CN117624684A - Bioactive membrane with sugar response and preparation method and application thereof - Google Patents

Abstract

The invention discloses a bioactive membrane with sugar response, and a preparation method and application thereof, and relates to the technical field of biological medicine. The preparation method comprises the following steps: s1: alternately depositing gelatin solution and tannic acid solution on the surface of the optical waveguide to obtain gelatin-tannic acid multilayer film; s2: the bovine serum albumin-phenylboronic acid conjugate is anchored on the gelatin-tannic acid multilayer film, so that the bioactive film with sugar response is obtained. The bioactive film can realize biological modification of the optical waveguide to obtain bioactivity, and can achieve the recycling of the biological coating through reversible combination of the phenylboronic acid group and the sugar, thereby enhancing the sensitivity and the specificity of detecting glucose.

Description

Bioactive membrane with sugar response and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to a bioactive membrane with sugar response, a preparation method and application thereof.

Background

The conventional blood glucose measuring method has various inconveniences, such as: the method is painful, inconvenient, and presents a risk of infection, etc., especially for patients requiring intensive insulin use, suggesting that blood glucose monitoring be performed at least 3 to 4 times per day. Thus, a reliable, comfortable, noninvasive method for measuring blood glucose, which is a clinical requirement, has prompted research and development of novel blood glucose measuring methods.

Based on this, a series of new generation glucose sensors have been developed. Among them, the optical waveguide glucose sensor is receiving attention more and more because of having not receiving electromagnetic interference, low cost, need not mark, small in size, easy operation etc. advantage. Optical waveguide biosensors are directed to the detection of substances by molecular recognition and sensing interactions involving the attachment of an interacting component to the surface of the sensor such that the analyte is both accessible and specific to the component immobilized on the optical waveguide surface, resulting in a detectable change in the refractive index of the local microenvironment.

Thus, in designing a glucose optical waveguide sensor, how to stably anchor a sufficient amount of components that interact with glucose to the surface of the optical waveguide is critical to the sensitivity and accuracy measurement of the optical waveguide sensor.

Disclosure of Invention

The invention provides a bioactive membrane with sugar response, a preparation method and application thereof, and aims to solve the problems in the background technology. Generating a stable and compact gelatin-tannic acid multilayer film on the surface of the optical waveguide in situ by an autonomous loading method so as to increase the specific surface area of the gelatin-tannic acid multilayer film; a lectin mimic of the bovine serum albumin-phenylboronic acid conjugate is prepared by a proper method, and the glucose receptor bovine serum albumin-phenylboronic acid conjugate is anchored on the porous structure of the gelatin-tannic acid multilayer film to determine the concentration of glucose, so that the biological coating can be recycled through reversible combination of phenylboronic acid groups and glucose.

In order to achieve the technical purpose, the invention mainly adopts the following technical scheme:

in a first aspect, the invention discloses a method for preparing a bioactive membrane with sugar response, comprising the steps of:

s1: alternately depositing gelatin solution and tannic acid solution on the surface of the optical waveguide to obtain gelatin-tannic acid multilayer film;

s2: the bovine serum albumin-phenylboronic acid conjugate is anchored on the gelatin-tannic acid multilayer film, so that the bioactive film with sugar response is obtained.

In a preferred embodiment of the present invention, in step S1, the gelatin solution is a gelatin solution having a concentration of 1mg/mL and a ph=6.5; the tannic acid solution is tannic acid solution with concentration of 1mg/mL and PH=7.0.

In a preferred embodiment of the present invention, in step S1, the preparation of the gelatin-tannic acid multilayer film specifically comprises the steps of:

s11: removing the outer coating of the optical waveguide by using dilute HNO ₃ Soaking, and then flushing the optical waveguide by using deionized water and absolute ethyl alcohol;

s12: soaking the optical waveguide obtained in the step S11 into NaOH solution, repeatedly flushing the optical waveguide with deionized water, and drying;

s13: immersing the optical waveguide obtained in the step S12 into a gelatin solution, and then rinsing with PBS buffer solution;

s14: immersing the optical waveguide obtained in the step S13 into a tannic acid solution, and then rinsing with PBS buffer solution;

s15: repeating steps S13 and S14 to obtain the gelatin-tannic acid multilayer film.

Further, in step S11, the diluted HNO ₃ Is diluted HNO with mass fraction of 5% ₃ The soaking time is 1 hour; in step S12, whereThe NaOH solution is 8mg/mL NaOH solution, which is soaked for 3.5 hours at 40 ℃ and then soaked for 30 minutes at room temperature.

Further, in step S13, the immersion time was 10 minutes, and the pH of the PBS buffer was 6.5; in step S14, the immersion time was 10 minutes, and the pH of the PBS buffer was 7.0.

In a preferred embodiment of the present invention, in step S2, the bovine serum albumin-phenylboronic acid conjugate is prepared by the following method:

dissolving succinic anhydride and 3-aminobenzene boric acid in anhydrous pyridine, and reacting to obtain succinylaminobenzeneboric acid;

and (3) carrying out condensation reaction on succinylaminobenzeneboronic acid and bovine serum albumin to prepare the bovine serum albumin-phenylboronic acid conjugate.

Further, the molar ratio of the succinic anhydride to the 3-aminophenylboronic acid is 1:1; succinimidyl phenylboronic acid and bovine serum albumin are condensed under the catalysis of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide.

In a preferred embodiment of the present invention, in step S2, the bovine serum albumin-phenylboronic acid conjugate is anchored to the gelatin-tannic acid multilayer film, specifically comprising the steps of:

s21: dissolving with deionized water to prepare lectin analog of bovine serum albumin-phenylboronic acid conjugate;

s22: immersing the gelatin-tannic acid multilayer film obtained in the step S1 into the solution prepared in the step S21, combining phenol groups in tannic acid with bovine serum albumin through hydrophobic action and hydrogen bonding action, and anchoring the bovine serum albumin-phenylboronic acid conjugate on the gelatin-tannic acid multilayer film.

In a second aspect, the invention discloses a biologically active membrane with a sugar response prepared by the method of the first aspect.

In a third aspect, the invention discloses an application of the bioactive film in the preparation of an optical waveguide biochemical sensor for detecting glucose.

Compared with the prior art, the invention has the following beneficial effects:

the bioactive film can realize biological modification of the optical waveguide to obtain bioactivity, and can achieve the recycling of the biological coating through reversible combination of the phenylboronic acid group and the sugar, thereby enhancing the sensitivity and the specificity of detecting glucose.

Drawings

FIG. 1 is a graph showing comparison of infrared spectrograms of 3-aminophenylboronic acid, succinic anhydride and BPOA, BSA, BSA-BA;

FIG. 2 is an SDS-PAGE electrophoresis of each group, wherein M.Marker,1.BSA,2, 3, 4, 5 are amino substitution degrees of 16%, 25%, 53%, 40% BSA-BA, respectively;

FIG. 3 is a TLC color chart of each group;

FIG. 4 is a graph comparing the determination of sugar capture capacity of each group using alizarin red method;

FIG. 5 is an electron microscope image of 1, 4, 5, 10 thin film scans, respectively;

FIG. 6 is a graph showing the effect of BSA-BA modified gelatin-tannin bioactive membranes on glucose.

Detailed Description

The invention and its embodiments are described below without limitation, and the actual embodiments are not limited thereto. In summary, if one of ordinary skill in the art is informed by this disclosure, a structural manner and an embodiment similar to the technical solution should not be creatively devised without departing from the gist of the present invention.

The preparation method mainly uses succinylaminobenzeneboronic acid as an intermediate and bovine serum albumin as a carrier, and prepares the bovine serum albumin-phenylboronic acid conjugate through amidation reaction under the catalysis of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide; the method comprises the steps of taking gelatin and tannic acid as skeletons, and constructing in situ based on hydrophobic interaction, electrostatic interaction between hydrogen bonds and adjacent layers by using a layer-by-layer self-assembly (LBL) technology to obtain a nano gelatin-tannic acid multilayer film; the lectin mimetic bovine serum albumin-phenylboronic acid conjugate is anchored on the gelatin-tannic acid multilayer film by utilizing the capability of phenolic groups in tannic acid to combine with proteins through hydrophobic action and hydrogen bonds, so as to prepare the gelatin-tannic acid bioactive film. The gelatin-tannic acid bioactive film can realize biological modification of the optical waveguide to obtain bioactivity, and can achieve the recycling of a biological coating through reversible combination of phenylboronic acid groups and sugar, so that the sensitivity and the specificity of detecting glucose are enhanced.

The present invention will be described in detail with reference to the accompanying drawings and examples.

Example 1

1. Synthesis and activation of succinimidyl phenylboronic acid (BPOA)

Succinic anhydride (5 g 0.05 mol) and 3-aminophenylboronic acid (7.75 g 0.05 mol) were added to 40ml of anhydrous pyridine, and the mixture was stirred overnight at room temperature on a magnetic stirrer. Adding 20ml of deionized water, standing for 1h, concentrating the solution to paste by a rotary evaporator, adding 10ml of glacial acetone into the concentrated solution, refrigerating overnight in a freezer, taking out the concentrate, adding 50ml of deionized water, adjusting the pH to 1.0 by hydrochloric acid, clarifying the solution to become turbid, and generating a large amount of precipitate. The solution was placed in a freezer overnight, crystals were precipitated, filtered with a buchner funnel and washed three times with deionized water, and the solids were collected. The solid was recrystallized in 200ml of boiling water to give succinylaminobenzeneboronic acid as a white needle-like solid.

The appropriate amount of succinimidyl phenylboronic acid is added to a PBS solution (pH=60.2M, 0.01M) (the volume of the solution is 3:500 according to the mass ratio of succinimidyl phenylboronic acid), and 94mg of succinimidyl phenylboronic acid is preferably completely dissolved in 15mL of a PBS buffer solution (0.01M) with pH of 6. The magnet was placed on a constant temperature magnetic stirrer and stirring (500 rpm) was turned on until the solid was completely dissolved. Taking 2.4 times of EDC and 2.5 times of NHS of the molar number of the butyryl diamido phenylboronic acid, adding the mixture into the solution, and activating the mixture for 2 hours under the condition of magnetic stirring (700 rpm).

2. Synthesis of bovine serum albumin-phenylboronic acid (BSA-BA) conjugates

100mg of Bovine Serum Albumin (BSA) was added to the above activated solution until complete dissolution, the pH of the solution was adjusted to 7 with 0.2M, 0.01M disodium hydrogen phosphate, respectively, placed on a magnetic stirrer overnight at room temperature, transferred to a dialysis filter bag (MW: 3500), dialyzed with 20 volumes of pH=7 of 0.01M PBS buffer at room temperature for 8 hours, each 3-4 hours replaced with buffer to remove unreacted succinimidyl phenylboronic acid, the solution was transferred to a clean petri dish, pre-frozen at-80℃for 8 hours, and lyophilized in a lyophilizer for 48 hours after the pre-freezing to give a white solid bovine serum albumin-phenylboronic acid BSA-BA conjugate.

BSA-BA with different amino substitution degrees was synthesized by changing the ratio of BPOA to BSA.

3. Gelatin tannic acid bioactive membrane construction

Gelatin solution and tannic acid solution with concentration of 1mg/mL were prepared, 100mg gelatin was taken in 100mL ph=6.5 PBS solution, and the seeds were placed on a magnetic stirrer and heated and stirred at 60 ℃ for 1h, at which time gelatin was completely dissolved. 100mg of tannic acid was dissolved in 100mL of deionized water, and the mixture was placed in a magnetic stirrer and stirred for 30 minutes, and the pH of the TA solution was adjusted to 7.0 using 0.5M sodium hydroxide solution.

The optical waveguide was divided into small segments of approximately 10cm, the outer cladding was removed, and first, 5% dilute HNO was used ₃ Soaking the optical waveguide modification region for 1h, and repeatedly flushing the optical waveguide modification region for 3 times by using deionized water and absolute ethyl alcohol; soaking the optical waveguide modification region in 100mL of NaOH solution with concentration of 8mg/mL for 3 half an hour (at 40 ℃) for 30 minutes, repeatedly washing the surface of the optical waveguide modification region with deionized water for 10 minutes to remove redundant impurities, and drying at 30 ℃ for 1 hour, wherein the aim of the step is to activate hydroxyl (-OH) of the optical waveguide modification region to enable the optical waveguide modification region to have functionalization. The light guide modification zone was immersed in the gelatin solution for 10 minutes and then rinsed with a pH 6.5PBS buffer for 6 minutes. The optical waveguide modification zone was immersed in the TA solution for 10 minutes and then rinsed with PBS buffer at pH 7.0 for 6 minutes, which is a bilayer fabrication step. The manufacturing steps are repeated until 1-10 bilayers of films of different thickness are obtained.

4. Embedding boric acid groups based on gelatin-tannic acid biofilm

1mg/mL BSA-BA lectin analog was dissolved and prepared with deionized water, the 10-layer gelatin-tannic acid optical waveguide constructed above, 10cm long, was immersed in the solution for 6 hours, and the BSA-BA lectin analog was anchored to the gelatin-tannic acid multilayer film by the ability of the phenolic groups in tannic acid to bind to proteins through hydrophobic interactions and hydrogen bonds, so that biological modification of the optical waveguide was achieved to obtain biological activity, and the boric acid groups were attached to the optical waveguide.

Example 2

Infrared determination the infrared spectra of 3-aminophenylboronic acid, succinic anhydride, BPOA, BSA, BSA-BA in example 1 compare the control molecular structures, verifying BPOA and BSA-BA.

The structure is confirmed as shown in fig. 1. The characteristic absorption peak of amino is 3500-3300cm ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Characteristic absorption peaks of benzene ring are 1600, 1580, 1500 and 1450cm ^-1 The method comprises the steps of carrying out a first treatment on the surface of the The characteristic absorption peak of carbonyl is 1900-1600cm ^-1 Within the region; the carbonyl group of succinic anhydride on succinimidophenylboronic acid is combined with the amino group of 3-aminophenylboronic acid, and the amino group gives an electron group, so that the c=o absorption shifts to the low frequency direction, and thus it is known that the prepared BPOA is a desired substance. According to the characteristic peaks it shows: bovine serum albumin BSA has a number of characteristic absorption bands in the infrared spectrum, with an amide band of 1600-1700cm ^-1 After covalent bonding succinimidyl phenylboronic acid, the reaction mixture was heated to 810cm ^-1 The left and right double peaks appear, and benzene rings can be obtained, so that the BPOA and the bovine serum albumin can be obtained to carry out amidation reaction, and the bovine serum albumin-phenylboronic acid conjugate can be obtained.

Example 3

20mg leucine was dissolved in a 100mL beaker with 80mL of PBS buffer at pH=8.2 to a volume of 100mL to prepare 1.525×10 ^-3 Standard solution. 4mg of BSA and 4mg of BSA-BA lectin analog were dissolved in 4mL of PBS buffer having pH=8.2, and the solution was subjected to ultrasonic dispersion to prepare a sample reagent of 1 mg/mL. 1g of Sodium Dodecyl Sulfate (SDS) was dissolved in 10mL of deionized water to prepare a 10% SDS solution. 80uL of 5% 3-nitrobenzenesulfonic acid (TNBS) solution was diluted to 20mL to prepare a 0.01% TNBS solution.

A blank group, a leucine control group, a BSA control group, and a sample group were set, respectively. 3mL of pH=8.2 PBS buffer, leucine solution, BSA solution, and sample solution were placed in 10mL tubes of the blank group, leucine control group, BSA control group, and sample group, and 2mL of 0.01% TNBS solution was added, respectively. The well mixed solutions were placed in a thermostatted shaker (temperature: 37 ℃ C., rotation speed: 200 rpm) and incubated for 4h, after which 2mL of 10% SDS solution and 1mL of 1M HCl were added to each group, respectively. Vortex stirring is carried out on a turbine stirrer, and stirring is uniform. Absorbance of each sample was measured at 340nm using a two-beam uv-vis spectrophotometer. The substitution degree of the amino group on BSA was calculated.

The results are shown in tables 1-3. The amino substitution was varied according to the pH of the reaction, the mass feed ratio, the concentration of PBS buffer, etc. during the experiment, as shown in tables 1 to 3 below.

Under the condition of the same feeding ratio, when the pH value of the solution is adjusted to 7 by adding the bovine serum albumin after BPOA activation, the amino substitution degree of the bovine serum albumin is obviously increased. As the feed ratio increases, the amino substitution degree of the bovine serum albumin also gradually increases, and the feed ratio 10:6 to 10: the substitution degree of 12 is increased by 17%, and the final feeding ratio is 10:12 to 46%. The results show that under certain reaction conditions, the bovine serum albumin-phenylboronic acid conjugate prepared under the same feed ratio has the amino substitution degree of the bovine serum albumin obtained by using the 0.01M PBS buffer solution which is 13% higher than that of the bovine serum albumin obtained by using the 0.2M PBS buffer solution.

TABLE 1 amino substitution of bovine serum albumin at reaction pH

TABLE 2 degree of amino substitution of bovine serum Albumin with different buffer concentrations

TABLE 3 amino substitution of bovine serum Albumin with different feed ratios (mass ratios)

Example 4

SDS-PAGE electrophoresis shows molecular weight. Group 1 is control group BSA, and groups 2, 3, 4 and 5 are sample groups BSA-BA with substitution degree of 16%, 25%, 53% and 40% respectively.

As shown in FIG. 2, the sample group and the control group have a height difference between 55-70kDa, and the amino substitution is not self-substitution (bovine serum albumin molecular weight 68 kDa), and the molecular weight of the BSA-BA protein of the sample group is larger than that of the BSA of the control group, so that the successful coupling of the succinimidyl phenylboronic acid and the bovine serum albumin can be obtained.

Example 5

Alizarin Red (ARS) fluorescent coloration. Alizarin can be used as a color reagent to carry out TLC color development on boric acid compounds with high selectivity and sensitivity. Alizarin is an anthraquinone natural product which is itself non-fluorescent under 366nm light, but when mixed with boric acid, the ortho-diphenol hydroxyl groups of alizarin complex with boron to form a complex which strongly shows yellow fluorescence.

The results are shown in FIG. 3. Yellow fluorescence was developed by 1mM alizarin acetone solution with boric acid groups under 365nm ultraviolet light. The BPOA of example 1 was used as a control group, and BSA-BA obtained in different batches and different feed ratios were used as a sample group in groups 1, 2, 3, 4, 5 and 6, and yellow fluorescence was observed in both the control group and the sample group. The prepared bovine serum albumin-boric acid conjugate is proved to contain boric acid groups.

Example 6

Alizarin Red (ARS) displacement method determines sugar capturing ability. 1.4mg of alizarin red was precisely weighed and dissolved in 40mL of PBS buffer at pH 7.4 to prepare 1.0X10 ^-4 Alizarin red solution; 5.0mg of BPOA, BSA and BSA-BA were precisely weighed and dissolved in 5mL of PBS buffer having a pH of 7.4 to prepare a 1mg/mL solution. 0.54g of glucose was weighed and dissolved in 1.5mL of PBS buffer at pH 7.4 to prepare a 2M glucose solution. Preparing 7 groups of solutions, the group numbers being respectively 1, 2, 3, 4, 5, 6, 7,1 groups to 6 groups being respectively added 200ul of 1.0X10 ^-4 Is a solution of ARS; 500ul of 1mg/mL of BPOA solution is added to the groups 2 and 3 respectively; 500ul of 1mg/mL BSA solution was added to each of the groups 4 and 7; 500ul of 1mg/mL BSA-BA solution was added to each of the 5 and 6 groups; 500ul of 2M glucose solution is added to each of the 3 groups and the 6 groups; groups 1, 2, 4, 5, and 7 were each supplemented with 1mL, 500ul, 700ul of PBS buffer at pH 7.4 to a total volume of 1.2mL. And (3) scanning the wavelength of 300-700nm by using a double-beam ultraviolet-visible spectrophotometer, and further analyzing.

As a result, as shown in FIG. 4 a-b, ARS solution was pink in color and absorbed at a wavelength of about 510 nm; the group 2 is changed from the pink of group 1 to yellow, and the ultraviolet absorption wavelength is changed from about 510nm to about 460 nm. The color of the 3 groups is slightly more than that of the 2 groups, and the absorption wavelength is rightwards shifted than that of the 2 groups, so that the BPOA can be proved to be combined with sugar; the group 5 is changed from the pink of group 1 to light yellow, and the ultraviolet absorption wavelength is changed from about 510nm to about 490 nm; the color of the group 6 is changed from pale yellow to pale pink compared with the group 5, and the absorption wavelength is shifted from about 490nm to about 510nm, so that ARS is released after glucose is added, and the bovine serum albumin-boric acid conjugate has the capacity of capturing sugar, and the color of the group 4 is not obviously changed compared with the color of the group 1, so that the color change of the group 6 is caused by the bound boric acid group.

Example 7

The surface morphology of the multilayer films was observed by scanning electron microscopy and compared.

The results are shown in FIG. 5. The image is that the hydroxyl enrichment of the optical waveguide is not carried out before the coating, the surface morphology of the multilayer film is observed through a scanning electron microscope, and when 1 layer can be observed, the surface of the optical waveguide is uneven and only a partial coating is arranged; when the optical waveguide is used for 4 layers, the surface of the optical waveguide is even compared with that of the optical waveguide of 1 layer, and partial coating is uneven; when the optical waveguide is used for 5 layers, the surface of the optical waveguide is relatively uniform, and a compact porous structure can be obviously observed; the coating is most uniform when 10 layers are formed, the porous structure is more obvious than when 5 layers are formed, and the thickness change of the gelatin tannic acid constructed optical waveguide can be observed. Thus, in subsequent experiments, the gelatin-tannin structured optical waveguides were modified by first using 5% HNO ₃ And 8mg/mLNaOH to activate the hydroxyl group (-OH) of the optical waveguide modification region to haveThe optical waveguide is constructed by using gelatin-tannic acid, because gelatin is a long-chain molecule composed of amino acids and the ratio of hydrophilic amino acids is large, wherein hydroxyproline and hydroxylysine are amino acids with hydroxyl groups, have a plurality of hydroxyl groups, can be covalently combined with the hydroxyl groups through hydrogen bonds, can improve the coating effect of 1 layer, and further can lead the surface coating of the optical waveguide to be uniform.

Example 8

The gelatin-tannic acid optical waveguide biofilm constructed in example 1 was subjected to a sugar response.

The results are shown in FIG. 6. The wavelength of transmitted light can shift leftwards, namely the wavelength is reduced, with the increase of concentration after the gelatin-tannic acid constructed optical waveguide biological membrane is embedded with the bovine serum albumin-boric acid conjugate. Although the concentration change is small, the wavelength change can be detected, and meanwhile, the comparison of the measurement range graphs of postprandial glucose of healthy and diabetic patients in body fluid is obvious, so that the sensitivity of the optical waveguide for detecting sugar is better; the detection was performed using a NaCl solution, and the obtained NaCl curve was overlapped with the 0mM curve, and the NaCl solution was not responsive, so that it was found that the specificity of the optical waveguide for detecting sugar was good.

The foregoing is merely a specific implementation of the disclosure, but the scope of the embodiments of the disclosure is not limited thereto, and any person skilled in the art may easily think of changes, substitutions or combinations within the technical scope of the embodiments of the disclosure or under the ideas of the embodiments of the disclosure, and all fall within the scope of the embodiments of the disclosure.

Claims (10)

1. A method for preparing a bioactive membrane having a sugar response, comprising the steps of:

s1: alternately depositing gelatin solution and tannic acid solution on the surface of the optical waveguide to obtain gelatin-tannic acid multilayer film;

s2: the bovine serum albumin-phenylboronic acid conjugate is anchored on the gelatin-tannic acid multilayer film, so that the bioactive film with sugar response is obtained.

2. The method for preparing a bioactive film having a sugar response of claim 1, wherein: in the step S1, the gelatin solution is gelatin solution with the concentration of 1mg/mL and the PH=6.5; the tannic acid solution is tannic acid solution with concentration of 1mg/mL and PH=7.0.

3. The method for preparing a bioactive film having a sugar response of claim 1, wherein: in step S1, the preparation of the gelatin-tannic acid multilayer film specifically includes the following steps:

s11: removing the outer coating of the optical waveguide by using dilute HNO ₃ Soaking, and then flushing the optical waveguide by using deionized water and absolute ethyl alcohol;

s12: soaking the optical waveguide obtained in the step S11 into NaOH solution, repeatedly flushing the optical waveguide with deionized water, and drying;

s13: immersing the optical waveguide obtained in the step S12 into a gelatin solution, and then rinsing with PBS buffer solution;

s14: immersing the optical waveguide obtained in the step S13 into a tannic acid solution, and then rinsing with PBS buffer solution;

s15: repeating steps S13 and S14 to obtain the gelatin-tannic acid multilayer film.

4. A method of preparing a bioactive film having a sugar response as claimed in claim 3, wherein: in step S11, the diluted HNO ₃ Is diluted HNO with mass fraction of 5% ₃ The soaking time is 1 hour; in the step S12, the NaOH solution is 8mg/mL NaOH solution, and is soaked for 3.5 hours at 40 ℃ and then soaked for 30 minutes at room temperature.

5. A method of preparing a bioactive film having a sugar response as claimed in claim 3, wherein: in step S13, the immersion time was 10 minutes, and the pH of the PBS buffer was 6.5; in step S14, the immersion time was 10 minutes, and the pH of the PBS buffer was 7.0.

6. The method for preparing a bioactive film having a sugar response of claim 1, wherein: in step S2, the bovine serum albumin-phenylboronic acid conjugate is prepared by the following method:

dissolving succinic anhydride and 3-aminobenzene boric acid in anhydrous pyridine, and reacting to obtain succinylaminobenzeneboric acid;

and (3) carrying out condensation reaction on succinylaminobenzeneboronic acid and bovine serum albumin to prepare the bovine serum albumin-phenylboronic acid conjugate.

7. The method for preparing a bioactive film having a sugar response of claim 6, wherein: the molar ratio of the succinic anhydride to the 3-aminophenylboronic acid is 1:1; succinimidyl phenylboronic acid and bovine serum albumin are condensed under the catalysis of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide.

8. The method for preparing a bioactive film having a sugar response of claim 1, wherein: in step S2, the bovine serum albumin-phenylboronic acid conjugate is anchored on the gelatin-tannic acid multilayer film, and specifically comprises the following steps:

s21: dissolving with deionized water to prepare lectin analog of bovine serum albumin-phenylboronic acid conjugate;

s22: immersing the gelatin-tannic acid multilayer film obtained in the step S1 into the solution prepared in the step S21, combining phenol groups in tannic acid with bovine serum albumin through hydrophobic action and hydrogen bonding action, and anchoring the bovine serum albumin-phenylboronic acid conjugate on the gelatin-tannic acid multilayer film.

9. A bioactive film having a sugar response produced by the method of any one of claims 1-8.

10. Use of a bioactive film as claimed in claim 9 in the manufacture of an optical waveguide biochemical sensor for detecting glucose.