Construction of klebsiella pneumoniae molecularly imprinted sensor based on dual recognition of magnetic mips and zif-8

Abstract

This invention constructs a sandwich-structured sensor based on the synergistic effect of magnetic molecular imprinting and boric acid affinity for rapid visual detection of Klebsiella pneumoniae. The sensor uses Fe3O4@SiO2 with surface-grafted double bonds as a carrier and Klebsiella pneumoniae as a template to prepare magnetic molecularly imprinted polymers (MIPs) with specific recognition cavities via free radical polymerization. Simultaneously, a ZIF-8 material loaded with curcumin was synthesized and modified with p-carboxyphenylboronic acid to construct a ZIF-8@Cur@PBA signal probe. The boric acid groups specifically bind to the cis-diol groups of bacterial surface glycoproteins. In the presence of the target bacteria, the MIPs and probe form a sandwich structure mediated by the bacteria. After magnetic separation, hydrochloric acid is added to cleave ZIF-8, releasing curcumin and amplifying the signal. The signal is then neutralized with sodium hydroxide to restore color, enabling visual semi-quantitative detection through visual observation, while quantitative analysis is performed by measuring ultraviolet absorbance. This sensor exhibits excellent selectivity, anti-interference ability, and stability, providing a new method for rapid and sensitive detection of Klebsiella pneumoniae, and has important practical application value.

Description

Technical Field

[0001] This invention belongs to the fields of biosensor technology and pathogen detection technology, specifically relating to a bacterial sandwich-type visual sensor based on magnetic molecularly imprinted polymers and ZIF-8 signal probes, its preparation method, and its applications. In particular, this invention is used for rapid, sensitive, and visual detection of Klebsiella pneumoniae. Background Technology

[0002] Hospital-acquired infections have become a significant challenge in global public health. Klebsiella pneumoniae, as an opportunistic pathogen, is one of the leading causes of ventilator-associated pneumonia, urinary tract infections, and sepsis. In recent years, drug resistance in Klebsiella pneumoniae has become increasingly prominent. The widespread transmission of carbapenem-resistant Klebsiella pneumoniae (CRKP) has hampered clinical treatment and has been listed by the World Health Organization as a priority pathogen for novel antibiotics. Therefore, establishing rapid, accurate, and highly sensitive detection methods for Klebsiella pneumoniae is crucial for guiding rational drug use in clinical practice, controlling infection outbreaks, and preventing the spread of drug-resistant strains.

[0003] Currently, the main detection methods for Klebsiella pneumoniae include traditional culture, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA). Traditional culture, as the gold standard, provides accurate results, but it is time-consuming (usually 2-5 days), failing to meet the needs of rapid diagnosis in the early stages of infection. PCR offers high sensitivity and specificity, but its operation relies on precise temperature control instruments and specialized molecular biology skills, limiting its application in rapid on-site testing scenarios. ELISA, based on the specific binding of antigen and antibody, offers good throughput; however, the high cost of biological antibody preparation, significant batch-to-batch variability, and susceptibility to environmental factors limit its widespread application in the point-of-care testing of complex samples.

[0004] To overcome the limitations of the aforementioned methods, sensing strategies based on molecular imprinting (MIT) and metal-organic frameworks (MOFs) have attracted widespread attention. MIT mimics the antibody-antigen recognition mechanism to prepare molecularly imprinted polymers (MIPs) with specific recognition cavities, offering advantages such as high selectivity, high stability, and low cost. For example, existing literature (e.g., Biosensors and Bioelectronics, 2021, 190, 113439) reports a bacterial capture and detection method based on magnetic MIPs, utilizing Fe3O4 carriers to achieve rapid magnetic enrichment of target bacteria. However, such sensors with a single recognition element often rely on large equipment such as electrochemical workstations or fluorescence spectrometers for signal output, and the signal reading process is difficult to interpret directly with the naked eye, limiting their visualization applications in grassroots or field environments.

[0005] On the other hand, metal-organic framework materials, especially the zeolite imidazole ester framework ZIF-8, have been explored as signal amplification platforms due to their high specific surface area, excellent modifiability, and unique pH-responsive decomposition characteristics. For example, some studies (such as ACS Applied Materials & Interfaces, 2020, 12, 45678) have used ZIF-8 to encapsulate dye molecules to construct signal probes, which release signal molecules through acid-triggered cleavage, thus achieving signal amplification. However, the effective integration of ZIF-8 signal probes with molecularly imprinted recognition elements, especially the construction of an integrated "recognition-capture-signal output" sandwich structure that does not require aptamers or antibody assistance, for the rapid and visual detection of Klebsiella pneumoniae, still requires further research and refinement.

[0006] To address the shortcomings of the existing technologies, there is an urgent need in the field to develop a novel sensing system that combines rapid magnetic separation, specific molecular imprint recognition, and ZIF-8 signal amplification. This invention aims to provide a Klebsiella pneumoniae detection sensor that requires no aptamer assistance, is easy to operate, and provides visualized results, thereby achieving highly sensitive and selective rapid on-site screening of the pathogen. Summary of the Invention

[0007] Purpose of the invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a bacterial sandwich visualization sensor based on magnetic molecularly imprinted polymers and ZIF-8 signal probes. This sensor does not require aptamer assistance and can achieve rapid, highly sensitive, and visual detection of Klebsiella pneumoniae. The invention also provides its preparation method and application.

[0009] Technical solution

[0010] In a first aspect, the present invention provides a bacterial sandwich visualization sensor based on magnetic molecular imprinting and ZIF-8 signal amplification, the sensor comprising:

[0011] (a) A magnetic molecularly imprinted polymer, wherein the magnetic molecularly imprinted polymer uses magnetic nanoparticles with surface-grafted double bonds as a carrier and target bacteria as a template, and forms a molecularly imprinted polymer layer on the surface of the magnetic nanoparticles through surface imprinting polymerization; the molecularly imprinted polymer layer has specific recognition cavities that are complementary to the target bacteria in terms of size, shape and functional groups.

[0012] (b) A signal probe, wherein the signal probe is a functionalized ZIF-8 material encapsulated with chromogenic molecules, and the surface of the functionalized ZIF-8 material is modified with phenylboronic acid groups that can specifically bind to bacterial surface glycosyl groups.

[0013] The magnetic molecularly imprinted polymer and the signal probe form a sandwich-structured complex of "magnetic molecularly imprinted polymer-bacteria-signal probe" mediated by the target bacteria.

[0014] In a preferred embodiment, the chromogenic molecule is curcumin; the functionalized ZIF-8 material is ZIF-8@Cur@PBA, which is prepared by amide bond condensation reaction of curcumin-loaded ZIF-8 and p-carboxyphenylboronic acid.

[0015] In another preferred embodiment, the target bacterium is Klebsiella pneumoniae; the magnetic nanoparticles are Fe3O4 nanoparticles, the surface of which is sequentially coated with a SiO2 layer and grafted with carbon-carbon double bonds; the molecularly imprinted polymer layer is formed by polymerization of the functional monomer acrylamide and the crosslinking agent N,N'-methylenebisacrylamide.

[0016] Secondly, the present invention provides a method for preparing the sensor, including the preparation of a magnetic molecularly imprinted polymer and the preparation of a ZIF-8@Cur@PBA signal probe.

[0017] Thirdly, the present invention provides a visual detection method for Klebsiella pneumoniae using the sensor for non-disease diagnostic purposes.

[0018] Beneficial effects

[0019] Compared with the prior art, the present invention has the following significant advantages:

[0020] No aptamer / antibody required: Artificial recognition sites can be directly constructed using molecular imprinting technology, resulting in lower costs and better stability.

[0021] Dual recognition, high selectivity: The magnetic molecularly imprinted polymer provides specific recognition through complementary shape / functional groups, while the ZIF-8@Cur@PBA probe provides chemical affinity recognition of boric acid and glycosyl groups. This dual recognition ensures high selectivity for Klebsiella pneumoniae.

[0022] Signal amplification and high sensitivity: Utilizing the acid response characteristics of ZIF-8, it rapidly cleaves and releases a large number of curcumin molecules under acidic conditions, achieving signal amplification. The detection limit is as low as 72.4 CFU / mL, with a wide linear range (0.5 × 10⁻⁶). 3 ~ 10 7 (CFU / mL).

[0023] Visual detection: semi-quantitative analysis can be performed by directly observing the color intensity of curcumin's natural yellow color with the naked eye, or by accurately quantifying it using an ultraviolet spectrophotometer, making it suitable for rapid on-site screening.

[0024] Rapid separation and enrichment: The magnetic carrier enables rapid magnetic separation of target bacteria after capture, simplifying the operation steps and shortening the detection time (total incubation time 90 minutes).

[0025] Good stability and reproducibility: It retains more than 84% of its initial performance after 6 weeks of storage at room temperature, with little batch-to-batch variation. Attached Figure Description

[0026] [ Figure 1 A schematic diagram of the sensor's design.

[0027] [ Figure 2 Feasibility study. (A) UV-Vis absorbance spectrum of Klebsiella pneumoniae detected by MIPs / NIPs; (B) Visualization results of MIPs / NIPs before and after the addition of Klebsiella pneumoniae.

[0028] [ Figure 3 The morphology and particle size of each material were characterized. (A) Fe3O4; (B) Fe3O4@SiO2; (C) Fe3O4@SiO2-CH=CH2; (D) MIPs; (E) Scanning electron microscopy (SEM) image of NIP; (F) Fe3O4; (G) Fe3O4@SiO2; (H) Fe3O4@SiO2-CH=CH2; (I) MIPs; (J) Dynamic light scattering (DLS) of NIP.

[0029] [ Figure 4 Morphology and particle size characterization of ZIF-8@Cur and ZIF-8@Cur@PBA. (A) ZIF-8@Cur; (B) Scanning electron microscopy (SEM) image of ZIF-8@Cur@PBA. (C) ZIF-8@Cur; (D) Dynamic light scattering (DLS) image of ZIF-8@Cur@PBA.

[0030] [ Figure 5 (A) Fe3O4; (B) Fe3O4@SiO2; (C) Fe3O4@SiO2-CH=CH2; (D) MIPs; (E) NIPs; (F) ZIF-8@Cur; (G) ZIF-8@Cur@PBA water contact angle

[0031] [ Figure 6 Infrared spectra of (A) Fe3O4; (B) Fe3O4@SiO2; (C) Fe3O4@SiO2-CH=CH2; (D) MIPs; (E) NIPs; (F) ZIF-8@Cur; (G) ZIF-8@Cur@PBA; Zeta potential diagram of (A) Fe3O4; (B) Fe3O4@SiO2; (C) Fe3O4@SiO2-CH=CH2; (D) MIPs; (E) NIPs; (F) ZIF-8@Cur; (G) ZIF-8@Cur@PBA

[0032] [ Figure 7 (A) Optimization of the amount of Cur added in the synthesis of ZIF-8-NH2@Cur; (B) Optimization of the reaction time in the synthesis of ZIF-8-NH2@Cur.

[0033] [ Figure 8 Orthogonal optimization of MIP functional monomers and reduction of crosslinking agent dosage.

[0034] [ Figure 9 (A) Orthogonally optimized UV absorption spectra of MIPs / NIPs functional monomers and crosslinking agents; (B) Corresponding visualization.

[0035] [ Figure 10 Optimization of the ratio of functional monomers and crosslinking agents in MIPs.

[0036] [ Figure 11 (A) Graph showing the change in absorbance values ​​of the optimized MIPs / NIPs functional monomer and crosslinking agent ratio in UV absorption spectra; (B) Visualization results of the corresponding MIPs / NIPs.

[0037] [ Figure 12 (A) Imprinting time / h; (B) Imprinting temperature / ℃

[0038] [ Figure 13 (A) Imprint template dosage / CFU·mL -1 (B) Optimization of eluents

[0039] [ Figure 14 Reduce nonspecific adsorption

[0040] [ Figure 15(A) Incubation temperature; (B) MIPs / NIPs dosage / mg·mL -1

[0041] [ Figure 16 (A) ZIF-8@Cur@PBA dosage / mg·mL -1 (B) Incubation pH

[0042] [ Figure 17 (A) Effect of MIPs / NIPs on absorbance changes after different incubation times for Klebsiella pneumoniae; (B) Effect of MIPs / NIPs on absorbance changes after different incubation times with ZIF-8@Cur@PBA.

[0043] [ Figure 18 (A) MIPs detection of different concentrations of Klebsiella pneumoniae (0, 10) 1 0.5×10 2 10 2 0.5×10 3 10 3 0.5×10 4 10 4 10 5 10 6 10 7 10 8 (A) Linearity of absorbance at CFU / mL; (B) Linear relationship between ∆Abs of MIPs and Klebsiella pneumoniae concentration; (C) Detection of different concentrations of Klebsiella pneumoniae (0, 10 CFU / mL) by NIPs. 1 0.5×10 2 10 2 0.5×10 3 10 3 0.5×10 4 10 4 10 5 10 6 10 7 10 8 (CFU / mL) linearity; (D) visualization results of MIPs / NIPs detection of different concentrations of Klebsiella pneumoniae.

[0044] [ Figure 19 (A) Selectivity of MIPs / NIPs sensors for TMV; (B) Competitiveness of MIPs / NIPs sensors for TMV.

[0045] [ Figure 20 (A) Reproducibility of MIPs sensors; (B) Temporal stability of MIPs sensors.

[0046] [ Figure 21The anti-interference capability of MIPs sensors.

[0047] [ Figure 22 This sensor is compared with other virus sensors. Detailed Implementation

[0048] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer. All reagents used are commercially available analytical grade.

[0049] Example: Sensor for detecting Klebsiella pneumoniae

[0050] 1. Material Synthesis

[0051] 1.1 Preparation of Fe3O4 magnetic nanoparticles

[0052] 1.68 g of FeCl3·6H2O was weighed and dissolved in 50 mL of ethylene glycol, and stirred until completely dissolved. 1.25 g of anhydrous sodium acetate and 1.25 g of polyethylene glycol (PEG) were added sequentially, and the mixture was stirred continuously at room temperature for 1 hour. The mixture was transferred to a polytetrafluoroethylene-lined high-pressure reactor and reacted at 200 °C for 10 hours. After cooling to room temperature, the product was collected by magnetic separation, washed three times each with deionized water and anhydrous ethanol, and dried under vacuum at 60 °C for 12 hours to obtain Fe3O4 magnetic nanoparticles.

[0053] 1.2 Synthesis of Fe3O4@SiO2

[0054] Weigh 300 mg of Fe3O4 particles and disperse them in a mixed solvent of 160 mL anhydrous ethanol and 40 mL deionized water. Sonicate the mixture for 30 minutes to ensure uniform dispersion. Then, add 5 mL of ammonia and 1 mL of tetraethyl orthosilicate (TEOS) sequentially, and stir the mixture at 40 °C for 12 hours. After the reaction is complete, collect the Fe3O4@SiO2 composite particles by magnetic separation, wash them three times with deionized water, and vacuum dry them at 60 °C for 12 hours.

[0055] 1.3 Synthesis of Fe3O4@SiO2-CH=CH2

[0056] 250 mg of Fe3O4@SiO2 composite microspheres were ultrasonically dispersed in 50 mL of anhydrous toluene, and 3 mL of methacryloxypropyltrimethoxysilane (MPS) was added. The mixture was stirred at 70 °C for 12 hours under nitrogen protection. After the reaction was completed, the product was collected by magnetic separation, washed with water and dried under vacuum to obtain modified magnetic nanoparticles with surface-grafted double bonds, denoted as Fe3O4@SiO2-CH=CH2.

[0057] 1.4 Preparation of Magnetic Molecularly Imprinted Polymers (MIPs)

[0058] Take 10 mL of solution with a concentration of 1.0 × 10⁻⁶. 8 CFU / mL Klebsiella pneumoniae bacterial suspension was centrifuged at 8000 r / min for 6 minutes, and the bacterial precipitate was collected and resuspended in 20 mmol / L Tris-HCl buffer solution (pH=8.5). 36 mg of acrylamide (functional monomer) and 12 mg of N,N'-methylenebisacrylamide (crosslinking agent) were added sequentially, and the mixture was stirred for 30 minutes until homogeneous. Separately, 40 mg of Fe3O4@SiO2-CH=CH2 was ultrasonically dispersed in 1 mL of Tris-HCl buffer solution for 1 minute. The monomer-containing bacterial suspension was then mixed with the magnetic nanoparticle dispersion and stirred continuously for 1 hour. Subsequently, 120 µL of tetramethylethylenediamine (TEMED) and an appropriate amount of ammonium persulfate (APS) were added to the reaction system, and the mixture was stirred for another 2 hours to initiate the polymerization reaction. After the reaction was completed, the product was collected using magnetic separation technology, yielding magnetically imprinted polymers (MIPs) with specific recognition ability against Klebsiella pneumoniae. As a control, the preparation steps for non-imprinted polymers (NIPs) are the same as above, except that the bacterial culture is replaced with an equal volume of Tris-HCl buffer solution.

[0059] 1.5 Synthesis of ZIF-8@Cur

[0060] Weigh 0.5 mmol of zinc nitrate hexahydrate into a 50 mL round-bottom flask, add 0.7 mL of ultrapure water and 0.58 mmol of triethylamine (TEA) sequentially, and stir until homogeneous. Separately, weigh 100 mg of curcumin (Cur) and dissolve it in 3.3 mL of anhydrous methanol. After ultrasonic-assisted dispersion, add it dropwise to the above zinc salt solution and stir continuously at 37 °C. Simultaneously, weigh 1.67 mmol of 2-aminobenzimidazole and 5.64 mmol of 2-methylimidazole and disperse them in a mixed solvent of 3.3 mL of ultrapure water and 1.7 mL of anhydrous methanol. After complete ultrasonic dissolution, slowly add it dropwise to the above reaction system, and add anhydrous methanol to bring the total volume to 16.7 mL. Continue stirring the mixture at 37 °C for 1 hour. After the reaction is complete, collect the product by centrifugation and wash repeatedly with anhydrous methanol until the supernatant is colorless to remove unloaded free curcumin. The obtained solid product was dried under vacuum at 60°C for 12 hours to obtain ZIF-8@Cur.

[0061] 1.6 Synthesis of ZIF-8@Cur@PBA

[0062] 83 mg (0.5 mmol) of p-carboxyphenylboronic acid was dissolved in 4 mL of anhydrous ethanol. 1 mL of 0.5 mmol EDC and 1 mL of 1 mmol NHS were added sequentially, and the mixture was stirred at 37 °C for 1 hour to activate the carboxyl groups of PBA. Separately, 200 mg of the prepared ZIF-8@Cur was dispersed in 4 mL of anhydrous ethanol and sonicated for 20 minutes to ensure uniform dispersion. The ZIF-8@Cur dispersion was added dropwise to the activated PBA solution, and the mixture was shaken in a 37 °C water bath for 12 hours. After the reaction, 30 mL of ultrapure water was added, and the mixture was allowed to stand for 4 hours to precipitate. The product was collected by centrifugation and repeatedly washed with ultrapure water until the supernatant was colorless and transparent. The product was then vacuum dried at 60 °C for 12 hours to obtain the phenylboronic acid-functionalized ZIF-8@Cur composite material, denoted as ZIF-8@Cur@PBA.

[0063] 2. Material Characterization

[0064] 2.1 Morphology and Particle Size Characterization

[0065] The morphology of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-CH=CH2, MIPs and NIPs was observed using scanning electron microscopy (SEM), and the results are as follows: Figure 3 As shown in (A)-(E), the Fe3O4 particles are spherical with a diameter of approximately 400 nm; the surface is slightly rough after coating with SiO2; the morphology does not change significantly after grafting double bonds; after forming the imprinted layer, a distinct polymer coating layer is visible on the surface of MIPs and NIPs, with the particle size increasing to approximately 520 nm. Dynamic light scattering (DLS) results ( Figure 3 (F)-(J)) were consistent with SEM observations.

[0066] SEM observations of ZIF-8@Cur and ZIF-8@Cur@PBA Figure 4 (A)-(B) show that both exhibit a regular dodecahedral structure, with a slight increase in particle size for ZIF-8@Cur@PBA. DLS results ( Figure 4 (C)-(D)) shows that the average particle size of ZIF-8@Cur is about 300 nm, and that of ZIF-8@Cur@PBA is about 330 nm.

[0067] 2.2 Chemical Structure Characterization

[0068] Fourier transform infrared spectroscopy (FT-IR) analysis confirmed that Fe3O4@SiO2 at 808 cm⁻¹ -1 and 1108 cm -1 Si-O characteristic peaks appear at 1650 cm⁻¹; MIPs and NIPs show peaks at 1650 cm⁻¹. -1A characteristic peak of an amide group appears nearby. ZIF-8@Cur at 1580 cm⁻¹ -1 (C=N), 1145 cm -1 and 990 cm -1 A characteristic peak is observed at the (imidazolium ring) at 3500 cm⁻¹. -1 A curcumin hydroxyl peak appears nearby. ZIF-8@Cur@PBA at 1610 cm⁻¹ -1 (C=O) and 1350 cm -1 A new peak appeared at (BO), confirming the successful grafting of PBA.

[0069] 2.3 Characterization of surface charge and hydrophilicity

[0070] Zeta potential measurements showed that Fe3O4 was +6.2 mV, Fe3O4@SiO2 decreased to -21.6 mV, Fe3O4@SiO2-CH=CH2 increased to -9.6 mV, and MIPs and NIPs changed to +2.5 mV and +4.6 mV, respectively, indicating successful imprint layer coating. ZIF-8@Cur was +12.9 mV, and ZIF-8@Cur@PBA decreased to -1.6 mV, confirming successful PBA modification. The water contact angle measurements were consistent with the changes in surface chemical properties.

[0071] 3. Detection performance evaluation

[0072] 3.1 Feasibility Verification

[0073] Take 1 mg / mL of MIPs and NIPs, and mix them with 1×10 7 CFU / mL Klebsiella pneumoniae was incubated at 37°C for 1 hour. After magnetic separation and washing, ZIF-8@Cur@PBA probe (1 mg / mL) was added and incubated for another 30 minutes. After magnetic separation and washing, ZIF-8 was lysed with 0.1 M hydrochloric acid to release curcumin. After neutralization with 0.1 M NaOH, the UV absorbance was measured. The results are as follows: Figure 2 As shown, the absorbance of the MIPs+ bacteria group was significantly higher than that of the MIPs blank group, while the NIPs group showed no significant change. Visualization images show that the MIPs+ bacteria group solution was a distinct orange, while the blank group was very pale. This indicates the feasibility of the sensor.

[0074] 3.2 Linear Range and Detection Limit

[0075] Under optimal detection conditions (MIPs dosage 1.5 mg, probe concentration 1 mg / mL, incubation pH 8.5, temperature 37℃, MIPs-bacteria incubation 60 min, probe incubation 30 min), MIPs were incubated with different concentrations of Klebsiella pneumoniae (0, 10, ... 1 0.5×10 210 2 0.5×10 3 10 3 0.5×10 4 10 4 10 5 10 6 10 7 10 8 The absorbance was measured using a reaction (CFU / mL). Results are as follows: Figure 18 As shown: the solution color gradually deepens with increasing bacterial concentration. Figure 18 (D)), the absorbance value increases ( Figure 18 (A)). At 0.5×10 3 ~ 10 7 Within the CFU / mL range, the absorbance change (∆Abs) showed a good linear relationship with the bacterial concentration (C). Here, ∆Abs = Absi - Abs0 represents the difference in absorbance before and after bacterial addition; C represents the concentration of Klebsiella pneumoniae, in CFU / mL. The fitted linear regression equation was ∆Abs = 0.06887 C - 0.04568, with a correlation coefficient R² = 0.99378. This equation indicates that, under the detection system of this invention, the absorbance change is directly and positively correlated with the absolute bacterial concentration (CFU / mL), rather than its logarithmic concentration. This makes the interpretation of the detection results more intuitive.

[0076] Determination of the limit of detection (LOD): Under the same detection conditions, 11 parallel determinations were performed on the blank sample (i.e., the sample without Klebsiella pneumoniae), and the standard deviation (δ) of the absorbance change (∆Abs) at the characteristic absorption wavelength was calculated. In this example, the standard deviation δ of the blank sample was determined to be 1.662 × 10⁻⁶. -3 (Retain reasonable decimal places). According to the detection limit calculation formula LOD = 3δ / S, where S is the slope of the standard curve mentioned above (S = 0.06887), the calculated detection limit (LOD) of the sensor of this invention for Klebsiella pneumoniae is: 3 × (1.662 × 10⁻⁶). -3 ) / 0.06887 ≈ 72.4 CFU / mL.

[0077] 3.3 Selectivity and Competitiveness

[0078] Salmonella enteritidis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were selected as interfering bacteria, all at a concentration of 1×10⁻⁶. 7 CFU / mL. Selectivity test ( Figure 19The results showed that the Δ value of MIPs against Klebsiella pneumoniae was significantly higher than that against other interfering bacteria. A competitive experiment (1:1 mixture of target bacteria and interfering bacteria) showed that the presence of interfering bacteria had no significant effect on the detection of Klebsiella pneumoniae, indicating that the sensor has good selectivity.

[0079] 3.4 Anti-interference capability

[0080] Common ions (Na+) were added to the detection system. + K + Mg 2+ Ca 2+ HCO3 - HPO4 2- The results showed that the coexisting substances had no significant impact on the detection results, and the sensor had good anti-interference ability.

[0081] 3.5 Reproducibility and Stability

[0082] Five batches (n=5) of independently synthesized magnetic molecularly imprinted polymers (MIPs) were tested under the same detection conditions for a concentration of 1×10⁻⁶. 7 The absorbance variation (∆Abs) of each batch was recorded using CFU / mL Klebsiella pneumoniae standards. The average ∆Abs (Mean) of the five batches was 0.643, and the standard deviation (SD) was 0.018. The relative standard deviation (RSD) of the sensor response signals for the five batches was calculated to be 2.8%. This RSD value is far below the acceptable 5% variation threshold for conventional biosensors, indicating that the sensor preparation method of this invention is stable and controllable, with minimal batch-to-batch differences and good reproducibility. Figure 20 (A)). The same batch of MIPs, after being sealed at room temperature for 2...0, 1, 2, 4, and 6 weeks, still retained 84.85% of their initial performance after 6 weeks. Figure 20 (B) indicates that the sensor has good reproducibility and storage stability.

[0083] 4. Comparison with existing technologies

[0084] Compared with existing methods for detecting Klebsiella pneumoniae, the sensor of this invention does not require aptamers or antibodies, has a low detection limit (72.4 CFU / mL), and a wide linear range (0.5 × 10⁻⁶ CFU / mL). 3 ~ 10 7 It has a concentration of CFU / mL, enabling visual detection and is magnetic for easy separation, making it suitable for rapid on-site detection.

[0085] Industrial applicability

[0086] The sensor preparation method of this invention is mature and stable, and the raw materials used are all common chemicals, resulting in low cost. The detection process requires no large instruments, is simple to operate, and is very suitable for making detection kits for rapid on-site screening of clinical samples, food, environment, and other fields, showing broad prospects for industrialization.

Claims

1. A bacterial sandwich-structured visual sensor based on magnetic molecular imprinting and ZIF-8 signal amplification, characterized in that, The sensor includes: (a) A magnetic molecularly imprinted polymer, wherein the magnetic molecularly imprinted polymer uses magnetic nanoparticles with surface-grafted double bonds as a carrier and Klebsiella pneumoniae as a template, and forms a molecularly imprinted polymer layer on the surface of the magnetic nanoparticles through surface imprinting polymerization; the molecularly imprinted polymer layer has specific recognition cavities that are complementary to the target bacteria in terms of size, shape and functional groups. (b) A signal probe, wherein the signal probe is a functionalized ZIF-8 material encapsulated with curcumin, and the surface of the functionalized ZIF-8 material is modified with p-hydroxyphenylboronic acid groups that can specifically bind to bacterial surface glycosyl groups. The magnetic molecularly imprinted polymer and the signal probe form a sandwich-structured complex of "magnetic molecularly imprinted polymer-bacteria-signal probe" mediated by the target bacteria.

2. The sensor of claim 1, wherein, The colorimetric molecule is curcumin; the functionalized ZIF-8 material is ZIF-8@Cur@PBA, which is prepared by amide bond condensation reaction of curcumin-loaded ZIF-8 and p-carboxyphenylboronic acid.

3. The sensor of claim 1, wherein, The target bacterium is Klebsiella pneumoniae; the magnetic nanoparticles are Fe3O4 nanoparticles, the surface of which is sequentially coated with a SiO2 layer and grafted with carbon-carbon double bonds; the molecularly imprinted polymer layer is formed by free radical polymerization of the functional monomer acrylamide and the crosslinking agent N,N'-methylenebisacrylamide, thereby constructing a specific recognition cavity on the surface of the magnetic nanoparticles that is complementary to Klebsiella pneumoniae in size, shape and functional groups.

4. A method of manufacturing a sensor according to any one of claims 1 to 3, characterized in that Includes the following steps: P1: Preparation of magnetically imprinted polymers: P1a: Synthesis of Fe3O4 magnetic nanoparticles; P1b: Coating Fe3O4 with SiO2 to obtain Fe3O4@SiO2; P1c: Fe3O4@SiO2 is reacted with methacryloyloxypropyltrimethoxysilane to obtain Fe3O4@SiO2-CH=CH2 with carbon-carbon double bonds grafted on the surface. P1d: Using Fe3O4@SiO2-CH=CH2 as a carrier and Klebsiella pneumoniae as a template, functional monomer acrylamide and crosslinking agent N,N'-methylenebisacrylamide were added, along with initiators tetramethylethylenediamine and ammonium persulfate. Free radical polymerization was carried out under the action of the initiators to form an imprinted layer. P1e: Elution removes the Klebsiella pneumoniae template, yielding a magnetically imprinted polymer; P2: Preparation of signal probes: P2a: Synthesis of ZIF-8@Cur loaded with curcumin; P2b: The p-carboxyphenylboronic acid was subjected to an amidation reaction with ZIF-8@Cur to obtain the ZIF-8@Cur@PBA signal probe; The magnetic molecularly imprinted polymer and the ZIF-8@Cur@PBA signal probe are packaged separately or in combination to obtain the final product.

5. The preparation method according to claim 4, characterized in that, In step P1d, the amount of acrylamide used is 0.5 mmol, the amount of N,N'-methylenebisacrylamide used is 0.08 mmol, and the amount of tetramethylethylenediamine used is 120 μL; the polymerization reaction temperature is 37℃, and the polymerization reaction time is 2 hours.

6. The preparation method according to claim 4, characterized in that, In step P1e, the eluent is a 1% sodium dodecyl sulfonate solution containing 5% acetic acid.

7. The preparation method according to claim 4, characterized in that, In step P2a, the mass ratio of curcumin to zinc nitrate hexahydrate is 0.27 mmol : 0.5 mmol; the reaction time is 1 hour.

8. A visual detection method for Klebsiella pneumoniae on the surface of hospital instruments, utilizing the sensor described in any one of claims 1-3, characterized in that, Includes the following steps: U1: The sample to be tested is brought into contact with the magnetic molecular imprinted polymer described in claim 1 and incubated at 37°C and pH 8.5, so that the magnetic molecular imprinted polymer can specifically capture the target bacteria through the imprinted cavity; U2: After magnetic separation, the signal probe described in claim 1 is added and incubation continues, so that the signal probe binds to the glycosyl groups on the surface of bacteria through the phenylboronic acid groups to form a sandwich structure complex of "magnetic molecularly imprinted polymer-bacteria-signal probe". U3: Magnetic separation and washing of the complex, addition of acidic solution to cleave the ZIF-8 structure, release of chromogenic molecules, observation of solution color change or measurement of its ultraviolet absorbance after neutralization; The darker the solution color or the higher the absorbance value, the higher the concentration of Klebsiella pneumoniae in the sample. This method is used to detect Klebsiella pneumoniae contamination on the surface of hospital instruments, and the results are not directly used for disease diagnosis.

9. The detection method according to claim 8, characterized in that, In step U1, the incubation time is 60 minutes; in step U2, the incubation time is 30 minutes; in step U3, the acidic solution is 0.1 M hydrochloric acid, and neutralization is done with 0.1 M sodium hydroxide.

10. The detection method according to claim 8, characterized in that, The chromogenic molecule is curcumin, and the detection wavelength is 425 nm. Multiple parallel measurements were performed on the blank sample, the standard deviation of the absorbance change value was calculated, and the detection limit was calculated to be less than 100 CFU / mL by combining the slope of the linear regression equation and the three-signal-to-noise ratio method (3σ / k).

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