Photonic crystal hydrogel sensor for visually detecting drug-resistant bacteria as well as preparation method and application of photonic crystal hydrogel sensor

Abstract

The invention relates to the technical field of biomedical materials, and discloses a photonic crystal hydrogel sensor for visually detecting drug-resistant bacteria as well as a preparation method and application of the photonic crystal hydrogel sensor. The preparation method comprises the following steps: immersing a glass sheet into an aqueous dispersion of colloidal photonic crystal microspheres, and depositing a photonic crystal structure to obtain a photonic crystal template; assembling the modified cover glass and a photonic crystal template into a sandwich structure, injecting the pre-polymerization liquid into the sandwich structure, and performing thermal polymerization to obtain photonic crystal hydrogel; and immersing the photonic crystal hydrogel into a reduction solution for a reduction reaction, washing a reaction product, and carrying out a cross-linking reaction on the reaction product and an enzyme response cross-linking agent solution to obtain the photonic crystal hydrogel sensor. According to the photonic crystal hydrogel sensor, the enzyme response cross-linking agent is broken through beta-lactamase secreted by metabolism of drug-resistant bacteria to cause swelling of hydrogel, so that the lattice spacing of the photonic crystal hydrogel is changed, visual color change is caused, and rapid identification and quantitative detection of the drug-resistant bacteria are realized.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical materials, and in particular to a photonic crystal hydrogel sensor for visually detecting drug-resistant bacteria, and a preparation method and application thereof. Background Art

[0002] Antibiotic resistance (AMR) is a major global public health challenge. The spread of drug-resistant bacteria makes many infectious diseases difficult to control, increasing the difficulty of treatment and the risk of death. Approximately 80% of drug-resistant bacteria (such as methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Enterobacteriaceae) secrete β-lactamases during colonization, which hydrolyze β-lactam antibiotics (such as penicillins and cephalosporins), rendering them ineffective, prolonging treatment, and increasing healthcare costs. Accurate detection of drug-resistant bacteria can provide clinical treatment guidance, avoid unnecessary antibiotic use, and curb the spread of drug-resistant bacteria. This is not only key to improving patient outcomes but also a vital strategy for addressing the global AMR crisis. However, traditional detection methods such as culture and PCR are time-consuming and complex, making them inadequate for rapid diagnosis in emergency situations. Therefore, the development of rapid, sensitive, and specific detection technologies for identifying drug-resistant bacteria is crucial.

[0003] In recent years, novel sensing strategies based on bacterial metabolic signatures have garnered significant attention. Enzymes, virulence factors, acidic metabolites released during bacterial colonization, or changes in pH and temperature within the infection microenvironment, can all serve as sensing targets for bacterial detection. β-lactamase, a hallmark metabolite of drug-resistant bacteria, provides an ideal target for specific detection of drug-resistant bacteria. By designing sensing materials that specifically interact with β-lactamase, rapid identification and quantitative detection of drug-resistant bacteria can be achieved.

[0004] Hydrogel materials, due to their stimulus-responsive properties, can achieve the capture and signal conversion of target metabolites through the dynamic reconstruction of cross-linked networks. At the same time, photonic crystals, as periodic nanostructured materials, can produce structural colors due to their photonic bandgap properties and produce sensitive optical responses to external stimuli (such as changes in refractive index and lattice period) through Bragg diffraction. Based on this, it is of great significance to combine the properties of photonic crystals with hydrogels to develop new materials that can specifically interact with β-lactamase, thereby achieving rapid identification and quantitative detection of drug-resistant bacteria. Summary of the Invention

[0005] The present application provides a photonic crystal hydrogel sensor for visual detection of drug-resistant bacteria, and its preparation method and application, aiming to solve the technical problem of the difficulty in rapid identification and quantitative detection of drug-resistant bacteria in the prior art.

[0006] In order to achieve the above objectives, this application adopts the following technical solutions.

[0007] In a first aspect of the present application, a method for preparing a photonic crystal hydrogel sensor for visually detecting drug-resistant bacteria is provided, comprising:

[0008] S1, immersing a glass slide in an aqueous dispersion of colloidal photonic crystal microspheres to deposit a photonic crystal structure and obtain a photonic crystal template;

[0009] S2, assembling the modified cover glass and the photonic crystal template into a sandwich structure, injecting the prepolymer solution into the sandwich structure, and obtaining the photonic crystal hydrogel through thermal polymerization;

[0010] The prepolymer solution comprises a polymerizable monomer, a disulfide bond crosslinking agent, N,N'-methylenebisacrylamide, an initiator, a catalyst and a solvent;

[0011] S3, immersing the photonic crystal hydrogel in a reducing solution to perform a reduction reaction, washing the reaction product, and then performing a cross-linking reaction with an enzyme-responsive cross-linking agent solution to obtain a photonic crystal hydrogel sensor.

[0012] Preferably, the colloidal photonic crystal comprises polystyrene or poly(styrene-methyl methacrylate-acrylic acid);

[0013] The content of the colloidal photonic crystal microspheres in the aqueous dispersion of the colloidal photonic crystal microspheres is 0.05 to 0.15 wt %;

[0014] The temperature for depositing the photonic crystal structure is 50° C. to 60° C., and the humidity is 50% to 60%.

[0015] Preferably, the modified cover glass is a cover glass with polymerizable methacrylate groups introduced on the surface;

[0016] The gap of the sandwich structure is 100 to 500 μm.

[0017] Preferably, the polymerizable monomer includes any one of acrylamide, N-isopropylacrylamide or methacrylic acid;

[0018] The disulfide bond cross-linking agent is N,N'-bisacryloyl-L-cystine;

[0019] The initiator is ammonium persulfate or potassium persulfate;

[0020] The catalyst is tetramethylethylenediamine or 3-dimethylaminopropionitrile;

[0021] The solvent is deionized water.

[0022] Further preferably, in the prepolymer solution, the concentration of acrylamide is 70-100 mg / mL, the concentration of N,N'-methylenebisacrylamide is 0.75-2.16 mg / mL, the concentration of N,N'-bisacryloyl-L-cystine is 5-30 mg / mL, the concentration of ammonium persulfate is 0.57-1.1 mg / mL, and the concentration of tetramethylethylenediamine is 0.28-0.57 mg / mL.

[0023] Further preferably, the preparation method of the N,N'-bisacryloyl-L-cystine comprises:

[0024] Sodium hydroxide and L-cystine were dissolved in methanol, acryloyl chloride was added at 0°C for reaction, and the liquid phase was collected by filtration after the reaction;

[0025] The liquid phase is added to cold ether for precipitation, and the solid is collected to obtain N,N'-bisacryloyl-L-cystine.

[0026] Preferably, the preparation method of the enzyme-responsive cross-linking agent comprises:

[0027] 7-Amino-3-chloromethyl-3-cephazoline-4-carboxylic acid p-methoxybenzyl hydrochloride was dissolved in anhydrous dichloromethane. Triethylamine was slowly added under an ice bath and nitrogen protection, followed by the addition of N-methylmorpholine and 4-aminothiophenol. The solvent was removed from the reaction solution, and the solution was purified by silica gel column chromatography to obtain compound 1.

[0028] Compound 1, 3-maleimidopropionic acid, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate were dissolved in anhydrous N,N-dimethylformamide. Under nitrogen protection, N,N-diisopropylethylamine was added to react. The reaction solution was extracted with dichloromethane and water, washed, dried, and purified by flash column chromatography to obtain compound 2.

[0029] Compound 2 was added to a mixture of trifluoroacetic acid, anisole, and dichloromethane in a volume ratio of 1:1:5, and the reaction was carried out under nitrogen protection in an ice bath. After the solvent was removed from the reaction solution, the product was dissolved in acetone and then precipitated with cold ether. The precipitate was collected and vacuum-dried to obtain an enzyme-responsive cross-linker.

[0030] Preferably, the solute of the reducing solution is tris(2-carboxyethyl)phosphine or dithiothreitol, and the solvent is PBS buffer; the concentration of the reducing solution is 5 to 50 mM;

[0031] and / or,

[0032] The solvent of the enzyme-responsive cross-linking agent solution is a mixed solution of dimethyl sulfoxide and PBS buffer, wherein the volume proportion of PBS buffer is 0% to 80%; and the concentration of the enzyme-responsive cross-linking agent solution is 2 to 16 mg / mL.

[0033] In a second aspect of the present application, a photonic crystal hydrogel sensor prepared by the above preparation method is provided.

[0034] The third aspect of the present application provides the use of the above-mentioned photonic crystal hydrogel sensor in the visual detection of drug-resistant bacteria that can produce β-lactamase.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] This application prepares a drug-resistant bacteria-responsive photonic crystal hydrogel sensor by combining a non-specifically responsive crosslinker with a drug-resistant bacteria-responsive crosslinker. The photonic crystal hydrogel sensor comprises a permanent crosslinking network composed of N,N'-methylenebisacrylamide (BIS) and an enzyme-responsive crosslinking network cleavable by β-lactamase. β-lactamase secreted by drug-resistant bacteria acts on the enzyme-responsive crosslinker, causing it to break, leading to hydrogel swelling and further changes in the lattice spacing of the photonic crystal hydrogel, resulting in a visual color change.

[0037] The photonic crystal hydrogel sensor described in this application exhibits excellent resistance to photobleaching and optical tunability, and is made from non-polluting raw materials. Its detection process does not require sophisticated instruments or specialized technical support, and intuitive structural color changes enable instant interpretation of test results, enabling rapid identification and quantitative detection of drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] To more clearly illustrate the technical solutions of the embodiments of this application, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some of the embodiments described in this application. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0039] Figure 1 This is a graph showing the β-lactamase response test results of the photonic crystal hydrogel sensor prepared in Example 1-3;

[0040] Figure 2 is the SEM image of the hydrogel of Comparative Examples 1-3;

[0041] Figure 3 Graph showing the bandgap change test results of the photonic crystal hydrogel of Example 2, the photonic crystal hydrogel reduced with tris(2-carboxyethyl)phosphine, and PCH4 obtained after cross-linking with an enzyme-responsive cross-linker;

[0042] Figure 4 The graph is a test result of the responsiveness of PCH4 in β-lactamase solution at different times;

[0043] Figure 5 This is a test diagram of the responsiveness of PCH4 in β-lactamase solutions with different concentrations;

[0044] Figure 6 This is the test result of PCH4's response to Pseudomonas aeruginosa bacterial solution. DETAILED DESCRIPTION

[0045] The following will be combined with the accompanying drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0046] In the following description of this embodiment, the terms "include", "comprising", "having" and "containing" are open-ended terms, meaning including but not limited to.

[0047] In the following description of this embodiment, the term "and / or" is used to describe the association relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, B exists alone, and both A and B exist. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0048] In the following description of this embodiment, the term "at least one" refers to one or more, and "plurality" refers to two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0049] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0050] Those skilled in the art should understand that in the following description of the embodiments of the present application, the order of serial numbers does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0051] It will be understood by those skilled in the art that the numerical ranges in the examples of the present application are to be understood as specifically disclosing each intermediate value between the upper and lower limits of the range. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also included in the present application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0052] Unless otherwise indicated, the technical / scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application belongs. Although this application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0053] In a first aspect, the present application provides a method for preparing a photonic crystal hydrogel sensor for visually detecting drug-resistant bacteria, comprising:

[0054] S1, immersing a glass slide in an aqueous dispersion of colloidal photonic crystal microspheres to deposit a photonic crystal structure and obtain a photonic crystal template;

[0055] In the present application, the colloidal photonic crystals include polystyrene or poly(styrene-methyl methacrylate-acrylic acid). The content of the colloidal photonic crystal microspheres in the aqueous dispersion is 0.05-0.15 wt%.

[0056] The present application deposits a photonic crystal structure on a glass sheet under constant temperature and constant humidity, wherein the temperature is preferably 50° C. to 60° C. and the humidity is preferably 50% to 60%.

[0057] S2, assembling the modified cover glass and the photonic crystal template into a sandwich structure, injecting a prepolymer solution into the sandwich structure, and obtaining a photonic crystal hydrogel through thermal polymerization; wherein the prepolymer solution comprises a polymerizable monomer, a disulfide bond crosslinking agent, N,N'-methylenebisacrylamide, an initiator, a catalyst, and a solvent;

[0058] In this application, the cover glass is modified by introducing polymerizable methacrylate groups onto the surface of the cover glass. Specifically, a glass slide that has been cleaned by plasma or ozone is immersed in a 1-5wt% aqueous solution of 3-(trimethoxysilyl)propyl methacrylate (TMSPMA). A small amount of acetic acid is added to adjust the pH to 5, and the reaction is carried out at room temperature for 2-24 hours to introduce polymerizable methacrylate groups onto the glass surface. By introducing polymerizable methacrylate groups, they can participate in the polymerization reaction and play a role in fixing the photonic crystal hydrogel.

[0059] In the present application, the modified cover glass and the photonic crystal template are assembled into a sandwich structure with a gap thickness of 100 to 500 μm; a prepolymer solution is injected into the sandwich structure until the pores of the photonic crystal template are completely penetrated, and thermal polymerization is initiated at room temperature to form a photonic crystal hydrogel.

[0060] The prepolymer solution described herein comprises the following components: the polymerizable monomer may be any one of acrylamide, N-isopropylacrylamide, or methacrylic acid; the disulfide crosslinker may be N,N'-bisacryloyl-L-cystine; the initiator may be ammonium persulfate or potassium persulfate; the catalyst may be tetramethylethylenediamine or 3-dimethylaminopropionitrile; and the solvent may be deionized water. As a preferred embodiment, the concentration of acrylamide in the prepolymer solution is 70-100 mg / mL, the concentration of N,N'-methylenebisacrylamide is 0.75-2.16 mg / mL, the concentration of N,N'-bisacryloyl-L-cystine is 5-30 mg / mL, the concentration of ammonium persulfate is 0.57-1.1 mg / mL, and the concentration of tetramethylethylenediamine is 0.28-0.57 mg / mL.

[0061] Wherein, the N,N'-bisacryloyl-L-cystine is prepared by the following method:

[0062] Sodium hydroxide and L-cystine were dissolved in methanol, acryloyl chloride was added at 0°C and reacted for 12 hours, and the liquid phase was collected by filtration after the reaction; wherein, the mass ratio of sodium hydroxide to L-cystine was 2:(1.8-2.7), and the mass volume ratio of L-cystine to acryloyl chloride was (1.8-2.7) g / (1.5-2.2) mL.

[0063] The liquid phase was added to cold ether for precipitation, and the solid was collected and dried under vacuum at 40° C. to obtain N,N′-bisacryloyl-L-cystine.

[0064] S3, immersing the photonic crystal hydrogel in a reducing solution to perform a reduction reaction, washing the reaction product, and then performing a cross-linking reaction with an enzyme-responsive cross-linking agent solution to obtain a photonic crystal hydrogel sensor.

[0065] In the present application, the disulfide bond is reduced to a sulfhydryl group through a reduction reaction. The solute of the reducing solution is tris(2-carboxyethyl)phosphine or dithiothreitol, and the solvent is PBS buffer; the concentration of the reducing solution is 5 to 50 mM;

[0066] In this application, the enzyme-responsive crosslinker solution is a mixture of dimethyl sulfoxide and PBS buffer, with the PBS buffer comprising 0% to 80% by volume. The concentration of the enzyme-responsive crosslinker solution is 2 to 16 mg / mL. Through the crosslinking reaction, the maleimide groups and thiol groups form a secondary crosslinked network, resulting in a dual-network photonic crystal hydrogel sensor.

[0067] In the present application, the enzyme-responsive cross-linking agent is responsive to β-lactamase, and its preparation method includes:

[0068] 7-Amino-3-chloromethyl-3-cephazoline-4-carboxylic acid p-methoxybenzyl hydrochloride (ACLE) was dissolved in anhydrous dichloromethane. Triethylamine was slowly added under an ice bath and nitrogen protection, followed by the addition of N-methylmorpholine and 4-aminothiophenol. The reaction progress was monitored by thin-layer chromatography (developing solvent: ethyl acetate / dichloromethane = 3:7, v / v). After the reaction, the reaction solution was freed from the solvent and purified by silica gel column chromatography (elution gradient: 20%-80% ethyl acetate / dichloromethane) to obtain compound 1.

[0069] The preferred usage ratio of ACLE, triethylamine, N-methylmorpholine and 4-aminothiophenol is (4.5-9) g: (3-6) mL: (1.5-3) mL: (2.25-4.5) g.

[0070] Compound 1, 3-maleimidopropionic acid (MPA), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate were dissolved in anhydrous N,N-dimethylformamide. Under nitrogen protection, N,N-diisopropylethylamine (DIPEA) was added and the reaction was carried out for 75 minutes. The endpoint was monitored by thin layer chromatography (developing solvent: ethyl acetate / dichloromethane = 4:1, v / v). The reaction solution was extracted with dichloromethane and water, washed, dried, concentrated, and purified by flash column chromatography (elution gradient: 40%-80% ethyl acetate / dichloromethane) to obtain compound 2.

[0071] Among them, the usage ratio of compound 1, MPA, HATU and DIPEA is preferably (3.6-7.2) g: (5.6-11.2) g: (12.6-25.2) g: (18-36) mL.

[0072] Compound 2 is added into a mixed solution of trifluoroacetic acid, anisole and dichloromethane with a volume ratio of 1:1:5, and the reaction is carried out under the protection of nitrogen in an ice bath, and the deprotection process is monitored by thin layer chromatography (developing agent: 100% ethyl acetate); after the solvent is removed from the reaction solution by rotary evaporation, the product is dissolved in acetone and then precipitated with cold ether, and the precipitate is collected and dried under vacuum to obtain the enzyme-responsive crosslinker;

[0073] The mass-volume ratio of compound 2 to the mixed solvent is 3-6 g / 105-210 mL.

[0074] The present application prepares a drug-resistant bacteria-responsive double-network photonic crystal hydrogel sensor by selecting a non-specific response crosslinker and a drug-resistant bacteria-responsive crosslinker. The drug-resistant bacteria-responsive crosslinker is acted on by β-lactamase secreted by drug-resistant bacteria, so that the enzyme-responsive crosslinker is broken, thereby causing the swelling of the hydrogel and further causing the change of the lattice spacing of the photonic crystal hydrogel, resulting in a visual color change.

[0075] Specifically, when the photonic crystal hydrogel sensor is co-incubated with β-lactamase, the β-lactamase-responsive crosslinker in the photonic crystal hydrogel sensor is broken, causing the swelling of the hydrogel to increase, thereby driving the increase of the photonic crystal lattice spacing, resulting in the red shift of the photonic band gap and the structural color, and the naked-eye visual detection can be realized within 2 hours.

[0076] In a second aspect, the present application provides a photonic crystal hydrogel sensor prepared by the above preparation method. The photonic crystal hydrogel sensor of the present application has excellent anti-photobleaching properties and optical performance tunability, and the raw materials are pollution-free. The detection process does not need to rely on precise instruments or professional technical support, and the detection result can be immediately interpreted through the intuitive structural color change, so that rapid identification and quantitative detection of drug-resistant bacteria can be realized.

[0077] The photonic crystal hydrogel sensor of the present application can be used for visual detection of drug-resistant bacteria that can produce β-lactamase. The photonic crystal hydrogel sensor of the present application comprises a permanent crosslinking network composed of N,N'-methylene bisacrylamide (BIS) and an enzyme-responsive crosslinking network that can be cleaved by β-lactamase. When the photonic crystal hydrogel is soaked in a β-lactamase solution or a drug-resistant bacteria solution that secretes β-lactamase, the structural color and diffraction peak wavelength of the sensor will change accordingly, achieving the purpose of visual detection of β-lactamase and drug-resistant bacteria. The detectable drug-resistant bacteria include methicillin-resistant Staphylococcus aureus MRSA, carbapenem-resistant enterobacteriaceae, Pseudomonas aeruginosa, Bacillus cereus or Enterobacter cloacae, etc.

[0078] The present application will be further described below through specific examples. In the examples of the present application, the correspondence between the raw materials used and their abbreviations is as follows:

[0079] ACLE is 7-amino-3-chloromethyl-3-cephazoline-4-carboxylic acid p-trimethoprim hydrochloride

[0080] HATU is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0081] DIPEA is N,N-diisopropylethylamine

[0082] BIS is N,N'-methylenebisacrylamide

[0083] BISS stands for N,N'-bisacryloyl-L-cystine

[0084] TMSPMA is 3-(trimethoxysilyl)propyl methacrylate

[0085] Example Synthesis of disulfide crosslinker N,N'-bisacryloyl-L-cystine (BISS)

[0086] Dissolve 2.0 g of sodium hydroxide and 2.7 g of L-cystine in 70 mL of methanol with continuous stirring. Gradually add 2.2 mL of acryloyl chloride to the solution at 0°C and allow to react for 12 hours. After the reaction, filter out the solid byproducts and precipitate the clear solution in cold ether. Filter the solid precipitate from the solution and vacuum dry at 40°C for 2 days to obtain N,N'-bisacryloyl-L-cystine, or BISS.

[0087] Example Synthetic Enzyme Responsive Cross-Linker

[0088] 4.5 g of ACLE was dissolved in 150 mL of anhydrous dichloromethane and stirred in an ice bath under nitrogen protection. 3 mL of triethylamine was added over 20 min, followed by 1.5 mL of N-methylmorpholine and 2.25 g of 4-aminothiophenol. The mixture was stirred in an ice bath for 3 hours, and the reaction progress was monitored by thin-layer chromatography (developing solvent: ethyl acetate / dichloromethane = 3:7, v / v). After the reaction, the solvent was removed by rotary evaporation, and the mixture was purified by silica gel column chromatography (elution gradient: 20%-80% ethyl acetate / dichloromethane) to obtain compound 1.

[0089] 3.6 g of compound 1, 5.6 g of 3-maleimidopropionic acid, and 12.6 g of HATU were dissolved in 36 mL of anhydrous N,N-dimethylformamide and stirred at room temperature under nitrogen for 15 minutes. 18 mL of DIPEA was then added, and the reaction was continued for 75 minutes. The end point was monitored by thin-layer chromatography (developing solvent: ethyl acetate / dichloromethane = 4:1, v / v). The reaction solution was extracted with dichloromethane-water, washed twice with water and once with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The mixture was purified by flash column chromatography (elution gradient: 40%-80% ethyl acetate / dichloromethane) and dried to obtain compound 2.

[0090] 3 g of compound 2 was dissolved in 105 mL of a mixed solvent (trifluoroacetic acid: anisole: dichloromethane = 1:1:5, v / v / v) and reacted for 1.5 hours under nitrogen protection in an ice bath. The deprotection progress was monitored by thin layer chromatography (developing solvent: 100% ethyl acetate). After removing the solvent by rotary evaporation, the product was dissolved in acetone and precipitated with cold ether three times. The precipitate was collected by centrifugation and dried in vacuo to obtain an enzyme-responsive cross-linker.

[0091] Example 1

[0092] This embodiment provides a method for preparing a photonic crystal hydrogel sensor, comprising:

[0093] S1, vertically immersing a cleaned glass sheet into an aqueous dispersion of polystyrene colloidal photonic crystal microspheres with a solid content of 0.05 wt%, and depositing a photonic crystal structure on the surface of the glass sheet at a temperature of 50° C. and a humidity of 50%, to obtain a photonic crystal template;

[0094] S2. Prepare a prepolymer solution using deionized water as solvent, which includes the following components at the following concentrations: acrylamide 100 mg / mL, BIS 1.08 mg / mL, BISS 14.72 mg / mL, ammonium persulfate 0.8 mg / mL, and tetramethylethylenediamine 4 μL / mL.

[0095] A TMSPMA-modified coverslip and a photonic crystal template were assembled into a sandwich structure with a gap thickness of 300 μm. Prepolymerization liquid was injected until it completely penetrated the pores of the photonic crystal template, and thermal polymerization was initiated at room temperature to form a photonic crystal hydrogel.

[0096] S3, the photonic crystal hydrogel was taken out from the sandwiched glass sheet, and was immersed in a PBS solution of tris(2-carboxyethyl)phosphine with a concentration of 50 mM and pH 7.0 at 37 °C for 2 h to reduce the disulfide bond to thiol; then it was washed with PBS buffer and was immersed in a dimethyl sulfoxide solution of enzyme-responsive crosslinker with a concentration of 2 mg / mL at 37 °C for 2 h to form a secondary crosslinking network between the maleimide group and the thiol group, thereby obtaining a photonic crystal hydrogel sensor, which was denoted as PCH2.

[0097] Example 2

[0098] Example 2 differed from Example 1 in that the concentration of the dimethyl sulfoxide solution of enzyme-responsive crosslinker in step S3 was 4 mg / mL, and the rest was the same as Example 1. The photonic crystal hydrogel sensor prepared in Example 2 was denoted as PCH4.

[0099] Example 3

[0100] Example 3 differed from Example 1 in that the concentration of the dimethyl sulfoxide solution of enzyme-responsive crosslinker in step S3 was 8 mg / mL, and the rest was the same as Example 1. The photonic crystal hydrogel sensor prepared in Example 2 was denoted as PCH8.

[0101] Comparative Example 1

[0102] Comparative Example 1 differed from Example 1 in that there was no step S1, and two clean glass sheets were used instead of the modified cover glass and the photonic crystal template to form a sandwich structure, and the rest was the same as Example 1. The hydrogel obtained in Comparative Example 1 was denoted as H2.

[0103] Comparative Example 2

[0104] Comparative Example 2 differed from Example 2 in that there was no step S1, and two clean glass sheets were used instead of the modified cover glass and the photonic crystal template to form a sandwich structure, and the rest was the same as Example 2. The hydrogel obtained in Comparative Example 1 was denoted as H4.

[0105] Comparative Example 3

[0106] Comparative Example 3 differed from Example 3 in that there was no step S1, and two clean glass sheets were used instead of the modified cover glass and the photonic crystal template to form a sandwich structure, and the rest was the same as Example 3. The hydrogel obtained in Comparative Example 3 was denoted as H8.

[0107] The photonic crystal hydrogel sensors PCH2, PCH4, and PCH8 prepared in Examples 1-3 were subjected to a β-lactamase response test, and the specific test method was as follows:

[0108] First, the initial photonic bandgap wavelength of the photonic crystal hydrogel sensor was recorded using a fiber optic spectrometer. Then, the photonic crystal hydrogel sensor was immersed in a 0.02U / mL β-lactamase solution at 37°C for 2 hours. The photonic crystal hydrogel was then taken out to record its photonic bandgap wavelength and structural color, and the bandgap shift was calculated. The test results are shown in Figure 2. Figure 1 shown.

[0109] from Figure 1 As can be seen, the photonic bandgap shifts of PCH2, PCH4, and PCH8 all increase, demonstrating significant β-lactamase responsiveness. This is due to the presence of β-lactamase-responsive crosslinks in these photonic crystal hydrogel sensors. In the presence of β-lactamase, these crosslinks break, causing the hydrogel to swell, increasing the photonic crystal lattice spacing and red-shifting the photonic bandgap and structural color. Compared to PCH2, PCH4 exhibits a larger bandgap shift at equilibrium; whereas, the bandgap shift in PCH8 decreases compared to PCH2 and PCH4. This is because increasing the enzyme-responsive crosslinker component provides more reactive sites, increasing the β-lactamase responsiveness of the photonic crystal hydrogel. However, further increasing the enzyme-responsive crosslinker content decreases the β-lactamase responsiveness of the photonic crystal hydrogel. This is because excessive crosslinker content densifies the hydrogel network, hindering enzyme access and weakening the enzyme response.

[0110] The morphology of hydrogels H2, H4, and H8 corresponding to PCH2, PCH4, and PCH8 was tested to verify the effect of the crosslinker on the micromorphology of the hydrogel. The SEM images are shown in Figure 2. Figure 2 As shown. Figure 2 It can be seen that as the content of enzyme-responsive cross-linker increases, the cross-linking degree of the hydrogel increases; the pores of hydrogels H2, H4, and H8 decrease in sequence, and H8 presents a denser network structure, which weakens the enzyme response of its corresponding photonic crystal hydrogel PCH8.

[0111] The test results show that PCH4 has an appropriate amount of enzyme-responsive crosslinker and an appropriate crosslinking density, and has better enzyme response ability. The photonic crystal hydrogel obtained in step S2 of Example 2, the photonic crystal hydrogel after tris(2-carboxyethyl)phosphine reduction in step S3, and the PCH4 obtained after crosslinking with the enzyme-responsive crosslinker were tested for photonic bandgap change. The test results are as follows: Figure 3 shown.

[0112] from Figure 3 The initial band gap of the photonic crystal hydrogel is around 520 nm. After treatment with tris(2-carboxyethyl)phosphine, the disulfide bonds break, the hydrogel swells, and the band gap red-shifts to around 576 nm. After forming a second cross-linked network, the hydrogel shrinks, and the band gap blue-shifts to around 560 nm. Correspondingly, the structural color shifts from green to orange, and finally to yellow-green.

[0113] PCH4 was tested for β-lactamase response as follows:

[0114] 1. Responsiveness test of PCH4 in β-lactamase solution at different times

[0115] The initial photon bandgap wavelength of PCH4 was recorded using a fiber spectrometer, and its initial structural color was recorded using a camera. PCH4 was immersed in a 0.02U / mL β-lactamase solution at 37°C, and the photon bandgap wavelength and structural color were recorded after 0min, 10min, 20min, 30min, 40min, 60min, 80min, 100min, 120min, 150min, 180min, and 240min, respectively. The shift of the bandgap was also calculated. The test results are shown in Figure 2. Figure 4 shown.

[0116] Depend on Figure 4 It can be seen that with the increase of immersion time, the photon bandgap red shift of PCH4 increases, and the structural color changes from yellow-green to orange-red, showing obvious β-lactamase responsiveness.

[0117] 3. Responsiveness test of PCH4 in β-lactamase solutions with different concentrations

[0118] The initial photonic bandgap wavelength of the photonic crystal hydrogel was recorded using a fiber spectrometer, and the initial structural color of the material was recorded using a camera. The photonic crystal hydrogel was placed in 0U / mL, 0.0125U / mL, 0.025U / mL, 0.05U / mL, 0.1U / mL, 0.2U / mL, 0.4U / mL, 0.6U / mL, 0.8U / mL, and 1.0U / mL β-lactamase solutions at 37°C, and the photonic crystal hydrogel was taken out after 2 hours to record its photonic bandgap wavelength and structural color, and the bandgap offset was calculated. The test results are as follows: Figure 5 shown.

[0119] Depend on Figure 5 It can be seen that with the increase of β-lactamase concentration, the photon bandgap red shift of PCH4 increases, and the structural color changes from yellow-green to orange-red, showing obvious β-lactamase responsiveness.

[0120] PCH4 was tested for its response to Pseudomonas aeruginosa bacterial solution as follows:

[0121] The initial photonic band gap wavelength of the photonic crystal hydrogel was recorded using a fiber optic spectrometer, and the initial structural color of the material was recorded using a camera. The photonic crystal hydrogel was placed in 0 CFU / mL, 5×10 2 CFU / mL, 5×10 3CFU / mL, 5×10 4 CFU / mL, 5×10 5 CFU / mL, 5×10 6 CFU / mL of β-lactamase solution, and took out the photonic crystal hydrogel after 2 hours to record its photonic bandgap wavelength and structural color, and calculated the bandgap offset. The test results are as follows Figure 6 shown.

[0122] from Figure 6 It can be seen that as the concentration of Pseudomonas aeruginosa increases, the red shift of the photon band gap of PCH4 increases, and the structural color changes from yellow-green to orange-red. 2 After 2 hours of incubation in a bacterial solution with a CFU / mL concentration, a visible color change occurs. The photonic crystal hydrogel sensor of this application can instantly interpret the test results through intuitive structural color changes, enabling rapid identification and quantitative detection of drug-resistant bacteria, and can be used to detect drug-resistant bacteria that secrete β-lactamase.

[0123] Although this specification has been used to fully describe the present application using general descriptions and specific embodiments, it is obvious to those skilled in the art that modifications or improvements may be made based on the present application. Therefore, such modifications or improvements made without departing from the spirit of the present application are within the scope of protection claimed in this application.

Claims

1. A method for preparing a photonic crystal hydrogel sensor for visual detection of drug-resistant bacteria, characterized in that: include: S1, immersing a glass slide in an aqueous dispersion of colloidal photonic crystal microspheres to deposit a photonic crystal structure and obtain a photonic crystal template; S2, assembling the modified cover glass and the photonic crystal template into a sandwich structure, injecting the prepolymer solution into the sandwich structure, and obtaining the photonic crystal hydrogel through thermal polymerization; The prepolymer solution comprises a polymerizable monomer, a disulfide bond crosslinking agent, N,N'-methylenebisacrylamide, an initiator, a catalyst and a solvent; S3, immersing the photonic crystal hydrogel in a reducing solution to perform a reduction reaction, washing the reaction product, and then performing a cross-linking reaction with an enzyme-responsive cross-linking agent solution to obtain a photonic crystal hydrogel sensor.

2. The preparation method according to claim 1, characterized in that The colloidal photonic crystal comprises polystyrene or poly(styrene-methyl methacrylate-acrylic acid); The content of the colloidal photonic crystal microspheres in the aqueous dispersion of the colloidal photonic crystal microspheres is 0.05 to 0.15 wt %; The temperature for depositing the photonic crystal structure is 50° C. to 60° C., and the humidity is 50% to 60%.

3. The preparation method according to claim 1, characterized in that The modified cover glass is a cover glass with polymerizable methacrylate groups introduced on the surface; The gap of the sandwich structure is 100 to 500 μm.

4. The preparation method according to claim 1, characterized in that The polymerizable monomer includes any one of acrylamide, N-isopropylacrylamide or methacrylic acid; The disulfide bond cross-linking agent is N,N'-bisacryloyl-L-cystine; The initiator is ammonium persulfate or potassium persulfate; The catalyst is tetramethylethylenediamine or 3-dimethylaminopropionitrile; The solvent is deionized water.

5. The preparation method according to claim 4, characterized in that In the prepolymer solution, the concentration of acrylamide is 70-100 mg / mL, the concentration of N,N'-methylenebisacrylamide is 0.75-2.16 mg / mL, the concentration of N,N'-bisacryloyl-L-cystine is 5-30 mg / mL, the concentration of ammonium persulfate is 0.57-1.1 mg / mL, and the concentration of tetramethylethylenediamine is 0.28-0.57 mg / mL.

6. The preparation method according to claim 4, characterized in that The preparation method of the N,N'-bisacryloyl-L-cystine comprises: Sodium hydroxide and L-cystine were dissolved in methanol, acryloyl chloride was added at 0°C for reaction, and the liquid phase was collected by filtration after the reaction; The liquid phase is added to cold ether for precipitation, and the solid is collected to obtain N,N'-bisacryloyl-L-cystine.

7. The preparation method according to claim 1, characterized in that The preparation method of the enzyme-responsive cross-linking agent comprises: 7-Amino-3-chloromethyl-3-cephazoline-4-carboxylic acid p-methoxybenzyl hydrochloride was dissolved in anhydrous dichloromethane. Triethylamine was slowly added under an ice bath and nitrogen protection, followed by the addition of N-methylmorpholine and 4-aminothiophenol. The solvent was removed from the reaction solution, and the solution was purified by silica gel column chromatography to obtain compound 1. Compound 1, 3-maleimidopropionic acid, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate were dissolved in anhydrous N,N-dimethylformamide. Under nitrogen protection, N,N-diisopropylethylamine was added to react. The reaction solution was extracted with dichloromethane and water, washed, dried, and purified by flash column chromatography to obtain compound 2. Compound 2 was added to a mixture of trifluoroacetic acid, anisole, and dichloromethane in a volume ratio of 1:1:5, and the reaction was carried out under nitrogen protection in an ice bath. After the solvent was removed from the reaction solution, the product was dissolved in acetone and then precipitated with cold ether. The precipitate was collected and vacuum-dried to obtain an enzyme-responsive cross-linker.

8. The preparation method according to claim 1, characterized in that The solute of the reducing solution is tris(2-carboxyethyl)phosphine or dithiothreitol, and the solvent is PBS buffer; the concentration of the reducing solution is 5 to 50 mM; and / or, The solvent of the enzyme-responsive cross-linking agent solution is a mixed solution of dimethyl sulfoxide and PBS buffer, wherein the volume proportion of PBS buffer is 0% to 80%; and the concentration of the enzyme-responsive cross-linking agent solution is 2 to 16 mg / mL.

9. A photonic crystal hydrogel sensor prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the photonic crystal hydrogel sensor according to claim 9 in visual detection of drug-resistant bacteria that can produce β-lactamase.

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