CN112577937A

CN112577937A - Preparation method and application of novel lysozyme fluorescence sensor

Info

Publication number: CN112577937A
Application number: CN202011437519.8A
Authority: CN
Inventors: 李雅萍; 陈欣然; 张一沫; 李紫燕; 韩洪亮; 张伊琳; 武冰冰
Original assignee: Capital Normal University
Current assignee: Capital Normal University
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2021-03-30

Abstract

The invention discloses a preparation method and application of a novel lysozyme fluorescence sensor, which comprises the following steps: (1) preparing and purifying carbon quantum dots; (2) and (3) preparing a lysozyme molecularly imprinted polymer. The molecular imprinting hydrogel fluorescence sensor with the specific recognition capability on the template molecule LYZ is synthesized by taking CQDs as a fluorescence signal, LYZ as a template molecule, NIPAAM as a temperature-sensitive monomer, MAA and HEMA as auxiliary monomers, MBA as a cross-linking agent, APS as an initiator and TEMED as a catalyst.

Description

Preparation method and application of novel lysozyme fluorescence sensor

Technical Field

The invention relates to the technical field of lysozyme detection. In particular to a preparation method of a novel lysozyme fluorescence sensor.

Background

Lysozyme (LYZ) is an important indicator for clinical diagnosis of several diseases: the LYZ concentration in the cerebrospinal fluid of patients with tuberculous meningitis, neurosyphilis and fungal meningitis can be obviously increased; changes in LYZ activity may also occur in serum and urine from patients with leukemia and some renal diseases. In addition, LYZ may be used as an effective substitute for some antibiotics, and may even be used for the treatment of some infections such as AIDS and ulcers. Therefore, it is of great interest in practical manufacturing life to rapidly, efficiently and simply detect LYZ from a complex matrix of a real biological sample. The existing common LYZ detection methods comprise: high performance liquid chromatography, capillary electrophoresis, enzyme-linked immunosorbent assay, ultraviolet spectrophotometry, and fluorescence probe. The method generally has the defects of high instrument cost, requirement on sample purity, complicated operation steps and the like, and the selectivity of the method is easily influenced because interference substances such as high-abundance proteins and the like are inevitably present in the sample during actual detection. Therefore, a detection method with high selectivity, convenient operation and moderate cost is urgently needed to be found.

The molecular imprinting technology is used for detecting specific molecules by a bionic principle, so that the technology has excellent specific recognition and sensitivity, especially in extremely complex samples. The synthesis of a Molecularly Imprinted Polymer (MIP) generally requires three steps: firstly, assembling a template molecule and a functional monomer through covalent bond or non-covalent bond; then adding an initiator, and polymerizing the functional monomer and the cross-linking agent to generate a fixed high polymer system; and finally, eluting the template molecule from the polymer by using an eluent to obtain a cavity which is matched with the template molecule and has multiple action points, and further accurately identifying the template molecule in subsequent detection. In practical detection, holes in the MIP can selectively adsorb template molecules through shape matching and interaction such as electrostatic interaction (including complexation), hydrogen bonding, hydrophobic interaction and the like between functional groups. Therefore, the working principle of MIP can be described by the 'key-lock principle' in an iconic way.

As early as 1972, Wulff and Sarhan published reports on molecular imprinting, one of the most common methods of molecular recognition in existence, which allows rapid, sensitive and selective recognition and detection of target molecules. Compared with a molecular recognition method in nature, the molecular imprinting method has the characteristics of low cost, high recognition capability, long-term durability and the like, is wide in application, and can be used in the fields of chromatography, medicine carrying, solid phase extraction, sensing technology and the like. In practical research and production, molecular imprinting techniques have been used in a variety of ways. In the aspect of small molecular imprinting, a photoluminescence probe is constructed by utilizing a molecular imprinting polymer in a Kochaporn Chulalalat topic group, the method has good repeatability, the relative standard deviation is less than 6%, the method can be successfully used for determining amoxicillin in an actual sample, the recovery rate is 85-102%, and the effective analysis of trace amoxicillin is realized. The method has the characteristics of high selectivity and repeatability, and is expected to be used for low-cost simple detection of the erythromycin. Besides antibiotics, MIPs also show very excellent potential in the specific detection of artificial dyes. The Suxiao \28635teamsuccessfully prepares MIP with high specificity and recognition capability by using rhodamine B as a template molecule, the method can be used as a solid phase extraction material, the recovery rate is between 78.47 and 101.6 percent, the relative standard deviation is less than 2 percent, and the method is expected to be used for detecting illegally added rhodamine B in food.

As can be seen from the above examples, the molecular imprinting technique is widely used. However, the conventional molecular imprinting technology has some problems to be solved and some defects which are not ignored: the traditional molecular imprinting technology has the defects of centrifugation, complex and complex operation process and the like during detection after adsorption, and template molecules used in the MIP preparation process generally mainly comprise small molecular compounds, but the imprinting of biological macromolecules such as proteins, viruses, cells and the like is still slow. In addition, the combination of the molecular imprinting technology and the on-line fluorescence detection to realize both the high selectivity of molecular imprinting and the high sensitivity of fluorescence detection is an important trend in the development of the molecular imprinting technology, and is also a popular field for the research of the molecular imprinting technology at present.

The molecular imprinting fluorescence sensor takes a molecular imprinting polymer as a recognition unit and a specific fluorescence signal as a signal unit, generates the change of the fluorescence signal by combining with a target product, and is further used for qualitative and quantitative online analysis of a target object, and the principle is shown in figure 1. Currently, the common fluorescent units include fluorescent molecules, heavy metal quantum dots, and carbon quantum dots. Carbon Quantum Dots (CQDs) generally refer to carbon nanocrystals with a size less than 10nm, and have many excellent optical properties, such as photoluminescence, photoinduced electron transfer and electrochemical luminescence (ECL), and in addition, compared with conventional semiconductor quantum dots containing heavy metals, they have some other significant advantages, such as chemical inertness, biocompatibility, low toxicity, emission spectrum controllability, good light stability, greater stokes shift, longer fluorescence lifetime, good water solubility, photobleaching resistance, etc., so that they have been widely noticed and researched by researchers, and have unparalleled advantages.

The prior application of the applicant (Chinese patent application publication No. CN110283275A) discloses a molecular imprinting nanogel fluorescence sensor which takes CQDs as a fluorescence signal, LYZ as a template molecule, NIPAAm as a temperature-sensitive monomer, AAm, HEMA and MAA as auxiliary monomers, MBA as a cross-linking agent, APS as an initiator and TEMED as a catalyst, and can realize selective recognition of the molecular imprinting nanogel fluorescence sensor, but the sensitivity and the reproducibility are not ideal.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide a preparation method of a novel lysozyme fluorescence sensor with good sensitivity and reproducibility.

In order to solve the technical problems, the invention provides the following technical scheme:

the preparation method of the novel lysozyme fluorescence sensor comprises the following steps:

(1) preparing and purifying carbon quantum dots;

(2) and (3) preparing a lysozyme molecularly imprinted polymer.

The preparation method of the novel lysozyme fluorescence sensor comprises the following steps in the step (1):

(1-1) accurately weighing 0.5g of polyethyleneimine BPEI and 0.1g of citric acid CA in a 20mL small beaker, fully dissolving the materials by using 10mL of 80 ℃ distilled water, heating the small beaker at 180 ℃ by using an oil bath pot to obtain light yellow viscous gel, adding 1mL of distilled water to obtain light yellow solution, and repeating the experimental operation until orange yellow gel is obtained; dissolving the prepared orange gel in 10mL of deionized water, and cooling to room temperature;

(1-2) using 0.01mol/L HCl as a mobile phase, passing the product through a silica gel column, collecting two effluent components, reserving the second component, collecting, performing rotary evaporation concentration, dissolving with 10mL deionized water, filling into a dialysis bag with molecular weight cutoff of 500Da, dialyzing until the pH is neutral, performing rotary evaporation concentration, fixing the volume of a carbon quantum dot CQDs solution with 0.0250g/mL, and storing in a refrigerator at 4 ℃ for later use.

The preparation method of the novel lysozyme fluorescence sensor comprises the following steps of (2):

(2-1) preparation of monomers: accurately weighing a certain amount of monomer N-isopropyl acrylamide NIPAAM;

(2-2) adding a PBS buffer solution with pH of 7.4, a carbon quantum dot CQDs and lysozyme LYZ into the monomer, and performing ultrasonic dissolution;

(2-3) adding a cross-linking agent N, N' -Methylene Bisacrylamide (MBA), then adding magnetons, fully mixing the reactants in a round-bottom flask, and fully stirring;

(2-4) adding initiators of ammonium persulfate APS and tetramethylethylenediamine TEMED, vacuumizing, and carrying out polymerization reaction under the protection of nitrogen to obtain porous hydrogel;

(2-5) freeze-drying the porous hydrogel in a freeze dryer, and then grinding the porous hydrogel into powder by using a mortar to obtain a molecularly imprinted polymer MIP capable of imprinting lysozyme LYZ;

(2-6) dissolving the molecularly imprinted polymer MIP in the mixed eluent of acetic acid HAc and sodium dodecyl sulfate SDS;

(2-7) putting the molecularly imprinted polymer MIP and the mixed eluent into a dialysis bag with the molecular weight cutoff of 15000Da, and eluting with the eluent; every 24 hours, taking a certain amount of the eluate, and carrying out ultraviolet spectrum detection by using an ultraviolet spectrophotometer until the protein absorption peak value of an ultraviolet spectrum LYZ tends to be stable, namely the template molecule is considered to be completely removed; then putting the dialysis gel bag into deionized water for dialysis to remove acetic acid and sodium dodecyl sulfate, and finally freeze-drying and grinding the gel.

In the step (2-1), the preparation method of the novel lysozyme fluorescence sensor further comprises an auxiliary monomer, wherein the auxiliary monomer is one or a mixture of hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA).

In the step (2-1), the addition amount of the monomer N-isopropylacrylamide NIPAAM is 3.955 mmol.

According to the preparation method of the novel lysozyme fluorescence sensor, the total substance amount of the monomer N-isopropyl acrylamide NIPAAM and the auxiliary monomer is 3.89mmol-4.02mmol, wherein the monomer N-isopropyl acrylamide NIPAAM is 3.560mmol, the hydroxyethyl methacrylate HEMA is 0.230mmol-0.460mmol, and the methacrylic acid MAA is 0.165mmol-0.330 mmol.

The preparation method of the novel lysozyme fluorescence sensor comprises the following steps of (2-2): adding 20mL of PBS buffer solution with pH value of 7.4, 1mL of 0.0250g/mL CQD and 0.1g LYZ into monomer N-isopropylacrylamide NIPAAM or a mixture of monomer N-isopropylacrylamide NIPAAM and auxiliary monomers, and ultrasonically dissolving;

in step (2-3): adding 28mg of cross-linking agent N, N' -methylene bisacrylamide MBA, then adding magnetons, fully mixing the reactants in a round-bottom flask, and fully stirring in a 298.15K oil bath for 4 hours;

in step (2-4): adding an initiator of 20mg APS and 20 mu L TEMED into a sample with sufficient mixed reaction, vacuumizing the sample under the protection of nitrogen, and polymerizing the sample at the ambient temperature of 298.15K for 16 hours to obtain porous hydrogel;

in step (2-6): dissolving 10mL HAc and 10g sodium dodecyl sulfate SDS in 90mL deionized water as mixed eluent of HAc acetate and sodium dodecyl sulfate SDS; the milled MIP was dissolved in 5mL of the combined eluates.

The application of the novel lysozyme fluorescence sensor in the detection of lysozyme.

The application of the novel lysozyme fluorescence sensor for detecting lysozyme comprises the following steps:

(1) the concentration ranges of the components are 6.138 multiplied by 10^-6g/mL-2.707×10^-5g/mL LYZ standard solution;

(2) taking 1mL MIP gel solution, placing the MIP gel solution in 1mL LYZ standard solution with different mass concentrations, and placing the MIP gel solution in a table type constant temperature oscillator for incubation: mixing and incubating fully for 50min-24 hours at 298.15K;

(3) measuring the fluorescence intensity of the solution by a fluorescence spectrophotometer under the conditions of excitation of 280nm, emission of 290nm-500nm, slit of 10.0nm and voltage of 700V, and carrying out parallel detection on the same sample at least twice.

In the application of the novel lysozyme fluorescence sensor in lysozyme detection, the concentration of MIP gel solution is 0.0003 g/mL; the incubation time was 70min at 298.15K.

The technical scheme of the invention achieves the following beneficial technical effects:

the molecular imprinting hydrogel fluorescence sensor with the specific recognition capability on the template molecule LYZ is synthesized by taking CQDs as a fluorescence signal, LYZ as a template molecule, NIPAAM as a temperature-sensitive monomer, MAA and HEMA as auxiliary monomers, MBA as a cross-linking agent, APS as an initiator and TEMED as a catalyst.

1. The LYZ molecular imprinting hydrogel is characterized by a scanning electron microscope and an ultraviolet spectrophotometer, and the successful preparation, imprinting and elution of the LYZ molecular imprinting hydrogel are proved. The obtained LYZ molecularly imprinted hydrogel is porous and has a larger specific surface area, so that the incubation time of LYZ is greatly reduced, and compared with a gel simultaneously containing monomer AAM and NIPAAM, the LYZ molecularly imprinted hydrogel only contains the monomer NIPAAM, and the stability and the reproducibility of detecting LYZ are better.

2. The molecularly imprinted hydrogel containing different auxiliary monomer components is prepared, and the hydrogel only containing NIPAAm temperature-sensitive monomers shows better selectivity on template molecules LYZ through investigation.

The NIPAAm is used as a temperature-sensitive monomer, and the result shows that the molecularly imprinted hydrogel has temperature response to the identification of LYZ, and the selectivity can be improved by the temperature rise within a certain range. Thus, the specific recognition of LYZ by the hydrogel can be effectively controlled by temperature.

Drawings

FIG. 1 shows the detection principle of a molecularly imprinted fluorescent sensor;

FIG. 2 is a route for preparing a lysozyme LYZ fluorescence sensor;

FIG. 3 a scanning electron microscope image of component B;

b scanning electron microscope images of component D;

FIG. 4 is a graph of fluorescence spectra of MIP before and after binding of component B to the template molecule;

FIG. 5298.15K is a graph of the change in fluorescence intensity of a sample with time at an incubation temperature;

FIG. 6 shows the linear curve of the fluorescence response of the MIP and NIP of fraction A to the template molecule LYZ at 298.15K, respectively;

FIG. 7 shows the linear curve of the fluorescence response of MIPs and NIPs of fraction B, respectively, at 298.15K to the template molecule LYZ;

FIG. 8 shows the linear curve of the fluorescence response of MIPs and NIPs of fraction C, respectively, to the template molecule LYZ at 298.15K;

FIG. 9 shows the linear curve of the fluorescence response of MIPs and NIPs of fraction D, respectively, to the template molecule LYZ at 298.15K;

FIG. 10 shows the linear response curves of MIP and NIP of fraction A at 318.15K to fluorescence of the template molecule LYZ, respectively;

FIG. 11 shows the linear response curves of MIP and NIP of fraction B at 318.15K to fluorescence of the template molecule LYZ, respectively;

FIG. 12 shows the linear response curves of MIP and NIP of fraction C to fluorescence of the template molecule LYZ at 318.15K, respectively;

FIG. 13 shows the linear response curves of MIP and NIP of fraction D to fluorescence of the template molecule LYZ at 318.15K, respectively;

FIG. 14 Effect of incubation temperature on selectivity.

Detailed Description

Example 1 preparation of Lysozyme imprinted Polymer MIP

First, experimental instrument and reagent

1. Laboratory apparatus

An ultrasonic cleaner (KQ118, kunshan ultrasonic instrument ltd); scanning electron microscope (SU8010, hitachi, japan); ultraviolet spectrophotometer (UV-vis, UV 2550, shimadzu, japan); fluorescence spectrometer (FL4500, hitachi, japan); electronic balance (BSA1245-CW, Saedodes, Germany); high speed centrifuges (H1650, Hunan instrument); a heat collection type constant temperature heating stirrer (Hengchi-1, Zhengzhou Hengchi); magnetic stirrers (model HZ85-2, Beijing Zhongwei industries, Ltd.); ultra pure water purification systems (PLA-CAXXBIOM2, Casada BIO USA); an electric heating constant temperature air-blast drying oven (DHG-9070A, Shanghai Baixin instruments and Equipment works); temperature control shaking table (TS-200B, Shanghai Tianzai Zhijie plant); oil pump (SHB-III, Zheng Changcheng science, industry and trade Co., Ltd.).

2. Experimental reagent

Citric acid (CA, 99%), polyethyleneimine (BPEI, 99%), methacrylic acid (MAA, 99%), hydroxyethyl methacrylate (HEMA, 99%), N-isopropylacrylamide (NIPAAm, 99%), N-methylenebisacrylamide (MBA, 99.8%), sodium dodecyl sulfate (SDS, ≧ 86%), ammonium persulfate (APS, 99.99%), Tetramethylethylenediamine (TEMED), LYZ (LYZ, 99%) purchased from Sigma-Aldrich; column chromatography silica gel (98%) was purchased from alatin (shanghai, china); HCl (guaranteed reagent) purchased from the beijing institute of chemical reagents; KH (Perkin Elmer)₂PO₄(98%) purchased from Alfa Aesar, usa; NaOH (more than or equal to 98.0%) is purchased from chemical reagents of national drug group, Inc.; CH (CH)₃COOH (HAc, super grade pure) from BeijingChemical reagent research institute; molecular weight cut-off 500Da, 15000Da dialysis bags purchased from MYM Biotechnology; the experimental water was ultrapure water.

Preparation of di-lysozyme imprinted polymer

1. Preparation of carbon quantum dots

In a 20mL small beaker, accurately weighing 0.5g BPEI and 0.1g CA, fully dissolving with 10mL 80 ℃ distilled water, heating the small beaker at about 180 ℃ by using an oil bath pan to obtain a light yellow viscous gel, adding 1mL distilled water to obtain a light yellow solution, and repeating the experimental operation to obtain the orange yellow gel. The resulting orange-yellow gel was dissolved in 10mL of deionized water and cooled to room temperature.

And (3) purification: using 0.01mol/L HCL as a mobile phase, passing the product through a silica gel column, collecting two effluent components, reserving the second component, collecting, performing rotary evaporation concentration, dissolving with 10mL deionized water, filling into a dialysis bag with molecular weight cutoff of 500Da, dialyzing until the pH is neutral, performing rotary evaporation concentration, fixing the volume of a CQDs solution of 0.0250g/mL, and storing in a refrigerator at 4 ℃ for later use.

Preparation of 2 lysozyme molecularly imprinted polymer

Accurately weighing a certain amount of monomers MAA, hydroxyethyl methacrylate HEMA and N-isopropyl acrylamide NIPAAM, wherein the specific weighing amount is shown in Table 1, and mixing to prepare four monomer mixtures with different components;

adding 20mL of PBS buffer solution with pH value of 7.4, 1mL of 0.0250g/mL CQD and 0.1g LYZ into monomer mixtures with different components respectively, performing ultrasonic complete dissolution, then adding 28mg of cross-linking agent MBA, subsequently adding magneton, fully mixing the reactants in a round-bottomed flask, and fully stirring in a 298.15K oil bath for 4 hours;

then, adding an initiator 20mg APS and 20 μ L TEMED into the sample with sufficient mixing reaction, then vacuumizing the sample under the protection of nitrogen, and then polymerizing the sample at the environmental temperature of 298.15K for 16 hours to obtain porous hydrogel;

the gel was lyophilized in a lyophilizer, and then ground into a powder with a mortar to obtain MIPs that can be blotted with LYZ.

A mixed eluate was prepared by dissolving 10mL of HAc and 10g of SDS in 90mL of deionized water. Dissolving a certain amount of ground MIP in 5mL of eluent, and putting the solution of MIP and the eluent into a dialysis bag with the cut-off molecular weight of 15000Da for elution by the eluent. And taking a certain amount of the eluate at intervals of 24 hours, and carrying out ultraviolet spectrum detection by using an ultraviolet spectrophotometer until the protein absorption peak value of an ultraviolet spectrum LYZ tends to be stable, namely the template molecule is considered to be completely removed. The dialyzed gel bag was then dialyzed against deionized water to remove HAc and SDS, and finally the gel was freeze-dried and ground.

A certain amount of ground gel solid is taken to prepare a gel stock solution with the concentration of 0.0003 g/mL. Diluting 0.2mL of the gel stock solution to 50mL to prepare 1.2X 10^-6g/mL of the gel solution, and stored at 4 ℃ in a refrigerator for later use.

The preparation steps and the reagent amount of the non-imprinted polymer (NIP) are the same as those for preparing MIP except that the template protein LYZ is not added.

Table 1: monomeric composition of different MIPs

Example 2 application of the novel lysozyme fluorescence sensor to the detection of lysozyme

The concentration ranges of the components are 6.138 multiplied by 10^-6g/mL-2.707×10^-5g/mL of a series of LYZ standard solutions of different mass concentrations; respectively putting 1mL of MIP and NIP gel solution into 1mL of LYZ standard solution with different mass concentrations for incubation, putting the prepared sample into a table type constant temperature oscillator, fully mixing for more than 24 hours at 298.15K, then measuring the fluorescence intensity of the solution by using a fluorescence spectrophotometer under the conditions of excitation of 280nm, emission of 290nm-500nm, a slit of 10.0nm and voltage of 700V, and carrying out parallel detection on the same sample at least twice.

Results and discussion

1. Preparation route of molecular imprinting hydrogel

FIG. 2 shows the preparation route of LYZ molecularly imprinted hydrogel. Firstly, CQDs is used as a fluorescence signal, LYZ is used as a template molecule, NIPAAm is used as a temperature-sensitive monomer, HEMA/MAA is used as an auxiliary monomer, MBA is used as a cross-linking agent, APS is used as an initiator, TEMED is used as a catalyst, and the molecularly imprinted hydrogel is synthesized, wherein the template molecule LYZ is combined with the temperature-sensitive monomer NIPAAm and the auxiliary monomer through weak bond action to form a hydrogel polymer, and after the template molecule LYZ is dialyzed and eluted in SDS-HAc eluent, a molecular cavity with specific binding sites, the shape and the size of which are consistent with those of LYZ is reserved.

2. Microstructure of molecularly imprinted hydrogels

As shown in FIG. 3, the prepared molecularly imprinted hydrogel has a three-dimensional porous structure. In contrast to the spherical structure of the prior application of the applicant (chinese patent application publication No. CN110283275A), molecularly imprinted hydrogel having a porous structure was obtained by changing the preparation conditions, although the components were prepared similarly. Compared with the gel with a spherical structure, the porous hydrogel has larger specific surface area, so that the porous hydrogel has better stability and specificity in detecting LYZ.

3. Elution of LYZ

Detecting the ultraviolet absorption peak of template molecule LYZ in the eluent by using an ultraviolet spectrophotometer, and if the ultraviolet absorption spectrum of the eluent has an absorption peak at the wavelength of about 280nm, namely the absorption peak is consistent with the absorption peak of LYZ, proving that the imprinting and the elution are successful. Collecting the eluate every 24h, and repeatedly detecting until the LYZ absorption peak does not increase, which proves that the elution reaches the end point

4. Fluorescence spectroscopy

By using fluorescence spectrum, it can be observed that the fluorescence intensity changes when the molecularly imprinted hydrogel and LYZ are adsorbed, as shown in FIG. 4, compared with the blank group, the molecularly imprinted hydrogel polymer has obvious fluorescence response to the template molecule, and the fluorescence intensity is obviously enhanced after combination.

5. Investigation of incubation time of molecularly imprinted hydrogel and lysozyme

In the molecular imprinting recognition, the incubation time is an extremely important reaction condition. When the incubation time is short, the functional monomer, the template molecule and the cross-linking agent cannot form sufficient binding sites, and when the incubation time is too long, non-specific adsorption in the solution may be formed. Therefore, this experiment examined the optimal incubation time for the MIPs prepared. The mass concentration of the preparation is 1.43 multiplied by 10^-6Taking 1mL LYZ standard solution, placing in 1mL MIP gel solution of component B, incubating in a table type constant temperature oscillator at 298.15K, and taking out a mixed sample at intervals of 5 minutes to determine the fluorescence intensity of the solution in a fluorescence spectrophotometer. The results are shown in FIG. 5.

As can be seen from the graph, the concentration at 1.43X 10^-6The fluorescence intensity obviously rises within 41min under g/mL LYZ; then, the rising trend of the fluorescence intensity of the sample begins to be slow; the inflection point begins to appear on the scattergram about 50min, and the overall trend gradually keeps stable and tends to be constant, which shows that the MIP has response to LYZ and is basically stable for 70 min. The microstructure of the prepared gel is porous, so that the specific surface area is increased, and the incubation time is greatly reduced. (incubation time of prior application required more than 29 h.)

From this, the experiment finally determined that the optimal incubation time was around 70 min.

6. Composition inspection of molecularly imprinted hydrogels

The molecularly imprinted polymer and the template molecule are mainly combined through intermolecular weak bond action, and different auxiliary monomers have different functional groups due to different structures, so that the molecularly imprinted polymer and the template molecule may show different degrees of specificity when interacting with each other. Therefore, we explored their selectivity for the template molecule LYZ by synthesizing four different compositions of molecularly imprinted hydrogel polymers (compositions as in Table 1 monomer compositions) for two auxiliary monomers, MAA and HEMA. The following figures (FIGS. 6-9) show the linear curves of the fluorescence response of MIPs and NIPs composed of different auxiliary monomers to the template molecule LYZ at 298.15K, respectively. The abscissa is LYZ concentration (g/mL) and the ordinate is F/F₀. Wherein, F₀Is fluorescence in the absence of LYZIntensity, F is the fluorescence intensity after binding to LYZ.

The imprinted molecule IF ═ K (mip)/K (nip), (K is the slope of the fluorescence response linear curve). By experiment, from fig. 6 to fig. 9, the four components of the imprinted molecule at 298.15K are: IF (a, 298.15K) ═ 0.844; IF (B, 298.15K) ═ 1.301; IF (C, 298.15K) ═ 1.051; IF (D, 298.15K) ═ 1.311. As can be seen from the data, at 298.15K, component B and component D are more selective for the target analyte.

Although the value of the imprinted molecule IF of the present application is not as high as that of the prior application, the stability and reproducibility of component D from the present application are superior to those of the prior application, as shown in table 2.

TABLE 2

7. Study of temperature-sensitive Properties

In the experiment, NIPAAm with temperature responsiveness is used as a functional monomer, so that the specific selectivity of gel blotting is regulated and controlled by incubation temperature. Therefore, this experiment examined the incubation temperature of the MIPs prepared. In this experiment, a comparative experiment of the incubation temperature of 318.15K was performed, in which the temperature of the tabletop constant temperature oscillator was changed to 318.15K, and the rest of the preparation steps, the detection steps and the conditions were not changed. At the same temperature, at least two experiments are carried out in parallel, and the fluorescence response linear curves of MIP and NIP composed of different auxiliary monomers to the template molecule LYZ at 318.15K are shown as the following graphs (FIG. 10-FIG. 13).

As can be seen from FIG. 14, when the blotting factor IF of each gel was compared at two incubation temperatures under a constant condition, the remaining three fractions were all IF except for fraction B (the functional monomer was HEMA + MAA)_298.15 _K<IF_318.15 _K(ii) a By further comparing the IF values of the remaining three groups and the standard deviation of the two parallel experiments, it was found that the blot factor IF values of the three groups of gels were IF at an incubation temperature of 318.15K_D>IF_C>IF_AWhen the functional monomer composition is NIPAAM, hatchingWhen the incubation temperature was 318.15K, the IF was at the maximum and was 1.38. Therefore, the experimental result also proves that the imprinting effect of the hydrogel can be regulated to a certain extent through the temperature.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are possible which remain within the scope of the appended claims.

Claims

1. The preparation method of the novel lysozyme fluorescence sensor is characterized by comprising the following steps:

(1) preparing and purifying carbon quantum dots;

(2) and (3) preparing a lysozyme molecularly imprinted polymer.

2. The method for preparing a novel lysozyme fluorescence sensor according to claim 1, wherein in the step (1), the method comprises the following steps:

3. The method for preparing a novel lysozyme fluorescence sensor according to claim 1, wherein in the step (2):

4. The method for preparing a novel lysozyme fluorescence sensor according to claim 3, wherein in the step (2-1), an auxiliary monomer is further included, and the auxiliary monomer is one or a mixture of hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA).

5. The method for preparing a novel lysozyme fluorescence sensor according to claim 3, wherein in the step (2-1), the amount of the monomer N-isopropylacrylamide NIPAAM added is 3.955 mmol.

6. The method for preparing a novel lysozyme fluorescence sensor according to claim 4, wherein the total amount of the monomer N-isopropylacrylamide NIPAAM and the auxiliary monomer is 3.89mmol to 4.02mmol, wherein the monomer N-isopropylacrylamide NIPAAM is 3.560mmol, the hydroxyethyl methacrylate HEMA is 0.230mmol to 0.460mmol, and the methacrylic acid MAA is 0.165mmol to 0.330 mmol.

7. The method for preparing a novel lysozyme fluorescence sensor according to claim 3, wherein in the step (2-2): adding 20mL of PBS buffer solution with pH value of 7.4, 1mL of 0.0250g/mL CQD and 0.1g LYZ into monomer N-isopropylacrylamide NIPAAM or a mixture of monomer N-isopropylacrylamide NIPAAM and auxiliary monomers, and ultrasonically dissolving;

8. Use of the novel lysozyme fluorescence sensor of any one of claims 1 to 7 for the detection of lysozyme.

9. The use of the novel lysozyme fluorescence sensor of claim 8 for the detection of lysozyme, comprising the steps of:

(1) respectively prepared in a concentration range of 6.138×10^-6g/mL-2.707×10^-5g/mL LYZ standard solution;

(2) taking 1mL MIP gel solution, placing the MIP gel solution in 1mL LYZ standard solution with different mass concentrations, and placing the MIP gel solution in a table type constant temperature oscillator for incubation: mixing and incubating fully for 10min-24 hours at 298.15K;

10. The use of the novel lysozyme fluorescence sensor of claim 9 to detect lysozyme, wherein the concentration of the MIP gel solution is 0.0003 g/mL; the incubation time was 70min at 298.15K.