A nanoprobe, its preparation method and application

Abstract

The application 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 magnetic nano materials and long afterglow nano materials. The magnetic nano materials comprise magnetic nano particles modified with concanavalin A, and are used for recognizing, capturing and separating bacteria. The long afterglow nano materials comprise green long afterglow nano particles modified with polymyxin B and red long afterglow nano particles modified with vancomycin. The green long afterglow nano particles modified with polymyxin B and the red long afterglow nano particles modified with vancomycin are respectively used for recognizing and outputting anti-interference signals. The nano probe can be applied to identifying gram-negative and gram-positive bacteria, can improve the anti-interference capability of detection, and has the advantages of rapidness, simplicity, low cost and high stability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanoprobes, and particularly relates to a nanoprobe and a preparation method and application thereof. BACKGROUND

[0002] Bacterial gram staining helps doctors quickly identify the possible type of pathogenic bacteria in the preliminary diagnosis of infectious diseases, so as to select a suitable antibiotic treatment plan in advance. The method for bacterial gram staining used in the clinic mainly depends on professional operators, and the technical requirements for personnel are high. It is particularly important to develop a new method for obtaining test results that is not dependent on the professionalism of personnel, fast, simple, and has low cost and high stability.

[0003] Molecular recognition is carried out by using a molecular recognition agent that specifically binds to bacteria without destroying the cells. The molecular recognition agent includes an antibody, an aptamer, a bacteriophage, and a specific protein. However, these recognition agents based on biological materials have poor stability, high cost, and difficult preparation methods; in addition, some of them are not commercially available. SUMMARY

[0004] In order to solve the problems in the prior art, the application provides a nanoprobe and a preparation method and application thereof, so as to improve the anti-interference ability of detection, and achieve the purposes of rapidity, simplicity, low cost, and high stability.

[0005] The technical problem of the application is solved by using the following technical scheme:

[0006] The application aims to provide a nanoprobe, which comprises a magnetic nanomaterial and a long-afterglow nanomaterial, the magnetic nanomaterial comprises magnetic nanoparticles modified with concanavalin A, and is used for recognizing, capturing, and separating bacteria, the long-afterglow nanomaterial comprises green long-afterglow nanoparticles modified with polymyxin B and red long-afterglow nanoparticles modified with vancomycin, and the green long-afterglow nanoparticles modified with polymyxin B and the red long-afterglow nanoparticles modified with vancomycin are respectively used for recognizing and outputting anti-interference signals.

[0007] The magnetic nanoparticles modified with concanavalin A are separation and enrichment nanoparticles with a recognition function, the recognition molecule is concanavalin A (Con A), which has the function of recognizing the carbohydrate structure on the bacterial cell wall, the magnetic nanoparticles provide the function of separation and enrichment, small-molecule antibiotics show obvious selectivity to bacterial strains, and at the same time have excellent stability, good tolerance, cost-effectiveness, and ready availability. Experimental research shows that vancomycin (Van) has the ability to selectively target gram-positive (G + ) bacteria, and vancomycin-functionalized magnetic nanoparticles (MNPs) can be applied to G +Targeted detection of bacteria, polymyxin B (PMB) has strong affinity to Gram-negative (G - ) bacteria, and polymyxin B (PMB) modified quantum dots or gold nanoparticles can be used for detection of G - The interaction between polymyxin B and Gram-negative bacteria is promoted by a synergistic combination of electrostatic and hydrophobic interactions, which emphasizes the specificity and effectiveness of the molecules in this case, green long afterglow nanoparticles have green afterglow performance, red long afterglow nanoparticles have red afterglow performance, long afterglow nanoparticles (PLNPs) have a remarkable ability to capture excited energy and dissipate this energy through the slow release of trapped carriers, resulting in persistent 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, resulting in superior signal modulation ratios that facilitate more accurate and sensitive analytical results. The present application takes advantage of the different recognition of bacteria by vancomycin and polymyxin B, and discriminates by the emission band difference of long afterglow nanoparticles.

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

[0009] The present application aims to provide a preparation method of nanoprobes, including the preparation of magnetic nanoparticles modified with concanavalin A, the preparation of green long afterglow nanoparticles modified with polymyxin B on the surface, and the preparation of red long afterglow nanoparticles modified with vancomycin on the surface.

[0010] The magnetic nanoparticles are carboxylated, then the carboxyl group is activated and coupled with a small molecule compound concanavalin A to obtain magnetic nanoparticles modified with concanavalin A. The green long afterglow nanoparticles are hydroxylated, then the hydroxylated green long afterglow nanoparticles are aminated, then 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, then 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 concanavalin A modified magnetic nanoparticles, vancomycin modified red long afterglow nanoparticles, and polymyxin B modified green long afterglow nanoparticles are stored at low temperature for standby use.

[0011] Further, the preparation method of the magnetic nanoparticles modified with concanavalin A includes the following steps:

[0012] The magnetic nanomaterials were dispersed in anhydrous citric acid buffer solution and ultrasonicated for 10 min, stirred overnight at room temperature, then washed with deionized water three times to obtain carboxylated magnetic nanoparticles; then the carboxylated magnetic nanoparticles were dissolved in 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution, 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and N-hydroxysuccinimide sulfonic acid sodium salt were added, and the carboxyl group was activated at room temperature for 2 h. After washing with PBS for 3 times, 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 h, and the concanavalin A modified magnetic nanoparticles (Con A-MNP) were collected magnetically. After washing with binding buffer (10 mM PBS containing 1 mM CaCl2 and 1 mM MnCl2) for three times, the preparation was stored. After the preparation of the concanavalin A modified magnetic nanoparticles, 1% BSA was added and blocked at 37°C for 2 h.

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

[0014] Further, the preparation method of the 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) are dispersed in sodium hydroxide solution (1-5 mmol / L), stirred at room temperature for 24 h, washed with deionized water for three times, and dried in vacuum to obtain hydroxylated green long afterglow nanoparticles. The hydroxylated long afterglow nanoparticles are dissolved in N,N-dimethylformamide, 3-aminopropyltriethoxysilane (5-20 μL per mL of reaction solution) is added under electromagnetic stirring, and the mixture is placed in an oil bath at 80°C and stirred electromagnetically for 24 h. After centrifugal separation, the product is washed with N,N-dimethylformamide and anhydrous ethanol, and dried in vacuum to obtain aminated green long afterglow nanoparticles. The aminated green long afterglow nanoparticles are dissolved in N,N-dimethylformamide, and succinic anhydride solution in N,N-dimethylformamide (2-8 mg / mL) and 4-dimethylaminopyridine solution in N,N-dimethylformamide (0.5-2.0 mg / mL) are added in sequence under stirring. After stirring at room temperature for 12 h, the product is centrifugally separated, washed with 50% anhydrous ethanol for three times, and dried in vacuum to obtain carboxylated green long afterglow nanoparticles. The carboxylated green long afterglow nanoparticles are dissolved in PBS buffer, and ultrasonic treatment is performed for 10 min. 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and N-hydroxysuccinimide sulfonic acid sodium salt are added and incubated for 10 min. Subsequently, polymyxin B is added, and the pH is adjusted to 7.1-7.6 with sodium bicarbonate. After incubation at 30°C for 12 h, the unbound polymyxin B is removed by centrifugal separation. Methoxypolyethylene glycol amine (M.W. 750) is added and sealed for 2 h at 37°C to obtain polymyxin B-modified green long afterglow nanoparticles.

[0016] Further, the preparation method of the green long afterglow nanoparticles is as follows: germanium oxide powder is dissolved in sodium hydroxide solution to obtain a sodium germanate solution (Na2GeO3). Then, aqueous solutions of zinc nitrate (Zn(NO3)2), manganese chloride (MnCl2) and praseodymium nitrate (Pr(NO3)3) are mixed under magnetic stirring, and nitric acid solution is added to obtain a precursor mixture solution. Subsequently, the sodium germanate (Na2GeO3) is added dropwise to the precursor mixture solution under continuous stirring. The pH of the mixed precursor solution is adjusted to alkaline, preferably 7.4-7.8. The solution is ultrasonically treated at room temperature for 10 min in an ultrasonic instrument, and then stirred at room temperature for 30 min in a magnetic stirrer. The obtained solution is transferred into a high-pressure reaction kettle with a polytetrafluoroethylene liner, and subjected to hydrothermal reaction at 220°C for 16 h. After the completion of the hydrothermal reaction, the solution is naturally cooled to room temperature, and the precipitate is washed with 50% ethanol for three times after centrifugal separation, and dried in vacuum 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] Further, the method for preparing the red long afterglow nanoparticles modified with vancomycin comprises the following steps: dispersing red long afterglow nanoparticles (1-5 mg / mL) in sodium hydroxide solution (1-5 mg / mL), stirring at room temperature for 24 h, washing with deionized water for 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℃ oil bath, and stirring under electromagnetic stirring for 24 h; centrifugal separation, washing with N,N-dimethylformamide and anhydrous ethanol, and vacuum drying to obtain aminated red long afterglow nanoparticles; dissolving 20-50 μg of vancomycin in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 5.5) containing EDC (5-30 mg) and NHS (10-50 mg) for 2 h; then adding the aminated red long afterglow nanoparticles (1-5 mg / mL) to the above mixture and stirring for another 6 h; centrifugal cleaning of the obtained nanoparticles to remove unattached carboxylated materials, and adding methoxypolyethylene glycol amine (M.W. 750) for blocking at 37℃ for 2 h to obtain vancomycin-modified red long afterglow nanoparticles.

[0019] Further, the method for preparing the red long afterglow nanoparticles comprises the following steps: mixing zinc nitrate (Zn(NO3)2), gallium nitrate (Ga(NO3)3), and chromium nitrate (Cr(NO3)3) and stirring; adding deionized water, adjusting the total volume to 15 mL, adding concentrated ammonia solution (28%) (about 1 mL) to adjust the pH, preferably adjusting the pH to 8.5-9.5, forming a white precipitate, and continuing to stir for 0.5 h; then transferring the mixture to a polytetrafluoroethylene-lined hydrothermal reactor (25 mL) and sealing; placing the hydrothermal reactor in a 220℃ reaction for 10 h, and then naturally cooling to room temperature; centrifugal separation of the obtained white precipitate dispersed in dilute hydrochloric acid to form a transparent solution, which can remove possible zinc oxide impurities; and then washing with isopropanol and vacuum drying to obtain the red long afterglow nanoparticles.

[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 application also aims to provide the use of a nanoprobe or a method for preparing a nanoprobe in identifying gram-negative and gram-positive bacteria.

[0022] Further, the method for identifying gram-negative and gram-positive bacteria comprises the following steps: mixing gram-negative bacteria and gram-positive bacteria together, mixing with concanavalin A modified magnetic nanoparticles, vancomycin modified red long afterglow nanoparticles and polymyxin B modified green long afterglow nanoparticles for 5 minutes, performing magnetic separation for 5 minutes, detecting the phosphorescence intensity (time resolution, delay time: 20 mu s) of the magnetic absorption precipitate group and the magnetic absorption supernatant group by using an enzyme-labeled instrument, calculating the intensity ratio, and using the difference between the green afterglow (excitation: 250 nm, emission: 529 nm) and the red afterglow (excitation: 250 nm, emission: 697 nm) emission bands to type gram-positive bacteria and gram-negative bacteria.

[0023] The mixed bacteria of gram-negative bacteria and gram-positive bacteria are diluted to different concentrations, mixed with concanavalin A modified magnetic nanoparticles, vancomycin modified red long afterglow nanoparticles and polymyxin B modified green long afterglow nanoparticles for 5 minutes, and then subjected to magnetic separation for 5 minutes; the phosphorescence intensity (time resolution, delay time: 20 mu s) of the magnetic absorption precipitate group and the magnetic absorption supernatant group is detected by using an enzyme-labeled instrument, and the intensity ratio is calculated to establish a standard curve of the intensity ratio and the concentration of the target analyte.

[0024] The collected sample is mixed with concanavalin A modified magnetic nanoparticles, vancomycin modified red long afterglow nanoparticles and polymyxin B modified green long afterglow nanoparticles for 5 minutes, and then subjected to magnetic separation for 5 minutes; the phosphorescence intensity (time resolution, delay time: 20 mu s) of the magnetic absorption precipitate group and the magnetic absorption supernatant group is detected by using an enzyme-labeled instrument, and the intensity ratio is calculated to establish a standard curve of the intensity ratio and the concentration of the target analyte.

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

[0026] 1. The present application uses small molecule compound concanavalin A to modify magnetic nanoparticles as recognition and separation enrichment particles, simplifies the detection process and replaces the traditional centrifugation method; at the same time, vancomycin and polymyxin B are used to modify nanoparticles with different long afterglows as recognition molecules and signal output molecules, which utilize the characteristics of long afterglow, anti-interference and low background to improve the anti-interference ability of detection, and the whole process is fast and simple.

[0027] 2. The present application uses 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 cost, greatly improves the convenience of detection and widens the application scenarios.

[0028] The above description is only a summary of the technical scheme of the present application. In order to make the technical means of the present application more clearly understood, the present application can be implemented according to the content of the description, and in order to make the above content and purposes, characteristics and advantages of the present application more apparent and easy to understand, the specific embodiments of the present application are described below. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 TEM image of the magnetic nanoparticle in the present application.

[0030] Figure 2 TEM image of the green long afterglow nanoparticle in the present application.

[0031] Figure 3 Phosphorescence spectrum of the green long afterglow nanoparticle in the present application.

[0032] Figure 4 TEM image of the red long afterglow nanoparticle in the present application.

[0033] Figure 5 Phosphorescence spectrum of the red long afterglow nanoparticle in the present application.

[0034] Figure 6 Potential change graph of the magnetic nanoparticle before and after modification in the present application.

[0035] Figure 7 Potential change graph of the green long afterglow nanoparticle before and after modification in the present application.

[0036] Figure 8 Potential change graph of the red long afterglow nanoparticle before and after modification in the present application. DETAILED DESCRIPTION

[0037] The technical scheme of the present application is further described in detail below in combination with specific embodiments. It should be understood that the following embodiments are only illustrative and explanatory of the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology implemented based on the above content of the present application is covered within the scope of protection intended by the present application.

[0038] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or prepared by existing methods.

[0039] Example 1: Preparation of magnetic nanoparticle

[0040] The preparation method of the magnetic nanoparticle 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. The mixture was vigorously stirred for 30 min, hydrothermally reacted at 200°C 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 2. 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 in 0.1-0.6 mol / L anhydrous citric acid buffer, sonicate 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, and react at room temperature for 2 h to activate the carboxyl groups. After washing three times with PBS, the activated MNPs (1 mg / mL) were mixed with 1-5 mg / mL ConA in PBS containing 1 mM CaCl2 and 1 mM MnCl2, stirred for 1 h, and magnetically collected to obtain ConA-MNPs. The ConA-MNPs were washed three times with binding buffer (1 mM CaCl2 and 1 mM MnCl2 in 10 mM PBS) and stored at low temperatures.

[0049] 3. After the preparation of 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 blocked Concanavalin A modified magnetic nanoparticles.

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

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

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

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

[0054] 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 under magnetic stirring, and 1.0-1.5 mmol of Na2GeO3 is added dropwise to the mixture under continuous stirring;

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

[0056] The transmission electron microscopy (TEM) image of the prepared green long afterglow nanoparticles is as shown in Figure 2 The phosphorescence spectrum is as shown in Figure 3 , Figure 2 and Figure 3 The prepared green long afterglow nanoparticles, 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 hydrothermally reacted at 220°C for 8-24 h.

[0057] Example 4: Preparation of polymyxin B modified green long afterglow nanoparticles

[0058] The method for preparing the 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 with deionized water for three times, and vacuum dry to obtain hydroxylated green long afterglow nanoparticles;

[0060] 2. Dissolve 20-100 mg of the hydroxylated green nanoparticles in 20 mL of N,N-dimethylformamide, slowly add 3-aminopropyltriethoxysilane 50-200 μL under electromagnetic stirring, place in an 80°C oil bath, and stir electromagnetically for 24 h, then centrifugally separate, wash twice with N,N-dimethylformamide and once with anhydrous ethanol, and vacuum dry to obtain aminated green long afterglow nanoparticles;

[0061] 3. Dissolve 20-100 mg of the aminated 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 under vigorous stirring, stir at room temperature for 12 h, centrifugally separate, wash with 50% anhydrous ethanol for three times, and vacuum dry to obtain carboxylated green long afterglow nanoparticles;

[0062] 4. Dissolve 1-5 mg of the carboxylated green long afterglow nanoparticles in 2-10 mL of PBS buffer, ultrasonicate for 10 min, add 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole 2-10 mg and N-hydroxysuccinimide sulfonate sodium salt 5-30 mg, 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, centrifugally remove the unbound polymyxin B, add 1% of PEG-NH2 (M.W. 750) to block the unbound sites at 37°C for 2 h, and obtain the blocked polymyxin B modified green long afterglow nanoparticles.

[0063] The potential changes of the green long afterglow nanoparticles (G-PLNP) before and after modification are as shown in Figure 7 , Figure 7Poly-myxin B modified green long afterglow nanoparticles, in step 1, 30-100 mg green long afterglow nanoparticles were dispersed in 10-50 mL of 5 mmol / L sodium hydroxide solution; in step 2, 20-100 mg of hydroxylated green nanoparticles were dissolved in 20 mL of N,N-dimethylformamide, and 50-200 μL of 3-aminopropyltriethoxysilane was slowly added under electromagnetic stirring; in step 3, 1-5 mg of aminated green long afterglow nanoparticles were dissolved in 2-10 mL of N,N-dimethylformamide solution, and 1-5 mg / mL of succinic anhydride and 0.5-2.0 mg / mL of 4-dimethylaminopyridine in N,N-dimethylformamide solution were slowly added in turn under vigorous stirring; in step 4, 1-5 mg of carboxylated green long afterglow nanoparticles were dissolved in 2-10 mL of PBS buffer solution, and 2-10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 5-30 mg of N-hydroxysuccinimide sulfonate sodium salt were added after ultrasonic treatment for 10 min, and the mixture was incubated at room temperature for 10 min, followed by the addition of 1-5 mg of polymyxin B.

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

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

[0066] 1. 1 mmol of Zn(NO3)2, 2 mmol of Ga(NO3)3, and 0.001-0.01 mmol of Cr(NO3)3 were mixed together and stirred vigorously, and the total volume was adjusted to 15 mL by adding deionized water;

[0067] 2. Concentrated ammonium hydroxide (28%) solution (about 1 mL) was quickly added to adjust the pH to 9-9.5, and a white precipitate was immediately formed. After stirring for another 0.5 h, the mixture was transferred to a polytetrafluoroethylene-lined hydrothermal reactor and sealed;

[0068] 3. The hydrothermal reactor was placed in a 220°C reactor for 10 h, and then naturally cooled until room temperature was reached. The white precipitate obtained after centrifugation was dispersed in dilute hydrochloric acid to form a transparent solution, which could remove possible zinc oxide impurities in this step, and then washed with an excess of isopropanol, and finally vacuum dried to obtain red long afterglow nanoparticles.

[0069] The transmission electron microscopy image of the prepared red long afterglow nanoparticles is shown in Figure 4 , and the phosphorescence spectrum is shown in Figure 5 , Figure 4 and 5 The red long afterglow nanoparticles prepared in

[0070] Example 6: Preparation of vancomycin-modified red long afterglow nanoparticles

[0071] The method for preparing 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 with deionized water three times, and vacuum dry to obtain hydroxylated red long afterglow nanoparticles;

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

[0074] 3. Activate the carboxyl group: dissolve 10-50 μg of vancomycin in 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 5.5) containing EDC (3-10 mg) and NHS (10-50 mg) and stir for 2 h, then add 2 mg / mL of aminated red long afterglow nanoparticles to the above mixture and stir for another 6 h, centrifuge the obtained nanoparticles, wash to remove unattached carboxylated materials, add mPEG-NH2 (M.W. 750) at a concentration of 0.5-5.0%, and seal at 37°C for 2 h to obtain vancomycin-modified red long afterglow nanoparticles.

[0075] The potential change of red long afterglow nanoparticles (R-PLNP) before and after modification is as follows Figure 8 , Figure 8 In the vancomycin-modified red long afterglow nanoparticles, 10-100 mg of red long afterglow nanoparticles are dispersed in 20-50 mL of 5 mmol / L sodium hydroxide solution in step 1; 10-50 mg of hydroxylated red nanoparticles are dissolved in 20-40 mL of N,N-dimethylformamide in step 2, and 3-aminopropyltriethoxysilane 10-100 μL is slowly added under electromagnetic stirring; in step 3, 10-50 μg of vancomycin is dissolved in 2-(N-morpholino) ethanesulfonic acid buffer (pH 5.5) containing EDC (3-10 mg) and NHS (10-50 mg) and stirred for 2 h, and mPEG-NH2 (M.W. 750) is added and sealed at 37°C for 2 h.

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

[0077] The method for rapidly identifying gram-negative and positive bacteria by the nanoprobe of the present application

[0078] 1. Take the bacterial suspension mixture of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) 100 μL (50 μL each) as the experimental group, and the control group is 100 μL H2O group, and 40 μg / mL-0.5 mg / mL ConA-MNP, 40 μg / mL-0.5 mg / mL R-PLNP-Van, 40 μg / mL-0.5 mg / mL G-PLNP-PMB mixture 300 μL (three kinds of nanoparticles each 100 μL) are added to the two groups, and after 5 min, magnetic separation (5 min) is performed;

[0079] 2. The supernatant is sucked out and reserved, the precipitate is added with the same volume of H2O as the supernatant, and the phosphorescence intensity (time resolution) of the magnetic absorption precipitate group and the magnetic absorption supernatant group is detected by the enzyme label instrument, the intensity ratio is calculated, the difference between the green afterglow (excitation: 250 nm, emission: 529 nm) and the red afterglow (excitation: 250 nm, emission: 697 nm) emission band is used to type the gram-positive and gram-negative bacteria; At the same time, the target bacteria are diluted to different concentrations, mixed with concanavalin A modified magnetic nanoparticles, vancomycin modified red long afterglow nanoparticles, and polymyxin B modified green long afterglow nanoparticles, reacted for 5 min, and then separated by magnetism for 5 min. The phosphorescence intensity (time resolution, delay time: 20 μs) of the magnetic absorption precipitate group and the magnetic absorption supernatant group is detected by the enzyme label instrument, and the intensity ratio is calculated, and a standard curve of the intensity ratio and the concentration of the target analyte is established;

[0080] 3. The clinical sample is mixed with concanavalin A modified magnetic nanoparticles, vancomycin modified red long afterglow nanoparticles, and polymyxin B modified green long afterglow nanoparticles, reacted for 5 min, and then separated by magnetism for 5 min. The phosphorescence intensity (time resolution, delay time: 20 μs) of the magnetic absorption precipitate group and the magnetic absorption supernatant group is detected by the enzyme label instrument, and the intensity ratio is calculated, and the concentration of the target analyte in the sample is obtained by comparing with the standard curve.

[0081] The application provides a scheme for rapidly identifying and typing gram-positive and gram-negative bacteria based on long afterglow nanomaterials, which mainly comprises green long afterglow nanoparticles, red long afterglow nanoparticles and magnetic nanoparticles; the magnetic nanoparticles are modified by concanavalin A, the red long afterglow nanoparticles are modified by vancomycin and the green long afterglow nanoparticles are modified by polymyxin B; the bacteria are captured by concanavalin A, separated by the magnetic nanoparticles, and then identified by vancomycin and polymyxin B, wherein vancomycin identifies gram-positive bacteria and polymyxin B identifies gram-negative bacteria, and the bacteria are identified by the emission band difference of the long afterglow nanoparticles.

[0082] The above-mentioned serial numbers of the embodiments of the application are only for description, and do not represent the advantages and disadvantages of the embodiments.

[0083] The embodiments of the application are described above in combination with the drawings, but the application is not limited to the above-mentioned specific embodiments, and the above-mentioned specific embodiments are only illustrative but not restrictive, and those skilled in the art can make many forms under the inspiration of the application without departing from the purpose of the application and the scope protected by the claims, which all belong to the protection of the application.

Claims

1. A nanoprobe, characterized in that: The invention comprises magnetic nanomaterials and long-lasting glow nanomaterials. The magnetic nanomaterials include magnetic nanoparticles modified with concanavalin A, which are used to identify, capture and separate bacteria. The long-lasting glow nanomaterials include green long-lasting glow nanoparticles modified with polymyxin B on the surface and red long-lasting glow nanoparticles modified with vancomycin on the surface. The green long-lasting glow nanoparticles modified with polymyxin B on the surface and the red long-lasting glow nanoparticles modified with vancomycin on the surface are used to identify anti-interference signal output respectively. The preparation method of green long-lasting glow nanoparticles modified with polymyxin B on the surface 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. The mixture is 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 stirring and then centrifuging, washing with anhydrous ethanol and drying to obtain carboxylated green long-lasting glow nanoparticles, dissolving the carboxylated green long-lasting glow 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, incubating and centrifuging to remove unbound polymyxin B, adding methoxypolyethylene glycol amine group for blocking, and obtaining polymyxin B-modified green long-lasting glow nanoparticles; The preparation method of green long-lasting glow nanoparticles comprises: dissolving germanium oxide powder in a sodium hydroxide solution to obtain a sodium germanate solution; then mixing aqueous solutions of zinc nitrate, manganese chloride, and praseodymium nitrate under magnetic stirring; and adding a nitric acid solution to obtain a precursor mixed solution; then, dropwise adding sodium germanate to the precursor mixed solution under continuous stirring; adjusting the pH of the mixed solution to alkaline, ultrasonicating, and stirring on a magnetic stirrer; placing the obtained solution in a high-pressure reactor for hydrothermal reaction; cooling and centrifuging, washing the precipitate with ethanol, and drying to obtain the green long-lasting glow nanoparticles; The preparation method of red long-lasting nanoparticles with vancomycin surface modification includes: dispersing red long-lasting nanoparticles in a sodium hydroxide solution and stirring, washing with deionized water and drying to obtain hydroxylated red long-lasting nanoparticles, dissolving the hydroxylated red long-lasting nanoparticles in N,N-dimethylformamide, adding 3-aminopropyltriethoxysilane under electromagnetic stirring, placing in an oil bath, electromagnetic stirring, centrifuging, washing with N,N-dimethylformamide and anhydrous ethanol, and drying to obtain amino-modified red long-lasting nanoparticles, dissolving vancomycin in a 2-(N-morpholino)ethanesulfonic acid buffer containing EDC and NHS; then adding the amino-modified red long-lasting nanoparticles to the above mixture and stirring again, washing the obtained nanoparticles by centrifugation, and adding methoxypolyethylene glycol to block the amine groups to obtain vancomycin-modified red long-lasting nanoparticles. 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 the solution is mixed and washed with isopropyl alcohol. After drying, the red long-afterglow nanoparticles are obtained.

2. The method for preparing a nanoprobe according to claim 1, wherein: 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-lasting glow nanoparticles with surface modification of polymyxin B: The green long-lasting glow nanoparticles are hydroxylated, aminated, and carboxylated, and finally the carboxylated green long-lasting glow nanoparticles are coupled with polymyxin B to obtain polymyxin B-modified green long-lasting glow nanoparticles; Preparation of red long-lasting nanoparticles modified with vancomycin on the surface: After the red long-lasting nanoparticles are hydroxylated and aminated, the aminated red long-lasting nanoparticles are coupled with vancomycin to obtain vancomycin-modified red long-lasting nanoparticles.

3. The method for preparing a nanoprobe according to claim 2, wherein: 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 groups. 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 binding buffer and stored.

4. The method for preparing a nanoprobe according to claim 3, wherein: 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. After the mixture is stirred, it is 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. Use of a nanoprobe according to claim 1 or a method for preparing a nanoprobe according to any one of claims 2 to 4 in identifying Gram-negative and Gram-positive bacteria.

6. The use according to claim 5, characterized in that: The method for identifying Gram-negative and Gram-positive bacteria includes: mixing Gram-negative bacteria and Gram-positive bacteria together, reacting the mixture with concanavalin A-modified magnetic nanoparticles, vancomycin-modified red long-afterglow nanoparticles, and polymyxin B-modified green long-afterglow nanoparticles, performing magnetic separation, detecting the phosphorescence intensity of the magnetic precipitation group and the magnetic supernatant group using a microplate reader, calculating the intensity ratio, and utilizing the difference in the emission bands of the green afterglow and the red afterglow to identify and quantitatively detect Gram-positive and Gram-negative bacteria; A mixture of Gram-negative and Gram-positive bacteria was diluted to different concentrations and reacted with concanavalin A-modified magnetic nanoparticles, vancomycin-modified red long-lasting nanoparticles, and polymyxin B-modified green long-lasting nanoparticles. The mixture was then magnetically separated and the phosphorescence intensity of the magnetic precipitation group and the magnetic supernatant group was measured using a microplate reader. The intensity ratio was calculated, and a standard curve of the intensity ratio and the target analyte concentration was established. 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. After magnetic separation, the phosphorescence intensity of the magnetic precipitation group and the magnetic supernatant group was detected by a microplate reader. 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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