Polymyxin B functionalized nanoprobe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nanoprobes, and particularly relates to a polymyxin B functionalized nanoprobe and a preparation method and application thereof.The nanoprobe comprises magnetic nanoparticles and long-afterglow nanoparticles, the surfaces of the magnetic nanoparticles and the long-afterglow nanoparticles are modified with polymyxin B respectively, and the polymyxin B functionalized nanoprobe is formed by modifying polymyxin B on the surfaces of the magnetic nanoparticles and the long-afterglow nanoparticles. The preparation method of the nanoprobe comprises a method for modifying long afterglow nanoparticles with polymyxin B and a method for modifying magnetic nanoparticles with polymyxin B. According to the nanoprobe disclosed by the invention, the magnetic nanoparticles with separation and enrichment effects and the long afterglow nanoparticles with afterglow luminescence and interference resistance are prepared on the basis of taking antibiotics as recognition molecules; through the enriched and separated magnetic nanoparticles and the long afterglow nanoparticles for providing signals, a new detection means is provided for rapid and quantitative background-free detection of gram-negative bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of nanoprobes, and in particular relates to a polymyxin B functionalized nanoprobe and a preparation method and application thereof. Background Art

[0002] Escherichia coli (E. coli), also known as Escherichia coli, is a Gram-negative short rod that accounts for 50% of hospital-acquired urinary tract infections (UTIs). Therefore, early pathogen detection in clinical urine samples can effectively control disease progression. Currently, bacterial culture is the gold standard method for diagnosing pathogens in patients. However, it has low sensitivity, long time consumption (16-18h), high requirements for working parameters (temperature and pH), and lacks timely clinical diagnostic information. Pathogens can also be identified using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction. However, they have several limitations, including time-consuming and costly pretreatment and sample susceptibility to contamination. The above methods all have certain requirements for testing personnel and instruments and equipment, and their implementation in grassroots hospitals is somewhat difficult. At present, the identification of pathogens mostly relies on antibody aptamers, which have strong specificity, but poor stability, easy deformation, and high price. The development of simple, rapid and low-cost detection methods is particularly important in grassroots hospitals. Summary of the invention

[0003] In order to solve the problems in the prior art, the present invention provides a polymyxin B functionalized nanoprobe and a preparation method and application thereof. Based on antibiotics as recognition molecules, magnetic nanoparticles with separation and enrichment effects, as well as long afterglow nanoparticles with afterglow luminescence and anti-interference are prepared. By enriching and separating the magnetic nanoparticles and providing long afterglow nanoparticles for signal provision, a new detection method is provided for rapid quantitative background-free detection of Gram-negative bacteria.

[0004] The present invention solves the technical problem by adopting the following technical solutions:

[0005] The present invention aims to provide a polymyxin B functionalized nanoprobe, comprising magnetic nanoparticles and long afterglow nanoparticles, wherein the surfaces of the magnetic nanoparticles and the long afterglow nanoparticles are respectively modified with polymyxin B.

[0006] Small molecule antibiotics show significant selectivity against bacterial strains, while having excellent stability, good tolerability, cost-effectiveness and ready accessibility. Studies have found that polymyxin B (PMB) is effective against Gram-negative (Gram-negative, G -) bacteria, polymyxin B (PMB)-modified quantum dots or gold nanoparticles can be used to detect G - Bacteria. The interaction between polymyxin B and Gram-negative bacteria is facilitated by a synergistic combination of electrostatic and hydrophobic interactions, underscoring the specificity and effectiveness of the molecule in this context.

[0007] Long-lasting phosphorescent nanoparticles (PLNPs) have a remarkable ability to capture excitation energy and dissipate this energy through the slow release of captured carriers, resulting in sustained phosphorescence even in the absence of continuous excitation. This unique property of PLNPs enables them to effectively circumvent the confounding effects of autofluorescence in complex biological matrices. As a result, PLNP-based detection systems produce superior signal-to-noise ratios, which is conducive to obtaining more accurate and sensitive analytical results.

[0008] Further, the magnetic nanoparticles include Fe3O4, and the long afterglow nanoparticles include Zn2GeO 4: Mn, Pr.

[0009] A method for preparing a polymyxin B functionalized nanoprobe includes a method for modifying long afterglow nanoparticles with polymyxin B and a method for modifying magnetic nanoparticles with polymyxin B.

[0010] Furthermore, the method for modifying long afterglow nanoparticles with polymyxin B comprises:

[0011] 1-50 mg / mL long afterglow nanoparticles were dispersed in 1-50 mg / mL sodium hydroxide solution, stirred at room temperature for 8-72 h, washed with deionized water three times, and vacuum dried to obtain hydroxylated long afterglow nanoparticles. The hydroxylated long afterglow nanoparticles were then dissolved in N,N-dimethylformamide, and 3-aminopropyltriethoxysilane was added under electromagnetic stirring. The amount of 3-aminopropyltriethoxysilane was 10 μL per milliliter of reaction solution. The mixture was placed in an 80°C oil bath, electromagnetically stirred for 8-24 h, and then centrifuged and separated. The obtained nanoparticles were dissolved in N,N-dimethylformamide, and N,N-dimethylformamide solution containing succinic anhydride and 4-dimethylaminopyridine were added in sequence under stirring, stirred at room temperature for 4-36 hours, centrifuged, washed three times with 50% anhydrous ethanol, and vacuum dried to obtain carboxylated long afterglow nanoparticles, and polymyxin B was added for coupling to obtain polymyxin B modified long afterglow nanoprobes.

[0012] Further, the method for coupling polymyxin B with carboxylated long afterglow nanoparticles includes:

[0013] The carboxylated long afterglow nanoparticles were dissolved in PBS buffer, sonicated for 5-30 minutes, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide sulfonic acid sodium salt were added and incubated at room temperature for 5-30 minutes, then polymyxin B was added, the pH was adjusted to 7.0-8.5 with sodium bicarbonate, incubated at 25-40°C for 4-36 hours, and the unbound polymyxin B was removed by centrifugation. mPEG-NH2 was added and blocked at 25-40°C for 1-8 hours to obtain polymyxin B modified long afterglow nanoparticles.

[0014] Furthermore, the preparation method of the long afterglow nanoparticles includes:

[0015] Germanium oxide is dissolved in sodium hydroxide solution to obtain sodium germanate solution (Na2GeO3), and then, the aqueous solutions of precursors Zn(NO3)2, MnCl2 and Pr(NO3)3 are fully mixed under magnetic stirring, and the molar ratio of Zn(NO3)2, MnCl2 and Pr(NO3)3 is 1.98:(0.005-0.04):(0.005-0.04), and nitric acid solution is added to obtain a precursor mixed solution, and the mixture is stirred under continuous stirring. 0.8-2 mmol of sodium germanate solution is added dropwise to the precursor mixed solution, and then the pH of the precursor mixed solution is adjusted to 7.0-8.2, placed in an ultrasonicator for room temperature ultrasound for 1-30 minutes, stirred at room temperature for 10 minutes to 8 hours, and then the resulting solution is placed at 220°C for hydrothermal reaction for 4-72 hours. After the hydrothermal reaction is completed, it is naturally cooled to room temperature. After centrifugation, the precipitate is washed three times with 50% ethanol, and vacuum dried to obtain long afterglow nanoparticles (ZGMP).

[0016] Furthermore, the method for modifying magnetic nanoparticles with polymyxin B includes:

[0017] The magnetic nanomaterials were dispersed in anhydrous citric acid buffer, ultrasonicated for 1-30 min, stirred overnight at room temperature, and then washed three times with deionized water to obtain carboxylated magnetic nanoparticles. The carboxylated magnetic nanoparticles were then dissolved in PBS buffer, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and sodium salt of N-hydroxysuccinimide sulfonate were added, reacted at room temperature for 10 min-3 h, and polymyxin B (PMB) was added for coupling. The final concentration of PMB was 1:1 with the final concentration of magnetic nanoparticles. The mixed solution was adjusted to pH 7.4 with sodium bicarbonate, incubated at 25-40 ° C for 18 h, magnetically separated, washed three times with deionized water, and dispersed in deionized water for storage. After the preparation of the polymyxin B-modified magnetic nanoparticles, mPEG-NH2 (MW750) was added and blocked at 37 ° C for 2 h.

[0018] Furthermore, the preparation method of magnetic nanoparticles includes:

[0019] FeCl3 and trisodium citrate were dissolved in ethylene glycol, and then sodium acetate was added under stirring. The mixture was stirred for 30 minutes and then sealed in a stainless steel autoclave lined with polytetrafluoroethylene. The autoclave was heated at 200°C and maintained for 10 hours and then cooled to room temperature. The black product was washed with ethanol and deionized water and vacuum dried to obtain magnetic nanoparticles.

[0020] A polymyxin B functionalized nanoprobe or a method for preparing a polymyxin B functionalized nanoprobe is used in detecting gram-negative bacteria, wherein the gram-negative bacteria include Escherichia coli.

[0021] Further, the method for detecting Gram-negative bacteria includes:

[0022] Taking Escherichia coli (E. coli) as a representative of Gram-negative bacteria, the target bacteria were diluted to different concentrations, mixed with polymyxin B-modified magnetic nanoparticles and polymyxin B-modified long afterglow nanoparticles, reacted for 1-20 minutes, and then magnetically separated for 1-20 minutes. The phosphorescence intensity (time resolution) of the magnetic precipitation group and the magnetic supernatant group was detected by an ELISA instrument, and the intensity ratio was calculated to establish a standard curve of the intensity ratio and the concentration of the target analyte.

[0023] The clinical samples were mixed with polymyxin B-modified magnetic nanoparticles and polymyxin B-modified long afterglow nanoparticles. After reacting for 1-20 minutes, they were magnetically separated for 1-20 minutes. The phosphorescence intensity (time resolution) of the magnetic precipitation group and the magnetic supernatant group was detected by an enzyme marker. The intensity ratio was calculated and compared with the standard curve to obtain the concentration of the target detection object in the sample.

[0024] Compared with the prior art, the beneficial technical effects of the present invention are:

[0025] 1. The present invention simplifies the detection process by modifying the small molecule compound polymyxin B on magnetic nanoparticles as identification and separation enrichment particles, replacing the traditional centrifugation method. Polymyxin B is modified on long-lasting nanoparticles, and can also be used as a recognition molecule and a signal output molecule. The anti-interference ability of the detection is improved by using the characteristics of afterglow, anti-interference, and low background. The whole process is fast and simple.

[0026] 2. The present invention realizes the use of the small molecule compound polymyxin B to modify magnetic nanoparticles and long afterglow nanoparticles, which significantly improves the detection speed, reduces the detection time and detection cost, greatly improves the convenience of detection and broadens its application scenarios.

[0027] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 TEM image of the magnetic nanoparticles in the present invention.

[0029] Figure 2 This is a TEM image of the long afterglow nanomaterial of the present invention.

[0030] Figure 3 It is the Fourier infrared spectrum of the polymyxin B modified magnetic nanomaterial in the present invention.

[0031] Figure 4 This is the Fourier infrared spectrum of the polymyxin B modified long afterglow nanoparticles of the present invention.

[0032] Figure 5 This is a diagram of the optimized conditions in the present invention, wherein: a. PMB concentration, b. PMB-MNP concentration, c. magnetic separation time, d. PMB concentration, e. PMB-PLNP concentration, f. incubation time.

[0033] Figure 6 This is a feasibility analysis diagram of the present invention, wherein: a. fluorescence intensity of the magnetic separation group, b. phosphorescence intensity of the magnetic separation group, c. fluorescence intensity of the magnetic separation supernatant group, d. phosphorescence intensity of the magnetic separation supernatant group.

[0034] Figure 7 These are performance analysis diagrams of the present invention, including: a. TEM diagram of bacterial capture, b. sensitivity analysis, and c. specificity analysis. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0036] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0037] Example 1

[0038] The preparation method of magnetic nanoparticles is as follows:

[0039] 1. Dissolve FeCl3 (0.3-1.0 g) and trisodium citrate (0.1-0.4 g) in ethylene glycol.

[0040] 2. Then, NaAc (0.5-2.5 g) was added under stirring, and the mixture was stirred vigorously for 30 min. Finally, the solution was sealed and heated at 200°C for 10 h, and then cooled to room temperature.

[0041] 3. Wash the black product with ethanol and deionized water several times.

[0042] The transmission electron microscopy images of the prepared magnetic nanoparticles are shown in Figure 1 , Figure 1 The magnetic nanoparticles prepared in the method include 0.65 g of FeCl3, 0.20 g of trisodium citrate, and 1.2 g of NaAc.

[0043] Example 2

[0044] The preparation method of long afterglow nanoparticles is:

[0045] 1. GeO2 powder was dissolved in NaOH solution to prepare Na2GeO3 solution.

[0046] 2. Mix 1.98 mmol of Zn(NO3)2, 0.005-0.04 mmol of MnCl2, 0.005-0.04 mmol of Pr(NO3)3 and 300 μL of concentrated HNO3 under magnetic stirring, and add 0.8-2 mmol of Na2GeO3 dropwise to the mixture under continuous stirring.

[0047] 3. The pH of the mixed precursor solution was adjusted to 7.0-8.2, and the solution was ultrasonically treated and stirred at room temperature for 10 min-8 h. Finally, the mixture was transferred to a polytetrafluoroethylene-lined reactor and subjected to a hydrothermal reaction at 220 ° C for 4-72 h. The synthesized ZGMP was collected after centrifugation and washed three times with 50% ethanol.

[0048] The transmission electron microscope image of the obtained long afterglow nanoparticles is shown in Figure 2 , Figure 2 To prepare long afterglow nanoparticles, in step 2, MnCl2 is 0.005-0.04 mmol, Pr(NO3)3 is 0.005-0.04, and the added Na2GeO3 is 0.8-2 mmol. In step 3, the pH of the mixed precursor solution is adjusted to 7.0-8.2, and a hydrothermal reaction is carried out at 220°C for 4-72 hours.

[0049] Example 3

[0050] Preparation of Polymyxin B Modified Magnetic Nanoparticles

[0051] 1. Disperse 10 mg / mL magnetic nanomaterials in citric acid buffer, ultrasonicate for 1-30 min, stir overnight at room temperature, and then wash three times with deionized water to obtain carboxylated magnetic nanoparticles.

[0052] 2. Dissolve 4 mg of carboxylated magnetic nanoparticles in 2 mL of PBS buffer, add 2-10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 5-30 mg of N-hydroxysuccinimide sulfonic acid sodium salt, react at room temperature for 10 min-3 h, and add 4 mg of small molecule PMB for coupling. Adjust the pH of the mixture to 7.0-8.5, incubate at 25-40°C for 4-36 h, separate magnetically, wash three times with deionized water, disperse in deionized water, and store at 4°C.

[0053] 3. After the preparation of the polymyxin B-modified magnetic nanoprobe, mPEG-NH2 (MW750) was added and blocked at 37°C for 2 hours to block the unbound sites and obtain the blocked polymyxin B-modified magnetic nanoprobe (PMB-MNP).

[0054] The Fourier transform infrared results before and after magnetic nanoparticle modification are shown in Figure 3 , Figure 3 Polymyxin B modified magnetic nanoparticles, in step 1, ultrasonication for 1 to 30 minutes; in step 2, adding 2 to 20 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 5 to 30 mg N-hydroxysuccinimide sulfonic acid sodium salt, reacting at room temperature for 10 minutes to 3 hours; incubating at 25-40°C for 4 to 36 hours.

[0055] Example 4

[0056] Preparation of long-lasting glow nanoparticles modified with polymyxin B

[0057] 1. Disperse 20-1000 mg of long afterglow nanomaterial in 20 ml of 1-50 mg / mL sodium hydroxide solution, stir at room temperature for 8-72 h, wash three times with deionized water, and vacuum dry to obtain hydroxylated long afterglow nanoparticles.

[0058] 2. Dissolve 5-50 mg of hydroxylated nanoparticles in 20 mL of N,N-dimethylformamide, slowly add 200 μL of 3-aminopropyltriethoxysilane under electromagnetic stirring, place in an 80°C oil bath, stir electromagnetically for 24 hours, then centrifuge, wash twice with N,N-dimethylformamide, once with anhydrous ethanol, and vacuum dry to obtain amino-modified long afterglow nanoparticles.

[0059] 3. Dissolve 5-50 mg of amino-modified long afterglow nanoparticles in 10-30 mL of N,N-dimethylformamide solution, and slowly add 1-5 mg / mL of succinic anhydride in N,N-dimethylformamide solution and 0.1-0.5 mg / mL of 4-dimethylaminopyridine in N,N-dimethylformamide solution in sequence under vigorous stirring. Stir at room temperature for 4-36 hours, centrifuge, wash three times with 50% anhydrous ethanol, and obtain carboxylated long afterglow nanoparticles after vacuum drying.

[0060] 4. Dissolve 1-5 mg of carboxylated long afterglow nanoparticles in 5-10 mL of PBS buffer, sonicate for 1-30 min, add 3-9 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10-30 mg of N-hydroxysuccinimide sulfonic acid sodium salt, incubate at room temperature for 1-30 min, then add 1-6 mg of polymyxin B, adjust the pH to 7.0-8.5 with sodium bicarbonate, incubate at 25-40°C for 4-36 h, remove unbound polymyxin B by centrifugation, add mPEG-NH2 (MW750) with a final concentration of 1% at 25-40°C for blocking for 0.5-8 h, block the unbound sites, and obtain blocked polymyxin B modified long afterglow nanoparticles (PMB-PLNP).

[0061] The Fourier infrared results of long afterglow nanoparticles before and after modification are shown in Figure 4 , Figure 4 Long afterglow nanoparticles modified with polymyxin B, in step 1, 20-1000 mg of long afterglow nanomaterials are dispersed in 20 ml of 5 mg / mL sodium hydroxide solution, and stirred at room temperature for 8-72 hours; in step 3, 5-50 mg of amino-modified long afterglow nanoparticles are dissolved in 20 mL of N, N-dimethylformamide solution, and N, N-dimethylformamide solution containing 1-5 mg / mL succinic anhydride and 0.1-0.5 mg / mL 4-dimethylaminopyridine is slowly added in sequence under vigorous stirring, and stirred at room temperature for 4-36 hours; in step 4, 1-5 mg of carboxylated long afterglow nanoparticles are dispersed in 4 mL of PBS buffer, ultrasonicated for 1-30 minutes, and 3-9 mg 1-Ethyl-(3-dimethylaminopropyl) carbodiimide, 10-30 mg N-hydroxysuccinimide sulfonic acid sodium salt, incubate at room temperature for 1-30 min, then add 1-6 mg polymyxin B, adjust the pH to 7.0-8.5 with sodium bicarbonate, incubate at 25-40°C for 4-36 h, remove unbound polymyxin B by centrifugation, and add mPEG-NH2 (MW750) to block at 37°C for 2 h.

[0062] Example 5

[0063] Application of Polymyxin B Functionalized Nanoprobes

[0064] 1. Take Escherichia coli (E.coli) suspension, dilute to different concentrations, take 50 μl of each as the experimental group, and the control group is 50 μl of H2O group. Add 50 μl of the mixture of 0.2-1.0 mg / mL MNP-PMB and 0.2-1.0 mg / mL PLNP-PMB to the two groups respectively, react for 1-20 minutes, and then magnetically separate for 1-20 minutes. The specific conditions are as follows: Figure 5 As shown, the reaction time and magnetic separation time are both 5 min.

[0065] 2. The supernatant was aspirated and retained, and the precipitate was added with an equal volume of H2O as the supernatant. The ELISA instrument was used to detect the phosphorescence intensity (time resolution) of the magnetic precipitation group and the magnetic supernatant group, and the intensity ratio was calculated to establish a standard curve of the intensity ratio and the concentration of the target analyte. The standard curve equation is expressed as Y = 11.87x + 24.63, and the coefficient R = 0.9964.

[0066] Method validation and performance analysis are shown in Figure 6 and Figure 7 .

[0067] 3. Mix the clinical samples with polymyxin B-modified magnetic nanoparticles and long afterglow nanoparticles, react for 1-20 minutes, and then perform magnetic separation for 1-20 minutes. Use an ELISA instrument to detect the phosphorescence intensity (time resolution) of the magnetic precipitation group and the magnetic supernatant group, calculate the intensity ratio, and compare it with the standard curve to obtain the concentration of the target detection object in the sample.

[0068] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0069] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A polymyxin B functionalized nanoprobe, characterized in that: The invention comprises magnetic nanoparticles and long afterglow nanoparticles, and the surfaces of the magnetic nanoparticles and the long afterglow nanoparticles are respectively modified with polymyxin B.

2. A polymyxin B functionalized nanoprobe as claimed in claim 1, characterized in that: The magnetic nanoparticles include Fe3O4, and the long afterglow nanoparticles include Zn2GeO4:Mn,Pr.

3. A method for preparing a polymyxin B functionalized nanoprobe according to claim 1 or 2, characterized in that: The invention comprises a method for modifying long afterglow nanoparticles with polymyxin B and a method for modifying magnetic nanoparticles with polymyxin B.

4. The method for preparing a polymyxin B functionalized nanoprobe according to claim 3, characterized in that: The method of modifying long afterglow nanoparticles with polymyxin B comprises: 1-50 mg / mL long afterglow nanoparticles are dispersed in 1-50 mg / mL sodium hydroxide solution, stirred at room temperature for 8-72 hours to obtain hydroxylated long afterglow nanoparticles, then the hydroxylated long afterglow nanoparticles are dispersed in N,N-dimethylformamide, 3-aminopropyltriethoxysilane is added under electromagnetic stirring, and the mixture is placed in an 80°C oil bath and stirred for 24 hours, separated, washed, and dried to obtain amino-modified long afterglow nanoparticles; the obtained nanoparticles are dissolved in N,N-dimethylformamide, and N,N-dimethylformamide solution containing succinic anhydride and N,N-dimethylformamide solution containing 4-dimethylaminopyridine are added in sequence under stirring, stirred at room temperature for 4-36 hours, and carboxylated long afterglow nanoparticles are obtained after centrifugation and washing; then, polymyxin B is added for coupling to obtain polymyxin B-modified long afterglow nanoparticle probes.

5. The method for preparing a polymyxin B functionalized nanoprobe according to claim 4, characterized in that: The method of coupling polymyxin B with carboxylated long-lasting nanoparticles includes: The carboxylated long afterglow nanoparticles were dissolved in PBS buffer, sonicated for 1-30 min, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide sulfonic acid sodium salt were added and incubated at room temperature for 1-30 min, then polymyxin B was added, the pH was adjusted to 7.0-8.5 with sodium bicarbonate, and the mixture was incubated at 25-40°C for 4-36 h. The unbound polymyxin B was removed by centrifugation, and mPEG-NH2 was added and blocked at 25-40°C for 0.5-8 h to obtain polymyxin B modified long afterglow nanoparticles.

6. The method for preparing a polymyxin B functionalized nanoprobe according to claim 4, characterized in that: The preparation method of long afterglow nanoparticles comprises: Germanium oxide is dissolved in sodium hydroxide solution to obtain sodium germanate solution, then, an aqueous solution of Zn(NO3)2, MnCl2 and Pr(NO3)3 in a molar ratio of 1.98:(0.005-0.04):(0.005-0.04) is fully mixed under magnetic stirring, and a nitric acid solution is added to obtain a precursor mixed solution, and 0.8-2 mmol of sodium germanate solution is added dropwise to the precursor mixed solution under continuous stirring, and then the pH of the precursor mixed solution is adjusted to 7.0-8.5, ultrasonicated for 1-30 minutes, stirred at room temperature for 10 minutes to 8 hours, and then the obtained solution is transferred to a polytetrafluoroethylene-lined high-pressure reactor, and hydrothermally reacted at 220°C for 4-72 hours. After the hydrothermal reaction is completed, it is naturally cooled to room temperature, and the precipitate is washed 3 times with 50% ethanol after centrifugation, and vacuum dried to obtain long afterglow nanoparticles.

7. The method for preparing a polymyxin B functionalized nanoprobe according to claim 3, characterized in that: The method of modifying magnetic nanoparticles with polymyxin B comprises: The magnetic nanomaterial is dispersed in anhydrous citric acid buffer, ultrasonicated for 1-30 minutes, stirred overnight at room temperature, and then washed three times with deionized water to obtain carboxylated magnetic nanoparticles. The carboxylated magnetic nanoparticles are then dissolved in PBS buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and sodium salt of N-hydroxysuccinimide sulfonate are added, reacted at room temperature for 10 minutes to 3 hours, and polymyxin B is added for coupling. The pH of the mixture is adjusted with sodium bicarbonate, incubated at 25-40°C for 18 hours, magnetically separated, washed three times with deionized water, and stored for later use.

8. The method for preparing a polymyxin B functionalized nanoprobe according to claim 7, characterized in that: The method for preparing magnetic nanoparticles comprises: FeCl3 and trisodium citrate were dissolved in ethylene glycol, and then sodium acetate was added under stirring, the mixture was stirred until dissolved, and placed under high temperature and high pressure at 200°C for 10 hours, and then cooled to room temperature. The product was washed with ethanol and deionized water to obtain magnetic nanoparticles.

9. Use of a polymyxin B functionalized nanoprobe according to claim 1 or 2 or a method for preparing a polymyxin B functionalized nanoprobe according to any one of claims 3 to 8 in detecting gram-negative bacteria, characterized in that: Gram-negative bacteria include Escherichia coli.

10. The use according to claim 9, characterized in that: Methods for detecting gram-negative bacteria include: The target bacteria were diluted to different concentrations, mixed with polymyxin B-modified magnetic nanoparticles and polymyxin B-modified long afterglow nanoparticles, reacted for 1-20 minutes, and then subjected to magnetic separation for 1-20 minutes. The phosphorescence intensity of the magnetic precipitation group and the magnetic supernatant group was detected by an ELISA instrument, and the intensity ratio was calculated to establish a standard curve of the intensity ratio and the concentration of the target analyte; The clinical samples were mixed with polymyxin B-modified magnetic nanoparticles and polymyxin B-modified long afterglow nanoparticles. After reacting for 1-20 minutes, they were magnetically separated for 1-20 minutes. The phosphorescence intensity of the magnetic precipitation group and the magnetic supernatant group was detected by an enzyme marker. The intensity ratio was calculated and compared with the standard curve to obtain the concentration of the target detection object in the sample.

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