A magnetic zif-90-phage composite probe and a preparation method and application thereof

Abstract

The present application relates to the technical field of microbial biosensing rapid detection, and particularly relates to a magnetic ZIF-90-phage composite probe and a preparation method and application thereof.The present application provides a magnetic ZIF-90-phage composite probe, the composite probe is a Fe3O4@ZIF-90@phage core-shell structure, and comprises: an aminated Fe3O4 magnetic bead core, an aldehyde group functionalized ZIF-90 porous shell layer coated on the surface of the aminated Fe3O4 magnetic bead, and an MRSA specific phage outer layer loaded on the surface of the ZIF-90 porous shell layer by covalent grafting.The present application solves the pain points of low loading and easy inactivation of traditional probes.The MP probe is magnetically fixed on the magnet on the back of the screen-printed carbon electrode, so that the same electrode simultaneously has three functions of stirring enrichment, electrochemical impedance total bacteria detection and fluorescent live bacteria detection, and realizes the integration of "enrichment-detection" on-site portable operation.

Description

Technical Field

[0001] This invention relates to the field of rapid detection technology of microbial biosensing, and in particular to a magnetic ZIF-90-phage composite probe, its preparation method and application. Background Technology

[0002] MRSA (methicillin-resistant Staphylococcus aureus) is a leading cause of hospital-acquired infections, characterized by highly pathogenic, multidrug-resistant, Gram-positive bacteria. It can cause critical illnesses such as pneumonia and bloodstream infections, with an infection mortality rate exceeding 40% in ICU patients. The World Health Organization has listed it as one of the priority superbugs for developing new diagnostic technologies. In clinical infection assessment, live bacteria possess active proliferation and toxin-producing capabilities, while dead bacteria release endotoxins that induce inflammatory responses. Merely detecting the total bacterial count cannot accurately assess the actual infection risk; therefore, it is essential to differentiate between live and dead bacteria and quantify them separately to provide a reliable basis for anti-infective treatment and prognosis.

[0003] Currently, mainstream detection methods all have significant shortcomings. While plate culture is the gold standard, it takes 24–72 hours and can only detect live bacteria, failing to reflect the number of dead bacteria, making it unsuitable for rapid emergency diagnosis. PCR and LAMP isothermal amplification techniques are highly sensitive but cannot distinguish between live and dead bacteria, and nucleic acid amplification easily generates aerosol contamination, heavily relying on large laboratory equipment and unsuitable for on-site, real-time testing. Single electrochemical sensors based on antibodies or aptamers can only capture all bacteria, lacking specific live bacteria recognition signals, and similarly cannot distinguish between live and dead states. ATP bioluminescence relies on exogenous insect luciferase, with expensive reagents and requiring continuous low-temperature cold chain storage and transportation, resulting in high detection costs and making it difficult to promote in primary healthcare institutions. Traditional phage-modified magnetic bead probes lack a protective layer; phages become significantly inactive at room temperature after about 3 months, and the low phage loading on the magnetic bead surface leads to insufficient enrichment efficiency and detection sensitivity.

[0004] Although a few dual-mode sensors have been reported, none have achieved an integrated electrode design for enrichment and detection. The enrichment, cleaning, and detection steps are separate operations, resulting in cumbersome and time-consuming processes. Furthermore, existing dual-mode systems generally rely on exogenous labeled enzymes or fluorescent molecules as signal sources, increasing detection costs and operational complexity. A low-cost strategy for distinguishing live and dead bacteria using endogenous fluorescent signals generated by bacterial metabolic enzymes has yet to emerge. Therefore, there is an urgent need to develop an integrated sensing system with high probe stability, portability, rapid detection, controllable cost, and the ability to simultaneously quantify live and dead MRSA bacteria on-site, in order to overcome the current technical bottlenecks in clinical and field testing. Summary of the Invention

[0005] The purpose of this invention is to provide a magnetic ZIF-90-phage composite probe, its preparation method, and its application. This addresses several shortcomings of existing MRSA detection technologies, including low phage probe loading and poor room temperature stability; cumbersome and inconvenient operation due to the separation of enrichment and detection; inability to distinguish between live and dead bacteria and a high risk of false positives due to a single signal mode; reliance on expensive exogenous enzyme reagents for live bacteria quantification; and low enrichment efficiency in complex matrices leading to high detection limits.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a magnetic ZIF-90-phage composite probe, wherein the composite probe has a Fe3O4@ZIF-90@phage core-shell structure, comprising: Aminated Fe3O4 magnetic bead core; An aldehyde-functionalized ZIF-90 porous shell coating the surface of the aminated Fe3O4 magnetic beads; and The MRSA-specific phage outer layer is covalently grafted onto the surface of the ZIF-90 porous shell.

[0007] Preferably, the ZIF-90 porous shell is an aldehyde-functionalized zeolite imidazole ester framework structure, providing aldehyde sites for covalent grafting of bacteriophages and forming a porous microenvironment protective layer.

[0008] Preferably, the ZIF-90 shell has a thickness of 60–80 nm and a specific surface area of ​​216 m² / g; after coating the phage, the probe has a specific surface area of ​​168 m² / g, and the mesoporous channels are retained to ensure contact between MRSA and the phage.

[0009] Preferably, the composite probe is superparamagnetic, and a single probe carries 40 to 50 phages.

[0010] This invention provides a stirred integrated electrochemical / fluorescence dual-mode sensor, comprising: A disposable screen-printed carbon electrode, wherein a button magnet is provided on the back side of the disposable screen-printed carbon electrode; The magnetic ZIF-90-phage composite probe is magnetically fixed to the surface of the disposable screen-printed carbon electrode working electrode. A handheld miniature stirrer is assembled and connected to the disposable screen-printed carbon electrode; An electrochemical detection module electrically connected to the disposable screen-printed carbon electrode; and Fluorescence detection module.

[0011] Preferably, the disposable screen-printed carbon electrode has both stirring and enrichment functions and electrochemical sensing and detection functions; the button magnet is a neodymium magnet with a diameter of 3 mm; the composite probe is magnetically attached to the working electrode surface of the disposable screen-printed carbon electrode; and the fluorescence detection module is a 365nm LED ultraviolet excitation light source and a mobile phone visualization fluorescence imaging component.

[0012] This invention provides a method for simultaneous quantitative detection of live and dead MRSA bacteria using the composite probe or the sensor, comprising the following steps: (1) The magnetic ZIF-90-phage composite probe was fixed on the working electrode surface of the screen-printed carbon electrode, the electrode was immersed in the sample to be tested, and the sample was stirred and enriched so that the composite probe could specifically capture MRSA in the sample. (2) Electrochemical impedance spectroscopy was used to detect the change in charge transfer impedance at the electrode interface, and the total concentration of all MRSA in the sample was quantified based on the impedance signal; (3) Add micrococcal nuclease substrate MUP to the electrode surface and incubate to allow the micrococcal nuclease secreted by live MRSA bacteria to catalyze the hydrolysis of MUP to generate fluorescent substance MU. Excite and detect the fluorescence intensity using 365nm ultraviolet light, and quantify the concentration of live MRSA bacteria in the sample based on the fluorescence signal. (4) Subtract the concentration of live bacteria obtained in step (3) from the total bacterial concentration obtained in step (2) to calculate the concentration of dead MRSA bacteria in the sample.

[0013] Preferably, the conditions for stirring enrichment in step (1) are: stirring speed 400~500 rpm, enrichment time 15~25 min; after enrichment, the electrode is rinsed with PBS buffer to remove free bacteria.

[0014] Preferably, the detection frequency range of the electrochemical impedance spectroscopy in step (2) is 10² to 10⁻¹⁰. 5 Hz, the normalized impedance value is linearly correlated with the total log concentration of MRSA bacteria; the concentration of MUP substrate in step (3) is 90~110μM, and the incubation time is 13~17min.

[0015] Preferably, in step (2), 40 μL of 0.01 mol / L K3Fe(CN)6-PBS detection solution is added to the electrode, and the electrochemical workstation collects 10-10 samples. 9 The CFU / mL impedance spectrum was fitted to a standard curve Y = 1.670X + 4.37 R with the impedance value normalized to the Nyquist semicircle radius Rct (Y = (ZZ) / Z). 2 =0.991, calculate the total MRSA concentration in the sample ( Figure 9 ).

[0016] Preferably, in step (3) quantitative fluorescence of live bacteria: 40 μL of PBS-LB substrate solution containing 100 μM MUP is added to the same SPCE and incubated at room temperature for 10-20 min; the electrode is irradiated with a 365 nm LED, and the fluorescence intensity is read using a portable fluorometer. The standard curve is Y = 0.32X - 0.20R. 2 = 0.992 to calculate viable bacterial concentration; The total detection time is ≤50 min, and the detection linearity range is 10-10. 9 CFU / mL, detection limit 30 CFU / mL.

[0017] This invention provides the application of the magnetic ZIF-90-phage composite probe in the separation and purification of MRSA in blood samples for non-diagnostic purposes.

[0018] This invention provides the application of the aforementioned stirred integrated electrochemical / fluorescence dual-mode sensor or the method described therein in the on-site simultaneous rapid screening of live and dead MRSA bacteria in clinical ICU wastewater, milk, or blood samples for non-diagnostic purposes.

[0019] The two standard curve detection principles and coordinate definitions provided by this invention are distinguished as follows: ① The equation Y=1.670X+4.37 is the electrochemical impedance spectroscopy standard curve: the horizontal axis is log(MRSA) (CFU / mL), and the vertical axis is the relative impedance change value (Z-Z0) / Z0; MRSA concentration of 10-10 is used. 9 The CFU / mL was calibrated at 9 gradient points, and the least squares linear fitting method was used, with R²=0.991.

[0020] ② The equation Y = 0.32X - 0.20 is the standard curve for fluorescence detection: the horizontal axis is log(MRSA) (CFU / mL), and the vertical axis is the relative change in fluorescence (F - F0) / F0; MRSA concentration of 10⁻¹⁰ is used. 9 The CFU / mL was calibrated at 9 gradient points, and the least squares linear fitting method was used, with R²=0.992.

[0021] For all concentration gradient samples on the two standard curves, three biological parallels and three instrumental replicates were set up. The number of parallel determinations for a single concentration was uniformly set to n=6, and all samples were fitted using the least squares univariate linear regression method commonly used in quantitative analysis chemistry.

[0022] This invention discloses an integrated dual-mode sensor based on a magnetic ZIF-90-phage composite probe (MP probe) for the simultaneous on-site quantitative detection of live / dead methicillin-resistant Staphylococcus aureus (MRSA). The probe employs a Fe3O4@ZIF-90@phage core-shell structure, where the porous aldehyde framework of ZIF-90 not only significantly increases the phage loading capacity but also provides long-term protection of its biological activity at room temperature, completely solving the problems of low loading and easy inactivation of traditional probes. The MP probe is magnetically fixed to a magnet on the back of a screen-printed carbon electrode (SPCE), enabling the same electrode to simultaneously perform three functions: stirring enrichment, electrochemical impedance spectroscopy for total bacterial detection, and fluorescent viable bacterial detection, achieving integrated on-site portable operation of "enrichment-detection".

[0023] In terms of detection mechanism, this invention utilizes micrococcal nuclease (MN) secreted by live MRSA bacteria. This enzyme specifically catalyzes the hydrolysis of MUP substrate to produce fluorescent substance MU, and the intrinsic fluorescence intensity directly quantifies live bacteria. Meanwhile, the electrochemical impedance signal is used to quantify all MRSA (live bacteria + dead bacteria) captured by the MP probe; the difference between the two is the number of dead bacteria. The entire process requires no exogenous expensive enzyme markers, has a detection limit as low as 30 CFU / mL, a total detection time of ≤50 minutes, and the dual-mode signals can be cross-validated, effectively reducing the risk of false positives. It combines low cost, high specificity, and strong resistance to matrix interference.

[0024] This invention is widely applicable to rapid on-site screening of complex samples such as ICU wastewater, milk, and blood. It is particularly effective in separating and purifying MRSA from red blood cell suspensions, combining detection and bacterial removal functions. The MP probe is stable at room temperature for over 6 months, significantly superior to existing reagent storage and transportation methods. Compared to industry challenges such as long culture cycles, the inability of PCR to distinguish between live and dead bacteria, and the high cost of ATP luminescent reagents, this invention provides an efficient, convenient, and reliable solution for clinical infection control, food safety monitoring, and blood pathogen purification, with broad prospects for translational applications.

[0025] Compared with the prior art, the present invention has the following beneficial effects: Firstly, at the probe material level, this invention constructs a Fe3O4@ZIF-90@phage core-shell structure using a porous aldehyde-based ZIF-90 framework. This fully utilizes the large specific surface area and abundant aldehyde functional groups of ZIF-90, providing numerous covalent grafting sites for the phage, thus significantly increasing the phage loading per unit probe and enhancing its ability to capture target bacteria. Simultaneously, the porous ZIF-90 shell forms a physical microenvironment barrier on the magnetic bead surface, effectively isolating it from external oxygen, humidity, and temperature fluctuations, providing long-term protection for phage activity. This greatly extends the probe's storage stability at room temperature, fundamentally solving the technical problem of traditional phage probes having low loading capacity and short-term inactivation at room temperature due to the lack of a protective layer.

[0026] Secondly, regarding the detection device and operation process, this invention directly integrates a screen-printed carbon electrode into the handheld stirring component, allowing the same electrode to simultaneously function as a stirring paddle and a sensing interface. After specific capture is completed during the enrichment stage, electrochemical detection can be performed directly without transfer, eliminating the need for separate enrichment equipment and cumbersome sample transfer and cleaning steps, greatly simplifying the operation process. The entire device is compact, lightweight, and highly integrated, requiring no external large auxiliary equipment, truly breaking through the limitations of fixed laboratory settings. It can be conveniently applied to on-site, real-time testing scenarios such as ICU wards, emergency rooms, and field emergencies, meeting the urgent clinical demand for portable and easy-to-operate equipment in rapid testing.

[0027] Thirdly, regarding detection signal and performance, this invention utilizes the micrococcal nuclease secreted by the live MRSA bacteria to catalyze the substrate and generate an endogenous fluorescence signal. The detection signal directly originates from the metabolic activity of the target live bacteria, completely eliminating the dependence on expensive exogenous enzyme-labeled reagents. This not only significantly reduces the reagent cost per test but also eliminates the additional burden of cold chain storage and transportation. Simultaneously, the electrochemical impedance signal is used for quantifying total bacteria, and the endogenous fluorescence signal is used for specific quantification of live bacteria. These two signals are independent and mutually corroborative, effectively eliminating false positive or false negative results caused by matrix interference in single signal patterns. The stirring enrichment operation further amplifies the target bacteria capture amount at the electrode interface, significantly improving detection sensitivity. The overall detection time is significantly shorter than traditional culture methods, truly achieving a unity of "rapid detection" and "accurate detection."

[0028] Fourth, in terms of application breadth and functional expansion, this invention demonstrates excellent anti-interference ability and detection reliability in various complex sample matrices such as milk, ICU wastewater, whole blood, and red blood cell suspensions. It can effectively cope with the influence of interfering components such as proteins, lipids, blood cells, and bacteria in actual samples, and has broad sample adaptability. It is worth mentioning that the MP probe has dual functions of detection and separation and purification. While completing the quantitative detection of MRSA, it can also efficiently remove pathogens from samples such as blood through magnetic separation without damaging normal formed elements such as red blood cells, providing a feasible solution for the "detection-purification integration" of clinical infection samples. In addition, the platform has highly flexible scalability—simply by changing the specific bacteriophage species, it can be easily extended to the detection of other important foodborne and clinical pathogens such as Salmonella, Escherichia coli, and Pseudomonas aeruginosa, demonstrating its huge application potential and promotional value as a general-purpose rapid detection platform. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0030] Figure 1 : Schematic diagram of the overall process of MP probe preparation, stirring enrichment, and dual-mode detection; (a) MP material preparation; (b) MRSA stirring enrichment treatment; (c) On-site electrochemical and fluorescence detection of MRSA live and dead bacteria; (d) Fluorescence response mechanism based on MRSA endogenous MN enzyme.

[0031] Figure 2 MP probe operation flowchart ((A) Complete system setup; (B–E) Preparation and enrichment of target bacteria with magnetic stirring electrode; (H) Electrochemical quantification of total MRSA bacteria; (I–K) MRSA live bacteria fluorescence detection).

[0032] Figure 3 MP probe XRD, FTIR, XPS, particle size Zeta potential characterization spectra; (a) XRD values ​​of different magnetic particle (MP) precursors and their ability to trap MRSA; hysteresis loop analysis; (b) Infrared spectra of iron oxide, ZIF-90, mesoporous material MP, and MP samples after MRSA capture, respectively; (c) X-ray photoelectron spectroscopy (XPS) of MP material and Fe3O4@ZIF-90 composite; (d) Zeta potential test results of MP and its precursors.

[0033] Figure 4 Electrochemical impedance spectroscopy (EIS) total bacterial response, fluorescence-specific response curves of live bacteria, and comparison of interference from different bacteria; (A) EIS impedance response at different bacterial strain concentrations: 10³ CFU / mL MRSA (live and dead), 10³ CFU / mL Escherichia coli, 10 5 CFU / mL MRSA live bacteria and Escherichia coli; (B) Fluorescent signal response: 10³ CFU / mL MRSA live / dead bacteria (yellow), 10³ CFU / mL Escherichia coli, 10 5 CFU / mL Escherichia coli (red), blank control group.

[0034] Figure 5 : Differential pulse voltammetry (DPV) response curve of MRSA.

[0035] Figure 6 : The survival rate of MRSA live bacteria on the electrode surface changes over time.

[0036] Figure 7 Enzyme kinetic analysis (MN–MUP); (A) Michaelis–Menten kinetic model; (B) Lineweaver-Burk double reciprocal plotting analysis.

[0037] Figure 8 Principle of live and dead bacteria determination.

[0038] Figure 9 MRSA electrochemical-fluorescence dual-signal quantitative detection performance; (A) MRSA gradient concentration Nyquist impedance map (10–10 9 (CFU / mL), with a magnified view of the local curve in the illustration; (B) The relative rate of change of impedance is logarithmically linear with bacterial concentration; (C) Gradient concentration fluorescence spectra of live bacterial MRSA; (D) The relative change rate of fluorescence is logarithmically linear with bacterial concentration. Illustration: Real-world SPCE detection using a portable device. Detailed Implementation

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] All reagents and materials used in this invention were purchased from Aladdin.

[0041] Example 1: Preparation of magnetic MP composite probe ( Figure 1 , Figure 3 )

[0042] 1. Preparation of aminated Fe3O4 magnetic beads: 2.0 g FeCl3 6H2O and 0.8 g FeCl2 Mix and coprecipitate with 4H2O, adjust pH to 10 with ammonia, and react at 80℃ for 10 min; modify silane with APTES (γ-aminopropyltriethoxysilane) ethanol solution (1 mL) at 60℃ for 60 min, wash and dry to obtain amino magnetic beads.

[0043] 2. Synthesis of Fe3O4@ZIF-90 core-shell: DMF was dispersed in amino magnetic beads, and 0.1975 g zinc acetate and 0.2 g 2-formaldehyde imidazole solution were added dropwise. The mixture was stirred at room temperature for 12 h and then grown as a solvothermal porous shell of ZIF-90.

[0044] 3. Phage grafting preparation of MP: MB@ZIF-90 was dispersed in pH 8.0 Tris-HCl buffer, and MRSA-specific phage (IME-SA118; Staphylococcus phage IME-SA118, complete genome; GenBank: KR902361.1) was added. The mixture was shaken in the dark for 4 h, and the free phage was washed several times with PBS. The phage was then freeze-dried to obtain the MP probe.

[0045] Characterization results: DLS particle size analysis showed that the average particle size of MP was 203.6 nm ± 4.2 nm, and a single probe was loaded with 47 phage strains; after 6 months of storage at room temperature, the capture activity was retained by 93%.

[0046] Example 2: Assembly of an integrated portable stirring sensor ( Figure 2 )

[0047] 1. A 3 mm button magnet is attached to the back of the disposable carbon screen printed electrode SPCE; 2. Take 50 μL of 1 mg / mL MP probe (prepared in Example 1) and coat it evenly on the SPCE working electrode, then fix it with a magnet. 3. The SPCE inserts into a handheld micro stirring head, and is equipped with a portable electrochemical workstation, a 365 nm LED fluorescence detection module, and a mobile phone imaging component; the entire set of equipment weighs less than 1.2 kg and can be directly carried to the ICU and wards for on-site use.

[0048] Example 3: On-site synchronous detection of MRSA in ICU wastewater

[0049] 1. Sample pretreatment: Solid impurities were removed from the ICU wastewater from Southern Medical University Southern Hospital by filter paper, and 500 mL of filtrate was collected; 2. Stirring enrichment: The assembled SPCE was immersed in the sample, stirred at 450 rpm for 20 min, and washed 5 times with PBS to remove free bacteria; 3. Electrochemical determination of total bacteria: Add potassium ferricyanide PBS electrolyte and collect samples at 10²~10⁻⁶. 5 Hz impedance spectrum, substituted into the standard curve, yielded a total MRSA concentration of 161 CFU / mL ( Figure 5 ); 4. Fluorescence assay for viable bacteria: Add substrate solution containing 100 μM MUP and incubate at room temperature for 15 min. Excite with UV light at 365 nm and read the fluorescence. Calculate viable bacteria count as 137 CFU / mL. Figure 7 , Figure 9 ); 5. Dead bacteria calculation: Total bacterial concentration - Viable bacterial concentration = 24 CFU / mL ( Figure 8 ); 6. Spike recovery validation: With the addition of 10² CFU / mL MRSA, the detection recovery rate was 91.0% and the RSD was 7.54%, which meets the requirements for on-site quantitative detection.

[0050] Example 4 Specificity Experiment ( Figure 4 )

[0051] To verify the specific recognition ability of the dual-mode sensor of this invention for MRSA, three non-MRSA Staphylococcus aureus standard strains (S. aureus ATCC 25928, ATCC 43300, and ATCC 29213) were selected as controls. Under optimized experimental conditions (MP 30 μg, stirring at 400 rpm for 20 min; MUP 100 mM, reaction for 20 min, room temperature), the concentrations of all strains were 10 μg. 4 CFU / mL of each strain and a blank control were detected simultaneously. MRSA was also mixed with the three control strains in equal volumes before detection to evaluate its recognition performance in complex samples.

[0052] Electrochemical impedance spectroscopy (EIS) mode-specific results

[0053] Table 1

[0054] The results showed that the normalized impedance response induced by MRSA ((Z-Z0) / Z0 = 12.10) was significantly higher than that of each control strain (0.20–0.75), with a signal ratio approximately 16–60 times that of the control group. The signal of the mixed sample (12.30) was close to that of the pure MRSA sample, indicating that other coexisting strains did not significantly interfere with the impedance detection of MRSA, and the sensor had good specificity.

[0055] Fluorescence (FL) pattern specificity results

[0056] Table 2

[0057] In fluorescence mode, the normalized fluorescence signal of MRSA ((F-F0) / F0 = 2.45) was approximately 10-12 times that of the control strains (0.20-0.25). The signal of the mixed sample (2.50) was basically consistent with that of pure MRSA, further confirming that the MN enzyme-mediated fluorescence signal was only generated by live MRSA. Other Staphylococcus aureus strains do not express this enzyme and therefore do not produce false positive signals.

[0058] Example 5: Anti-interference test

[0059] To investigate the impact of common interfering bacteria in real-world complex samples on MRSA detection, three common foodborne / opportunistic pathogens—Escherichia coli (E. coli), Listeria monocytogenes (L. monocytogenes, LM), and Salmonella typhimurium (S. typhimurium, ST)—were selected as interfering bacteria and detected both individually and in coexistence with MRSA. The concentration of each bacteria was 10-1. 4 CFU / mL.

[0060] EIS mode anti-interference results

[0061] Table 3

[0062] In EIS mode, the normalized impedance signals of the three interfering bacteria when they existed alone were all below 0.30, which was significantly different from the MRSA signal (10.80). (p < 0.0001). The signal (10.50) of the mixture of MRSA and interfering bacteria was not significantly different from that of pure MRSA, indicating that the presence of interfering bacteria does not affect the specific enrichment of MRSA on the electrode surface and the impedance response.

[0063] FL mode anti-interference results

[0064] Table 4

[0065] In FL mode, the normalized fluorescence signals of the three interfering bacteria when present individually were all below 0.20, which was significantly different from the MRSA signal (2.10). (p < 0.0001). The fluorescence signal of the mixed sample of MRSA and interfering bacteria (2.15) was not significantly different from that of pure MRSA (ns, p > 0.05), indicating that the interfering bacteria do not express MN enzyme and will not cause cross-interference in the fluorescence detection of live MRSA.

[0066] Example 6 Reproducibility and Stability Experiment ( Figure 6 )

[0067] Using optimized detection conditions, a concentration of 10 was... 4 MRSA samples of CFU / mL were repeatedly measured intra-batch and inter-batch, with each batch measured in parallel 6 times. The reproducibility of the method was evaluated by relative standard deviation (RSD).

[0068] EIS pattern reproducibility

[0069] Table 5

[0070] FL mode reproducibility

[0071] Table 6

[0072] In EIS mode, the inter-batch and intra-batch RSDs are 0.46% and 1.30%, respectively, and in FL mode, they are 2.49% and 3.12%, respectively, all less than 5%, indicating that the dual-mode sensor of the present invention has good reproducibility and stability, and meets the actual detection requirements.

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A magnetic ZIF-90-phage composite probe, characterized in that, The composite probe is a Fe3O4@ZIF-90@phage core-shell structure, comprising: Aminated Fe3O4 magnetic bead core; An aldehyde-functionalized ZIF-90 porous shell coating the surface of the aminated Fe3O4 magnetic beads; and The MRSA-specific phage outer layer is covalently grafted onto the surface of the ZIF-90 porous shell.

2. The magnetic ZIF-90-phage composite probe according to claim 1, characterized in that, The ZIF-90 porous shell is an aldehyde-functionalized zeolite imidazole ester framework structure, providing aldehyde sites for covalent grafting of bacteriophages and forming a porous microenvironment protective layer.

3. The magnetic ZIF-90-phage composite probe according to claim 1, characterized in that, The composite probe is superparamagnetic, and a single probe can load 40-50 phages.

4. A stirred integrated electrochemical / fluorescence dual-mode sensor, characterized in that, include: A disposable screen-printed carbon electrode, wherein a button magnet is provided on the back side of the disposable screen-printed carbon electrode; The magnetic ZIF-90-phage composite probe as described in any one of claims 1-3 is magnetically fixed to the surface of the working electrode of the disposable screen-printed carbon electrode. A handheld miniature stirrer is assembled and connected to the disposable screen-printed carbon electrode; An electrochemical detection module electrically connected to the disposable screen-printed carbon electrode; and Fluorescence detection module.

5. The stirred integrated electrochemical / fluorescence dual-mode sensor according to claim 4, characterized in that, The disposable screen-printed carbon electrode has both stirring and enrichment functions and electrochemical sensing and detection functions. The button magnet is a neodymium magnet with a diameter of 3 mm. The composite probe is magnetically attached to the working electrode surface of the disposable screen-printed carbon electrode. The fluorescence detection module is a 365nm LED ultraviolet excitation light source and a mobile phone visualization fluorescence imaging component.

6. A method for simultaneous quantitative detection of live and dead MRSA bacteria using the composite probe according to any one of claims 1-3 or the sensor according to claim 4 or 5, characterized in that, Includes the following steps: (1) The magnetic ZIF-90-phage composite probe was fixed on the working electrode surface of the screen-printed carbon electrode, the electrode was immersed in the sample to be tested, and the sample was stirred and enriched so that the composite probe could specifically capture MRSA in the sample. (2) Electrochemical impedance spectroscopy was used to detect the change in charge transfer impedance at the electrode interface, and the total concentration of all MRSA in the sample was quantified based on the impedance signal; (3) Add micrococcal nuclease substrate MUP to the electrode surface and incubate to allow the micrococcal nuclease secreted by live MRSA bacteria to catalyze the hydrolysis of MUP to generate fluorescent substance MU. Excite and detect the fluorescence intensity using 365nm ultraviolet light, and quantify the concentration of live MRSA bacteria in the sample based on the fluorescence signal. (4) Subtract the concentration of live bacteria obtained in step (3) from the total bacterial concentration obtained in step (2) to calculate the concentration of dead MRSA bacteria in the sample.

7. The method for simultaneous quantitative detection of live and dead MRSA bacteria according to claim 6, characterized in that, The conditions for stirring enrichment in step (1) are: stirring speed 400~500 rpm, enrichment time 15~25 min; after enrichment, the electrode is rinsed with PBS buffer to remove free bacteria.

8. The method for simultaneous quantitative detection of live and dead MRSA bacteria according to claim 6, characterized in that, The detection frequency range of the electrochemical impedance spectroscopy method described in step (2) is 10² to 10⁻⁶. 5 Hz, the normalized impedance value is linearly correlated with the total log concentration of MRSA bacteria; the concentration of MUP substrate in step (3) is 90~110μM, and the incubation time is 13~17min.

9. The application of the magnetic ZIF-90-phage composite probe according to any one of claims 1-3 in the separation and purification of MRSA in blood samples for non-diagnostic purposes.

10. The application of the stirred integrated electrochemical / fluorescence dual-mode sensor as described in claim 4 or 5, or the method described in any one of claims 6 to 8, in the on-site simultaneous rapid screening of live and dead MRSA bacteria in clinical ICU wastewater, milk, or blood samples for non-diagnostic purposes.

Images