A visual molecularly imprinted sensor for detecting Salmonella enteritidis

Abstract

This invention discloses a visual molecularly imprinted sensor for detecting Salmonella enteritidis, comprising: magnetic molecularly imprinted polymers (MIPs), a signal amplification probe ZIF-8@CPBA, and gold nanoparticles (AuNPs). The MIPs are prepared by surface imprinting polymerization using Fe3O4@SiO2 with double bonds as a carrier and Salmonella enteritidis as a template. The signal amplification probe ZIF-8@CPBA uses ZIF-8 as a carrier, and its surface is modified with p-carboxyphenylboronic acid. During detection, after the MIPs capture the target bacteria, they form a sandwich structure with the probe. Acid cleavage releases Zn²⁺, inducing the AuNPs to change from red to blue, achieving visual detection. This sensor requires no complex instruments, has a detection limit of 54 CFU / mL, and completes detection in approximately 16 minutes, making it suitable for rapid on-site screening of food products.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically relating to a visual molecular imprint sensor for detecting Salmonella enteritidis. Background Technology

[0002] Salmonella enteritidis, a foodborne pathogen, is one of the leading causes of foodborne illnesses globally and has been listed as a key target for international public health surveillance by the World Health Organization. Achieving rapid, sensitive, and specific early diagnosis of this pathogen is crucial for ensuring food safety and effective public health control. Traditional culture methods, considered the "gold standard" for detection, provide reliable results but take 3-5 days, making them unsuitable for rapid testing. Immunological assays (such as ELISA) are relatively simple to perform and are currently one of the commonly used methods for Salmonella screening. For example, the indirect ELISA method based on flagellin FliC has a concordance rate of up to 94.1% with commercially available kits, demonstrating good application potential. However, these methods still have inherent limitations: dependence on antibody quality, risk of cross-reactivity, long processing time, limited sensitivity, inability to distinguish between dead and live bacteria, and susceptibility to interference from complex matrices. Molecular biology methods are developing rapidly; PCR technology can achieve rapid detection at the gene level, and multiplex PCR can simultaneously detect multiple pathogens. In recent years, new technologies such as CRISPR-Cas, digital PCR, and microfluidics have emerged, but they still face challenges such as cumbersome operation and low adaptability to various scenarios. Therefore, the development of a bacterial detection method that is low-cost, highly selective, sensitive, and rapidly visualized is highly anticipated.

[0003] Molecular imprinting technology (MIT) has shown great potential in the field of pathogen detection due to its ability to prepare polymers with specific recognition sites, predictable structure, recognition specificity, and excellent physicochemical stability. In recent years, researchers have attempted to apply molecularly imprinted polymers (MIPs) to Salmonella detection. For example, one study used lipopolysaccharide (LPS), a characteristic component of the Salmonella outer membrane, as an imprint template to construct a polydopamine-based molecularly imprinted electrochemical sensor, achieving a sensitive detection of 10 CFU / mL while avoiding live bacteria handling [Lee S, Kim H, Kim M, et al. Rapid and simple on-site salmonella detection in food via direct sample loading using alipopolysaccharide-imprinted polymer [J]. Journal of Nanobiotechnology, 2025,23: 279.]. However, most existing MIP sensors rely on fluorescence or electrochemical signal output, still requiring specialized instruments to read the results, making it difficult to achieve truly real-time, on-site visualization detection.

[0004] Based on this, this invention combines the high selectivity of molecular imprinting technology with a visualization signal amplification strategy that uses acid cleavage of ZIF-8 to release Zn²⁺-induced aggregation of gold nanoparticles (AuNPs). Using Fe3O4@SiO2@C=C as a carrier (i.e., Fe3O4@SiO2 surface-modified with carbon-carbon double bonds) and Salmonella enterica as a template, magnetic molecularly imprinted polymers (MIPs) are prepared via surface free radical polymerization as capture units; simultaneously, ZIF-8@CPBA surface-modified with p-carboxyphenylboronic acid (4-CPBA) is synthesized as a signal amplification probe. During detection, the MIPs selectively capture the target bacteria, and after magnetic separation, ZIF-8@CPBA is added, forming a "MIPs-bacteria-ZIF-8@CPBA" sandwich structure; the addition of acid cleavage of ZIF-8 releases a large amount of Zn. 2+ This induces the rapid aggregation of negatively charged AuNPs, causing the solution to change from red to blue, thus enabling rapid, sensitive, and visual detection of Salmonella enteritidis. Summary of the Invention

[0005] To address the shortcomings of existing methods for detecting Salmonella enteritidis, such as complex operation, reliance on large instruments, long detection cycles, and difficulty in achieving visualized on-site detection, this invention provides a visualized molecular imprint sensor for detecting Salmonella enteritidis, enabling highly sensitive, highly specific, and rapid visualized on-site detection of the target bacteria.

[0006] Technical solution: The present invention provides a method for preparing a visual molecularly imprinted sensor for detecting Salmonella enteritidis, comprising the following steps:

[0007] (1) Using Fe3O4@SiO2@C=C as a carrier, Salmonella enterica as a template molecule, acrylamide (AM) as a functional monomer, and N,N'-methylenebisacrylamide (MBA) as a crosslinking agent, an imprinted layer was formed on the carrier surface through free radical polymerization. After eluting the template, magnetic MIPs (such as...) that specifically recognize Salmonella enterica were obtained. Figure 1 (As shown in A). These MIPs can be rapidly separated and enriched using an external magnetic field.

[0008] (2) An amino-containing ZIF-8 (ZIF-8-NH2) was synthesized, and p-carboxyphenylboronic acid (4-CPBA) was grafted onto its surface to obtain ZIF-8@CPBA functionalized material. This material utilizes the property that the phenylboronic acid group can reversibly form a cyclic ester with the vicinal diol structure of bacterial surface lipopolysaccharide (LPS) under weakly alkaline conditions to achieve selective recognition and binding to Salmonella enteritidis (e.g., ...). Figure 1 (As shown in B in the diagram).

[0009] (3) Magnetic microinjectors (MIPs) were used to selectively capture Salmonella enteritidis in complex samples, and matrix interference was removed by magnetic separation. Then, ZIF-8@CPBA was added to the system and incubated under weakly alkaline conditions to allow it to fully bind to the surface of the bacteria already captured on the MIPs, forming a sandwich structure of "MIPs-bacteria-ZIF-8@CPBA". After removing the unbound ZIF-8@CPBA by applying an external magnetic field, hydrochloric acid was added to the enriched product to cleave the ZIF-8 structure, releasing a large amount of Zn²⁺.

[0010] (4) Negatively charged gold nanoparticles (AuNPs) were synthesized using the citrate reduction method. The Zn²⁺ supernatant released from the pyrolysis in step (3) was added to the AuNPs solution. Zn²⁺ induced the aggregation of negatively charged AuNPs, causing a change in their localized surface plasmon resonance (LSPR) properties, and the solution color rapidly changed from red to blue (e.g., ...). Figure 1 (As shown in C). The degree of color change is related to the concentration of the target bacteria. Semi-quantitative or quantitative detection of Salmonella enteritidis can be achieved by visual observation or measurement with a UV-Vis spectrophotometer.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] (1) This invention utilizes ZIF-8 material as a high-density Zn²⁺ signal storage device. After sandwich recognition, Zn is released by acid cleavage of ZIF-8@CPBA bound to the surface of the target bacteria. 2+ And Zn 2+ The release amount is directly related to the number of bacteria bound; compared to directly adding free Zn 2+ This design achieves the effect of bacterial concentration on Zn 2+ Precise control of the release amount transforms the identification signal of trace bacteria into a visible color change corresponding to the bacterial concentration, greatly improving detection sensitivity. Experimental data confirms that the sensor has a detection limit as low as 54 CFU / mL for Salmonella enteritidis.

[0013] (2) This invention combines shape / functional group recognition using molecular imprinting technology with boric acid affinity recognition using ZIF-8@CPBA. Selectivity and competition experiments demonstrate that the MIPs' capture ability for target bacteria is significantly higher than that of common interfering bacteria such as Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae, effectively eliminating non-specific adsorption interference. This dual recognition mechanism ensures accurate anchoring of target bacteria even in complex bacterial environments, greatly improving the selectivity and anti-interference capability of the detection.

[0014] (3) The sensor constructed in this invention has a good linear range (1×10³ to 1×10³). 8 With its high CFU / mL capacity, extremely low detection limit (54 CFU / mL), good reproducibility and time stability (retaining over 90% activity after one month at room temperature), it is suitable for long-term use.

[0015] (4) The sensor constructed in this invention is suitable for rapid on-site screening without the need for large instruments. The results can be visually interpreted by the change in solution color (red → blue), making it very suitable for rapid on-site food safety testing and applications in resource-limited areas. Attached Figure Description

[0016] [ Figure 1 [Schematic diagram of the construction and detection principle of molecularly imprinted sensors. (A) shows the construction process of magnetic molecularly imprinted polymers (MIPs); (B) shows the preparation of ZIF-8@CPBA signal probes; (C) shows the visualization detection process based on the "MIPs-bacteria-ZIF-8@CPBA" sandwich structure.]

[0017] [ Figure 2 Sensor principle verification spectrum (inset from left to right: MIPs, MIPs+SE, NIPs, NIPs+SE)

[0018] [ Figure 3 (a) Zeta potential diagrams of AuNPs, ZIF-8, ZIF-8@CPBA, Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@C=C, MIPs, and NIPs. (b) Infrared spectra of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@C=C, MIPs, NIPs, ZIF-8, and ZIF-8@CPBA.

[0019] [ Figure 4 X-ray diffraction (XRD) patterns of ZIF-8 and ZIF-8-NH2@CPBA.

[0020] [ Figure 5 Dynamic light scattering particle size distribution of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@C=C, MIPs, NIPs, ZIF-8, and ZIF-8@CPBA.

[0021] [ Figure 6 (ac) SEM and EDS elemental distribution and EDS plot of Fe3O4; (df) SEM and EDS elemental distribution of Fe3O4@SiO2@C=C; (gi) SEM and EDS elemental distribution and EDS plot of MIPs; (jl) SEM and EDS elemental distribution and EDS plot of NIPs.

[0022] [ Figure 7 (a) UV-Vis absorption spectra of AuNPs at reaction time; (b) DLS plot of AuNPs after 16 min of reaction.

[0023] [ Figure 8 (a) Detection of different concentrations of Salmonella enteritidis (0, 1×10⁻⁶, 1×10⁻⁶) using MIPs 2 1×10 3 1×10 4 1×10 5 1×10 6 1×10 7 1×10 8 (a) Linearity of CFU / mL; (b) Linear fitting of MIPs; (c) Detection of different concentrations of Salmonella enteritidis (0, 1×10, 1×10) by NIPs. 2 1×10 3 1×10 4 1×10 5 1×10 6 1×10 7 1×10 8(d) Linearity of CFU / mL); (d) Detection of different concentrations of Salmonella enteritidis (0, 1×10, 1×10) by MIPs / NIPs. 2 1×10 3 1×10 4 1×10 5 1×10 6 1×10 7 1×10 8 A visualization of (CFU / mL).

[0024] [ Figure 9 (a) Colorimetric cards with different concentrations of S. enteritidis extracted from a smartphone; (b) Linearity between B / R and S. enteritidis concentration in the colorimetric image.

[0025] [ Figure 10 (a) Selectivity of MIPs against different bacteria (Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae); (b) Competitiveness of MIPs against different bacteria (Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae).

[0026] [ Figure 11 The anti-interference capability of MIPs.

[0027] [ Figure 12 (a) Reproducibility of MIPs; (b) Temporal stability of MIPs.

[0028] [ Figure 13 The sensor detects bacteria in milk.

[0029] [ Figure 14 The sensor rapidly detects Salmonella enteritidis, providing visualized images and RGB color readings. Detailed Implementation Plan

[0030] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments described below are for illustrative purposes only and do not limit the scope of application or extension of the present invention.

[0031] Example 1: Preparation method of visual molecular imprinted sensor for detecting Salmonella enteritidis and performance, morphology and structural characterization of intermediate products.

[0032] 1. Fabrication method of visual molecularly imprinted sensor

[0033] (1) Synthesis of Fe3O4@SiO2@C=C: 0.5 g of Fe3O4 nanoparticles were weighed and dispersed in 50 mL of isopropanol containing 2 mL of ultrapure water. 5 mL of NH3·H2O was slowly added dropwise under mechanical stirring at 700 r / min. After stirring for 5 min, 2.5 mL of TEOS was slowly added dropwise. After the addition was complete, the reaction was carried out at 45 ℃ with stirring for 6 h. After the reaction was completed, the product was washed with ultrapure water and anhydrous ethanol until the supernatant was clear and transparent. It was then dried in a vacuum oven at 60 ℃ for 12 h to obtain Fe3O4@SiO2. 500 mg of the dried Fe3O4@SiO2 was dispersed in 50 mL of toluene and ultrasonically dispersed for 30 min. 5 mL of MPS was added under mechanical stirring at 700 r / min, and the reaction was carried out at 70 ℃ for 12 h. After the reaction was completed, the product was collected with a magnet, washed several times with anhydrous ethanol, and dried under vacuum at 60 ℃ overnight.

[0034] (2) Synthesis of ZIF-8@CPBA: 0.1 mL of triethylamine (TEA) was added to 0.8 mL of an aqueous solution containing Zn(NO3)2·6H2O (0.67 mmol), and the solution was magnetically stirred at 25 °C until completely dissolved. Subsequently, 2-methylimidazole (4.83 mmol) dissolved in 4 mL of ultrapure water and 2-aminobenzimidazole (2.07 mmol) dissolved in 2 mL of anhydrous methanol were added sequentially while stirring. After thorough mixing, 17.4 mL of anhydrous methanol was added to the system, and stirring was continued for 30 min. After the reaction was completed, the resulting suspension was centrifuged at 10000 rpm / min for 5 min, and the supernatant was discarded to collect the precipitate. The precipitate was washed with ethanol until it was colorless, and the resulting solid product was placed in a vacuum drying oven and dried at 60 °C for 12 h to obtain ZIF-8-NH2. 41.5 mg (0.25 mmol) of p-carboxyphenylboronic acid (4-CPBA) was weighed and dissolved in 4 mL of anhydrous ethanol. 1 mL of 0.25 mM LEDC solution was added at 37 °C and stirred for 5 min, followed by the addition of 1 mL of 1 mM NHS solution and stirring for 1 h to activate the carboxyl groups in p-carboxyphenylboronic acid. Separately, 200 mg of ZIF-8-NH2 was weighed and dispersed in 4 mL of anhydrous ethanol, and sonicated for 20 min to ensure uniform dispersion. The ZIF-8-NH2 dispersion was added dropwise to the activated p-carboxyphenylboronic acid solution. After the addition was complete, the reaction was carried out at 37 °C with stirring for 12 h. After the reaction was complete, the product was collected by centrifugation and washed repeatedly with deionized water several times until the supernatant became colorless and transparent. The washed solid product was dried in a vacuum drying oven at 60 °C for 12 h.

[0035] (3) Synthesis of AuNPs: Colloidal AuNPs were synthesized by citrate reduction. In short, 100 mL of HAuCl4 (0.01% w / w) solution was heated to boiling with vigorous stirring, and 10 mL of 38.8 mM trisodium citrate was quickly added. The solution was then heated to a wine-red color and the reaction was stopped. After cooling to room temperature, the solution was stored at 4 °C.

[0036] (4) Preparation of molecularly imprinted and non-imprinted polymers: Take 5 mL of a 1.0×10⁻⁶ polymer solution. 8 CFU / mL Salmonella enteritidis culture was placed in a centrifuge tube and centrifuged at 8000 r / min for 5 min. The supernatant was discarded, and the lower precipitate was collected. 20 mmol / L Tris-HCl buffer (pH 8.5) was added to the precipitate to resuspend the bacteria until completely dispersed. Then, 36 mg AM and 12 mg MBA were added sequentially, and the mixture was stirred for 1 h. Next, 1 mL of Fe3O4@SiO2@C=C (containing 30 mg nanoparticles) dispersed in Tris-HCl buffer was added, and the mixture was stirred for another 1 h. Under argon protection, 120 μL TEMED and 20 mg APS were added to the system, and polymerization was initiated at 37 °C for 2 h. After the reaction, magnetic separation was performed using an external magnetic field. The supernatant was discarded, and the mixture was washed several times with ultrapure water. Subsequently, the mixture was eluted five times with a 2% SDS and 5% HAc solution, and then washed with ultrapure water until neutral. The resulting product was placed in a vacuum drying oven and dried overnight at 60 °C to obtain MIPs. The preparation process of NIPs is the same as that of MIPs, except that the Salmonella enteritidis bacterial culture is replaced with an equal volume of 20 mmol / L Tris-HCl buffer at pH 8.5.

[0037] 2. Visualization of the performance, morphology, and structure of molecularly imprinted sensors and intermediate products

[0038] To verify the successful preparation of the intermediate products and the final polymer, this invention used Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), dynamic light scattering particle size analyzer (DLS), and Zeta potential analyzer to characterize the properties, structure, morphology, and size of the prepared Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@C=C, MIPs, NIPs, ZIF-8, ZIF-8@CPBA, and AuNPs.

[0039] Figure 3(a) shows the Zeta potential diagrams for each material. The potential of gold nanoparticles (AuNPs) is -41.0 mV, which is attributed to the adsorption of citrate anions on their surface, giving them a large negative charge and thus exhibiting excellent colloidal stability. The potential of ZIF-8 is 11.0 mV, exhibiting positive charge. This is due to the protonation of the exposed zinc sites and amino functional groups on the surface after the zinc ions in the ZIF-8 framework coordinate with the imidazole ligands. After modification with 4-carboxyphenylboronic acid, the potential of ZIF-8@CPBA drops to -0.5 mV, approaching electroneutrality. This is mainly because the carboxyl groups are successfully grafted onto the ZIF-8 surface, and the negative charge from their deprotonation partially neutralizes the original positive charge of ZIF-8. The potential of Fe3O4 is -19.0 mV, exhibiting negative charge, which is related to the deprotonation of a large number of hydroxyl groups on its surface. After coating with SiO2, the potential of Fe3O4@SiO2 further decreased to -39.0 mV, with a significant increase in electronegativity. This is due to the abundant Si-OH groups on the SiO2 layer surface undergoing deprotonation in aqueous solution. The potential of MIPs was 3.8 mV, which is due to the partial protonation of the amide groups of the functional monomer AM used in the polymerization system under the test conditions, as well as the adsorption of cations on the surface of the cross-linked polymerization network. The potential of NIPs was 2.2 mV, similar to that of MIPs, also exhibiting a weak positive charge. This indicates that in the absence of template molecules, their polymerization network structure is similar to that of MIPs, and their surface chemical properties are basically the same, hence the small difference in their potentials.

[0040] like Figure 3 As shown in (b), in the infrared spectrum of ZIF-8, 3134 cm⁻¹ -1 and 2923 cm -1 The two peaks are the stretching vibration peaks of CH, at 1579 cm⁻¹. -1 and 1460 cm -1 The peaks at 1145 cm⁻¹ are characteristic of the C=N and C=C rings of the imidazole ring. -1 In addition to the characteristic peaks of Zn-N observed at 3382 cm⁻¹, characteristic peaks of Zn-N can also be observed at 3382 cm⁻¹. -1 and 3463 cm -1 The presence of NH stretching vibration peaks at 1650 cm⁻¹ and NH bending vibration peaks at 1621 cm⁻¹ and 1268 cm⁻¹ indicates the successful synthesis of ZIF-8 containing NH₂. In the spectrum of ZIF-8@CPBA, the peaks at 1650 cm⁻¹ and 1268 cm⁻¹ further confirm the successful synthesis of ZIF-8 containing NH₂. -1 The stretching vibration peak at C=O and at 1340 cm⁻¹ -1 The nearby BO characteristic peaks prove that the phenylboronic acid functional group was successfully introduced, indicating that CPBA was successfully modified on the ZIF-8 surface.

[0041] Furthermore, the formation of Fe3O4@SiO2, Fe3O4@SiO2@C=C, MIPs, and NIPs was also verified using infrared spectroscopy. Figure 3 In (b), Fe3O4 at 570 cm⁻¹ -1 Characteristic absorption was observed at 800 cm⁻¹ in the Fe₃O₄@SiO₂ spectrum. -1 and 1100 cm -1 The corresponding asymmetric stretching of O-Si-O and symmetric vibrations of Si-O indicate successful SiO2 coating. At 1680-1620 cm⁻¹ -1 The occurrence of C=C stretching vibrations indicates that the C=C modification was successful.

[0042] Figure 4 The X-ray diffraction (XRD) patterns of ZIF-8 and ZIF-8@CPBA are shown. The XRD pattern of ZIF-8 is consistent with the theoretical characteristic diffraction peaks. The position of the characteristic diffraction peaks of CPBA is basically unchanged, only the intensity is slightly different, indicating that the crystal form of ZIF-8 has not changed significantly after modification, and the material grafting is successful.

[0043] The size of each material was characterized by its hydration particle size, and the results are as follows: Figure 5 As shown, the Fe3O4 size distribution ranges from 160 to 250 nm. The modified Fe3O4@SiO2@C=C particles exhibit a significant increase in size after two coating processes, with the particle size mainly distributed in the 260-460 nm range. The particle size further increases after free radical polymerization, with MIPs and NIPs mainly distributed in the 400-600 nm range. Their particle size distributions are similar, and there is no significant difference between the imprinted MIPs and NIPs particles, proving the successful synthesis of the imprinted layer.

[0044] The morphological evolution and elemental distribution at each stage of material preparation were characterized using SEM and EDS, and the results are as follows: Figure 6 As shown. By Figure 6 (ac) As can be seen, commercially sourced Fe3O4 exhibits a typical octahedral structure. EDS surface scanning analysis shows that C, N, O, Fe, and Si elements are uniformly distributed within the particles. After SiO2 coating and C=C double bond modification (df), the resulting Fe3O4@SiO2@C=C particles gradually become more spherical and their particle size increases. Simultaneously, the signal intensities of C and Si elements in the EDS spectrum significantly increase, preliminarily confirming the successful grafting of the silicon layer and carbon-carbon double bonds. Further, MIPs and NIPs were prepared via surface-imprinted polymerization. Figure 6As shown in (gl), the surface of the polymerized particles is coated with a dense imprinted layer, exhibiting a smoother and more uniform spherical morphology, with the particle size further increasing to approximately 500 nm. Notably, the MIPs particles show obvious concave structures (gi) on their surface, while the NIPs surface is a complete and smooth sphere (jl). This morphological difference preliminarily confirms that the MIPs have formed imprinted cavities with specific recognition sites for Salmonella enteritidis. Furthermore, EDS elemental analysis shows that the characteristic element Zn appears in the imprinted polymerized material and is evenly distributed. This element originates from the functional monomer AM, providing direct structural evidence for the successful construction of the imprinted layer.

[0045] Figure 7 (b) is the DLS diagram of AuNPs after 16 min of reaction. As shown in the figure, the AuNPs have a uniform particle size distribution with an average particle size of 18-20 nm and a Zeta potential of -42 mV (e.g., Figure 3 (a) indicates that the colloid has excellent stability.

[0046] Example 2: Feasibility Analysis of the Visual Molecular Imprint Sensor for Detecting Salmonella Enteritidis

[0047] To evaluate the feasibility of the prepared MIPs in detecting Salmonella enteritidis, the capture ability of MIPs and NIPs against the target bacteria was investigated under the same experimental conditions. The detection results are as follows: Figure 2 As shown. In the experimental system, the concentration of MIPs or NIPs was 2 mg / mL, and 100 μL (1×10⁻⁶) of Salmonella enteritidis bacterial suspension was added. 8 The ZIF-8@CPBA dispersion (1 mg / mL) was replenished to a total volume of 1 mL with Tris-HCl buffer and incubated at 37 °C for a certain period of time. After magnetic separation, the magnetic complex was washed 2-3 times with ultrapure water to remove unbound bacteria. Then, 1 mL of ZIF-8@CPBA dispersion (1 mg / mL) was added to the system, and the mixture was incubated under weakly alkaline conditions to allow it to fully bind to the bacterial surface. After washing the unbound material with ultrapure water, 200 μL of HCl solution (0.1 M) was added to lyse ZIF-8@CPBA and release Zn. 2+ Take 200 μL of the lysis supernatant, add 500 μL of AuNPs solution, and observe the color change.

[0048] When MIPs are co-incubated with Salmonella enteritidis, the MIPs, due to the recognition cavities formed during preparation that specifically match the characteristic structures on the bacterial surface, can efficiently capture the target bacteria and form a "MIPs-bacteria-ZIF-8@CPBA" sandwich structure with ZIF-8@CPBA. Zn is released through acid cleavage. 2+AuNPs aggregated, causing the solution to change from red to blue, exhibiting a clear visual signal. In contrast, NIPs lacked specific recognition sites and relied solely on non-specific adsorption of small amounts of ZIF-8@CPBA, failing to effectively capture bacteria; therefore, the color of the supernatant showed no significant change after the addition of AuNPs. The effects of MIPs and NIPs without bacterial incubation for the same time were also tested, showing that the supernatant colors of both were essentially identical, indicating that AuNPs could not change color in the absence of bacteria. The experimental results demonstrate that the prepared sensor possesses good specific recognition ability for Salmonella enteritidis, and with signal amplification, it can achieve visual detection of bacteria.

[0049] Example 3: System optimization of the synthesis and detection conditions of the visual molecular imprinting sensor for detecting Salmonella enteritidis.

[0050] (1) Optimization of synthesis time of gold nanoparticles (AuNPs)

[0051] AuNPs were synthesized using the classic sodium citrate reduction method. Trisodium citrate was added to the chloroauric acid solution after boiling. Samples were taken at 11, 12, 13, 14, 15, 16, and 17 mins of reaction. The surface plasmon resonance (SPR) absorption peaks were monitored using UV-Vis absorption spectroscopy, and the color change of the solution was observed. As the reaction time increased from 11 min to 16 min, the SPR absorption peaks of AuNPs gradually strengthened and narrowed, indicating increased nanoparticle yield and a more uniform particle size distribution. At 16 min, the AuNPs solution exhibited a characteristic wine-red color, with the SPR absorption peak located at 520 nm, showing the highest absorption intensity and symmetrical peak shape. Further extending the reaction to 17 min resulted in a slight decrease in absorption peak intensity, possibly due to slight agglomeration of nanoparticles or detachment of stabilizers caused by prolonged heating (e.g., ...). Figure 7 (as shown in a). Therefore, 16 min was chosen as the optimal synthesis time for AuNPs and used in all subsequent experiments.

[0052] (2) Orthogonal optimization of functional monomers and crosslinking agents

[0053] The dosages of the functional monomer acrylamide (AM), the crosslinking agent N,N'-methylenebisacrylamide (MBA), and the catalyst tetramethylethylenediamine (TEMED) in the imprinting polymerization process were optimized using an L9(3³) orthogonal experiment. In the experiment, AM was set at three levels: 0.4 mmol, 0.5 mmol, and 0.6 mmol; MBA at three levels: 0.07 mmol, 0.08 mmol, and 0.09 mmol; and TEMED at three levels: 110 μL, 120 μL, and 130 μL. The experimental results were evaluated by comparing the imprinting factor IF (IF = ΔAbsMIPs / ΔAbsNIPs), where ΔAbs represents the difference in UV absorption of the supernatant before and after the addition of bacteria. Each experiment was repeated three times. Range analysis showed that the order of influence of each factor on the imprinting effect was: AM dosage > MBA dosage > TEMED dosage. By comparing the average IF values ​​of different factors and combining them with the actual visualized color change effects, the optimal dosages of AM, MBA, and TEMED were determined to be 0.5 mmol, 0.09 mmol, and 110 μL, respectively. Under these optimized conditions, the prepared molecularly imprinted polymer achieved an IF of 11.7 against Salmonella enteritidis, and the most significant visualized color change was observed under the aforementioned optimal synthesis conditions, indicating that it has good specific recognition ability against the target bacteria.

[0054] (3) Optimization of the ratio of functional monomers to crosslinking agents

[0055] To obtain better specific recognition and selectivity, based on the orthogonal optimization results, the dosage of functional monomers and crosslinking agents was further investigated: AM, MBA, and TEMED were halved (0.25 mmol, 0.045 mmol, 55 μL) and doubled (1.0 mmol, 0.18 mmol, 220 μL) respectively for comparative experiments. The results showed that when all three were halved, the IF was only 3.9, indicating a significant decrease in recognition ability. This was because the functional monomer, crosslinking agent, and catalyst were insufficient to form an effective crosslinked polymerization network, resulting in a lack of specific recognition sites. When all three were doubled, the IF dropped to 8.2, presumably due to the excessively thick imprinted layer leading to difficulty in template elution, while excessive crosslinking enhanced non-specific adsorption. Therefore, the orthogonally optimized ratio (AM 0.5 mmol, MBA 0.09 mmol, TEMED 110 μL) was confirmed as the optimal dosage combination, and this ratio was used in subsequent experiments.

[0056] (4) Optimization of imprinting time and temperature

[0057] Under constant synthetic conditions, the imprinting effect was investigated at polymerization times of 1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h. The results showed that the IF (intercalation efficiency) was 6.1 at a polymerization time of 1.0 h. This short reaction time resulted in incomplete free radical polymerization, insufficient cross-linking network formation, and difficulty in forming a structurally stable imprinted layer around the bacterial template. When the polymerization time was extended to 1.5 h, the IF increased to 7.9, reaching a maximum of 11.7 at 2.0 h. Further extension to 2.5 h and 3.0 h resulted in a decrease in the IF to 7.4 and 6.9, respectively. This may be due to excessive growth of the imprinted layer, with the polymer accumulating too thickly on the bacterial surface. This not only increases the difficulty of subsequent template elution but may also cause the imprinted cavities to collapse or deform due to excessive cross-linking, thus reducing the effectiveness of the recognition sites. Therefore, the optimal polymerization time was 2.0 h.

[0058] Under constant synthetic conditions, the imprinting effect was investigated at polymerization temperatures of 30 ℃, 37 ℃, and 45 ℃. The results showed that the inertial fraction (IF) was 10.5 at 30 ℃; the IF reached its maximum value of 11.7 at 37 ℃, a temperature that ensured stable polymerization and the formation of a regular imprinted network while avoiding potential damage to the template bacterial structure from high temperatures. Further increasing the temperature to 45 ℃ resulted in a decrease in IF to 9.5. This is because excessively high temperatures lead to rapid free radical generation, potentially causing localized burst polymerization and resulting in an uneven imprinted layer structure. Furthermore, excessive heat may cause denaturation or conformational changes in bacterial surface proteins, disrupting the original structural features of the template molecules and thus affecting recognition ability. Therefore, the optimal polymerization temperature is 37 ℃.

[0059] (5) Optimization of template usage

[0060] Under fixed synthetic conditions, the concentration of Salmonella enteritidis template at 1×10⁻⁶ was investigated. 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL and 1×10 8 Imprinting effect at CFU / mL. When the template concentration is 1×10⁻⁶... 5 At CFU / mL, the IF was 7.2; increasing to 1×10⁻⁶ CFU / mL resulted in an IF of 7.2. 6 At CFU / mL, the IF increased to 11.2; further increases to 1×10⁻⁶ 7 At CFU / mL, the IF was 10.9; when the template concentration reached 1×10⁻⁶, the IF was 10.9. 8 At CFU / mL, the IF reached its maximum value of 11.7. Therefore, the optimal template dosage was determined to be 1×10⁻⁶. 8 CFU / mL.

[0061] (6) Optimization of elution solvent

[0062] The choice of eluent has a significant impact on the removal efficiency of template molecules and the regeneration ability of imprinted sites. This invention investigated the effects of six different eluent compositions on the recognition performance of MIPs. When 5% HAc was used alone as the eluent, the IF was 10.0; when 1% SDS was used alone, the IF was 11.0. Combining both significantly improved the elution effect; the IF for 5% HAc + 1% SDS was 11.7, and the IF for 5% HAc + 2% SDS reached 12.7, the highest among all experimental groups. Further increasing the HAc concentration to 10% and combining it with 1% SDS resulted in an IF of 12.3; the IF for 10% HAc + 2% SDS was 12.5, both lower than that of 5% HAc + 2% SDS. These results indicate that the combination of 5% HAc + 2% SDS most effectively removes template molecules while better preserving the structural integrity of the imprinted cavity, thus determining it as the optimal elution condition.

[0063] (7) Optimization of the amount of MIPs / NIPs particles and ZIF-8@CPBA particles

[0064] The amount of MIPs / NIPs particles directly determines the number of recognition sites in the system, thus affecting the capture efficiency and non-specific adsorption level of the target bacteria. With other detection conditions fixed, the detection signal response was examined at MIPs / NIPs particle concentrations of 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, and 2.5 mg / mL. At a particle concentration of 1.0 mg / mL, the IF was 7.3, indicating a limited number of recognition sites and low target bacteria capture efficiency. Increasing the concentration to 1.5 mg / mL raised the IF to 9.3. Further increasing to 2.0 mg / mL, the IF reached its maximum value of 12.7, indicating that the imprinted cavities on the MIPs surface could most effectively capture the target bacteria at this concentration. Further increasing to 2.5 mg / mL, the IF decreased to 9.3, possibly because the high particle concentration led to increased aggregation, reducing the dispersibility and accessibility of effective recognition sites, while non-specific adsorption also increased accordingly. Therefore, the optimal MIPs / NIPs concentration was determined to be 2.0 mg / mL.

[0065] With the MIPs / NIPs concentration fixed at 2.0 mg / mL and other detection conditions unchanged, the detection signal response was investigated at ZIF-8@CPBA particle concentrations of 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL. When the ZIF-8@CPBA concentration was 0.5 mg / mL, the IF was 6.0, indicating a limited number of particles dispersed in the solution and fewer probes capable of recognizing and adsorbing onto the bacterial surface. Increasing the concentration to 1.0 mg / mL, the IF reached its maximum value of 12.7, indicating that at this concentration, ZIF-8@CPBA could fully bind to the bacteria captured by the MIPs, forming a stable sandwich structure. Further increasing the concentration to 1.5 mg / mL and 2.0 mg / mL, the IF decreased to 7.2 and 5.3, respectively. This is because excess ZIF-8@CPBA particles in the solution were non-specifically carried down during magnetic separation, leading to an increase in background signal. Therefore, the optimal concentration of ZIF-8@CPBA was determined to be 1.0 mg / mL.

[0066] (8) Optimization of incubation solvent

[0067] The experiment further investigated the effects of different incubation solvent systems on the recognition performance of MIPs in order to screen the media environment most conducive to specific binding. This invention examined the effects of five incubation solvents on the detection performance: water, Tris-HCl buffer (pH 8.5), PBS buffer (pH 8.5), Tris-HCl / water (1:1, v / v), and PBS / water (1:1, v / v). When water was used as the incubation solvent, the IF was only 5.7, indicating significant non-specific adsorption. When Tris-HCl buffer (pH 8.5) was used as the incubation solvent, the IF increased to 12.7. When PBS buffer (pH 8.5) was used as the incubation solvent, the IF reached the highest value of 14.1, indicating the best specific recognition performance. In the mixed solvent system, the IF of Tris-HCl / water (1:1, v / v) was 9.3, and the IF of PBS / water (1:1, v / v) was 13.3, both lower than the corresponding pure buffer systems, indicating that the introduction of water enhances non-specific adsorption, leading to a decrease in IF. Therefore, the optimal incubation solvent was determined to be PBS buffer (pH 8.5).

[0068] (9) Optimization of incubation time

[0069] The detection signal response of MIPs / NIPs with bacteria was examined at incubation times of 0, 5, 10, 20, 30, 40, and 60 min. The results showed that the ΔAbs value gradually increased with increasing incubation time, reaching a plateau at 30 min and then remaining unchanged, indicating that the adsorption of bacteria by the imprinted cavities on the MIPs surface had reached saturation. Therefore, the optimal incubation time was 30 min. The detection signal response of ZIF-8@CPBA with secondary incubation times of 0, 5, 10, 20, 30, 40, and 60 min was also examined. The results showed that ΔAbs reached equilibrium at 20 min and then stopped increasing. Therefore, the optimal secondary incubation time was 20 min.

[0070] Example 4: Application of the described visual molecular imprinting sensor for detecting Salmonella enteritidis

[0071] The experimental conditions in this embodiment were as follows: the amount of molecularly imprinted polymers (MIPs) was 2 mg / mL, the amount of ZIF-8@CPBA was 1 mg / mL, and the incubation solvent was PBS buffer (pH 8.5). The incubation time of MIPs with Salmonella enteritidis was 30 min, and the secondary incubation time with ZIF-8@CPBA was 20 min, with an incubation temperature of 37 ℃. Specifically, 1 mL of a 2 mg / mL MIPs dispersion (PBS, pH 8.5) was taken, and standard bacterial solutions of different concentrations were added. The mixture was incubated at 37 ℃ with shaking for 30 min. Magnetic separation was performed, and the mixture was washed with ultrapure water to remove unbound bacteria. 1 mL of 1 mg / mL ZIF-8@CPBA dispersion was added to the resulting magnetic complex, and the mixture was incubated at 37 ℃ with shaking for another 20 min. After magnetic separation and washing with ultrapure water, 200 μL of 0.1 M hydrochloric acid was added to the precipitate to lyse ZIF-8@CPBA and release zinc ions. After magnetic separation, 200 μL of the supernatant was taken and 500 μL of AuNPs solution was added, then mixed thoroughly. The color changes of each reaction system were recorded, and the absorbance was measured at 520 nm, denoted as Abs. i The change in absorbance due to the addition of bacteria in the system is expressed by the formula ∆Abs = Abs i -Abs0 (Abs0 is the absorbance before the addition of bacteria, Abs...) i (The absorbance after adding bacteria) is calculated.

[0072] (1) Linear and visual analysis of the described visual molecular imprinting sensor for detecting Salmonella enteritidis

[0073] like Figure 8 As shown, bacterial concentrations at 0, 1×10¹, 1×10², 1×10³, and 1×10¹ were investigated. 4 1×105 1×10 6 1×10 7 1×10 8 Within the CFU / mL range, the changes in UV absorption intensity of the MIPs and NIPs detection systems with bacterial concentration are shown in the following figures. Figure 8 As shown in (a), when the bacterial concentration is 1×10 3 -1×10 8 Within the CFU / mL range, the UV absorption intensity of the MIPs detection system showed a linear change. The linear regression equation was calculated based on the relationship between the change in absorbance of the supernatant, ΔAbs, and the bacterial concentration. The calculated linear regression equation was ΔAbs = 0.0280 Lgc - 0.0499 (…). Figure 8 (b) According to the formula for calculating the limit of detection (LOD), LOD = 3δ / k (where δ is the standard deviation of the results obtained from 10 blank sample tests, and k is the slope of the linear regression equation), the calculated limit of detection is 54 CFU / mL. However, because the NIPs detection system lacks a blot cavity for specific bacterial recognition, its UV absorption intensity did not show a clear and regular change when different concentrations of bacteria were present. Figure 8 c). From Figure 8 (d) Visualization of MIPs and NIPs shows that as the bacterial concentration increases, the color of MIPs also changes. The higher the bacterial concentration, the bluer the color of MIPs. However, the color of NIPs is not significantly related to the change in bacterial concentration, indicating that MIPs have good visualization detection capabilities.

[0074] To increase the convenience of the developed sensor, it was integrated with smartphone RGB color extraction software. The software was used to extract the RGB (red, green, blue) values ​​of the solution color when MIPs detected different concentrations of *S. enteritidis*, creating a colorimetric chart corresponding to the concentration. For example... Figure 9 The experimental results shown demonstrate the feasibility of using a smartphone combined with a colorimetric card for bacterial detection. As bacterial concentration increases, the ratio of the blue channel to the red channel (B / R) in the colorimetric image exhibits a significant upward trend. Within the range of 10⁻¹⁰... 8 CFU / mL range fit R 2 =0.9920, and the linear equation is B / R = 0.0563Lgc + 0.625. The results indicate that using a smartphone to acquire colorimetric signals enables quantitative detection of *S. enteritidis*, offering advantages such as ease of operation, rapid response, and suitability for on-site detection, providing an effective method for portable detection of pathogenic microorganisms.

[0075] (2) Selectivity and competition analysis of the visual molecular imprinting sensor used for detecting Salmonella enteritidis.

[0076] To evaluate the specific recognition ability of the prepared MIPs against Salmonella enteritidis, this invention selected four non-target strains, including Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae, for interference testing (bacterial concentration in solution was 1×10⁻⁶). 7 (CFU / mL), with NIPs as a control. Figure 10 As shown in (a), the response values ​​of MIPs to the target bacterium *Salmonella enterica* were significantly higher than their responses to the four competing bacteria. This result clearly indicates that recognition sites matching the spatial configuration of *Salmonella enterica* are formed on the surface of MIPs, enabling effective capture of the target bacterium. Notably, the response of MIPs to *Escherichia coli* was slightly higher than that to the other three interfering bacteria. This may be due to non-specific adsorption caused by the similar functional groups and physical sizes on the surfaces of *Salmonella enterica* and *Escherichia coli*, but its response value was still less than half that of the target bacteria, confirming the good selectivity of MIPs.

[0077] To further simulate the impact of multi-bacterial coexistence environment in actual samples on the recognition effect, this invention tested the capture ability of MIPs for Salmonella enteritidis in a mixed system of five bacteria. Figure 10 (b) Experimental data show that in mixed bacterial solutions containing all competing bacteria, MIPs maintained a high response value to Salmonella enteritidis, which was completely consistent with the response value in the single target bacteria system. The experimental results indicate that the specific sites on MIPs were not occupied or blocked by other competing bacteria, enabling them to accurately identify target bacteria in complex bacterial communities; meanwhile, the response of NIPs in mixed bacterial solutions was much lower than that of MIPs, further eliminating the interference of substrate non-specific adsorption.

[0078] (3) Analysis of the anti-interference, reproducibility and time stability of the visual molecular imprinting sensor used for detecting Salmonella enteritidis.

[0079] To investigate the ability of the prepared MIPs sensor to detect real samples, Mg, a common material in the environment, was selected. 2+ Ca 2 + Na + K + HCO3 - SO4 2- L-proline and glucose were used as interfering ions, with each ion concentration set at 50 μM and the bacterial concentration at 1×10⁻⁶. 7 Its anti-interference performance was investigated using CFU / mL. The results are as follows: Figure 11As shown, the detection results of bacteria by MIPs fluctuate slightly in the presence of interfering ions, but overall they are almost unaffected. This indicates that the sensor has good anti-interference performance in the presence of complex ions and is expected to complete the detection of Salmonella enteritidis in complex matrices.

[0080] To investigate the differences between the prepared sensors, five batches of MIPs were prepared simultaneously for the detection of Salmonella enteritidis. The results are as follows: Figure 12 As shown in (a), the ΔAbs values ​​of the five different batches of MIPs remained basically unchanged when detecting bacteria, indicating that the sensor has good reproducibility.

[0081] To investigate the time stability of the prepared sensor, MIPs prepared in the same batch were stored for 0, 1, 2, 3, 4, and 5 weeks and then used to detect bacteria at the same concentration. The results are as follows: Figure 12 As shown in (b), the ΔAbs signal response value of the sensor remains basically unchanged as the placement time increases. After one month of placement, its signal response value is still more than 90% of the initial value, indicating that the sensor has little impact on stability after long-term placement.

[0082] (4) Spiking and recovery experiment of the visual molecularly imprinted sensor for detecting Salmonella enteritidis in actual food samples.

[0083] To evaluate the detection performance of the developed molecularly imprinted sensor in a real food matrix, milk was selected as a representative sample for a spiked recovery experiment. First, different concentrations of Salmonella enteritidis standard bacterial solutions were added to milk samples diluted 10 times without the target bacteria, with three spiking levels (low, medium, and high) set. 5 10 6 10 7 (CFU / mL). Subsequently, each spiked sample was detected using a MIP sensor, with each concentration measured three times. The experimental results are as follows: Figure 13 As shown, the sensor achieved spiked recoveries of Salmonella enteritidis in milk matrix ranging from 93% to 105%, with relative standard deviations (RSDs) all below 5%, indicating good accuracy and repeatability. Furthermore, the recovery rate remained stable at different concentration levels, suggesting that other components in milk did not significantly interfere with the recognition and response of the MIPs sensor, demonstrating good matrix adaptability and reliability in practical food sample detection.

[0084] Example 5: Rapid detection and analysis of the visual molecular imprinting sensor for detecting Salmonella enteritidis.

[0085] To address the limitations of traditional bacterial detection methods, which are complex, time-consuming, and reliant on large instruments, this invention constructs a rapid bacterial detection method based on smartphone image recognition (RGB channel analysis), aiming to achieve on-site, immediate, and low-cost detection of pathogenic microorganisms.

[0086] Experimental conditions: MIPs dosage was 2.0 mg / mL, ZIF-8@CPBA dosage was 1.0 mg / mL, incubation solvent was PBS buffer (pH 8.5), and incubation temperature was 37℃. Due to the high target bacterial concentration (1×10⁻⁶) in rapid detection... 7 (CFU / mL) MIPs bind to bacteria rapidly in their surface-imprinted cavities. While maintaining distinguishable color signals, the incubation time can be appropriately shortened without affecting the interpretation results. Adding MIPs to the sample (1×10⁻⁶ CFU / mL) allows for specific identification of target bacteria. 7 After adding CFU / mL, a short incubation period (e.g., 5-20 minutes) is sufficient for efficient capture of target bacteria through specific binding to pre-existing pores on the surface of MIPs. Subsequently, ZIF-8@CPBA is added to the system, and incubation continues for several minutes to allow the bacteria to bind. Figure 14 Experimental results showed that when MIPs were incubated with bacteria for 10 minutes, followed by co-incubation with ZIF-8@CPBA for 6 minutes, the AuNPs solution in the system exhibited a clear and distinguishable color difference, gradually changing from red under conditions of no bacteria or low bacterial concentration to blue-purple under conditions of high bacterial concentration. The rapid detection time was only 16 minutes. This strategy provides a simple, rapid, and easily scalable technical approach for food safety monitoring, rapid clinical diagnosis, and environmental microbial contamination assessment.

Claims

1. A visual molecularly imprinted sensor for detecting Salmonella enteritidis, characterized in that, The sensor comprises: magnetic molecularly imprinted polymers (MIPs), a signal amplification probe ZIF-8@CPBA, and gold nanoparticles (AuNPs). The MIPs are prepared via surface free radical polymerization using Fe3O4@SiO2 with carbon-carbon double bonds as a carrier, Salmonella enterica as a template, acrylamide as a functional monomer, and N,N'-methylenebisacrylamide as a crosslinking agent. The signal amplification probe ZIF-8@CPBA uses ZIF-8 as a carrier, with its surface modified with p-carboxyphenylboronic acid. During detection, after the MIPs capture the target bacteria, they form a "MIPs-bacteria-ZIF-8@CPBA" sandwich structure with ZIF-8@CPBA, releasing Zn via acid cleavage. 2+ The Zn 2+ The aggregation of negatively charged AuNPs on the surface is induced, causing the solution to change from red to blue, thus enabling visual detection.

2. A visual method for detecting Salmonella enteritidis, characterized in that, Includes the following steps: (a) Provides magnetic molecularly imprinted polymers (MIPs), ZIF-8@CPBA probes with p-carboxyphenylboronic acid modified on the surface, and gold nanoparticles (AuNPs); wherein the MIPs are prepared by surface free radical polymerization reaction using Fe3O4@SiO2 with carbon-carbon double bonds modified on the surface as a carrier, Salmonella enterica as a template, acrylamide as a functional monomer, and N,N'-methylenebisacrylamide as a crosslinking agent; (b) Incubate MIPs with a sample containing the target bacteria to allow the MIPs to capture Salmonella enterica in the sample. After magnetic separation, add ZIF-8@CPBA probe for incubation to form a "MIPs-bacteria-ZIF-8@CPBA" sandwich structure. (c) After magnetic separation and washing, acid is added to cleave ZIF-8@CPBA and release Zn. 2+ Take the lysate and mix it with AuNPs. Based on the color change of the solution from red to blue caused by the aggregation of AuNPs, determine whether the sample contains Salmonella enteritidis. (d) Optionally, the absorbance at 520 nm is measured using a UV-Vis spectrophotometer to quantitatively detect Salmonella enteritidis.

3. A signal amplification probe, characterized in that, This includes metal-organic framework materials based on ZIF-8, whose surface is modified with p-carboxyphenylboronic acid. The probe cleaves and releases Zn under acidic conditions. 2+ It is used to induce the aggregation of negatively charged AuNPs on the surface to produce a visible color change.

4. The sensor as described in claim 1, characterized in that, The method for preparing the MIPs includes the following steps: (1) Fe3O4 nanoparticles were dispersed in isopropanol, ammonia and tetraethyl silicate were added, and the reaction was carried out at 45 °C for 6 h. After washing and drying, Fe3O4@SiO2 was obtained. Fe3O4@SiO2 was dispersed in toluene, 3-methacryloyloxypropyltrimethoxysilane (MPS) was added, and the reaction was carried out at 70 °C for 12 h. After magnetic separation, washing and drying, Fe3O4@SiO2 with carbon-carbon double bonds on the surface was obtained. (2) Using Salmonella enteritidis as a template, it was pre-assembled with acrylamide (AM) and N,N'-methylenebisacrylamide (MBA) in Tris-HCl buffer, and Fe3O4@SiO2 with carbon-carbon double bonds on its surface was added. Tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) were added under inert gas protection, and free radical polymerization was initiated at 37 °C for 2 hours. (3) After the reaction is completed, the product is separated by an external magnetic field, the template bacteria are eluted with an eluent, washed with ultrapure water until neutral, and dried under vacuum to obtain magnetic molecularly imprinted polymers (MIPs); the preparation method of non-imprinted polymers (NIPs) is the same except that template bacteria are not added.

5. The sensor as described in claim 1, characterized in that, The preparation method of the signal amplification probe ZIF-8@CPBA includes the following steps: Zn(NO3)2·6H2O was dissolved in water, and triethylamine was added. The mixture was stirred at 25 °C until completely dissolved. 2-Methylimidazole aqueous solution and 2-aminobenzimidazole methanol solution were added sequentially, and stirring was continued for 30 min. After centrifugation, washing, and drying, ZIF-8-NH2 was obtained. The carboxyl group of p-carboxyphenylboronic acid was activated with EDC / NHS at 37 °C for 1 h. The activated p-carboxyphenylboronic acid was reacted with ZIF-8-NH2 at 37 °C for 12 h. After centrifugation, washing, and drying, ZIF-8@CPBA was obtained.

6. The sensor as described in claim 1, characterized in that, The gold nanoparticles (AuNPs) were prepared by citrate reduction: 100 mL of 0.01% HAuCl4 solution was heated to boiling under vigorous stirring, 10 mL of 38.8 mM trisodium citrate was quickly added, and the reaction was stopped when the solution turned wine red. After cooling to room temperature, the solution was stored at 4 °C.

7. The method as described in claim 2, characterized in that, In step (b), the concentration of the MIPs in the incubation system is 1.0~2.5 mg / mL, preferably 2.0 mg / mL; the concentration of the ZIF-8@CPBA in the incubation system is 0.5~2.0 mg / mL, preferably 1.0 mg / mL; the incubation solvent is PBS buffer; the incubation time of the MIPs and the sample is 5~60 min, preferably 30 min; the incubation time of the ZIF-8@CPBA is 5~60 min, preferably 20 min; more preferably, when the incubation time of the MIPs and the sample is 10 min and the incubation time of the ZIF-8@CPBA is 6 min, the total detection time is about 16 min, which is suitable for rapid on-site visual screening.

8. The sensor as described in claim 4, characterized in that, In step (2), the amount of the functional monomer acrylamide is 0.4~0.6 mmol, preferably 0.5 mmol; the amount of the crosslinking agent N,N'-methylenebisacrylamide is 0.07~0.09 mmol, preferably 0.09 mmol; the amount of the catalyst tetramethylethylenediamine is 110~130 μL, preferably 110 μL; and the concentration of the template bacteria is 1×10⁻⁶. 5 ~1×10 8 CFU / mL, preferably 1×10⁻⁶ 8 CFU / mL.

9. The sensor as described in claim 4, characterized in that, The eluent in step (3) is a mixed solution of acetic acid and sodium dodecyl sulfate, the composition of which is selected from: 5% acetic acid, 1% sodium dodecyl sulfate, 5% acetic acid + 1% sodium dodecyl sulfate, 5% acetic acid + 2% sodium dodecyl sulfate, 10% acetic acid + 1% sodium dodecyl sulfate, 10% acetic acid + 2% sodium dodecyl sulfate, preferably 5% acetic acid + 2% sodium dodecyl sulfate.

10. The visual molecularly imprinted sensor for detecting Salmonella enteritidis as described in claim 1, characterized in that, The sensor has a linear detection range of 1×10³ ~ 1×10⁻⁶ for Salmonella enteritidis. 8 The detection limit is 54 CFU / mL, and the rapid detection takes only 16 minutes.

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