CN109283333B - Method for quantitatively analyzing drug resistance of escherichia coli based on gold shell-up-conversion chiral dimer - Google Patents
Method for quantitatively analyzing drug resistance of escherichia coli based on gold shell-up-conversion chiral dimer Download PDFInfo
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Abstract
A method for quantitatively analyzing the drug resistance of escherichia coli based on a gold shell-up-conversion chiral dimer belongs to the technical field of material chemistry. The method comprises the steps of firstly preparing gold shell nanoparticles, modifying polymyxin B antibodies on the surfaces of the gold shell nanoparticles, modifying polymyxin B on the surfaces of the upconversion nanoparticles, mixing and culturing the upconversion nanoparticles modified by the polymyxin B with escherichia coli with different drug resistance degrees respectively, adding the gold shell nanoparticles modified by the polymyxin B antibodies after reacting for a period of time, and forming gold shells and unbound upconversion into a dimer structure through antigen-antibody reaction. The determination of the drug resistance degree of the escherichia coli can be realized through fluorescence and circular dichroism spectroscopy. The gold shell-up-conversion chiral dimer structure prepared by the invention has a uniform structure and good biocompatibility, provides a method for simultaneously detecting the polymyxin B resistance degree of escherichia coli through fluorescence and CD signals, establishes a standard curve between the polymyxin B resistance concentration of the escherichia coli and the fluorescence signals and the CD signals, and has the advantages of high sensitivity, good selectivity, low detection limit and short time consumption.
Description
Technical Field
The invention relates to a method for quantitatively analyzing drug resistance of escherichia coli based on a gold shell-up-conversion chiral dimer, and belongs to the technical field of material chemistry.
Background
Improper use of antibacterial drugs accelerates the global spread of drug-resistant bacteria, which are one of the greatest threats to public health. Detection techniques for bacteria in food, medical and public environments have long been developed. Traditional methods, such as Minimum Inhibitory Concentration (MIC) and Polymerase Chain Reaction (PCR), detect and identify drug-resistant bacteria based on their phenotype and genotype, respectively, although effective, are laborious and time-consuming. With the rapid development of nanoscience and nanotechnology, nanomaterial probes have been used to rapidly identify bacterial pathogens. Previous studies have focused on the specific detection and imaging of bacterial infections through fluorescence, surface enhanced raman scattering, colorimetric nanosensors, and the like. However, the widespread use of antibacterial drugs has limited the clinical utility of these diagnostic tools. Extensive research has recently been conducted on optical activities at the nanoscale, focusing not only on the preparation of chiral structures, but also on the high signal-to-noise ratio of plasma Circular Dichroism (CD) spectroscopy in the visible range, showing great potential for the detection of biomolecules.
Polymyxin B is a broad-spectrum cationic polypeptide antibiotic commonly recognized as the last line of defense against bacterial infections, particularly gram-negative pathogens. However, recent studies have found that the plasmid-borne drug resistance gene mcr-1 can be easily transferred between different pathogens, resulting in the development of drug resistant strains. N-ethylethanolamine modification of lipid a groups in Lipopolysaccharide (LPS) is a key metabolic factor in the polymyxin resistance mechanism, as the encoded factors reduce the affinity between polymyxin and the bacterial surface. It remains a challenge to develop nanomaterial-based strategies to efficiently detect polymyxin-resistant bacteria to meet advanced clinical needs. Therefore, the development of the chiral assembly-based quantitative analysis of the drug resistance of the escherichia coli is particularly important.
Disclosure of Invention
The invention aims to provide a method for quantitatively analyzing the drug resistance of escherichia coli based on a gold shell-up-conversion chiral dimer.
According to the technical scheme, firstly, gold shell nanoparticles are prepared, an antibody of polymyxin B is modified on the surface of the gold shell nanoparticles, polymyxin B is modified on the surface of the upconversion nanoparticles, the upconversion nanoparticles modified by the polymyxin B are mixed and cultured with escherichia coli with different drug resistance degrees respectively, after reaction for a period of time, the gold shell nanoparticles modified by the polymyxin B antibody are added, and through antigen-antibody reaction, a gold shell and uncombined upconversion are formed to form a dimer structure. The determination of the drug resistance degree of the escherichia coli can be realized through fluorescence and circular dichroism spectroscopy.
A method for quantitatively analyzing the drug resistance of Escherichia coli based on gold shell-up-conversion chiral dimer comprises the following steps:
(1) and (3) synthesis of gold nanoparticles: reducing chloroauric acid by using tannic acid to synthesize gold nanoparticles with the diameter of 10 +/-2 nm;
(2) preparing gold shell nanoparticles: dissolving the gold nanoparticles prepared in the step (1) in a polyvinylpyrrolidone solution, and then adding silver nitrate and ascorbic acid solution to synthesize gold-coated silver nanoparticles; then adding chloroauric acid solution to prepare gold shell nano particles;
(3) surface modification of polymyxin B by upconversion nanoparticles: firstly, coupling cysteine on the surface of an up-conversion nanoparticle, and connecting the up-conversion nanoparticle with polymyxin B through amino-carboxyl reaction;
upconversion nanoparticles: purchased by high-tech development ltd, wangde, beijing;
(4) the surface of the gold-shell nanoparticle is modified with a polymyxin B antibody: connecting the gold shell nanoparticles obtained in the step (2) with polymyxin B antibody through Au-S bond;
(5) preparation of gold shell-up-converting chiral dimer: preparing a gold shell-up-conversion chiral dimer through an antigen-antibody reaction;
(6) bacterial origin and culture conditions: taking five polymyxin B resistant escherichia coli strains with drug resistance degrees of 4, 8, 16, 20 and 24mg/mL respectively;
polymyxin B-resistant E.coli strain (E15017, degree of resistance 24 MG/mL), polymyxin B-sensitive E.coli strain (MG 1655, degree of resistance 4 MG/mL) were supplied by Zhejiang university. Polymyxin B-resistant E.coli strains (EYAC 08-25-1, degree of resistance 20mg/mL and GZP11-11, degree of resistance 16 mg/mL) were provided by the university of agriculture in China. Polymyxin B-resistant E.coli strain (JN-PBR-1, drug resistance degree 8 mg/mL) was obtained from southern Jiangnan university. Individual colonies on solid agar plates were inoculated into 10mL of LB medium and cultured overnight at 37 ℃ with constant shaking at 200 rpm. Obtaining bacteria in exponential growth phase, OD600Is 0.7 (5X 10)7CFU/mL). Then, the bacteria were diluted to a final concentration of 1X 10 by PBS buffer (pH 7.4) 3CFU/mL, used in the following experiments.
(7) Detection of drug resistance of the gold shell-up-conversion chiral dimer to escherichia coli: and (4) incubating the escherichia coli with different drug resistance degrees cultured in the step (6) and the polymyxin B modified up-conversion nanoparticles obtained in the step (3) for 8 h. Because of different drug resistance degrees and different affinities of the bacterial membrane and polymyxin B, the drug resistance degree can be detected through the change of converted fluorescence on the surface of the bacterial membrane; at this time, the polymyxin B antibody modified gold shell in step (4) is added, and through antigen-antibody reaction, the gold shell is combined with the unbound up-conversion nanoparticles to form a dimer, so as to generate a CD signal, and the drug resistance degree can be detected through the change of the up-conversion fluorescence on the surface of the bacterial membrane.
(8) And (3) characterization of the gold shell-up-conversion chiral dimer on detection of the drug resistance of escherichia coli, and establishment of a standard curve: and (5) carrying out fluorescence signal characterization on the bacteria in the step (7) and establishing a standard curve. Meanwhile, bacteria are removed by centrifugation, and the supernatant is subjected to CD signal characterization to establish a standard curve.
The method comprises the following specific steps:
(1) preparation of gold-shell-up-converting chiral dimers
a. And (3) synthesis of gold nanoparticles: gold nanoparticles with a diameter of 10 + -2 nm were synthesized by reducing chloroauric acid with tannic acid, 0.1mL of 1% aqueous tannic acid and 80mL of 0.0125% aqueous chloroauric acid were heated to 60 ℃, and then the tannic acid solution was rapidly added to the chloroauric acid solution under vigorous stirring. After 2h reaction, the solution was cooled to room temperature until the color did not change, and then the solution was stored at 4 ℃ until use.
b. Preparing gold shell nanoparticles: first, gold nanoparticles prepared as described above were centrifuged at 13,000 rpm for 10min, the pellet was concentrated 5-fold and resuspended in phosphate buffer (10 mM, pH 7.4), and 1mL of the pellet was added with 1. mu.L of 10mM mPEG-SH (M)W= 5000), obtaining AuNP-PEG, fully mixing the AuNP-PEG with 0.5 mL of 1% polyvinylpyrrolidone solution, then adding 40 muL of 20 mM silver nitrate and 20 muL of 0.1M ascorbic acid solution respectively, uniformly mixing at room temperature, reacting for 10min, centrifuging at 6000 rpm for 5min, and suspending the precipitate in 1mL of 1% polyvinylpyrrolidone solution to obtain the gold-coated silver nanoparticles. Finally, 200. mu.L of 10mM chloroauric acid solution was added to the above solution, and the reaction was stirred at room temperature for 10min to obtain gold shell nanoparticles.
c. Surface modification of polymyxin B by upconversion nanoparticles:
preparation of upconversion nanoparticles UCNP: purchased from Beijing Wande high-tech development Co., Ltd, diluted 100-fold with Tris buffer of 10mM concentration and pH7.4 for use;
the water-soluble UCNP was diluted to 10 nM with Tris buffer (10 mM, pH 7.4). Cysteine Cys was then added, allowing Cys: molar concentration ratio of UCNP is 100: 1 UCNP was functionalized and after 4 h reaction at room temperature, ultrafiltration was performed using a 30kDa molecular weight ultrafiltration tube to remove unconjugated Cys. 4mg of polymyxin B was dissolved in 1mL of deionized water and activated by the addition of 4. mu.L of 2.5% glutaraldehyde (> 98%) for 30 min, and finally, 20 nM of Cys-UCNP solution was reacted with activated polymyxin B at room temperature for 6 h. The final product was purified by ultrafiltration (30 kDa molecular weight) and stored at 4 ℃ until use.
d. The surface of the gold-shell nanoparticle is modified with a polymyxin B antibody: the prepared gold shell nanoparticles were concentrated 10-fold, and then the polymyxin B antibody was added as a polymyxin B antibody: the molar concentration ratio of the gold shell is 100: 1 is modified. The mixture was reacted at room temperature for 6 h. The modified gold shells were centrifuged at 6000 rpm for 5min to remove any excess polymyxin B antibody and then resuspended in water.
e. Preparation of gold shell-up-converting chiral dimer: purified polymyxin B-modified UCNP and polymyxin B antibody-modified gold shell nanoparticles were purified in a 1: 1 in Tris buffer (pH 7.4) for 2h, and the mixture was centrifuged at 4000 rpm for 10min and resuspended in PBS.
(2) Detection of drug resistance of gold shell-up-conversion chiral dimer to escherichia coli
a. Bacterial origin and culture conditions: polymyxin B-resistant E.coli strain (E15017, degree of resistance 24 MG/mL), polymyxin B-sensitive E.coli strain (MG 1655, degree of resistance 4 MG/mL) were supplied by Zhejiang university. Polymyxin B-resistant E.coli strains (EYAC 08-25-1, degree of resistance 20mg/mL and GZP11-11, degree of resistance 16 mg/mL) were provided by the university of agriculture in China. Polymyxin B resistant Escherichia coli strain (JN-PBR-1, drug resistance degree 8mg/mL) was obtained from the university of south of the Yangtze river. Individual colonies on solid agar plates were inoculated into 10mL of LB medium and cultured overnight at 37 ℃ with constant shaking at 200 rpm. Obtaining bacteria in exponential growth phase, OD600Is 0.7 (5X 10) 7CFU/mL). Then, the bacteria were diluted to a final concentration of 1X 10 by PBS buffer (pH 7.4)3CFU/mL, used in the following experiments.
b. Detection of drug resistance of the gold shell-up-conversion chiral dimer to escherichia coli: and (3) incubating the escherichia coli with different drug resistance degrees cultured in the step (2 and a) and the polymyxin B modified up-conversion nanoparticles obtained in the step (1 and c) for 8 h. Because of different drug resistance degrees and different affinities of the bacterial membrane and polymyxin B, the drug resistance degree can be detected through the change of converted fluorescence on the surface of the bacterial membrane; at this time, the polymyxin B antibody-modified gold shell in step (1, d) is added, and the gold shell binds to the unbound up-conversion nanoparticles to form a dimer through an antigen-antibody reaction, thereby generating a CD signal.
(3) Characterization of the gold shell-up-conversion chiral dimer for detection of drug resistance of escherichia coli: performing electron microscope characterization on the formation process of the chiral dimer; and (3) performing fluorescent signal and CD signal characterization on bacterial drug resistance detection, and establishing a standard curve.
Description of the biological Material samples: polymyxin B-resistant E.coli strain (E15017, degree of resistance 24 MG/mL), polymyxin B-sensitive E.coli strain (MG 1655, degree of resistance 4 MG/mL) were supplied by Zhejiang university. Polymyxin B-resistant E.coli strains (EYAC 08-25-1, degree of resistance 20mg/mL and GZP11-11, degree of resistance 16 mg/mL) were provided by the university of agriculture in China. Polymyxin B-resistant E.coli strain (JN-PBR-1, drug resistance degree 8 mg/mL) was obtained from southern Jiangnan university.
The invention has the beneficial effects that: the gold shell-up-conversion chiral dimer structure prepared by the invention has a uniform structure and good biocompatibility, provides a method for simultaneously detecting the polymyxin B resistance degree of escherichia coli through fluorescence and CD signals, establishes a standard curve between the polymyxin B resistance concentration of escherichia coli and the fluorescence signals and the CD signals, has the advantages of high sensitivity, good selectivity, low detection limit and short time consumption, and has a very good practical application prospect.
Drawings
FIG. 1 is a transmission electron micrograph of a gold shell-up-converting chiral dimer of the present invention. a. Up-converting the nanoparticles; b. gold shell nanoparticles; c. gold shell-up-conversion chiral dimer.
FIG. 2 is a fluorescence spectrum and a standard curve of the gold shell-up-conversion chiral dimer for detecting drug-resistant bacteria. a. Fluorescence spectra of the dimer; b. fluorescence standard curve.
FIG. 3 is a CD spectrum and a standard curve of the gold shell-up-conversion chiral dimer detection drug-resistant bacteria of the present invention. a. CD spectrogram of dimer; b. CD standard curve.
Detailed Description
Example 1 preparation of gold shell-up-converting chiral dimer
All glassware was soaked in aqua regia, washed with double distilled water, and air dried for use. The water used in the experiment was 18.2 M.OMEGA.Milli-Q ultrapure water.
(1) And (3) synthesis of gold nanoparticles: gold nanoparticles with a diameter of 10 + -2 nm were synthesized by reducing chloroauric acid with tannic acid, 0.1mL of 1% aqueous tannic acid and 80mL of 0.0125% aqueous chloroauric acid were heated to 60 ℃, and then the tannic acid solution was rapidly added to the chloroauric acid solution under vigorous stirring. After 2h reaction, the solution was cooled to room temperature until the color did not change, and then the solution was stored at 4 ℃ until use.
(2) Preparing gold shell nanoparticles: first, the gold nanoparticles prepared above were centrifuged at 13,000 rpm for 10min, the precipitate was concentrated 5 times and resuspended in phosphate buffer (10 mM, pH 7.4), 1mL was taken and added with 10mM mPEG-SH (Mw = 5000) 1 μ L to obtain AuNP-PEG, which was then mixed well with 0.5 mL of 1% polyvinylpyrrolidone solution, then 40 μ L of 20 mM silver nitrate and 20 μ L of 0.1M ascorbic acid solution were added respectively, mixed well at room temperature, reacted for 10min, centrifuged at 6000 rpm for 5min, and the precipitate was resuspended in 1mL of 1% polyvinylpyrrolidone solution to obtain gold-coated silver nanoparticles. Finally, 200. mu.L of 10mM chloroauric acid solution was added to the above solution, and the reaction was stirred at room temperature for 10min to obtain gold shell nanoparticles. As shown in FIG. 1b, the prepared gold shell nanoparticles have uniform structure and good dispersibility.
(3) Preparation of upconversion nanoparticles: purchased from high tech, Inc. of Vanda, Beijing, diluted 100-fold with Tris buffer of 10mM concentration, p H of 7.4. The electron micrograph is shown in FIG. 1 a.
(4) Surface modification of polymyxin B by upconversion nanoparticles: the water-soluble UCNP was diluted to 10 nM with Tris buffer (10 mM, pH 7.4). Cys is then added, allowing Cys: molar concentration ratio of UCNP is 100: 1 UCNP was functionalized and after 4 h reaction at room temperature, ultrafiltration was performed using a 30kDa molecular weight ultrafiltration tube to remove unconjugated Cys. 4mg of polymyxin B was dissolved in 1mL of deionized water and activated by the addition of 4. mu.L of 2.5% glutaraldehyde (> 98%) for 30 min, and finally, 20 nM of Cys-UCNP solution was reacted with activated polymyxin B at room temperature for 6 h. The final product was purified by ultrafiltration (30 kDa molecular weight) and stored at 4 ℃ until use.
(5) The surface of the gold-shell nanoparticle is modified with a polymyxin B antibody: the prepared gold shell nanoparticles were concentrated 10-fold, and then the polymyxin B antibody was added as a polymyxin B antibody: the molar concentration ratio of the gold shell is 100: 1 is modified. The mixture was reacted at room temperature for 6 h. The modified gold shells were centrifuged at 6000 rpm for 5min to remove any excess polymyxin B antibody and then resuspended in water.
(6) Preparation of gold shell-up-converting chiral dimer: purified polymyxin B-modified UCNP and polymyxin B antibody-modified gold shell nanoparticles were purified in a 1: 1 in Tris buffer (pH 7.4) for 2h, and the mixture was centrifuged at 4000 rpm for 10min and resuspended in PBS. As shown in FIG. 1c, the chiral dimer has high yield, complete structure and good dispersibility.
Example 2 detection of resistance of gold shell-up-converting chiral dimer to E.coli
(1) Bacterial origin and culture conditions: polymyxin B-resistant Escherichia coli strain (E15017, drug resistance degree 24 MG/mL), polymyxin B-sensitive Escherichia coli strain (MG 1655, drug resistance degree 4 MG/mL) from ZhejiangProvided by river university. Polymyxin B-resistant E.coli strains (EYAC 08-25-1, degree of resistance 20mg/mL and GZP11-11, degree of resistance 16 mg/mL) were provided by the university of agriculture in China. Polymyxin B-resistant E.coli strain (JN-PBR-1, drug resistance degree 8 mg/mL) was obtained from southern Jiangnan university. Individual colonies on solid agar plates were inoculated into 10mL of LB medium and cultured overnight at 37 ℃ with constant shaking at 200 rpm. Obtaining bacteria in exponential growth phase, OD600Is 0.7 (5X 10)7CFU/mL). Then, the bacteria were diluted to a final concentration of 1X 10 by PBS buffer (pH 7.4)3CFU/mL, used in the following experiments.
(2) Detection of drug resistance of the gold shell-up-conversion chiral dimer to escherichia coli: and (3) incubating the escherichia coli with different drug resistance degrees cultured in the step (1) with the polymyxin B modified up-conversion nanoparticles obtained in the step (4) of the example 1 for 8 h. As the drug resistance degrees are different, the affinity of the bacterial membrane and polymyxin B is different, and the drug resistance degree can be detected through the change of converted fluorescence on the surface of the bacterial membrane, as shown in figure 2a, the binding force between the bacterial membrane and polymyxin is weakened along with the increase of the drug resistance degree of escherichia coli, so that the fluorescence signal is continuously reduced; at this time, the polymyxin B antibody-modified gold shell of example 1, step (5), was added and combined with the unbound upconverting nanoparticles to form a dimer by an antigen-antibody reaction, resulting in a CD signal. As shown in FIG. 3a, the binding force between the bacterial membrane and polymyxin is weakened due to the increase of the drug resistance of Escherichia coli, so that more and more free upconversion nanoparticles and gold shells are assembled into a dimer, and the CD signal is enhanced.
Example 3 characterization of the gold shell-up-conversion chiral dimer for detection of e.coli resistance: performing electron microscope characterization on the formation process of the chiral dimer; and (3) performing fluorescence signal and CD signal characterization on the bacterial drug resistance detection, and respectively establishing a standard curve. As shown in FIGS. 2b and 3b, the drug resistance of E.coli showed good linear relationship with the ratio of the number of bacteria to the CD signal and the ratio of the number of bacteria to the fluorescence intensity, respectively. The method can be used for detecting the drug resistance degree of the Escherichia coli.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (2)
1. A method for quantitatively analyzing the drug resistance of escherichia coli based on a gold shell-up-conversion chiral dimer is characterized by comprising the following steps:
(1) and (3) synthesis of gold nanoparticles: reducing chloroauric acid by using tannic acid to synthesize gold nanoparticles with the diameter of 8-12 nm;
(2) preparing gold shell nanoparticles: dissolving the gold nanoparticles prepared in the step (1) in a polyvinylpyrrolidone solution, adding a silver nitrate solution and an ascorbic acid solution to synthesize gold-coated silver nanoparticles, and then adding a chloroauric acid solution to prepare gold shell nanoparticles;
(3) surface modification of polymyxin B by upconversion nanoparticles: firstly, coupling cysteine on the surface of an up-conversion nanoparticle, and connecting the up-conversion nanoparticle with polymyxin B through amino-carboxyl reaction;
preparation of upconversion nanoparticles: taking up the UCNP (upconverting nanoparticles), and diluting the UCNP by 100 times by using a Tris buffer solution with the concentration of 10mM and the pH value of 7.4 for use;
the upconversion nanoparticles modify polymyxin B: diluting the water-soluble UCNP to 10 nM by using 10mM Tris buffer solution with pH7.4; cysteine Cys was then added, allowing Cys: molar concentration ratio of UCNP is 100: 1, after reacting for 4 hours at room temperature, performing ultrafiltration by using an ultrafiltration tube with a molecular weight of 30kDa to remove uncoupled Cys; dissolving 4mg polymyxin B in 1mL deionized water, adding 4 μ L2.5% glutaraldehyde for 30 min for activation, finally, reacting 20 nM Cys-UCNP solution with activated polymyxin B at room temperature for 6h, purifying the final product by 30kDa molecular weight ultrafiltration and storing at 4 ℃ for later use;
(4) the surface of the gold-shell nanoparticle is modified with a polymyxin B antibody: connecting the gold shell nanoparticles obtained in the step (2) with polymyxin B antibody through Au-S bond; concentrating the gold shell nanoparticles prepared in step (2) by 10 times, and then adding a polymyxin B antibody to obtain a polymyxin B antibody: the molar ratio of the gold shell nano particles is 100: 1 is modified; the mixture is reacted for 6h at room temperature; the modified gold shells were centrifuged at 6000 rpm for 5min to remove any excess polymyxin B antibody and then resuspended in water;
(5) preparation of gold shell-up-converting chiral dimer: preparing a gold shell-up-conversion chiral dimer through an antigen-antibody reaction;
(6) bacterial origin and culture conditions: taking five polymyxin B resistant escherichia coli strains with drug resistance degrees of 4, 8, 16, 20 and 24mg/mL respectively;
the single colony was inoculated into 10mL of LB medium and cultured overnight at 37 ℃ with constant shaking at 200 rpm; obtaining an exponential growth phase of 5 × 107Bacteria of CFU/mL, OD thereof6000.7, the bacteria were then diluted to a final concentration of 1X 10 by PBS buffer pH7.4 3CFU/mL, used for the following experiment;
(7) detection of drug resistance of the gold shell-up-conversion chiral dimer to escherichia coli: co-incubating escherichia coli with different drug resistance degrees cultured in the step (6) and the polymyxin B modified up-conversion nanoparticles obtained in the step (3) for 8 h; adding the polymyxin B antibody modified gold shell in the step (4), and combining the gold shell and the uncombined upconversion nanoparticles to form a dimer through an antigen-antibody reaction to generate a CD signal; detecting the drug resistance degree through the change of converted fluorescence on the surface of the bacterial membrane;
(8) and (3) characterization of the gold shell-up-conversion chiral dimer on detection of the drug resistance of escherichia coli, and establishment of a standard curve: performing fluorescence signal characterization on the bacteria in the step (7), and establishing a standard curve; meanwhile, bacteria are removed by centrifugation, and the supernatant is subjected to CD signal characterization to establish a standard curve.
2. The method for quantitatively analyzing the drug resistance of the chiral dimer to escherichia coli based on gold shell-up-conversion according to claim 1, which is characterized by comprising the following steps:
(1) and (3) synthesis of gold nanoparticles: reducing chloroauric acid by using tannic acid to synthesize gold nanoparticles with the diameter of 8-12nm, heating 0.1mL of 1% aqueous solution of tannic acid and 80mL of 0.0125% aqueous solution of chloroauric acid to 60 ℃, rapidly adding the solution of the tannic acid into the solution of the chloroauric acid under vigorous stirring, reacting for 2 hours, cooling the solution to room temperature when the color is unchanged, and storing the solution at 4 ℃ for later use;
(2) preparing gold shell nanoparticles: first, the gold nanoparticles prepared in step (1) were centrifuged at 13000 rpm for 10min, the pellet was concentrated 5 times and resuspended in 10mM phosphate buffer at pH7.4, and 1mL of 10mM mPEG-SH, M was added to 1. mu.L of 10mM mPEG-SHW= 5000, obtain AuNP-PEG; then fully mixing the gold-coated silver nanoparticle with 0.5 mL of 1% polyvinylpyrrolidone solution, then respectively adding 40 μ L of 20 mM silver nitrate and 20 μ L of 0.1M ascorbic acid solution, uniformly mixing at room temperature, reacting for 10min, centrifuging at 6000 rpm for 5min, and suspending the precipitate in 1mL of 1% polyvinylpyrrolidone solution to obtain gold-coated silver nanoparticles; finally, adding 200 mu L of 10mM chloroauric acid solution into the solution, and stirring and reacting for 10min at room temperature to obtain gold shell nano particles;
(3) surface modification of polymyxin B by upconversion nanoparticles:
preparation of upconversion nanoparticles: taking up the UCNP (upconverting nanoparticles), and diluting the UCNP by 100 times by using a Tris buffer solution with the concentration of 10mM and the pH value of 7.4 for use;
the upconversion nanoparticles modify polymyxin B: diluting the water-soluble UCNP to 10 nM by using 10mM Tris buffer solution with pH7.4; cysteine Cys was then added, allowing Cys: molar concentration ratio of UCNP is 100: 1, after reacting for 4 hours at room temperature, performing ultrafiltration by using an ultrafiltration tube with a molecular weight of 30kDa to remove uncoupled Cys; dissolving 4mg polymyxin B in 1mL deionized water, adding 4 μ L2.5% glutaraldehyde for 30 min for activation, finally, reacting 20 nM Cys-UCNP solution with activated polymyxin B at room temperature for 6h, purifying the final product by 30kDa molecular weight ultrafiltration and storing at 4 ℃ for later use;
(4) the surface of the gold-shell nanoparticle is modified with a polymyxin B antibody: concentrating the gold shell nanoparticles prepared in step (2) by 10 times, and then adding a polymyxin B antibody to obtain a polymyxin B antibody: the molar ratio of the gold shell nano particles is 100: 1 is modified; the mixture is reacted for 6h at room temperature; the modified gold shells were centrifuged at 6000 rpm for 5min to remove any excess polymyxin B antibody and then resuspended in water;
(5) preparation of gold shell-up-converting chiral dimer: purified polymyxin B-modified UCNP and polymyxin B antibody-modified gold shell nanoparticles were purified in a 1: 1 in Tris buffer at pH7.4 for 2h, and the mixture is centrifuged at 4000 rpm for 10min and resuspended in PBS;
(6) bacterial origin and culture conditions: taking five polymyxin B resistant escherichia coli strains with drug resistance degrees of 4, 8, 16, 20 and 24mg/mL respectively;
a single colony on a solid agar plate was inoculated into 10mL of LB medium and cultured overnight at 37 ℃ under constant shaking at 200 rpm;
obtaining an exponential growth phase of 5 × 107Bacteria of CFU/mL, OD thereof6000.7, the bacteria were then diluted to a final concentration of 1X 10 by PBS buffer pH7.4 3CFU/mL, used for the following experiment;
(7) detection of drug resistance of the gold shell-up-conversion chiral dimer to escherichia coli: taking 1mL of escherichia coli with different drug resistance degrees cultured in the step (6) and 100 mu L of polymyxin B modified upconversion nanoparticles obtained in the step (3) to incubate together for 8 h, then adding 100 mu L of polymyxin B antibody modified gold shell obtained in the step (4), after mixed culture for 2h, centrifuging at 2000 rpm for 5min, and determining upconversion fluorescence in a sediment resuspension 100 mu L of PBS buffer solution, wherein due to different drug resistance degrees, the bacterial membrane has different affinity with polymyxin B, and the drug resistance degree is detected through the change of the upconversion fluorescence on the surface of the bacterial membrane;
simultaneously, taking the supernatant to measure a CD signal, combining the gold shell and the uncombined up-conversion nano particles to form a dimer through antigen-antibody reaction, and generating the CD signal;
(8) and (3) characterization of the gold shell-up-conversion chiral dimer on detection of the drug resistance of escherichia coli, and establishment of a standard curve: performing fluorescence signal characterization on the bacteria in the step (7), and establishing a standard curve;
meanwhile, bacteria are removed by centrifugation, and the supernatant is subjected to CD signal characterization to establish a standard curve.
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