Nanoprobe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano-probes, and particularly relates to a nano-probe and a preparation method and application thereof.The nano-probe comprises a magnetic nano-material and a long afterglow nano-material, the magnetic nano-material comprises magnetic nano-particles modified with concanavalin A and is used for recognizing, capturing and separating bacteria, and the long afterglow nano-material is a long afterglow nano-material. The long-afterglow nano-material comprises green long-afterglow nano-particles of which the surfaces are modified with polymyxin B and red long-afterglow nano-particles of which the surfaces are modified with vancomycin, the green long-afterglow nanoparticles with the surfaces modified with polymyxin B and the red long-afterglow nanoparticles with the surfaces modified with vancomycin are respectively used for identifying anti-interference signal output, the nano-probe is applied to identification of gram-negative and positive bacteria, the anti-interference capability of detection can be improved, the whole process is rapid and simple, and the cost is low. And the cost is low, and the stability is high.

Description

Technical Field

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

[0002] Bacterial Gram typing helps doctors quickly identify possible pathogenic bacteria in the initial diagnosis of infectious diseases, so as to select appropriate antibiotic treatment plans in advance. The current clinical method of bacterial Gram typing mainly relies on professional operators and has high technical requirements for personnel. It is particularly important to develop new methods that do not rely on the professionalism of personnel and can quickly and easily obtain test results.

[0003] Molecular recognition is performed using molecular recognition agents that specifically bind to bacteria without destroying the cells. Molecular recognition agents include antibodies, aptamers, bacteriophages, and specific proteins. However, these biomaterial-based recognition agents have poor stability, high cost, and difficult preparation methods; in addition, some of them are not commercially available. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention provides a nanoprobe and a preparation method and application thereof, so as to improve the anti-interference ability of detection, and the whole process is fast, simple, low-cost and high-stability.

[0005] The present invention solves the technical problem by adopting the following technical solutions:

[0006] The present invention aims to provide a nanoprobe, comprising a magnetic nanomaterial and a long afterglow nanomaterial. The magnetic nanomaterial comprises magnetic nanoparticles modified with concanavalin A, which are used to identify, capture and separate bacteria. The long afterglow nanomaterial comprises green long afterglow nanoparticles modified with polymyxin B on the surface and red long afterglow nanoparticles modified with vancomycin on the surface. The green long afterglow nanoparticles modified with polymyxin B on the surface and the red long afterglow nanoparticles modified with vancomycin on the surface are used to identify anti-interference signal output, respectively.

[0007] Magnetic nanoparticles modified with concanavalin A are separation and enrichment nanoparticles with recognition function. The recognition molecule is concanavalin A (Con A), which has the ability to recognize carbohydrate structures on bacterial cell walls. Magnetic nanoparticles provide separation and enrichment functions. Small molecule antibiotics show obvious selectivity for bacterial strains, and have excellent stability, good tolerance, cost-effectiveness and ready accessibility. Experimental studies have found that vancomycin (Van) shows significant selective targeting of Gram-positive (G + ) bacteria, vancomycin-functionalized magnetic nanoparticles (MNPs) can be applied to G +Targeted detection of bacteria, polymyxin B (PMB) for Gram-negative - ) 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 promoted by a synergistic combination of electrostatic and hydrophobic interactions, emphasizing the specificity and effectiveness of the molecule in this case, green long afterglow nanoparticles have green afterglow performance, red long afterglow nanoparticles have red afterglow performance, long afterglow nanoparticles (PLNP) have a significant ability to capture excitation energy and dissipate this energy by slowly releasing captured carriers, and can produce continuous phosphorescence even in the absence of continuous excitation. This unique property of PLNPs enables it to effectively circumvent the confounding effect of autofluorescence in complex biological matrices, produce a superior signal modulation ratio, and is conducive to obtaining more accurate and sensitive analysis results. The present invention utilizes the difference between vancomycin and polymyxin B in recognizing bacteria, and distinguishes through the difference in the emission band of long afterglow nanoparticles.

[0008] Preferably, the magnetic nanomaterial is FeCl3, the green long afterglow nanoparticles are Zn2GeO4:Mn,Pr, and the red long afterglow nanomaterial is ZnGa2O4:Cr.

[0009] The present invention aims to provide a method for preparing a nanoprobe, including the preparation of magnetic nanoparticles modified with concanavalin A, the preparation of green long-lasting nanoparticles surface-modified with polymyxin B, and the preparation of red long-lasting nanoparticles surface-modified with vancomycin.

[0010] The magnetic nanoparticles are carboxylated, and then the carboxyl groups are activated and coupled with the small molecule compound concanavalin A to obtain magnetic nanoparticles modified with concanavalin A, the green long afterglow nanoparticles are hydroxylated, the hydroxylated green long afterglow nanoparticles are aminated, the aminated green long afterglow nanoparticles are carboxylated, and finally the carboxylated green long afterglow nanoparticles are coupled with polymyxin B (PMB) to obtain PMB-modified green long afterglow nanoparticles, the red long afterglow nanoparticles are hydroxylated, the hydroxylated red long afterglow nanoparticles are aminated, and the aminated red long afterglow nanoparticles are coupled with vancomycin to obtain vancomycin-modified red long afterglow nanoparticles. The magnetic nanoparticles modified with concanavalin A, the red long afterglow nanoparticles modified with vancomycin, and the green long afterglow nanoparticles modified with polymyxin B are stored at low temperatures for later use.

[0011] Furthermore, the method for preparing magnetic nanoparticles modified with concanavalin A comprises the following steps:

[0012] The magnetic nanomaterials were dispersed in anhydrous citric acid buffer and ultrasonically treated for 10 minutes, 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 2-(N-morpholino)ethanesulfonic acid (MES) buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and sodium salt of N-hydroxysuccinimide sulfonate were added, and the carboxyl groups were activated at room temperature for 2 hours. After washing three times with PBS, the activated magnetic nanoparticles (1 mg / mL) were mixed with 1 mg / mL concanavalin A in PBS containing 1 mM calcium chloride and 1 mM manganese chloride, stirred for 1 hour, and magnetically collected to obtain concanavalin A-modified magnetic nanoparticles (Con A-MNPs), which were washed three times with binding buffer (1 mM CaCl2 and 1 mM MnCl2 in 10 mM PBS) and then stored. After preparation, the concanavalin A-modified magnetic nanoparticles were blocked with 1% BSA at 37°C for 2 hours.

[0013] Furthermore, the preparation method of magnetic nanoparticles is as follows: ferric chloride and trisodium citrate are dissolved in ethylene glycol, and then sodium acetate is added under stirring, and the mixture is stirred for 30 minutes, and then the resulting solution is transferred to a stainless steel hydrothermal reactor lined with polytetrafluoroethylene, heated at 200°C and maintained for 10 hours, and then cooled to room temperature; the black product is washed with ethanol and deionized water, and the magnetic nanoparticles are obtained after vacuum drying.

[0014] Furthermore, the preparation method of green long afterglow nanoparticles modified with polymyxin B on the surface comprises the following steps:

[0015] The green long afterglow nanoparticles (1-5 mg / mL) were dispersed in a sodium hydroxide solution (1-5 mmol / L), stirred at room temperature for 24 h, washed with deionized water three times, and vacuum dried to obtain hydroxylated green long afterglow nanoparticles. The hydroxylated long afterglow nanoparticles were dissolved in N,N-dimethylformamide, and 3-aminopropyltriethoxysilane (5-20 μL per mL of reaction solution) was added under electromagnetic stirring. The mixture was placed in an 80°C oil bath, and electromagnetically stirred for 24 h before centrifugation. The mixture was washed with N,N-dimethylformamide and anhydrous ethanol, and vacuum dried to obtain amino-containing green long afterglow nanoparticles. The amino-containing green long afterglow nanoparticles were dissolved in N,N-dimethylformamide, and a N,N-dimethylformamide solution of succinic anhydride (2-8 mg / mL) was added in sequence under stirring. mL), N,N-dimethylformamide solution of 4-dimethylaminopyridine (0.5-2.0 mg / mL), stirred at room temperature for 12 h, centrifuged, washed three times with 50% anhydrous ethanol, and vacuum dried to obtain carboxylated green long afterglow nanoparticles, dissolved the carboxylated green long afterglow nanoparticles in PBS buffer, ultrasonicated for 10 min, added 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide sulfonic acid sodium salt and incubated for 10 min, then added polymyxin B, adjusted the pH to 7.1-7.6 with sodium bicarbonate, incubated at 30 ° C for 12 h, centrifuged to remove unbound polymyxin B, added methoxypolyethylene glycol amine (MW750) and blocked at 37 ° C for 2 h to obtain polymyxin B modified green long afterglow nanoparticles.

[0016] Furthermore, the preparation method of green long afterglow nanoparticles is as follows: dissolving germanium oxide powder in sodium hydroxide solution to obtain sodium germanate solution (Na2GeO3), then mixing aqueous solutions of zinc nitrate (Zn(NO3)2), manganese chloride (MnCl2) and praseodymium nitrate (Pr(NO3)3) under magnetic stirring, adding nitric acid solution to obtain a precursor mixed solution; subsequently, adding sodium germanate (Na2GeO3) dropwise to the precursor mixed solution under continuous stirring, adjusting the pH of the mixed precursor solution to alkaline, preferably pH 7.4-7.8, placing it in an ultrasonic instrument for room temperature ultrasonication for 10 minutes, then placing it on a magnetic stirrer and stirring at room temperature for 30 minutes, transferring the resulting solution to a polytetrafluoroethylene-lined high-pressure reactor, hydrothermally reacting at 220°C for 16 hours, and after the hydrothermal reaction is completed, naturally cooling to room temperature, centrifuging, washing the precipitate with 50% ethanol 3 times, and vacuum drying to obtain green long afterglow nanoparticles (ZGMP).

[0017] Preferably, the molar ratio of Zn(NO3)2, MnCl2 and Pr(NO3)3 is 1.98:0.01-0.05:0.01-0.05, and the amount of Na2GeO3 added is 1.0-1.5 mmol.

[0018] Furthermore, a method for preparing red long afterglow nanoparticles modified with vancomycin on the surface comprises: dispersing red long afterglow nanoparticles (1-5 mg / mL) in a sodium hydroxide solution (1-5 mg / mL), stirring at room temperature for 24 hours, washing with deionized water three times, and vacuum drying to obtain hydroxylated red long afterglow nanoparticles; dispersing the hydroxylated red long afterglow nanoparticles in N,N-dimethylformamide, adding 3-aminopropyltriethoxysilane (5-20 μL per mL of reaction solution) under electromagnetic stirring, placing in an 80° C. oil bath, electromagnetically stirring for 24 hours, centrifuging, washing with N,N-dimethylformamide and anhydrous ethanol, and vacuum drying to obtain amino red long afterglow nanoparticles; dissolving 20-50 μg of vancomycin in a 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 5.0) containing EDC (5-30 mg) and NHS (10-50 mg). 5.5) for 2 hours; then, amino-modified red long afterglow nanoparticles (1-5 mg / mL) were added to the above mixture and stirred for another 6 hours, the obtained nanoparticles were centrifuged and washed to remove unattached carboxylated materials, and methoxypolyethylene glycol amine (MW750) was added to block at 37°C for 2 hours to obtain vancomycin-modified red long afterglow nanoparticles.

[0019] Furthermore, the preparation method of red long afterglow nanoparticles is as follows: zinc nitrate (Zn(NO3)2), gallium nitrate (Ga(NO3)3), and chromium nitrate (Cr(NO3)3) are mixed and stirred; deionized water is added to adjust the total volume to 15 mL, and concentrated ammonia (28%) solution (about 1 mL) is added to adjust the pH. Preferably, the pH is adjusted to 8.5-9.5 to form a white precipitate. After continuing to stir for 0.5 h, the mixture is transferred to a polytetrafluoroethylene-lined hydrothermal reactor (25 mL) and sealed. The hydrothermal reactor is placed at 220°C for reaction for 10 h, and then naturally cooled to room temperature. The white precipitate obtained after centrifugation is dispersed in dilute hydrochloric acid to form a transparent solution. In this step, possible zinc oxide impurities can be removed, and then the mixture is mixed and washed with isopropanol, and the red long afterglow nanoparticles are obtained after vacuum drying.

[0020] Preferably, the molar ratio of Zn(NO3)2, Ga(NO3)3, and Cr(NO3)3 is 1:2:0.001-0.01.

[0021] The present invention also aims to provide a nanoprobe or a method for preparing the nanoprobe for use in identifying Gram-negative and Gram-positive bacteria.

[0022] Furthermore, the method for identifying Gram-negative and Gram-positive bacteria includes: mixing Gram-negative bacteria and Gram-positive bacteria together, reacting with magnetic nanoparticles modified with concanavalin A, red long-afterglow nanoparticles modified with vancomycin, and green long-afterglow nanoparticles modified with polymyxin B for 5 minutes, performing magnetic separation for 5 minutes, detecting the phosphorescence intensity of the magnetic precipitation group and the magnetic supernatant group with an enzyme marker (time resolution, delay time: 20 μs), and calculating the intensity ratio, and typing Gram-positive and Gram-negative bacteria by using the difference in emission bands of green afterglow (excitation: 250 nm, emission: 529 nm) and red afterglow (excitation: 250 nm, emission: 697 nm);

[0023] The mixed bacteria of Gram-negative bacteria and Gram-positive bacteria were diluted to different concentrations, mixed with magnetic nanoparticles modified with concanavalin A, red long-afterglow nanoparticles modified with vancomycin, and green long-afterglow nanoparticles modified with polymyxin B for 5 minutes, and then magnetically separated for 5 minutes. The phosphorescence intensity of the magnetic precipitation group and the magnetic supernatant group was detected by an ELISA instrument (time resolution, delay time: 20μs), and the intensity ratio was calculated to establish a standard curve of the intensity ratio and the concentration of the target analyte.

[0024] The collected samples were mixed with concanavalin A-modified magnetic nanoparticles, vancomycin-modified red long-afterglow nanoparticles, and polymyxin B-modified green long-afterglow nanoparticles for reaction for 5 minutes. After magnetic separation for 5 minutes, the phosphorescence intensity of the magnetic precipitation group and the magnetic supernatant group was detected by an enzyme marker (time resolution, delay time: 20μs), and the intensity ratio was calculated and compared with the standard curve to obtain the concentration of the target detection object in the sample.

[0025] Compared with the prior art, the beneficial technical effects of the present invention are:

[0026] 1. The present invention modifies magnetic nanoparticles with a small molecule compound concanavalin A as identification and separation enrichment particles, simplifies the detection process, and replaces traditional centrifugation methods; at the same time, vancomycin and polymyxin B are respectively modified on nanoparticles with different long afterglows, which can also be used as recognition molecules and signal output molecules. The long afterglow characteristics, anti-interference, and low background characteristics are utilized to improve the anti-interference ability of the detection, and the entire process is fast and simple.

[0027] 2. The present invention realizes the use of small molecule compound concanavalin A to modify magnetic nanoparticles and vancomycin and polymyxin B to modify 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.

[0028] 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

[0029] Figure 1 TEM image of the magnetic nanoparticles in the present invention.

[0030] Figure 2 This is a TEM image of the green long afterglow nanoparticles of the present invention.

[0031] Figure 3 This is the phosphorescence spectrum of the green long afterglow nanoparticles in the present invention.

[0032] Figure 4 This is a TEM image of the red long afterglow nanoparticles of the present invention.

[0033] Figure 5 This is the phosphorescence spectrum of the red long afterglow nanoparticles of the present invention.

[0034] Figure 6 This is a graph showing the potential changes before and after the magnetic nanoparticles are modified in the present invention.

[0035] Figure 7 This is a graph showing the potential change before and after modification of the green long afterglow nanoparticles in the present invention.

[0036] Figure 8 This is a graph showing the potential change before and after modification of the red long afterglow nanoparticles in the present invention. DETAILED DESCRIPTION

[0037] 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 descriptions 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.

[0038] 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.

[0039] Example 1: Preparation of magnetic nanoparticles

[0040] The preparation method of magnetic nanoparticles is as follows:

[0041] 1. Dissolve FeCl3 (0.2-1.0 g) and trisodium citrate (0.1-0.5 g) in ethylene glycol (10-50 mL);

[0042] 2. Then, NaAc (0.5-2.0 g) was added under stirring, and the mixture was vigorously stirred for 30 min, placed in a 200°C hydrothermal reaction for 10 h, and then cooled to room temperature;

[0043] 3. Wash the product with ethanol and deionized water several times.

[0044] Transmission electron microscopy images of the prepared magnetic nanoparticles are shown in Figure 1 . Figure 1 The prepared magnetic nanoparticles were prepared by adding 0.65 g of FeCl3, 0.2 g of trisodium citrate, 20 mL of ethylene glycol, and 1.2 g of NaAc.

[0045] Example 2: Preparation of Concanavalin A-modified Magnetic Nanoparticles

[0046] The preparation method of concanavalin A modified magnetic nanoparticles comprises:

[0047] 1. Disperse 10 mg / mL magnetic nanoparticles into 0.1-0.6 mol / L anhydrous citric acid buffer, ultrasonicate for 10 min, stir overnight at room temperature, and then wash three times with deionized water to obtain carboxylated magnetic nanoparticles;

[0048] 2. Dissolve 1-10 mg of carboxylated magnetic nanoparticles in 1-10 mL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer, add 5-15 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10-30 mg of N-hydroxysuccinimide sulfonic acid sodium salt, react at room temperature for 2 h to activate the carboxyl group, wash three times with PBS, mix the activated MNPs (1 mg / mL) with 1-5 mg / mL ConA in PBS containing 1 mM CaCl2 and 1 mM MnCl2, stir for 1 h, collect ConA-MNPs by magnetic collection, wash three times with binding buffer (10 mM PBS with 1 mM CaCl2 and 1 mM MnCl2), and store the obtained ConA-MNPs at low temperature;

[0049] 3. After the preparation of the Concanavalin A modified magnetic nanoparticles, BSA with a final concentration of 1% was added and blocked at 37°C for 2 hours to block the unbound sites and obtain the blocked Concanavalin A modified magnetic nanoparticles.

[0050] The potential change of magnetic nanoparticles before and after MNP modification is shown in the figure Figure 6 , Figure 6Con A modified magnetic nanoparticles, in step 1, the anhydrous citric acid buffer is 0.1-0.6 mol / L, in step 2, 1-10 mg of carboxylated magnetic nanoparticles are dissolved in 1-10 ml of 2-(N-morpholino)ethanesulfonic acid buffer, 5-15 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10-30 mg of N-hydroxysuccinimide sulfonic acid sodium salt are added; the activated MNPs (1 mg / mL) and 1-5 mg / mL Con A are mixed in PBS containing 1 mM CaCl2 and 1 mM MnCl2.

[0051] Example 3: Preparation of green long afterglow nanoparticles

[0052] The preparation method of green long afterglow nanoparticles is as follows:

[0053] 1. GeO2 powder is dissolved in NaOH solution to prepare Na2GeO3 solution;

[0054] 2. Mix 1.98 mmol of Zn(NO3)2, 0.01-0.05 mmol of MnCl2, 0.01-0.05 mol of Pr(NO3)3 and 100-500 μL of concentrated HNO3 under magnetic stirring, and add 1.0-1.5 mmol of Na2GeO3 dropwise to the mixture under continuous stirring;

[0055] 3. The pH of the mixed precursor solution was adjusted to 7.2-7.8, placed in an ultrasonicator for 10 min at room temperature, and stirred on a magnetic stirrer at room temperature for 30 min. Finally, the mixture was transferred to a polytetrafluoroethylene-lined reactor and hydrothermally reacted at 220°C for 8-24 h. The synthesized ZGMP was collected after centrifugation and washed three times with 50% ethanol.

[0056] The transmission electron microscope image of the green long afterglow nanoparticles is as follows: Figure 2 . Phosphorescence spectra such as Figure 3 , Figure 2 and Figure 3 The green long afterglow nanoparticles are prepared. In step 2, 1.98 mmol of Zn(NO3)2, 0.01-0.05 mmol of MnCl2, 0.01-0.05 mol of Pr(NO3)3 and 100-500 μL of concentrated HNO3 are mixed, and 1.0-1.5 mmol of Na2GeO3 is added dropwise to the mixture under continuous stirring. In step 3, the pH of the mixed precursor solution is adjusted to 7.2-7.8, and a hydrothermal reaction is carried out at 220°C for 8-24 hours.

[0057] Example 4: Preparation of green long-lasting glow nanoparticles modified with polymyxin B

[0058] The preparation method of polymyxin B modified green long afterglow nanoparticles comprises:

[0059] 1. Disperse 30-100 mg of green long afterglow nanoparticles in 10-50 mL of 5 mmol / L sodium hydroxide solution, stir at room temperature for 24 h, wash three times with deionized water, and vacuum dry to obtain hydroxylated green long afterglow nanoparticles;

[0060] 2. Dissolve 20-100 mg of hydroxylated green nanoparticles in 20 mL of N,N-dimethylformamide, slowly add 50-200 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 green long-lasting nanoparticles;

[0061] 3. Dissolve 20-100 mg of amino green long afterglow nanoparticles in 10-50 mL of N,N-dimethylformamide solution, slowly add N,N-dimethylformamide solution containing 1-5 mg / mL of succinic anhydride and 0.5-2.0 mg / mL of 4-dimethylaminopyridine in turn under vigorous stirring, stir at room temperature for 12 h, centrifuge, wash three times with 50% anhydrous ethanol, and vacuum dry to obtain carboxylated green long afterglow nanoparticles;

[0062] 4. Dissolve 1-5 mg of carboxylated green long afterglow nanoparticles in 2-10 mL of PBS buffer, sonicate for 10 min, add 2-10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 5-30 mg of sodium salt of N-hydroxysuccinimide sulfonate, incubate at room temperature for 10 min, then add 1-5 mg of polymyxin B, adjust the pH to 7.4 with sodium bicarbonate, incubate at 30°C for 12 h, remove unbound polymyxin B by centrifugation, add 1% PEG-NH2 (MW750) at 37°C for 2 h to block the unbound sites, and obtain blocked polymyxin B-modified green long afterglow nanoparticles.

[0063] The potential changes of green long afterglow nanoparticles (G-PLNP) before and after modification are shown in Figure 7 , Figure 7Green long afterglow nanoparticles modified with polymyxin B, in step 1, 30-100 mg of green long afterglow nanoparticles are dispersed in 10-50 mL of 5 mmol / L sodium hydroxide solution; in step 2, 20-100 mg of hydroxylated green nanoparticles are dissolved in 20 mL of N,N-dimethylformamide, and 50-200 μL of 3-aminopropyltriethoxysilane is slowly added under electromagnetic stirring; in step 3, 1-5 mg of aminolated green long afterglow nanoparticles are dissolved in 2-10 mL of N,N-dimethylformamide solution, and N,N-dimethylformamide solution containing 1-5 mg / mL of succinic anhydride and 0.5-2.0 mg / mL of 4-dimethylaminopyridine is slowly added in sequence under vigorous stirring; in step 4, 1-5 mg of carboxylated green long afterglow nanoparticles are dissolved in 2-10 mL In PBS buffer, ultrasonicate for 10 min, add 2-10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 5-30 mg of sodium salt of N-hydroxysuccinimide sulfonate, incubate at room temperature for 10 min, and then add 1-5 mg of polymyxin B.

[0064] Example 5: Preparation of red long afterglow nanoparticles

[0065] The preparation method of red long afterglow nanoparticles is as follows:

[0066] 1. Mix 1mmol Zn(NO3)2, 2mmol Ga(NO3)3, and 0.001-0.01mmol Cr(NO3)3 together, stir vigorously, and adjust the total volume to 15mL by adding deionized water;

[0067] 2. Rapidly add concentrated ammonium hydroxide (28%) solution (about 1 mL) to adjust the pH to 9-9.5. A white precipitate will form immediately. After stirring for another 0.5 h, transfer the mixture to a polytetrafluoroethylene-lined hydrothermal reactor and seal it.

[0068] 3. Place the hydrothermal reactor at 220°C for 10 hours, then cool naturally until it reaches room temperature. The white precipitate obtained after centrifugation is dispersed in dilute hydrochloric acid to form a transparent solution. In this step, possible zinc oxide impurities can be removed, and then mixed and washed with excess isopropanol, and finally vacuum dried to obtain red long afterglow nanoparticles.

[0069] The transmission electron microscope image of the obtained red long afterglow nanoparticles is as follows Figure 4 , phosphorescence spectrum such as Figure 5 , Figure 4 and 5 The red long afterglow nanoparticles prepared in step 1 have 0.004 mmol of Cr(NO3)3 and are adjusted to pH 9.2 in step 2.

[0070] Example 6: Preparation of Vancomycin-modified Red Long-Afterglow Nanoparticles

[0071] The preparation method of vancomycin modified red long afterglow nanoparticles comprises:

[0072] 1. Disperse 10-100 mg of red long afterglow nanoparticles in 20-50 mL of 5 mmol / L sodium hydroxide solution, stir at room temperature for 24 h, wash three times with deionized water, and vacuum dry to obtain hydroxylated red long afterglow nanoparticles;

[0073] 2. Dissolve 10-50 mg of hydroxylated red nanoparticles in 20-40 mL of N,N-dimethylformamide, slowly add 10-100 μL of 3-aminopropyltriethoxysilane under electromagnetic stirring, place in an 80°C oil bath, stir for 24 hours, then centrifuge, wash twice with N,N-dimethylformamide, once with anhydrous ethanol, and vacuum dry to obtain amino-containing red long-lasting nanoparticles;

[0074] 3. Activation of carboxyl groups: 10-50 μg of vancomycin was dissolved in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 5.5) containing EDC (3-10 mg) and NHS (10-50 mg) and stirred for 2 h. Then, 2 mg / mL of amino-modified red long afterglow nanoparticles were added to the above mixture and stirred for another 6 h. The obtained nanoparticles were centrifuged and washed to remove unattached carboxylated materials. 0.5-5.0% mPEG-NH2 (MW750) was added and blocked at 37°C for 2 h to obtain vancomycin-modified red long afterglow nanoparticles.

[0075] The potential changes before and after modification of red long afterglow nanoparticles (R-PLNP) are as follows Figure 8 , Figure 8 The method comprises the following steps: preparing red long afterglow nanoparticles modified with vancomycin, wherein in step 1, 10 to 100 mg of red long afterglow nanoparticles are dispersed in 20 to 50 mL of 5 mmol / L sodium hydroxide solution; in step 2, 10 to 50 mg of hydroxylated red nanoparticles are dissolved in 20 to 40 mL of N,N-dimethylformamide, and 10 to 100 μL of 3-aminopropyltriethoxysilane is slowly added under electromagnetic stirring; in step 3, 10 to 50 μg of vancomycin is dissolved in 2-(N-morpholino)ethanesulfonic acid buffer (pH 5.5) containing EDC (3 to 10 mg) and NHS (10 to 50 mg), stirred for 2 h, and mPEG-NH2 (MW750) is added to block at 37°C for 2 h.

[0076] Example 7: Rapid identification of Gram-negative and Gram-positive bacteria by the nanoprobe of the present invention

[0077] Method for rapidly identifying Gram-negative and Gram-positive bacteria using nanoprobes of the present invention

[0078] 1. Take 100 μL (50 μL each) of bacterial suspension mixture of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) as the experimental group and 100 μL H2O group as the control group. Add 300 μL (100 μL each) of the mixture of 40 μg / mL~0.5 mg / mL ConA-MNP, 40 μg / mL~0.5 mg / mL R-PLNP-Van, and 40 μg / mL~0.5 mg / mL G-PLNP-PMB to the two groups respectively. After 5 minutes of action, magnetic separation (5 minutes) was performed;

[0079] 2. The supernatant was aspirated and retained, and the same volume of H2O as the supernatant was added to the precipitate. 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. The difference in the emission bands of green afterglow (excitation: 250nm, emission: 529nm) and red afterglow (excitation: 250nm, emission: 697nm) was used to type Gram-positive and negative bacteria; at the same time, the target bacteria were diluted to different concentrations, mixed with magnetic nanoparticles modified with concanavalin A, red long afterglow nanoparticles modified with vancomycin, and green long afterglow nanoparticles modified with polymyxin B, reacted for 5 minutes, and magnetically separated for 5 minutes. The phosphorescence intensity of the magnetic precipitation group and the magnetic supernatant group was detected by an ELISA instrument (time resolution, delay time: 20μs), and the intensity ratio was calculated to establish a standard curve of the intensity ratio and the concentration of the target analyte;

[0080] 3. The clinical samples were mixed with magnetic nanoparticles modified with concanavalin A, red long-afterglow nanoparticles modified with vancomycin, and green long-afterglow nanoparticles modified with polymyxin B, and the mixture was reacted for 5 minutes. The mixture was magnetically separated for 5 minutes, and the phosphorescence intensity of the magnetic precipitation group and the magnetic supernatant group was detected by an ELISA instrument (time resolution, delay time: 20 μs). The intensity ratio was calculated and compared with the standard curve to obtain the concentration of the target detection substance in the sample.

[0081] The present invention provides a scheme for rapid identification and typing of Gram-positive and Gram-negative bacteria based on long-lasting nanomaterials, which mainly includes green long-lasting nanoparticles, red long-lasting nanoparticles and magnetic nanoparticles; the magnetic nanoparticles are modified with concanavalin A, the red long-lasting nanoparticles are modified with vancomycin and the green long-lasting nanoparticles are modified with polymyxin B; the bacteria are captured by concanavalin A, separated by magnetic nanoparticles, and then the difference in the recognition of bacteria by vancomycin and polymyxin B is used, vancomycin recognizes Gram-positive bacteria, polymyxin B recognizes Gram-negative bacteria, and identification is performed by the difference in the emission band of long-lasting nanoparticles. Compared with the prior art, the present invention saves time and is simple to operate.

[0082] 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.

[0083] 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 nanoprobe, characterized in that: It includes magnetic nanomaterials and long afterglow nanomaterials. The magnetic nanomaterials include magnetic nanoparticles modified with concanavalin A, which are used to identify, capture and separate bacteria. The long afterglow nanomaterials include green long afterglow nanoparticles modified with polymyxin B on the surface and red long afterglow nanoparticles modified with vancomycin on the surface. The green long afterglow nanoparticles modified with polymyxin B on the surface and the red long afterglow nanoparticles modified with vancomycin on the surface are respectively used to identify anti-interference signal output.

2. A method for preparing a nanoprobe according to claim 1, characterized in that: include Preparation of magnetic nanoparticles modified with concanavalin A: carboxylating the magnetic nanoparticles, activating the carboxyl groups, and coupling with concanavalin A to obtain magnetic nanoparticles modified with concanavalin A; Preparation of green long afterglow nanoparticles with surface modification of polymyxin B: hydroxylating, aminating and carboxylating the green long afterglow nanoparticles, and finally coupling the carboxylated green long afterglow nanoparticles with polymyxin B to obtain green long afterglow nanoparticles modified with polymyxin B; Preparation of red long afterglow nanoparticles modified with vancomycin on the surface: After the red long afterglow nanoparticles are hydroxylated and aminated, the aminated red long afterglow nanoparticles are coupled with vancomycin to obtain vancomycin-modified red long afterglow nanoparticles.

3. A method for preparing a nanoprobe as claimed in claim 2, characterized in that: The preparation method of magnetic nanoparticles modified with concanavalin A comprises the following steps: The magnetic nanomaterial is dispersed in anhydrous citric acid buffer, ultrasonically stirred, and then washed with deionized water to obtain carboxylated magnetic nanoparticles; the carboxylated magnetic nanoparticles are then dissolved in 2-(N-morpholino)ethanesulfonic acid buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and sodium salt of N-hydroxysuccinimide sulfonate are added to react and activate the carboxyl group, and after washing with PBS, the activated magnetic nanoparticles are mixed with concanavalin A in PBS containing calcium chloride and manganese chloride, stirred, and magnetically collected to obtain concanavalin A-modified magnetic nanoparticles, which are then washed with a binding buffer and stored.

4. A method for preparing a nanoprobe as claimed in claim 3, characterized in that: The preparation method of magnetic nanoparticles is as follows: ferric chloride and trisodium citrate are dissolved in ethylene glycol, and then sodium acetate is added under stirring, the mixture is stirred and sealed in a polytetrafluoroethylene-lined hydrothermal reactor for heating, and then cooled; the black product is washed with ethanol and deionized water and dried to obtain magnetic nanoparticles.

5. The method for preparing a nanoprobe according to claim 2, characterized in that: The preparation method of green long afterglow nanoparticles with surface modified with polymyxin B comprises the following steps: The green long afterglow nanoparticles are dispersed in a sodium hydroxide solution and stirred, washed with deionized water, and dried to obtain hydroxylated green long afterglow nanoparticles. The hydroxylated green long afterglow nanoparticles are dissolved in N,N-dimethylformamide, 3-aminopropyltriethoxysilane is added under electromagnetic stirring, and the mixture is placed in an oil bath. After electromagnetic stirring, the mixture is centrifuged and separated, washed with N,N-dimethylformamide and anhydrous ethanol, and dried to obtain amino-containing green long afterglow nanoparticles. The amino-containing green long afterglow nanoparticles are dissolved in N,N-dimethylformamide, and succinic anhydride and N,N-dimethylformamide are added in sequence under stirring. solution, a solution of 4-dimethylaminopyridine in N,N-dimethylformamide, continuing to stir and then centrifuging, washing with anhydrous ethanol and drying to obtain carboxylated green long afterglow nanoparticles, dissolving the carboxylated green long afterglow nanoparticles in PBS buffer, sonicating, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide sulfonic acid sodium salt for incubation, then adding polymyxin B, adjusting the pH with sodium bicarbonate, centrifuging after incubation to remove unbound polymyxin B, adding methoxypolyethylene glycol amine group for sealing, and obtaining polymyxin B modified green long afterglow nanoparticles.

6. A method for preparing a nanoprobe according to claim 5, characterized in that: The preparation method of green long afterglow nanoparticles is as follows: germanium oxide powder is dissolved in sodium hydroxide solution to obtain sodium germanate solution, and then aqueous solutions of zinc nitrate, manganese chloride and praseodymium nitrate are mixed under magnetic stirring, and nitric acid solution is added to obtain a precursor mixed solution; subsequently, sodium germanate is added dropwise to the precursor mixed solution under continuous stirring, the pH of the mixed solution is adjusted to alkaline, ultrasonicated, and then stirred on a magnetic stirrer, the obtained solution is placed in a high-pressure reactor for hydrothermal reaction, the precipitate is washed with ethanol after cooling and centrifugation, and the green long afterglow nanoparticles are obtained after drying.

7. The method for preparing a nanoprobe according to claim 2, characterized in that: The preparation method of red long afterglow nanoparticles modified with vancomycin on the surface comprises: dispersing the red long afterglow nanoparticles in a sodium hydroxide solution and stirring, washing with deionized water and drying to obtain hydroxylated red long afterglow nanoparticles, dissolving the hydroxylated red long afterglow nanoparticles in N,N-dimethylformamide, adding 3-aminopropyltriethoxysilane under electromagnetic stirring, placing in an oil bath, centrifuging after electromagnetic stirring, washing with N,N-dimethylformamide and anhydrous ethanol, drying to obtain amino red long afterglow nanoparticles, dissolving vancomycin in a 2-(N-morpholino)ethanesulfonic acid buffer containing EDC and NHS; then adding the amino red long afterglow nanoparticles to the above mixture and stirring again, washing the obtained nanoparticles by centrifugation, and adding methoxypolyethylene glycol amine group to seal to obtain vancomycin modified red long afterglow nanoparticles.

8. A method for preparing a nanoprobe according to claim 7, characterized in that: The preparation method of red long afterglow nanoparticles is as follows: zinc nitrate, gallium nitrate and chromium nitrate are mixed together and stirred; concentrated ammonium hydroxide solution is added to adjust the pH to form a white precipitate, and the stirring is continued. The mixture is placed in a reactor for high temperature and high pressure reaction, and then naturally cooled to room temperature. The white precipitate obtained after centrifugation is dispersed in hydrochloric acid to form a transparent solution, and then mixed and washed with isopropanol, and the red long afterglow nanoparticles are obtained after drying.

9. Use of a nanoprobe according to claim 1 or a method for preparing a nanoprobe according to any one of claims 2 to 8 in identifying Gram-negative and Gram-positive bacteria.

10. The use according to claim 9, characterized in that: The method for identifying Gram-negative and Gram-positive bacteria includes: mixing Gram-negative bacteria and Gram-positive bacteria together, reacting with magnetic nanoparticles modified with concanavalin A, red long-afterglow nanoparticles modified with vancomycin, and green long-afterglow nanoparticles modified with polymyxin B, performing magnetic separation, detecting the phosphorescence intensity of the magnetic precipitation group and the magnetic supernatant group with an enzyme marker, calculating the intensity ratio, and using the difference in the emission bands of green afterglow and red afterglow to identify and quantitatively detect Gram-positive and Gram-negative bacteria; The mixed bacteria of Gram-negative bacteria and Gram-positive bacteria were diluted to different concentrations, mixed with magnetic nanoparticles modified with concanavalin A, red long-afterglow nanoparticles modified with vancomycin, and green long-afterglow nanoparticles modified with polymyxin B, and then magnetically separated. 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 collected samples were mixed with magnetic nanoparticles modified with concanavalin A, red long-afterglow nanoparticles modified with vancomycin, and green long-afterglow nanoparticles modified with polymyxin B. The samples were separated by magnetic separation, and 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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