Preparation of boric acid-modified AIE nanoparticles and their application in rapid detection and efficient killing of Staphylococcus aureus

Abstract

The invention discloses a preparation method of boric acid-modified AIE nanoparticles and their application in rapid detection and efficient killing of Staphylococcus aureus. The invention belongs to the technical field of food safety immunoassay and photocatalytic sterilization, and specifically relates to a method for rapid detection and efficient killing of Staphylococcus aureus based on aggregation-induced luminescence materials. The purpose of the invention is to solve the problems that the existing fluorescent immunoassay method is prone to partial or complete quenching when detecting bacteria, which limits its application, and the traditional organic photosensitizers are limited by their planar rigid π-conjugated structure and poor water solubility, which lead to aggregation fluorescence quenching and insufficient ROS production, which limits their application. A boric acid-modified AIE nanoparticle is used in the rapid detection of Staphylococcus aureus. A boric acid-modified AIE nanoparticle is used in the efficient killing of Staphylococcus aureus. The invention proposes a multifunctional integrated platform that can not only provide early warning of the presence of bacteria, but also avoid the massive reproduction of bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety immunoassay and photocatalytic sterilization, and specifically relates to a method for rapid detection and efficient killing of Staphylococcus aureus based on aggregation-induced luminescence materials. Background Art

[0002] Staphylococcus aureus is a common foodborne pathogen that is widely present in nature. Food contaminated by Staphylococcus aureus not only causes waste of food resources, but also causes many food safety incidents. The rapid increase in bacterial infections requires convenient and cost-effective detection strategies.

[0003] At present, there are many strategies for bacterial detection, including polymerase chain reaction, plate culture, magnetic resonance, mass spectrometry, electrochemistry, and surface enhanced Raman scattering. Among them, fluorescence immunoassay is an effective tool for bacterial detection, but the fluorescence emission of traditional organic or inorganic fluorescent materials will be affected and partially or completely quenched in the aggregated state due to the ACQ effect, which greatly limits their practical application. Therefore, AIE (aggregation-induced emission) materials have attracted widespread attention. AIE materials have the advantages of high signal-to-noise ratio, high sensitivity, strong anti-bleaching ability, etc. with low background radiation, which overcomes the shortcomings of traditional fluorescent materials.

[0004] Recently, antimicrobial photodynamic therapy (aPDT), which uses photosensitizers to generate toxic reactive oxygen species (ROS), has become an alternative method to selectively eliminate bacteria or other pathogenic microorganisms. Due to the characteristics of aPDT that can photoswitch ROS generation and has good antibacterial effects on multidrug-resistant bacteria, it has attracted widespread attention and application in the treatment of bacterial infections. However, traditional organic photosensitizers (such as porphyrins, phthalocyanines, etc.) have a planar rigid π-conjugated structure and poor water solubility, resulting in aggregate fluorescence quenching (ACQ) and insufficient ROS production, which greatly affects their application in photodynamic therapy. Summary of the invention

[0005] The purpose of the present invention is to solve the problems that the existing fluorescent immunoassay method is prone to partial or complete quenching when detecting bacteria, which limits its application, and the traditional organic photosensitizers are prone to aggregation fluorescence quenching and insufficient ROS production due to their planar rigid π-conjugated structure and poor water solubility, which limits their application. A boric acid-modified AIE nanoparticle preparation and its application in rapid detection and efficient killing of Staphylococcus aureus are provided.

[0006] A method for preparing boric acid-modified AIE nanoparticles is specifically completed by the following steps:

[0007] 1. Dissolving 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene and distearoylphosphatidylethanolamine-polyethylene glycol 2000 in tetrahydrofuran, and then adding deionized water to obtain a mixed solution III; subjecting the mixed solution III to ultrasonic treatment, and then stirring in a dark fume hood to remove tetrahydrofuran, and then centrifuging, and washing the solid product obtained by centrifugation with deionized water, and then freeze-drying to obtain AIE nanoparticles; dispersing the AIE nanoparticles in deionized water to obtain an AIE nanoparticle solution, and storing it at 4°C;

[0008] 2. Mix the physiological saline containing EDC and NHS with the AIE nanoparticle solution, incubate at room temperature, and wash with physiological saline after incubation to obtain EDC / NHS-mediated AIE nanoparticles; resuspend the EDC / NHS-mediated AIE nanoparticles in physiological saline, add 3-aminophenylboronic acid to obtain a mixed solution IV; stir the mixed solution IV at room temperature, and finally wash with physiological saline to remove 3-aminophenylboronic acid to obtain APBA-AIE NPs, which are boronic acid-modified AIE nanoparticles.

[0009] A boric acid-modified AIE nanoparticle was applied in the rapid detection of Staphylococcus aureus.

[0010] A boric acid-modified AIE nanoparticle was applied in the efficient killing of Staphylococcus aureus.

[0011] Beneficial effects of the present invention:

[0012] The present invention can confirm that the material (immunoglobulin-functionalized Fe3O4 and boric acid-modified AIE nanoparticles) is successfully synthesized and has good detection and photocatalytic bactericidal properties through material characterization, Staphylococcus aureus detection and photocatalytic killing tests; further mechanism exploration can lead to the conclusion that the material can produce a large amount of activity (ROS) under light conditions, destroy the bacterial cell membrane, and cause leakage of cell contents, thereby achieving the purpose of sterilization; detection and bactericidal tests have proved that the material has achieved integrated detection and sterilization of Staphylococcus aureus, and the MTT cytotoxicity test has proved that the material has good biocompatibility and has great practical application value in the control of Staphylococcus aureus.

[0013] Innovations of the present invention:

[0014] (1) Most traditional food sterilization technologies have the disadvantages of high energy consumption, high equipment requirements, and environmental pollution. In the present invention, clean renewable energy solar energy is used, and nanomaterials are used as the medium for photocatalytic sterilization. This technology has significant advantages such as low energy consumption, simple operation, and complete degradation without secondary pollution.

[0015] (2) At present, traditional photosensitizer materials are often prone to produce ACQ effect, resulting in a significant reduction in fluorescence performance in the aggregated state. At the same time, they also have defects such as high dark toxicity, poor chemical stability and relatively low ROS efficiency in water, which greatly limits the application of PDT in the antibacterial field. In order to overcome these limitations, the AIE material selected in the present invention has enhanced fluorescence in the aggregated state and enhanced photodynamic performance when aggregated, which is an ideal material for fluorescent detection and photocatalytic killing of bacteria.

[0016] (3) The present invention proposes a multifunctional integrated platform that can not only provide early warning of the presence of bacteria, but also prevent the massive reproduction of bacteria from causing greater harm, providing new strategies and ideas for the development of a multifunctional sensing system that can target, detect and inactivate bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figures are effect diagrams of detecting Staphylococcus aureus at different dosages and different incubation times of immunoglobulin-functionalized Fe3O4 in Example 1, wherein a is a diagram showing the efficiency of capturing Staphylococcus aureus after changing the dosage of immunoglobulin-functionalized Fe3O4 when the solution containing Staphylococcus aureus and the immunoglobulin-functionalized Fe3O4 solution are incubated for 120 min, and b is a diagram showing the efficiency of capturing Staphylococcus aureus after changing the incubation time of the solution containing Staphylococcus aureus and the immunoglobulin-functionalized Fe3O4 solution when the dosage of immunoglobulin-functionalized Fe3O4 is 400 μg / mL;

[0018] Figure 2 This is a fluorescence signal diagram detected in Example 2, in which a is the fluorescence spectrum of the detection system after incubation of Staphylococcus aureus at different concentrations, and b is the standard curve of Staphylococcus aureus;

[0019] Figure 3 This is a plate diagram of the colony growth of Staphylococcus aureus under different light exposure times;

[0020] Figure 4 1 is a graph of the bactericidal efficiency of Staphylococcus aureus, in which 1 is a blank control, 2 is sunlight irradiation alone, 3 is sunlight irradiation and immunoglobulin-functionalized Fe3O4, 4 is sunlight irradiation and boric acid-modified AIE nanoparticles, and 5 is sunlight irradiation, immunoglobulin-functionalized Fe3O4 and boric acid-modified AIE nanoparticles in Example 3;

[0021] Figure 5 Figures 3 and 4 are morphological diagrams and laser confocal images of Staphylococcus aureus at different illumination times, wherein a is a morphological diagram of Staphylococcus aureus at different illumination times in Example 3, and b is a laser confocal image of live and dead cells of Staphylococcus aureus;

[0022] Figure 6 These are electron microscope images, where a is the SEM image of Fe3O4 and b is the SEM image of AIE nanoparticles. DETAILED DESCRIPTION

[0023] Specific implementation method 1: This implementation method is a method for preparing boric acid-modified AIE nanoparticles, which is specifically completed by the following steps:

[0024] 1. Dissolving 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene and distearoylphosphatidylethanolamine-polyethylene glycol 2000 in tetrahydrofuran, and then adding deionized water to obtain a mixed solution III; subjecting the mixed solution III to ultrasonic treatment, and then stirring in a dark fume hood to remove tetrahydrofuran, and then centrifuging, and washing the solid product obtained by centrifugation with deionized water, and then freeze-drying to obtain AIE nanoparticles; dispersing the AIE nanoparticles in deionized water to obtain an AIE nanoparticle solution, and storing it at 4°C;

[0025] 2. Mix the physiological saline containing EDC and NHS with the AIE nanoparticle solution, incubate at room temperature, and wash with physiological saline after incubation to obtain EDC / NHS-mediated AIE nanoparticles; resuspend the EDC / NHS-mediated AIE nanoparticles in physiological saline, add 3-aminophenylboronic acid to obtain a mixed solution IV; stir the mixed solution IV at room temperature, and finally wash with physiological saline to remove 3-aminophenylboronic acid to obtain APBA-AIE NPs, which are boronic acid-modified AIE nanoparticles.

[0026] Specific embodiment 2: The difference between this embodiment and specific embodiment 1 is that: the concentration of AIE nanoparticles in the AIE nanoparticle solution described in step 1 is 1 mg / mL; the mass volume ratio of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene, distearoylphosphatidylethanolamine-polyethylene glycol 2000, tetrahydrofuran and deionized water in the mixed solution III described in step 1 is 0.5g:1g:1mL:9mL; the ultrasound described in step 1 is ultrasonic treatment using a micro-tip probe ultrasonic pulverizer for 20min to 30min, and the output power of the micro-tip probe ultrasonic pulverizer is 180W; in step 1, stirring is performed in a dark fume hood at a stirring speed of 500r / min to 600r / min for 10h to 12h, tetrahydrofuran is removed, and then centrifuged, and the solid product obtained by centrifugation is washed 3 to 5 times with deionized water, and then freeze-dried at -50°C for 10h to obtain AIE NPs, i.e. AIE nanoparticles. The other steps are the same as those in the first embodiment.

[0027] Specific implementation method three: This implementation method is different from specific implementation method one or two in that: the concentration of EDC in the physiological saline containing EDC and NHS described in step 2 is 2 mg / mL, and the concentration of NHS is 2 mg / mL; the volume ratio of the physiological saline containing EDC and NHS to the AIE nanoparticle solution described in step 2 is 1:1; the mass volume ratio of EDC / NHS-mediated AIE nanoparticles, 3-aminophenylboronic acid and physiological saline in the mixed solution IV described in step 2 is 1 mg:2 mg:1 mL; in step 2, the mixed solution IV is stirred at room temperature for 30 minutes, and finally washed with physiological saline for 3 to 5 times to remove 3-aminophenylboronic acid to obtain APBA-AIE NPs, i.e., boric acid-modified AIE nanoparticles. The other steps are the same as specific implementation method one or two.

[0028] Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that: a boric acid-modified AIE nanoparticle is used in the rapid detection of Staphylococcus aureus. The other steps are the same as those of specific embodiments 1 to 3.

[0029] Specific embodiment 5: This embodiment differs from specific embodiments 1 to 4 in that the rapid detection of Staphylococcus aureus by boric acid-modified AIE nanoparticles is accomplished by the following steps:

[0030] The solution containing Staphylococcus aureus and the immunoglobulin functionalized Fe3O4 solution are uniformly mixed to obtain reaction system I; the reaction system I is incubated at 37°C for 30min to 120min, and then magnetic separation is performed to obtain a reaction solution; boric acid-modified AIE nanoparticles are added to the reaction solution to obtain reaction system II; the reaction system II is incubated at 37°C for 30min, and then magnetic separation is performed to obtain a solution to be tested; the fluorescence intensity of the solution to be tested and a blank solution not containing Staphylococcus aureus is respectively detected using a fluorescence spectrophotometer. If the fluorescence intensity of the solution to be tested is lower than the fluorescence intensity of the blank solution not containing Staphylococcus aureus, it indicates that the solution to be tested contains Staphylococcus aureus;

[0031] The concentration of Staphylococcus aureus in the reaction system I is 10-10 6 CFU / mL, the concentration of immunoglobulin-functionalized Fe3O4 was 400 μg / mL;

[0032] The concentration of the boric acid-modified AIE nanoparticles in the reaction system II is 200 μg / mL. The other steps are the same as those in the specific embodiments 1 to 4.

[0033] Specific embodiment 6: This embodiment differs from specific embodiments 1 to 5 in that: a boric acid-modified AIE nanoparticle is used in the efficient killing of Staphylococcus aureus. The other steps are the same as those of specific embodiments 1 to 5.

[0034] Specific embodiment 7: This embodiment differs from specific embodiments 1 to 6 in that: a boric acid-modified AIE nanoparticle efficiently kills Staphylococcus aureus by the following steps:

[0035] The immunoglobulin-functionalized Fe3O4 solution and the boric acid-modified AIE nanoparticles were added to a solution containing Staphylococcus aureus and stirred evenly to obtain a bacterial suspension; a 300W xenon lamp was used to simulate sunlight to irradiate the bacterial suspension for 20 minutes to 60 minutes to obtain a solution that kills Staphylococcus aureus;

[0036] The concentration of immunoglobulin functionalized Fe3O4 in the bacterial suspension is 400 μg / mL, and the concentration of boric acid modified AIE nanoparticles is 200 μg / mL;

[0037] The height of the 300W xenon lamp from the bacterial suspension is 20 cm. The other steps are the same as those of the first to sixth embodiments.

[0038] Specific embodiment eight: This embodiment differs from specific embodiments one to seven in that: the preparation method of the immunoglobulin functionalized Fe3O4 solution is specifically completed according to the following steps:

[0039] 1. Preparation of carboxylated Fe3O4:

[0040] FeCl3·6H2O is dissolved in ethylene glycol under stirring conditions, and then Na3C6H5O7·2H2O is added, and then stirred under heating conditions to obtain a mixed solution I; anhydrous NaOAc is added to the mixed solution I, and stirred until a clear solution is formed to obtain a mixed solution II; the mixed solution II is sealed in a Teflon-lined stainless steel high-pressure tank, and then a hydrothermal reaction is carried out, and the mixture is cooled to room temperature, and the generated black magnetite particles are collected; the generated black magnetite particles are washed to obtain carboxylated Fe3O4;

[0041] 2. Preparation of immunoglobulin functionalized Fe3O4 solution:

[0042] ①, washing the carboxylated Fe3O4 with sterile saline, and then resuspending it in saline containing EDC and NHS to obtain mixture I; stirring mixture I at room temperature, and then washing it with sterile saline to remove excess EDC and NHS to obtain activated Fe3O4;

[0043] ②, resuspend the activated Fe3O4 in sterile saline, and then add the porcine immunoglobulin solution to obtain mixture II; incubate mixture II at room temperature to obtain IgG-Fe3O4; wash IgG-Fe3O4 with sterile saline, and then resuspend it in sterile saline to a concentration of 1 mg / mL of IgG-Fe3O4 to obtain an immunoglobulin functionalized Fe3O4 solution, and store it at 4°C. The other steps are the same as those in specific embodiments one to seven.

[0044] Specific implementation method 9: The difference between this implementation method and specific implementation methods 1 to 8 is that the mass volume ratio of FeCl3·6H2O, Na3C6H5O7·2H2O, anhydrous NaOAc and ethylene glycol described in step 1 is 0.8g:0.47g:1.2g:40mL; in step 1, FeCl3·6H2O is dissolved in ethylene glycol under stirring, and then Na3C6H5O7·2H2O is added, and then stirred at 80℃~85℃ to obtain mixed solution I; anhydrous NaOAc is added to mixed solution I, and stirred until a clear solution is formed to obtain mixed solution II; mixed solution II is sealed in a Teflon-lined stainless steel high-pressure tank, and then hydrothermally reacted at 200℃ for 6h, cooled to room temperature, and the generated black magnetite particles are collected; the generated black magnetite particles are washed alternately with anhydrous ethanol and deionized water to remove ethylene glycol to obtain carboxylated Fe3O4. The other steps are the same as specific implementation methods 1 to 8.

[0045] Specific embodiment ten: This embodiment differs from specific embodiments one to nine in that: the concentration of EDC in the physiological saline containing EDC and NHS described in step 2① is 20 mg / mL, and the concentration of NHS is 20 mg / mL; the concentration of carboxylated Fe3O4 in the mixture I described in step 2① is 10 mg / mL; in step 2①, carboxylated Fe3O4 is washed 3 to 5 times with sterile physiological saline, and then resuspended in physiological saline containing EDC and NHS to obtain mixture I; mixture I is stirred at room temperature for 30 minutes, and then washed 3 to 5 times with sterile physiological saline to remove excess EDC and NHS to obtain activated Fe3O4; the concentration of the porcine immunoglobulin solution described in step 2② is 1mg / mL; the mass volume ratio of activated Fe3O4, porcine immunoglobulin solution and sterile saline in the mixture II described in step 2② is 10mg:300μL:10mL; in step 2②, the mixture II is cultured at room temperature for 2h to obtain IgG-Fe3O4; the IgG-Fe3O4 is washed 3 to 5 times with sterile saline, and then resuspended in sterile saline to a concentration of 1mg / mL of IgG-Fe3O4 to obtain an immunoglobulin functionalized Fe3O4 solution, and stored at 4°C; the saline described in step 2② is a sodium chloride aqueous solution with a mass fraction of 0.9%, and the preparation method is: weigh 9g of sodium chloride and dissolve it in 900mL of deionized water, and then dilute it to 1000mL with deionized water. The other steps are the same as the specific embodiments one to nine.

[0046] The following examples are used to verify the beneficial effects of the present invention:

[0047] Example 1: Application of boric acid-modified AIE nanoparticles in rapid detection of Staphylococcus aureus is accomplished by the following steps:

[0048] A solution containing Staphylococcus aureus (ATCC 29213) and an immunoglobulin functionalized Fe3O4 solution are mixed uniformly to obtain a reaction system I; the reaction system I is incubated at 37°C for 0 min, 30 min, 60 min, 90 min, 120 min or 150 min, and then separated by magnetic separation to obtain a reaction solution; boric acid-modified AIE nanoparticles are added to the reaction solution to obtain a reaction system II; the reaction system II is incubated at 37°C for 30 min, and then separated by magnetic separation to obtain a solution to be tested; a fluorescence spectrophotometer is used to detect the fluorescence intensity of the solution to be tested and a blank solution without Staphylococcus aureus, respectively. If the fluorescence intensity of the solution to be tested is lower than the fluorescence intensity of the blank solution without Staphylococcus aureus, it means that the solution to be tested contains Staphylococcus aureus;

[0049] The concentration of Staphylococcus aureus in the reaction system I is 10 3 CFU / mL, the concentrations of immunoglobulin-functionalized Fe3O4 were 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL;

[0050] The concentration of the boric acid-modified AIE nanoparticles in the reaction system II is 200 μg / mL.

[0051] Figure 1 The figures are effect diagrams of detecting Staphylococcus aureus at different dosages and different incubation times of immunoglobulin-functionalized Fe3O4 in Example 1, wherein a is a diagram showing the efficiency of capturing Staphylococcus aureus after changing the dosage of immunoglobulin-functionalized Fe3O4 when the solution containing Staphylococcus aureus and the immunoglobulin-functionalized Fe3O4 solution are incubated for 120 min, and b is a diagram showing the efficiency of capturing Staphylococcus aureus after changing the incubation time of the solution containing Staphylococcus aureus and the immunoglobulin-functionalized Fe3O4 solution when the dosage of immunoglobulin-functionalized Fe3O4 is 400 μg / mL;

[0052] from Figure 1 From a, we can see that the highest capture efficiency is achieved when the concentration of immunoglobulin functionalized Fe3O4 is 400 μg / mL; Figure 1 As shown in Figure b, the highest capture efficiency was obtained when the incubation time was 120 min.

[0053] Example 2: Application of boric acid-modified AIE nanoparticles in rapid detection of Staphylococcus aureus is accomplished by the following steps:

[0054] A solution containing Staphylococcus aureus (ATCC 29213) and an immunoglobulin-functionalized Fe3O4 solution were mixed evenly to obtain a reaction system I; the reaction system I was incubated at 37°C for 120 min, and then magnetically separated to obtain a reaction solution; boric acid-modified AIE nanoparticles were added to the reaction solution to obtain a reaction system II; the reaction system II was incubated at 37°C for 30 min, and then magnetically separated to obtain a solution to be tested; the fluorescence intensity of the solution to be tested was detected by a fluorescence spectrophotometer, and then compared with a standard curve of Staphylococcus aureus (fluorescence intensity versus Staphylococcus aureus concentration (0, 10, 10 2 , 10 3 , 10 4 , 10 5 and 10 6 cfu / mL) were compared; the lower the fluorescence intensity, the higher the concentration of Staphylococcus aureus;

[0055] The concentration of Staphylococcus aureus in the reaction system I is 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10, 0 CFU / mL, and the concentration of immunoglobulin-functionalized Fe3O4 was 400 μg / mL;

[0056] The concentration of the boric acid-modified AIE nanoparticles in the reaction system II is 200 μg / mL.

[0057] The detection effect of Staphylococcus aureus in Example 2 is shown in Table 1;

[0058] Table 1

[0059]

[0060] Figure 2 This is a fluorescence signal diagram detected in Example 2, in which a is the fluorescence spectrum of the detection system after incubation of Staphylococcus aureus at different concentrations, and b is the standard curve of Staphylococcus aureus;

[0061] Highly effective in killing Staphylococcus aureus:

[0062] Example 3: The photocatalytic bactericidal performance of the material was evaluated by inactivation of Staphylococcus aureus (ATCC29213). All experiments were carried out under sterile conditions. First, the strain was streaked on LB agar medium for activation and culture for 24 hours, and then a single colony was picked and inoculated in 30mL LB broth for shaking culture for 18 hours (37°C, 180r / min). The obtained bacterial suspension was washed with physiological saline and centrifuged to remove metabolites, and the bacterial suspension concentration was about 108CFU / mL. Take the immunoglobulin functionalized Fe3O4 solution and the boric acid modified AIE nanoparticles and add them to the solution containing Staphylococcus aureus, stir evenly, and obtain a bacterial suspension; use a 300W xenon lamp to simulate sunlight to irradiate the bacterial suspension for photocatalytic sterilization for 0min, 20min, 40min or 60min, and then perform a 10-fold serial dilution, and then take 100μL of the bacterial suspension sample and spread it on the nutrient agar plate, and then culture at 37℃ for 24h to count the surviving bacteria on the standard plate, and each group is parallel 3 times. The blank control test was carried out using the same method, and the killing effect of Staphylococcus aureus is shown in Table 2;

[0063] The concentration of immunoglobulin functionalized Fe3O4 in the bacterial suspension is 400 μg / mL, and the concentration of boric acid modified AIE nanoparticles is 200 μg / mL;

[0064] The height between the 300W xenon lamp and the bacterial suspension is 20cm.

[0065] Table 2

[0066] Lighting time / min 0 20 40 60 Viable bacteria count (CFU / mL) <![CDATA[2.6×10 7 ]]> <![CDATA[3.3×10 5 ]]> <![CDATA[6.6×10 3 ]]> 0

[0067] Figure 3 This is a plate diagram of the colony growth of Staphylococcus aureus under different light exposure times;

[0068] Figure 4 1 is a graph of the bactericidal efficiency of Staphylococcus aureus, in which 1 is a blank control, 2 is sunlight irradiation alone, 3 is sunlight irradiation and immunoglobulin-functionalized Fe3O4, 4 is sunlight irradiation and boric acid-modified AIE nanoparticles, and 5 is sunlight irradiation, immunoglobulin-functionalized Fe3O4 and boric acid-modified AIE nanoparticles in Example 3;

[0069] from Figure 4 It can be seen that the killing rate of Staphylococcus aureus in Example 3 can reach 100%.

[0070] Figure 5 Figures 3 and 4 are morphological diagrams and laser confocal images of Staphylococcus aureus at different illumination times, wherein a is a morphological diagram of Staphylococcus aureus at different illumination times in Example 3, and b is a laser confocal image of live and dead cells of Staphylococcus aureus;

[0071] Figure 5 From left to right in the middle, the illumination time is 0, 20, 40, and 60 min;

[0072] Figure 6 These are electron microscope images, where a is the SEM image of Fe3O4 and b is the SEM image of AIE nanoparticles.

Claims

1. Application of boric acid modified AIE nanoparticles, characterized in that The application of a boric acid-modified AIE nanoparticle in the rapid detection of Staphylococcus aureus is accomplished by the following steps: The solution containing Staphylococcus aureus and the immunoglobulin functionalized Fe3O4 solution are uniformly mixed to obtain reaction system I; the reaction system I is incubated at 37°C for 30min to 120min, and then magnetic separation is performed to obtain a reaction solution; boric acid-modified AIE nanoparticles are added to the reaction solution to obtain reaction system II; the reaction system II is incubated at 37°C for 30min, and then magnetic separation is performed to obtain a solution to be tested; the fluorescence intensity of the solution to be tested and a blank solution not containing Staphylococcus aureus is respectively detected using a fluorescence spectrophotometer. If the fluorescence intensity of the solution to be tested is lower than the fluorescence intensity of the blank solution not containing Staphylococcus aureus, it indicates that the solution to be tested contains Staphylococcus aureus; The concentration of Staphylococcus aureus in the reaction system I is 10-10 6 CFU / mL, the concentration of immunoglobulin-functionalized Fe3O4 was 400 μg / mL; The concentration of the boric acid-modified AIE nanoparticles in the reaction system II is 200 μg / mL.

2. Application of boric acid modified AIE nanoparticles, characterized in that The application of a boric acid-modified AIE nanoparticle in the efficient killing of Staphylococcus aureus is accomplished by the following steps: The immunoglobulin-functionalized Fe3O4 solution and the boric acid-modified AIE nanoparticles were added to a solution containing Staphylococcus aureus and stirred evenly to obtain a bacterial suspension; a 300W xenon lamp was used to simulate sunlight to irradiate the bacterial suspension for 20 minutes to 60 minutes to obtain a solution that kills Staphylococcus aureus; The concentration of immunoglobulin functionalized Fe3O4 in the bacterial suspension is 400 μg / mL, and the concentration of boric acid modified AIE nanoparticles is 200 μg / mL; The height between the 300W xenon lamp and the bacterial suspension is 20cm.

3. The use of a boric acid-modified AIE nanoparticle according to claim 1 or 2, characterized in that A method for preparing boric acid-modified AIE nanoparticles is specifically completed by the following steps:

1. Dissolving 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene and distearoylphosphatidylethanolamine-polyethylene glycol 2000 in tetrahydrofuran, and then adding deionized water to obtain a mixed solution III; subjecting the mixed solution III to ultrasonic treatment, and then stirring in a dark fume hood to remove tetrahydrofuran, and then centrifuging, and washing the solid product obtained by centrifugation with deionized water, and then freeze-drying to obtain AIE nanoparticles; dispersing the AIE nanoparticles in deionized water to obtain an AIE nanoparticle solution, and storing it at 4°C; 2. Mix the physiological saline containing EDC and NHS with the AIE nanoparticle solution, incubate at room temperature, and wash with physiological saline after incubation to obtain EDC / NHS-mediated AIE nanoparticles; resuspend the EDC / NHS-mediated AIE nanoparticles in physiological saline, add 3-aminophenylboronic acid to obtain a mixed solution IV; stir the mixed solution IV at room temperature, and finally wash with physiological saline to remove 3-aminophenylboronic acid to obtain APBA-AIE NPs, which are boronic acid-modified AIE nanoparticles.

4. The use of a boric acid-modified AIE nanoparticle according to claim 3, characterized in that The concentration of AIE nanoparticles in the AIE nanoparticle solution described in step one is 1 mg / mL; the mass volume ratio of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene, distearoylphosphatidylethanolamine-polyethylene glycol 2000, tetrahydrofuran and deionized water in the mixed solution III described in step one is 0.5g:1g:1mL:9mL; the ultrasound described in step one is ultrasonic treatment using a micro-tip probe ultrasonic pulverizer for 20min to 30min, and the output power of the micro-tip probe ultrasonic pulverizer is 180W; in step one, stirring is performed in a dark fume hood at a speed of 500r / min to 600r / min for 10h to 12h, tetrahydrofuran is removed, and then centrifuged, and the solid product obtained by centrifugation is washed 3 to 5 times with deionized water, and then freeze-dried at -50°C for 10h to obtain AIE NPs, i.e. AIE nanoparticles.

5. The use of a boric acid-modified AIE nanoparticle according to claim 3, characterized in that The concentration of EDC in the normal saline containing EDC and NHS described in step 2 is 2 mg / mL, and the concentration of NHS is 2 mg / mL; the volume ratio of the normal saline containing EDC and NHS to the AIE nanoparticle solution described in step 2 is 1:1; the mass volume ratio of EDC / NHS-mediated AIE nanoparticles, 3-aminophenylboronic acid and normal saline in the mixed solution IV described in step 2 is 1 mg:2 mg:1 mL; in step 2, the mixed solution IV is stirred at room temperature for 30 minutes, and finally washed with normal saline for 3 to 5 times to remove 3-aminophenylboronic acid to obtain APBA-AIE NPs, which are boronic acid-modified AIE nanoparticles.

6. The use of a boric acid modified AIE nanoparticle according to claim 1 or 2, characterized in that The preparation method of the immunoglobulin functionalized Fe3O4 solution is specifically completed according to the following steps:

1. Preparation of carboxylated Fe3O4: FeCl3·6H2O is dissolved in ethylene glycol under stirring conditions, and then Na3C6H5O7·2H2O is added, and then stirred under heating conditions to obtain a mixed solution I; anhydrous NaOAc is added to the mixed solution I, and stirred until a clear solution is formed to obtain a mixed solution II; the mixed solution II is sealed in a Teflon-lined stainless steel high-pressure tank, and then a hydrothermal reaction is carried out, and the mixture is cooled to room temperature, and the generated black magnetite particles are collected; the generated black magnetite particles are washed to obtain carboxylated Fe3O4; 2. Preparation of immunoglobulin functionalized Fe3O4 solution: ①, washing the carboxylated Fe3O4 with sterile saline, and then resuspending it in saline containing EDC and NHS to obtain mixture I; stirring mixture I at room temperature, and then washing it with sterile saline to remove excess EDC and NHS to obtain activated Fe3O4; ②. Resuspend the activated Fe3O4 in sterile saline, and then add porcine immunoglobulin solution to obtain mixture II; incubate mixture II at room temperature to obtain IgG-Fe3O4; wash IgG-Fe3O4 with sterile saline, and then resuspend it in sterile saline to a concentration of IgG-Fe3O4 of 1 mg / mL to obtain immunoglobulin functionalized Fe3O4 solution, and store it at 4°C.

7. The use of a boric acid-modified AIE nanoparticle according to claim 6, characterized in that The mass volume ratio of FeCl3·6H2O, Na3C6H5O7·2H2O, anhydrous NaOAc and ethylene glycol described in step one is 0.8g:0.47g:1.2g:40mL; in step one, FeCl3·6H2O is dissolved in ethylene glycol under stirring, and then Na3C6H5O7·2H2O is added, and then stirred at 80°C to 85°C to obtain a mixed solution I; anhydrous NaOAc is added to the mixed solution I, and stirred until a clear solution is formed to obtain a mixed solution II; the mixed solution II is sealed in a Teflon-lined stainless steel high-pressure tank, and then hydrothermally reacted at 200°C for 6h, cooled to room temperature, and the generated black magnetite particles are collected; the generated black magnetite particles are washed alternately with anhydrous ethanol and deionized water to remove ethylene glycol to obtain carboxylated Fe3O4.

8. The use of a boric acid-modified AIE nanoparticle according to claim 6, characterized in that The concentration of EDC in the physiological saline containing EDC and NHS described in step 2① is 20 mg / mL, and the concentration of NHS is 20 mg / mL; the concentration of carboxylated Fe3O4 in the mixture I described in step 2① is 10 mg / mL; in step 2①, carboxylated Fe3O4 is washed 3 to 5 times with sterile physiological saline, and then resuspended in the physiological saline containing EDC and NHS to obtain mixture I; the mixture I is stirred at room temperature for 30 minutes, and then washed 3 to 5 times with sterile physiological saline to remove excess EDC and NHS to obtain activated Fe3O4; the concentration of the porcine immunoglobulin solution described in step 2② is 1 mg / mL; the mixture described in step 2② The mass volume ratio of activated Fe3O4, porcine immunoglobulin solution and sterile saline in mixture II is 10 mg:300 μL:10 mL; in step 2②, mixture II is cultured at room temperature for 2 hours to obtain IgG-Fe3O4; IgG-Fe3O4 is washed 3 to 5 times with sterile saline, and then resuspended in sterile saline to a concentration of 1 mg / mL of IgG-Fe3O4 to obtain an immunoglobulin functionalized Fe3O4 solution, which is stored at 4°C; the saline described in step 2② is a sodium chloride aqueous solution with a mass fraction of 0.9%, and the preparation method is: weigh 9 g of sodium chloride and dissolve it in 900 mL of deionized water, and then dilute to 1000 mL with deionized water.

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