0045] S2, preparing various standard concentrations of O157:H7 Escherichia coli solutions;

[0046] S3, various standard concentrations of O157:H7 Escherichia coli solutions were treated with protein G purification magnetic beads and PCR amplified to obtain corresponding O157:H7 Escherichia coli PCR amplification products;

[0047] S4, assembling the electrochemical sensor of the claim through the PCR amplification product of O157:H7 Escherichia coli, performing electrochemiluminescence detection, and constructing a standard curve related to the electrochemiluminescence signal and the concentration of O157:H7 Escherichia coli;

[0048] S5. Extract a contaminated milk sample containing an unknown concentration of O157:H7 Escherichia coli, perform electrochemiluminescence detection in the same manner as steps S3 and S4, substitute the detected electrochemiluminescence signal into the standard curve, and the concentration of O157:H7 Escherichia coli in the contaminated milk sample can be obtained.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] 1. The present invention prepares Ru-MCOFs materials with excellent stability, uses them as the signal source of electrochemical sensors, and constructs a PEG-CS modified interface on the electrode to enhance the antifouling performance of the sensor in complex matrices. The Ru-MCOFs materials are immobilized on the electrode surface in large quantities by molecular amplification methods (polymerase chain reaction PCR and HCR), thereby developing and assembling a stable electrochemical sensor that can be used to detect common foodborne pathogens (such as O157:H7 Escherichia coli);

[0051] 2. The present invention uses Ru(dcbpy)3 2+ As a building block, Ru-MCOFs materials were synthesized and prepared. Ru-MCOFs materials inherit the advantages of MOF and COF, with high porosity, high specific surface area and adjustable structural functions. At the same time, they also provide excellent chemical stability and uniformly distributed metal active sites, which can significantly improve the stability of electrochemiluminescence performance.

[0052] 3. The present invention introduces PEG and CS at the electrode interface. The synergistic effect of PEG and CS exhibits excellent hydrophilicity, which can effectively resist nonspecific protein adsorption and further improve the stability of the sensor in a complex matrix, thereby maintaining the performance of the sensor.

[0053] 4. The present invention uses protein G purification magnetic beads to separate and capture the target (O157:H7 Escherichia coli DNA) in a complex substrate and performs PCR amplification, thereby ensuring the specificity and sensitivity of the measurement results of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is a schematic diagram of the present invention for detecting O157:H7 Escherichia coli using an electrochemiluminescent biosensor mediated by Ru-MCOFs material and PEG-CS modified interface.

[0055] Figure 2 Characterization of Ru-MCOFs materials: (A) TEM image of Ru-MCOFs, (B) STEM image of Ru-MCOFs, (CF) EDX elemental mapping of Ru-MOFs, (G) elemental analysis of Ru-MCOFs and (H) stability test of Ru-MCOF.

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

[0057] Figure 4 Feasibility study: EIS (A) and CV (B) characterizations of the electrochemical sensor at different modification stages, (C) ECL performance of the electrochemical sensor under different experimental conditions [a: bare electrode, b: blank group CS modified interface, c: blank group PEG modified interface, d: blank group PEG-CS modified interface, e: working group].

[0058] Figure 5 For the detection performance study: (A) The ECL measurement values ​​of the electrochemical sensor for O157:H7 Escherichia 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 5×10 4 CFU / mL; (B) Relationship between ECL intensity and target O157:H7 Escherichia coli concentration, inset: the relationship between ECL intensity and the logarithm of the target O157:H7 Escherichia coli concentration, error bars represent the standard deviation of three parallel tests.

[0059] Figure 6 Figures showing specificity and actual sample detection results: (A) ECL signal enhancement of different bacteria, (B) bar graph of the recovery rate of E. coli O157:H7 in milk samples at different dilution factors. DETAILED DESCRIPTION

[0060] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0061] 1. Reagents and instruments:

[0062] The bacterial strains used in the experiments were obtained from Anhui Product Quality Supervision and Inspection Institute. 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-Benzenetetramine tetrahydrochloride (BTA·4HCl) and polyphosphoric acid (PPA) were purchased from Adamas (Shanghai, China). Antibody against Escherichia coli O157:H7 (E. coli O157:H7) was from Thermo Fisher Scientific. Chloroauric acid (HAuCl4) was from J&K Scientific (Shanghai, China). Protein G purified magnetic beads (0.5 μm), streptavidin (SAV), bovine serum albumin (BSA), and HPLC-purified probes (Table S1) were purchased from Shanghai Shenggong Biotechnology Service Co., Ltd. (Shanghai, China). Fluorescein isothiocyanate (FITC) antibody was provided by Sinopharm Chemical Reagent Company (Shanghai, China).

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

[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 consisting of 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). The 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 composites

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

[0068] Add 50 mL of 0.01% HAuCl4 solution into a 150 mL flask, stir vigorously, boil, and quickly add 1.0 mL of 1% trisodium citrate. The color of the solution changes from dark red to purple and finally to wine red. Continue heating and stirring for 10 minutes. After cooling to room temperature, the obtained AuNPs solution is stored at 4°C for subsequent use.

[0069] 2.2. Preparation of gold nanoparticles (AuNPs) composites:

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

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

[0072] Mix 10 μL of 2 μM thiol primer (SH Primer), 2 μL of 1 mM TCEP, and 2 μL of 500 mM AB buffer, and incubate at room temperature for 1 hour for activation;

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

[0074] Subsequently, 10 μL of 100 μM dATP solution was added, and the mixture was shaken for 30 min 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 min;

[0076] Finally, the resulting gold nanoparticle (AuNPs) complex was collected by centrifugation at 7800 rpm for 10 min, and the resulting gold nanoparticle (AuNPs) complex was 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 on chamois leather with 0.05 μm alumina for 5 min to obtain a mirror surface; then, the electrode was thoroughly rinsed with ultrapure water and ultrasonically treated in ethanol, nitric acid (HNO3 / H2O, 1:1 volume ratio) and ultrapure water for 5 min to remove residual impurities; thereafter, the electrode was electrochemically cleaned in 0.5 M H2SO4 solution and a potential scan was performed 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 blown dry with nitrogen (N2).

[0079] 4. Bacterial culture

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

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

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

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

[0084] The precipitate was resuspended in 100 μL phosphate buffered saline (PBS, pH 7.4) containing 1% (w / v) bovine serum albumin (BSA) and 0.01% (v / v) Tween-20 to obtain antibody-functionalized magnetic beads, and stored in a brown bottle at 4°C;

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

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

[0087] Finally, the supernatant containing O157:H7 E. coli 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, including 2 μL of O157:H7 Escherichia coli DNA template, 12.5 μL of 2×PCR master mix (MasterMix), 1 μL of 10 μM FITC-labeled forward primer (FP), 1 μL of 10 μM biotin-labeled reverse primer (RP), and 8.5 μL of ddH2O; the amplification program was set as: 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 extension for another 5 minutes, and 35 cycles; after the amplification was completed, the O157:H7 Escherichia coli PCR amplification product was stored at 12°C for future use.

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

[0091] 7.1. Preparation of Ruthenium Metal Covalent Organic Frameworks (Ru-MCOFs):

[0092] 27 mg of 30 μM tris(4,4'-dicarboxylic acid-2,2'-bipyridyl)dichlororuthenium ([Ru(dcbpy)3]Cl2), 26 mg of 90 μM 1,2,4,5-benzenetetraamine tetrahydrochloride (BTA·4HCl), 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 resulting mixture was adjusted to 8.5 using a saturated NaHCO3 solution, a black powder was precipitated. The black powder was collected by centrifugation and washed with water, methanol and acetone in sequence for 12 hours using a Soxhlet extractor. The washed black powder was vacuum dried at 100°C for 6 hours to obtain a ruthenium metal covalent organic framework material (Ru-MCOFs).

[0094] 7.2. Preparation of Ruthenium Metal Covalent Organic Framework Material Modified with Streptavidin (Ru-MCOFs-SAV):

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

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

[0097] After centrifugation at 10000 rpm for 5 min and removal of the supernatant, a ruthenium metal covalent organic framework material modified with streptavidin was obtained. The ruthenium metal covalent organic framework material modified with streptavidin (Ru-MCOFs-SAV) was resuspended in PBS buffer (500 μL, pH 7.4) containing 1% (w / v) bovine serum albumin (BSA).

[0098] 8. Preparation of ECL electrochemical sensor

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

[0100] Next, 5 μL of 1% BSA blocking solution was added to the electrode surface and incubated for 1 h to block unbound sites;

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

[0102] The electrode was immersed in 10 μL of AuNPs complex and incubated for 30 min, then 10 μL of hybridization chain reaction (HCR) system containing 2 μL of 2 μM biotin-labeled probe H1, 2 μL of 2 μM biotin-labeled probe H2, and 6 μL of TE buffer was dropped on the electrode surface and incubated at 37 °C for 1 h;

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

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

[0105] The Ru-MCOFs were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS). Figure 2 TEM results ( Figure 2 ) and EDS images confirmed the successful synthesis of Ru-MCOFs and demonstrated 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+ Importantly, Ru-MCOFs exhibited excellent chemical and ECL stability during repeated testing, which is attributed to the Ru(bpy)3 2+ Strong covalent bonds between the unit and the benzimidazole unit.

[0106] 10. Characterization of electrochemical behavior

[0107] To carefully characterize the modification process, the stepwise assembly of the electrochemiluminescent biosensor on the electrode surface was verified using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measurements. Figure 4As shown in A, the EIS graph reflects the modifications made to the electrode at each immobilization stage. Initially, the EIS of the bare electrode exhibited an almost straight line (curve a), indicating excellent conductivity due to the absence of any significant electron transfer barrier. When the electrode was decorated with PEG / CS / SAV by drop coating, the charge transfer resistance (Ret) increased significantly (curve b), which was attributed to the attenuation of electron transfer induced by the SAV layer. After the introduction of O157:H7 Escherichia coli, the double-stranded amplification products were immobilized on the electrode surface and the electrode surface was further blocked using BSA, resulting in a continuous increase in Ret (curve c). Subsequently, when the AuNPs complex was added and HCR assembly was induced, a significant decrease in Ret was observed (curve d), thereby enhancing the electron transfer process. Finally, the continuous addition of Ru-MCOFs (curve e) resulted in a continuous increase in Ret, which can be attributed to their significant blocking effect on electron transfer at the electrode interface. The EIS changes observed in each modification step confirmed the occurrence of specific chemical and physical changes on the electrode surface, verifying the successful fabrication of a stable electrochemiluminescent biosensor. The trend of CV measurements ( Figure 4 B) is closely correlated with the EIS data, further verifying that the electrochemical biosensor was effectively constructed via a stepwise modification process.

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

[0109] The feasibility of the electrochemiluminescence biosensor was further evaluated by analyzing its ECL performance. Figure 4 As shown in Figure C, the bare electrode has almost no ECL signal response (curve a). In the absence of E. coli O157:H7, only a very weak ECL background response was observed, mainly due to the nonspecific adsorption of a very small amount of Ru-MCOFs on the electrode surface (curves b, c, and d). It is worth noting that 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, a significant enhancement in the ECL signal intensity (curve e) was observed after the introduction of the target E. coli O157:H7. This significant change is attributed to the large accumulation of Ru-MCOFs on the electrode surface through the classic SAV-biotin interaction. These results jointly verify the feasibility of the sensor from the perspective of electrochemiluminescence signal response.

[0110] 12. Study on the detection performance of Escherichia coli O157:H7

[0111] After the above studies and characterizations, the analytical performance of the electrochemiluminescence biosensor was evaluated by analyzing the dynamic range and linear relationship between the ECL intensity and different concentrations of O157:H7 E. coli under optimal conditions ( Figure 5). The ECL intensity of different concentrations of E. coli O157:H7 was measured as follows Figure 5 As shown in A. As expected, increasing the concentration of E. coli O157:H7 from 5 CFU / mL to 5×104 CFU / mL resulted in a gradual increase in the ECL intensity, demonstrating the effectiveness of the design method of the present invention. Based on 3σ / slope (σ = standard deviation of the blank sample), the detection limit was calculated to be 2.8 CFU / mL. In addition, the stability of the sensor was verified by repeatedly measuring the ECL signal values ​​of E. coli O157:H7 at the same concentration, which showed excellent relative standard deviation (RSD) values, as shown in Figure 1. Figure 5 These results confirm the advantages of the stability design and sensitivity enhancement strategy proposed in the present invention. The method of the present invention has superior pathogenic bacteria detection and analysis capabilities, especially in terms of sensitivity and linear range.

[0112] 13. Specificity and actual sample testing

[0113] Specificity and true sample evaluation are essential for accurate bacterial identification. Figure 6 As shown in A, by defining the ECL signal enhancement of the target as 100%, the relative signals for other bacteria (Listeria monocytogenes, Staphylococcus aureus, Enterobacter sakazakii, Pseudomonas aeruginosa, and Streptococcus enteritidis) and the blank are almost negligible. This substantial difference can be attributed to the magnetic bead enrichment and PCR amplification technology specifically used for the target bacteria, demonstrating that the method is highly specific for bacterial detection and identification. To further verify the practicality of this method, different concentrations of E. coli O157:H7 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-validated by traditional culture methods to ensure the absence of target bacteria). Figure 6 The recovery results shown in B indicate that the recovery of undiluted fresh milk was relatively low. However, the recovery of two-fold and five-fold diluted milk samples was significantly improved, indicating that the detection method has potential practical application value, especially when sample dilution is used. In addition, real milk samples were blindly analyzed and the relevant results are shown in Table S2. The results show that the detection consistency of the biosensor method of the present invention matches that of the traditional culture method, achieving a 100% consistency rate. This high degree of consistency highlights the robustness and reliability of the biosensor of the present invention and confirms its strong potential for practical application in pathogen detection, especially in complex sample matrices.

[0114] Table S2: Recoveries and RSDs in real samples

[0115]

[0116] The working principle of the invention is as follows: when the target Escherichia coli O157:H7 exists, protein G purification magnetic beads are used to separate and enrich the target bacteria, and then the specific sequence of Escherichia coli O157:H7 is amplified by PCR to generate a large number of double-stranded products modified by biotin and FITC. One end of the amplified product is fixed on the electrode interface modified by PEG-CS and SAV through SAV-biotin, and the other end is combined with the gold nanoparticle complex. Then, a hybridization chain reaction (HCR) is initiated, and a large number of Ru-MCOFs are combined with the hybridization product, thereby accumulating on the electrode surface and generating a significant ECL signal. As the concentration of Escherichia coli O157:H7 increases, the ECL signal becomes stronger, thereby realizing quantitative detection.

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

[0118] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A metal covalent organic framework material, characterized in that: Its preparation method is: Tris(4,4'-dicarboxylic acid-2,2'-bipyridyl)ruthenium dichloride, 1,2,4,5-phenyltetraamine tetrahydrochloride and polyphosphoric acid 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; After the pH of the resulting mixture was adjusted to 8.5 using a saturated NaHCO3 solution, a black powder was precipitated. The black powder was collected by centrifugation and washed with water, methanol and acetone in sequence for 12 hours using a Soxhlet extractor. The washed black powder was vacuum dried at 100°C for 6 hours to obtain a ruthenium metal covalent organic framework material.

2. A metal covalent organic framework-mediated steady-state electrochemiluminescence sensor, characterized in that: Its construction method is: Based on the ruthenium metal covalent organic framework material according to claim 1, preparing a ruthenium metal covalent organic framework material modified with streptavidin; A mixed solution of acetic acid solution containing polyethylene glycol, chitosan and streptavidin was 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 were blocked with BSA blocking solution; Next, after being thoroughly rinsed with ultrapure water and blown dry with nitrogen, the PCR amplification product of E. coli O157:H7 was dropped onto the glassy carbon electrode, and the PCR amplification product of E. coli O157:H7 was used to bind to the streptavidin on the glassy carbon electrode; The electrode is immersed in the gold nanoparticle complex for a period of time, the gold nanoparticle complex is used to bind to the PCR amplification product of O157:H7 Escherichia coli, and then the hybridization chain reaction system used to bind to the gold nanoparticle complex is dropped on the electrode surface and incubated for a period of time; Finally, the ruthenium metal covalent organic framework material modified with streptavidin is dropped onto the electrode surface and incubated for a period of time. The ruthenium metal covalent organic framework material is used to combine with the hybridization product of the hybridization chain reaction system. Finally, it is rinsed with deionized water to obtain an electrochemical sensor for detecting O157:H7 Escherichia coli in the matrix.

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

4.

4. The metal covalent organic framework-mediated steady-state electrochemiluminescence sensor according to claim 2, characterized in that: The O157:H7 Escherichia coli PCR amplification product is prepared by amplification of a PCR amplification reaction system, wherein the PCR amplification reaction system with a total volume of 25 μL includes 2 μL of O157:H7 Escherichia coli DNA template, 12.5 μL of 2×PCR premix, 1 μL of 10 μM FITC-labeled forward primer, 1 μL of 10 μM biotin-labeled reverse primer, and 8.5 μL of ddH2O; The hybridization chain reaction system includes 2 μL of a 2 μM biotin-labeled probe H1, 2 μL of a 2 μM biotin-labeled probe H2, and 6 μL of a TE buffer; The sequence of the FITC-labeled forward primer was: 5′-FITC-CGGACATCCATGTGATATGG-3′; The sequence of the biotin-labeled reverse primer was: 5′-Biotin-TTGCCTATCTACAGCTAATCC-3′; The sequence of the biotin-labeled probe H1 is: The sequence of the biotin-labeled probe H2 is: 5′-AGTCTAGGATTCGGCGTGGGTTAACACGCCGAATCCTAGACTACTTTG-biotin-3′.

5. The metal covalent organic framework-mediated steady-state electrochemiluminescence sensor according to claim 4, characterized in that: The gold nanoparticle complex was prepared by mixing 1 mL of Au NPs solution with 2.5 μL of anti-FITC antibody at a concentration of 2 mg / mL and incubating at room temperature for 1 hour; Then, the solution was centrifuged and concentrated tenfold to 100 μL to form a 10× AuNPs solution; Mix 10 μL of 2 μM thiol primer, 2 μL of 1 mM TCEP, and 2 μL of 500 mM AB buffer, and incubate at room temperature for 1 hour for activation; After activation, 100 μL of the aforementioned 10× AuNPs solution was added, and the mixture was shaken for 1 h to achieve coupling; Subsequently, 10 μL of 100 μM dATP solution was added, and the mixture was shaken for 30 min to block unreacted sites; Then, 20 μL of 0.1 M NaCl solution was added, and the mixture was aged at room temperature for 30 min; Finally, the resulting gold nanoparticle complexes were collected by centrifugation at 7800 rpm for 10 min, and the resulting gold nanoparticle complexes were resuspended in 1 mL of PBS buffer and stored at 4 °C until use; Wherein, the sequence of the thiol primer is:

6. The metal covalent organic framework-mediated steady-state electrochemiluminescence sensor according to claim 4, characterized in that: The extraction method of the O157:H7 Escherichia coli DNA template is as follows: Add 20 μL of 1 mg / mL anti-O157:H7 E. coli antibody to a solution containing 100 μL of 50 mg beads / mL protein G purified magnetic beads and incubate at room temperature for 30 minutes; Then, the mixture was centrifuged at 1000 rpm for 5 min, and the supernatant containing unbound antibody was discarded; The precipitate was 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, which were stored in a brown bottle at 4°C; 10 μL of antibody-functionalized magnetic beads and 1 mL of an unknown or known concentration of O157:H7 E. coli solution were placed in an EP tube, mixed, and incubated at 37°C for 30 minutes; After magnetic separation, the supernatant was removed; the suspension was resuspended in 100 μL of TE buffer and sonicated for 5 min to release the O157:H7 E. coli DNA into the supernatant; Finally, the supernatant containing O157:H7 E. coli DNA template was collected for subsequent PCR amplification.

7. The metal covalent organic framework-mediated steady-state electrochemiluminescence sensor according to claim 5, characterized in that: The preparation method of the AuNPs solution is as follows: 50 mL of 0.01% HAuCl4 solution is added to a 150 mL flask, and after boiling under vigorous stirring, 1.0 mL of 1% trisodium citrate is quickly added, and the color of the solution changes from dark red to purple and finally to wine red. Then, heating and stirring are continued for 10 minutes. After cooling to room temperature, the obtained AuNPs solution is stored at 4°C for subsequent use.

8. Application of a metal covalent organic framework-mediated steady-state electrochemiluminescence sensor in the field of Escherichia coli detection, characterized in that: The Escherichia coli is specifically O157:H7 Escherichia coli, and the detection includes the following steps: Culture of S1, O157:H7 Escherichia coli and positive control bacteria; S2, preparing various standard concentrations of O157:H7 Escherichia coli solutions; S3, various standard concentrations of E. coli O157:H7 solutions were treated with protein G purification magnetic beads and PCR amplified to obtain corresponding E. coli O157:H7 PCR amplification products; S4, assembling the electrochemical sensor according to any one of claims 2 to 7 by using the PCR amplification product of O157:H7 Escherichia coli, performing electrochemiluminescence detection, and constructing a standard curve related to the electrochemiluminescence signal and the concentration of O157:H7 Escherichia coli; S5. Extract a contaminated milk sample containing an unknown concentration of O157:H7 Escherichia coli, perform electrochemiluminescence detection in the same manner as steps S3 and S4, substitute the detected electrochemiluminescence signal into the standard curve, and the concentration of O157:H7 Escherichia coli in the contaminated milk sample can be obtained.

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