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CN119574856A - A nanoprobe and its preparation method and application - Google Patents

A nanoprobe and its preparation method and application Download PDF

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CN119574856A
CN119574856A CN202411680851.5A CN202411680851A CN119574856A CN 119574856 A CN119574856 A CN 119574856A CN 202411680851 A CN202411680851 A CN 202411680851A CN 119574856 A CN119574856 A CN 119574856A
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nanoparticles
magnetic
modified
long afterglow
afterglow
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CN119574856B (en
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李娟�
康月茜
程伟
陈锐
杨若彤
任文龙
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Chongqing Medical University
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Abstract

The invention belongs to the technical field of nanoprobes, and particularly relates to a nanoprobe and a preparation method and application thereof, wherein the nanoprobe comprises a magnetic nanoprobe and a long-afterglow nanoprobe, the magnetic nanoprobe comprises magnetic nanoparticles modified with canavalin A and used for identifying and capturing and separating bacteria, the long-afterglow nanoprobe 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 respectively used for identifying anti-interference signal output, and the nanoprobe can improve the anti-interference capability of detection when applied to identifying gram-negative positive bacteria.

Description

Nanometer probe and preparation method and application thereof
Technical Field
The invention belongs to the technical field of nano probes, and particularly relates to a nano probe and a preparation method and application thereof.
Background
Bacterial gram typing helps doctors to quickly identify the possible types of pathogenic bacteria in the primary diagnosis of infectious diseases, thereby leading to early selection of appropriate antibiotic treatment regimens. The existing method for clinically classifying bacteria mainly depends on professional operators, and has high technical requirements on the operators. Development of a new method for obtaining test results quickly and easily is important, which does not depend on the expertise of personnel.
Molecular recognition is performed with molecular recognition agents that specifically bind bacteria without destroying the cells. Molecular recognition agents include antibodies, aptamers, phages and specific proteins. However, these biomaterial-based identifiers are poor in stability, high in cost, difficult in preparation method, and, in addition, some are not commercially available.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides the nano probe, the preparation method and the application thereof, and the aims of improving the anti-interference capability of detection, along with quick and simple whole flow, low cost and high stability are fulfilled.
The invention solves the technical problems by adopting the following technical scheme:
The invention aims to provide a nano probe which comprises a magnetic nano material and a long-afterglow nano material, wherein the magnetic nano material comprises magnetic nano particles modified with canavalin A and is used for identifying and capturing and separating bacteria, the long-afterglow nano material comprises green long-afterglow nano particles with polymyxin B modified on the surface and red long-afterglow nano particles with vancomycin modified on the surface, and the green long-afterglow nano particles with polymyxin B modified on the surface and the red long-afterglow nano particles with vancomycin modified on the surface are respectively used for identifying anti-interference signal output.
The magnetic nanoparticles modified with the canavalin A are separation and enrichment nanoparticles with a recognition function, recognition molecules are canavalin A (Con A), the magnetic nanoparticles have a carbohydrate structure on the cell wall of bacteria, the magnetic nanoparticles provide the separation and enrichment function, small-molecule antibiotics show obvious selectivity on bacterial strains, meanwhile, the magnetic nanoparticles have excellent stability, good tolerance, cost effectiveness and ready accessibility, experimental research shows that Vancomycin (Vancomycin, van) shows obvious capability of selectively targeting Gram-positive (G +) bacteria, the Vancomycin functionalized Magnetic Nanoparticles (MNPs) can be applied to the targeted detection of G + bacteria, polymyxin B (polymyxin B, PMB) has strong affinity on Gram-negative (Gram-negative) bacteria, G -) modified quantum dots or gold nanoparticles can be used for detecting G - bacteria, the interaction between the polymyxin B and the Gram-negative bacteria is the capability of capturing the persistence of green particles through the synergistic interaction of static interaction and the persistence of the red particles, and the persistence of the persistence can be enhanced, and the persistence of the persistence can be continuously excited by the combination of the persistence of the red particles, and the persistence of the persistence can be sustained release of the green particles under the condition of long persistence of the persistence energy is not generated. PLNPs, which is a unique property, can effectively avoid the mixing effect of autofluorescence in complex biological matrixes, generate a superior signal modulation ratio, and are beneficial to obtaining more accurate and sensitive analysis results. The invention uses the difference of vancomycin and polymyxin B in bacterial identification and identifies the difference of the emission wave bands of the long afterglow nano particles.
Preferably, the magnetic nano material adopts FeCl 3, the green long afterglow nano particles are Zn 2GeO4:Mn and Pr, and the red long afterglow nano material is ZnGa 2O4:Cr.
The invention aims to provide a preparation method of a nano probe, which comprises the steps of preparing magnetic nano particles modified with canavalin A, preparing green long-afterglow nano particles modified with polymyxin B on the surfaces and preparing red long-afterglow nano particles modified with vancomycin on the surfaces.
Carboxylation of magnetic nanoparticles, then coupling with a micromolecular compound, namely, canavalin A, after activating carboxyl groups, obtaining magnetic nanoparticles modified with canavalin A, hydroxylating the hydroxylated green long-afterglow nanoparticles, aminating the hydroxylated green long-afterglow nanoparticles, carboxylating the aminated green long-afterglow nanoparticles, finally coupling the carboxylated green long-afterglow nanoparticles with polymyxin B (PMB) to obtain PMB modified green long-afterglow nanoparticles, hydroxylating the hydroxylated red long-afterglow nanoparticles, and coupling the aminated red long-afterglow nanoparticles with vancomycin to obtain vancomycin modified red long-afterglow nanoparticles. And (3) respectively storing the magnetic nano particles modified by the canavalin A, the red long afterglow nano particles modified by the vancomycin and the green long afterglow nano particles modified by the polymyxin B at low temperature for later use.
Further, the preparation method of the magnetic nanoparticle modified with the canavalin A comprises the following steps:
Dispersing magnetic nano material into anhydrous citric acid buffer solution, ultrasonic treating for 10min, stirring at room temperature overnight, washing with deionized water for three times to obtain carboxylated magnetic nano particles, dissolving carboxylated magnetic nano particles in 2- (N-morpholinyl) ethanesulfonic acid (MES) buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide sodium sulfonate salt, reacting at room temperature for 2h to activate carboxyl, washing with PBS for 3 times, mixing activated magnetic nano particles (1 mg/mL) with 1mg/mL of canavalin A in PBS containing 1mM calcium chloride and 1mM manganese chloride, stirring for 1h, magnetically collecting the magnetic nano particles modified by canavalin A (Con A-MNP), washing with 10mM PBS of binding buffer solution (1 mM CaCl 2 and 1mM MnCl 2) for three times, and storing. After the magnetic nano-preparation modified by the canavalin A, BSA with the concentration of 1% is added for blocking for 2 hours at 37 ℃.
The preparation method of the magnetic nano particles comprises the steps of dissolving ferric chloride and trisodium citrate in glycol, adding sodium acetate under stirring, stirring the mixture for 30min, transferring the obtained solution into a stainless steel water thermal reaction kettle with a polytetrafluoroethylene lining, heating and keeping the temperature at 200 ℃ for 10h, cooling to room temperature, washing a black product with ethanol and deionized water, and drying in vacuum to obtain the magnetic nano particles.
Further, the preparation method of the green long afterglow nanoparticle with the surface modified with polymyxin B comprises the following steps:
Dispersing green long afterglow nano particles (1-5 mg/mL) in a sodium hydroxide solution (1-5 mmol/L), stirring for 24 hours at room temperature, washing with deionized water for three times, vacuum drying to obtain hydroxylated green long afterglow nano particles, dissolving the hydroxylated long afterglow nano particles in N, N-dimethylformamide, adding 3-aminopropyl triethoxysilane (5-20 mu L per mL of reaction solution) under electromagnetic stirring, placing in an 80 ℃ oil bath, performing electromagnetic stirring for 24 hours, performing centrifugal separation, washing with N, N-dimethylformamide and absolute ethyl alcohol, vacuum drying to obtain aminated green long afterglow nano particles, dissolving the aminated green long afterglow nano particles in N, N-dimethylformamide, sequentially adding succinic anhydride N under stirring, N-dimethylformamide solution (2-8 mg/mL), N-dimethylformamide solution (0.5-2.0 mg/mL) of 4-dimethylaminopyridine, stirring at room temperature for 12h, centrifuging, washing with 50% absolute ethyl alcohol for three times, vacuum drying to obtain carboxylated green long afterglow nano particles, dissolving the carboxylated green long afterglow nano particles in PBS buffer solution, performing ultrasonic treatment for 10min, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide sulfonic acid sodium salt for incubation for 10min, then adding polymyxin B, adjusting pH to 7.1-7.6,30 ℃ by sodium bicarbonate for incubation for 12h, centrifuging to remove unbound polymyxin B, adding methoxy polyethylene glycol amino (M.W.750) for sealing for 2h at 37 ℃, obtaining the polymyxin B modified green long afterglow nano particles.
Further, the preparation method of the green long-afterglow nanoparticle comprises the steps of dissolving germanium oxide powder in a sodium hydroxide solution to obtain a sodium germanate solution (Na 2GeO3), mixing aqueous solutions of zinc nitrate (Zn (NO 3)2), manganese chloride (MnCl 2) and praseodymium nitrate (Pr (NO 3)3)) under magnetic stirring, adding a nitric acid solution to obtain a precursor mixed solution, then dropwise adding sodium germanate (Na 2GeO3) into the precursor mixed solution under a continuously stirring state, adjusting the pH of the mixed precursor solution to be alkaline, preferably to be 7.4-7.8, placing the mixed precursor solution in an ultrasonic instrument, performing ultrasonic treatment at room temperature for 10min, placing the mixed precursor solution on a magnetic stirrer, stirring the mixed solution for 30min at room temperature, transferring the obtained solution into a polytetrafluoroethylene lining high-pressure reaction kettle, performing a 220 ℃ hydrothermal reaction for 16h, naturally cooling to room temperature after the hydrothermal reaction is finished, washing the precipitate with 50% ethanol for 3 times, and performing vacuum drying to obtain the green long-afterglow nanoparticle (ZGMP).
Preferably, zn (NO 3)2、MnCl2 and Pr (molar ratio of NO 3)3 is 1.98:0.01-0.05:0.01-0.05), and Na 2GeO3 is added in an amount of 1.0-1.5 mmol.
Further, the preparation method of the red long afterglow nanoparticle with vancomycin modified on the surface comprises the steps of dispersing the red long afterglow nanoparticle (1-5 mg/mL) in a sodium hydroxide solution (1-5 mg/mL), stirring at room temperature for 24h, washing with deionized water for three times, vacuum drying to obtain hydroxylated red long afterglow nanoparticle, dispersing the hydroxylated red long afterglow nanoparticle in N, N-dimethylformamide, adding 3-aminopropyl triethoxysilane (5-20 mu L added to each mL of reaction solution) under electromagnetic stirring, placing in an 80 ℃ oil bath, electromagnetic stirring for 24h, centrifuging, washing with N, N-dimethylformamide and absolute ethyl alcohol, vacuum drying to obtain aminated red long afterglow nanoparticle, dissolving 20-50 mu g of vancomycin in a 2- (N-morpholinyl) ethanesulfonic acid (MES) buffer (pH 5.5) containing EDC (5-30 mg) and NHS (10-50 mg), adding the aminated red long afterglow nanoparticle (1-5 mg/mL) into the mixture, adding methoxy group-modified 10W to the mixture, washing with 5 ℃ to obtain the carboxyl group-modified long afterglow nanoparticle, washing with the carboxyl group of vancomycin, and washing to obtain the 5-37 ℃ long afterglow nanoparticle, and washing with the carboxyl group modified carboxyl group attached to the carboxyl group-containing vancomycin.
Further, the preparation method of the red long-afterglow nanoparticle comprises the steps of mixing and stirring zinc nitrate (Zn (NO 3)2), gallium nitrate (Ga (NO 3)3) and chromium nitrate (Cr (NO 3)3)), adding deionized water, adjusting the total volume to 15mL, adding concentrated ammonia water (28%) solution (about 1 mL) to adjust the pH, preferably adjusting the pH to 8.5-9.5, forming white precipitate, continuously stirring for 0.5h, transferring the mixture into a polytetrafluoroethylene-lined hydrothermal reaction kettle (25 mL), sealing, placing the hydrothermal reaction kettle in a 220 ℃ reaction kettle for 10h, naturally cooling to room temperature, dispersing the white precipitate obtained after centrifugation in dilute hydrochloric acid to form a transparent solution, removing possible zinc oxide impurities in the step, mixing and washing with isopropanol, and drying in vacuum to obtain the red long-afterglow nanoparticle.
Preferably, zn (molar ratio of NO 3)2、Ga(NO3)3、Cr(NO3)3 is 1:2:0.001-0.01).
The invention also aims to provide an application of the nano probe or the preparation method of the nano probe in identifying gram-negative and positive bacteria.
Further, the method for identifying gram-negative and positive bacteria comprises mixing gram-negative bacteria and gram-positive bacteria together, mixing with the magnetic nanoparticles modified by canavalin A, the red long afterglow nanoparticles modified by vancomycin and the green long afterglow nanoparticles modified by polymyxin B for 5min, performing magnetic separation for 5min, detecting the phosphorescence intensities (time resolution, delay time: 20 mu s) of a magnetic precipitation group and a magnetic supernatant group by using an enzyme-labeling instrument, calculating the intensity ratio, and typing the gram-positive bacteria and the negative bacteria by utilizing the difference of the emission wave bands of the green afterglow (excitation: 250nm, emission: 529 nm) and the red afterglow (excitation: 250nm, emission: 697 nm);
Diluting mixed bacteria of gram-negative bacteria and gram-positive bacteria to different concentrations, mixing and reacting with magnetic nanoparticles modified by canavalin A, red long afterglow nanoparticles modified by vancomycin and green long afterglow nanoparticles modified by polymyxin B for 5min, magnetically separating for 5min, detecting phosphorescence intensities (time resolution, delay time: 20 mu s) of a magnetic precipitation group and a magnetic supernatant group by using an enzyme-labeling instrument, calculating an intensity ratio, and establishing a standard curve of the intensity ratio and the concentration of a target analyte;
mixing the collected sample with the magnetic nano particles modified by the canavalin A, the red long afterglow nano particles modified by the vancomycin and the green long afterglow nano particles modified by the polymyxin B for reaction for 5min, detecting the phosphorescence intensities (time resolution, delay time: 20 mu s) of the magnetic precipitation group and the magnetic supernatant group by using an enzyme-labeled instrument through magnetic separation for 5min, calculating an intensity ratio, and comparing the intensity ratio with a standard curve to obtain the concentration of the target detection object in the sample.
Compared with the prior art, the invention has the beneficial technical effects that:
1. The invention uses vancomycin and polymyxin B to be respectively modified on the nano particles with different afterglow as identification molecules, can also be used as signal output molecules, and has the characteristics of interference resistance and low background by utilizing the characteristic of afterglow, thus improving the anti-interference capability of detection, and the whole process is quick and simple.
2. The invention realizes the modification of the magnetic nano particles by using the micromolecular compound canavalin A and the modification of the long afterglow nano particles by using vancomycin and polymyxin B, obviously improves the detection speed, reduces the detection time and the detection cost, greatly improves the convenience of detection and widens the application scene of the detection.
The foregoing description is only an overview of the present invention, and is intended to be illustrative of the present invention, as it is to be understood, and is to be accorded the widest scope consistent with the principles and features disclosed herein.
Drawings
Fig. 1 is a TEM image of magnetic nanoparticles in the present invention.
FIG. 2 is a TEM image of green long persistence nanoparticles of the invention.
FIG. 3 is a graph showing phosphorescence spectrum of the green long persistence nanoparticle of the present invention.
FIG. 4 is a TEM image of a red long afterglow nanoparticle of the present invention.
FIG. 5 is a graph showing phosphorescence spectrum of the red long afterglow nanoparticle of the present invention.
FIG. 6 is a graph showing the potential change before and after modification of magnetic nanoparticles according to the present invention.
FIG. 7 is a graph showing the potential change before and after modification of the green long persistence nanoparticle of the present invention.
FIG. 8 is a graph showing the potential change before and after modification of the red long afterglow nanoparticle in the present invention.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
In addition, unless otherwise specifically indicated, the various raw materials, reagents, instruments and equipment used in the present invention may be obtained commercially or prepared by existing methods.
Example 1 preparation of magnetic nanoparticles
The preparation method of the magnetic nano particles comprises the following steps:
1. FeCl 3 (0.2-1.0 g) and trisodium citrate (0.1-0.5 g) are dissolved in ethylene glycol (10-50 mL);
2. Then NaAc (0.5-2.0 g) is added under stirring, the mixture is vigorously stirred for 30min, placed at 200 ℃ for hydrothermal reaction for 10h, and then cooled to room temperature;
3. The product was washed several times with ethanol and deionized water.
The prepared magnetic nano particle transmission electron microscope chart is shown in figure 1. The magnetic nanoparticle prepared in FIG. 1 has an added amount of FeCl 3 of 0.65 g, trisodium citrate of 0.2 g, ethylene glycol of 20mL and NaAc of 1.2 g.
Example 2 preparation of Canavalia ectenes A modified magnetic nanoparticles
The preparation method of the canavalin A modified magnetic nanoparticle comprises the following steps:
1. Dispersing 10mg/mL of magnetic nanoparticles into 0.1-0.6 mol/L anhydrous citric acid buffer solution, carrying out ultrasonic treatment for 10min, stirring overnight at room temperature, and then washing with deionized water for three times to obtain carboxylated magnetic nanoparticles;
2. Dissolving 1-10 mg of carboxylated magnetic nanoparticles in 1-10 mL of 2- (N-morpholinyl) ethanesulfonic acid (MES) buffer, adding 5-15 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 10-30 mg of N-hydroxysuccinimide sulfonic acid sodium salt, reacting at room temperature for 2h to activate carboxyl groups, washing 3 times with PBS, mixing activated MNPs (1 mg/mL) with 1-5 mg/mL ConA in PBS containing 1mM CaCl 2 and 1mM MnCl 2, stirring for 1h, magnetically collecting ConA-MNP, washing three times with binding buffer (10 mM PBS of 1mM CaCl 2 and 1mM MnCl 2), and preserving the obtained ConA-MNP at low temperature;
3. And (3) adding BSA (BSA) with the final concentration of 1% into the prepared magnetic nano particles modified by the canavalin A for blocking for 2 hours at the temperature of 37 ℃ to block unbound sites, thereby obtaining the magnetic nano particles modified by the canavalin A after blocking.
The potential change patterns of the magnetic nanoparticles MNP before and after modification are shown in FIG. 6, the magnetic nanoparticles modified by the canavalin A in FIG. 6 are shown in the specification, the anhydrous citric acid buffer solution in the step 1 is 0.1-0.6 mol/L, 1-10 mg of carboxylated magnetic nanoparticles are dissolved in 1-10 mL of 2- (N-morpholinyl) ethanesulfonic acid buffer solution, 5-15 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 10-30 mg of N-hydroxysuccinimide sulfonic acid sodium salt are added, and activated MNPs (1 mg/mL) and 1-5 mg/mL Con A are mixed in PBS containing 1mM CaCl 2 and 1mM MnCl 2.
Example 3 preparation of Green Long persistence nanoparticles
The preparation method of the green long afterglow nanoparticle comprises the following steps:
the GeO 2 powder is dissolved in NaOH solution to prepare Na 2GeO3 solution;
2. Mixing 1.98mmol of Zn (NO 3)2, 0.01-0.05 mmol of MnCl 2, 0.01-0.05 mol of Pr (NO 3)3 and 100-500 mu L of concentrated HNO 3) under magnetic stirring, and dropwise adding 1.0-1.5 mmol of Na 2GeO3 into the mixture under continuous stirring;
3. And regulating the pH value of the mixed precursor solution to 7.2-7.8, placing the mixed precursor solution in an ultrasonic instrument, performing ultrasonic treatment at room temperature for 10min, and stirring the mixed precursor solution on a magnetic stirrer at room temperature for 30min. Finally, transferring the mixture into a polytetrafluoroethylene-lined reactor, performing hydrothermal reaction at 220 ℃ for 8-24 h, centrifuging, collecting the synthesized ZGMP, and washing with 50% ethanol three times.
The transmission electron microscope image of the prepared green long afterglow nano particles is shown in figure 2. The phosphorescence spectrum is shown in FIG. 3, the green long afterglow nanoparticle prepared in FIG. 2 and FIG. 3, in step 2, 1.98mmol of Zn (NO 3)2, 0.01-0.05 mmol of Mn Cl 2, 0.01-0.05 mol of Pr (NO 3)3 and 100-500. Mu.L of concentrated HNO 3) are mixed, 1.0-1.5 mmol of Na 2GeO3 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 hydrothermal reaction is carried out at 220 ℃ for 8-24 hours.
Example 4 preparation of polymyxin B-modified Green Long persistence nanoparticles
The preparation method of the polymyxin B modified green long afterglow nanoparticle comprises the following steps:
1. Dispersing 30-100 mg of green long-afterglow nano particles in 10-50 mL of 5mmol/L sodium hydroxide solution, stirring for 24 hours at room temperature, washing with deionized water for three times, and vacuum drying to obtain hydroxylated green long-afterglow nano particles;
2. Dissolving 20-100 mg of hydroxylated green nano particles in 20mL of N, N-dimethylformamide, slowly adding 50-200 mu L of 3-aminopropyl triethoxysilane under electromagnetic stirring, placing in an oil bath at 80 ℃, carrying out electromagnetic stirring for 24 hours, centrifuging, washing twice with N, N-dimethylformamide, washing once with absolute ethyl alcohol, and carrying out vacuum drying to obtain aminated green long-afterglow nano particles;
3. Dissolving 20-100 mg of the aminated green long-afterglow nano particles in 10-50 mL of N, N-dimethylformamide solution, sequentially and slowly adding 1-5 mg/mL of succinic anhydride and 0.5-2.0 mg/mL of 4-dimethylaminopyridine in N, N-dimethylformamide solution under intense stirring, stirring at room temperature for 12h, centrifugally separating, washing with 50% absolute ethyl alcohol for three times, and vacuum drying to obtain carboxylated green long-afterglow nano particles;
4. Dissolving 1-5 mg of carboxylated green long-afterglow nano particles in 2-10 mL of PBS buffer solution, carrying out ultrasonic treatment for 10min, adding 2-10 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 5-30 mg of N-hydroxysuccinimide sulfonic acid sodium salt, incubating for 10min at room temperature, then adding 1-5 mg of polymyxin B, adjusting pH to 7.4 by using sodium bicarbonate, incubating for 12h at 30 ℃, centrifuging to remove unbound polymyxin B, adding 1% of PEG-NH 2 (M.W.750) at 37 ℃ and sealing unbound sites for 2h, thus obtaining the sealed polymyxin B modified green long-afterglow nano particles.
The potential change before and after modification of the green long-afterglow nanoparticle (G-PLNP) is shown in FIG. 7, the polymyxin B modified green long-afterglow nanoparticle is shown in FIG. 7, 30-100 mg of the green long-afterglow nanoparticle is dispersed in 10-50 mL of 5mmol/L sodium hydroxide solution in step 1, 20-100 mg of the hydroxylated green nanoparticle is dissolved in 20mL of N, N-dimethylformamide, 50-200 mu L of 3-aminopropyl triethoxysilane is slowly added under electromagnetic stirring in step 2, 1-5 mg of the aminated green long-afterglow nanoparticle is dissolved in 2-10 mL of N, N-dimethylformamide solution, 1-5 mg of succinic anhydride and 0.5-2.0 mg/mL of 4-dimethylaminopyridine are slowly added in sequence under vigorous stirring, 1-5 mg of carboxylated green long-afterglow nanoparticle is dissolved in 2-10 mL of PBS buffer solution in step 4, 1-3-ethyl- (3-succinyl) carbodiimide sodium salt is added, and then 1-30 mg of polymyxin-10 mg of polymyxin B is added under vigorous stirring, and then the polymyxin 1-30 mg of room temperature is incubated.
Example 5 preparation of Red Long persistence nanoparticles
The preparation method of the red long afterglow nanoparticle comprises the following steps:
1. 1mmol Zn(NO3)2、2mmol Ga(NO3)3、0.001～0.01mmol Cr(NO3)3 are mixed together, stirred vigorously and the total volume is adjusted to 15mL by adding deionized water;
2. Concentrated ammonium hydroxide (28%) solution (about 1 mL) was added rapidly, the pH was adjusted to 9-9.5, a white precipitate formed immediately, after stirring for 0.5h again, the mixture was transferred to a teflon lined hydrothermal reaction vessel and sealed;
3. The hydrothermal reaction kettle is placed at 220 ℃ for reaction for 10 hours, and then naturally cooled until the room temperature is reached. And dispersing the white precipitate obtained after centrifugation in dilute hydrochloric acid to form a transparent solution, removing possible zinc oxide impurities in the step, mixing and washing with excessive isopropanol, and finally drying in vacuum to obtain the red long afterglow nano particles.
The transmission electron microscope diagram of the prepared red long afterglow nanoparticle is shown in figure 4, the phosphorescence spectrum is shown in figure 5, the red long afterglow nanoparticle prepared in figures 4 and 5 is Cr (NO 3)3 is 0.004mmol in step 1, and the pH is adjusted to 9.2 in step 2.
Example 6 preparation of vancomycin-modified Red Long persistence nanoparticle
The preparation method of the vancomycin-modified red long-afterglow nanoparticle comprises the following steps:
1. Dispersing 10-100 mg of red long afterglow nano particles in 20-50 mL of 5mmol/L sodium hydroxide solution, stirring at room temperature for 24h, washing with deionized water for three times, and vacuum drying to obtain hydroxylated red long afterglow nano particles;
2. Dissolving 10-50 mg of hydroxylated red nano particles in 20-40 mL of N, N-dimethylformamide, slowly adding 10-100 mu L of 3-aminopropyl triethoxysilane under electromagnetic stirring, placing in an oil bath at 80 ℃, carrying out electromagnetic stirring for 24 hours, centrifuging, washing twice with N, N-dimethylformamide, washing once with absolute ethyl alcohol, and carrying out vacuum drying to obtain aminated red long-afterglow nano particles;
3. Activating carboxyl, namely dissolving 10-50 mug of vancomycin in 2- (N-morpholinyl) ethanesulfonic acid (MES) buffer solution (pH 5.5) containing EDC (3-10 mg) and NHS (10-50 mg), stirring for 2 hours, adding 2mg/mL of aminated red long-afterglow nano particles into the mixture, stirring for 6 hours, centrifuging the obtained nano particles, cleaning to remove unattached carboxylated materials, adding mPEG-NH 2 (M.W.750) with the concentration of 0.5-5.0%, and sealing for 2 hours at 37 ℃ to obtain the vancomycin modified red long-afterglow nano particles.
The potential change before and after modification of the red long afterglow nanoparticle (R-PLNP) is shown in FIG. 8, 10-100 mg of the vancomycin modified red long afterglow nanoparticle is dispersed in 20-50 ml of 5mmol/L sodium hydroxide solution in step 1, 10-50 mg of the hydroxylated red nanoparticle is dissolved in 20-40 ml of LN, N-dimethylformamide, 10-100 mu L of 3-aminopropyl triethoxysilane is slowly added under electromagnetic stirring, 10-50 mu g of vancomycin is dissolved in 2- (N-morpholinyl) ethanesulfonic acid buffer (pH 5.5) containing EDC (3-10 mg) and NHS (10-50 mg) in step 3, stirring is carried out for 2h, mPEG-NH 2 (M.W.750) is added for 37 ℃ and sealing is carried out for 2h.
Example 7 rapid identification of gram-negative-positive bacteria by the nanoprobes of the present invention
The method for rapidly identifying gram-negative and positive bacteria by using the nano probe
1. Taking 100 mu L (50 mu L each) of a bacterial suspension mixture of escherichia coli (ESCHERICHIA COLI, E.coli) and staphylococcus aureus (Staphylococcus aureus, S.aureus) as an experimental group, wherein a control group is a 100 mu L H 2 O group, and adding 40 mu G/mL-0.5 mg/mL ConA-MNP,40 mu G/mL-0.5 mg/mL R-PLNP-Van and 40 mu G/mL-0.5 mg/mL G-PLNP-PMB of a mixture of 300 mu L (100 mu L each of three types of nanoparticles) into the experimental group, and performing magnetic separation (5 min) after the experimental group and the control group are subjected to action for 5min;
2. Sucking out supernatant, reserving, adding sediment into H 2 O with the same volume as the supernatant, detecting the phosphorescence intensity (time resolution) of a magnetic attraction sedimentation group and a magnetic attraction supernatant group by an enzyme label instrument, calculating an intensity ratio, typing gram-positive bacteria by using the difference of green afterglow (excitation: 250nm, emission: 529 nm) and red afterglow (excitation: 250nm, emission: 697 nm) emission wavebands, diluting target bacteria to different concentrations, mixing with magnetic nanoparticles modified by canavalin A, red long afterglow nanoparticles modified by vancomycin and green long afterglow nanoparticles modified by polymyxin B, reacting for 5min, detecting the phosphorescence intensity (time resolution, delay time: 20 mu s) of the magnetic attraction sedimentation group and the magnetic attraction supernatant group by the enzyme label instrument, and calculating the intensity ratio to establish a standard curve of the intensity ratio and the concentration of the target analyte;
3. Mixing the clinical sample with the magnetic nano particles modified by the canavalin A, the red long afterglow nano particles modified by the vancomycin and the green long afterglow nano particles modified by the polymyxin B, reacting for 5min, detecting the phosphorescence intensities (time resolution, delay time: 20 mu s) of the magnetic precipitation group and the magnetic supernatant group by using an enzyme-labeled instrument through magnetic separation for 5min, calculating an intensity ratio, and comparing with a standard curve to obtain the concentration of the target detection object in the sample.
The invention provides a scheme for rapidly identifying and typing gram-positive and negative bacteria based on a long-afterglow nanomaterial, which mainly comprises green long-afterglow, red long-afterglow nanoparticles and magnetic nanoparticles, wherein the magnetic nanoparticles are modified by using canavanin A, the red long-afterglow nanoparticles are modified by using vancomycin and the green long-afterglow nanoparticles are modified by using polymyxin B, bacteria are captured by using the canavanin A, the magnetic nanoparticles are used for separation, the vancomycin and polymyxin B are used for identifying the gram-positive bacteria, and the polymyxin B is used for identifying the gram-negative bacteria through the difference of emission wave bands of the long-afterglow nanoparticles. Compared with the prior art, the invention saves time and has simple operation.
The foregoing embodiment numbers of the present invention are merely for the purpose of description, and do not represent the advantages or disadvantages of the embodiments.
The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention.

Claims (10)

1.一种纳米探针，其特征在于，包括磁性纳米材料和长余辉纳米材料，磁性纳米材料包括修饰有刀豆蛋白A的磁性纳米粒子，用于识别捕获及分离细菌，长余辉纳米材料包括表面修饰有多粘菌素B的绿色长余辉纳米粒子和表面修饰有万古霉素的红色长余辉纳米粒子，表面修饰有多粘菌素B的绿色长余辉纳米粒子和表面修饰有万古霉素的红色长余辉纳米粒子分别用于识别抗干扰信号输出。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, and 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 used to identify anti-interference signal output respectively. 2.如权利要求1所述一种纳米探针的制备方法，其特征在于：包括2. A method for preparing a nanoprobe according to claim 1, characterized in that: 修饰有刀豆蛋白A的磁性纳米粒子的制备：将磁性纳米粒子羧基化，然后活化羧基后与刀豆蛋白A偶联，得到修饰有刀豆蛋白A的磁性纳米粒子；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; 表面修饰有多粘菌素B的绿色长余辉纳米粒子的制备：将绿色长余辉纳米粒子经过羟基化、氨基化和羧基化后，最后将羧基化的绿色长余辉纳米粒子与多粘菌素B进行偶联，得到多粘菌素B修饰的绿色长余辉纳米粒子；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; 表面修饰有万古霉素的红色长余辉纳米粒子的制备：将红色长余辉纳米粒子经过羟基化和氨基化后，氨基化的红色长余辉纳米粒子与万古霉素进行偶联，得到万古霉素修饰的红色长余辉纳米粒子。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.如权利要求2所述一种纳米探针的制备方法，其特征在于：修饰有刀豆蛋白A的磁性纳米粒子的制备方法包括以下步骤：3. A method for preparing a nanoprobe according to claim 2, characterized in that the method for preparing magnetic nanoparticles modified with concanavalin A comprises the following steps: 将磁性纳米材料分散到无水柠檬酸缓冲液中超声，搅拌，然后用去离子水洗涤，得到羧基化的磁性纳米粒子；然后将羧基化的磁性纳米粒子溶解在2-(N-吗啉基)乙磺酸缓冲液中，加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺、N-羟基琥珀酰亚胺磺酸钠盐，反应活化羧基，用PBS洗涤后，将活化的磁性纳米粒子与刀豆蛋白A在含有氯化钙和氯化锰的PBS中混合，搅拌，磁性收集得到刀豆蛋白A修饰的磁性纳米粒子，用结合缓冲液洗涤后保存。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.如权利要求3所述一种纳米探针的制备方法，其特征在于：磁性纳米粒子的制备方法为：将氯化铁和柠檬酸三钠溶于乙二醇中，然后在搅拌下加入醋酸钠，将混合物搅拌后密封在聚四氟乙烯内衬的水热反应釜中加热，然后冷却；将黑色产物用乙醇和去离子水洗涤干燥后得到磁性纳米粒子。4. A method for preparing a nanoprobe as described in claim 3, characterized in that: the preparation method of magnetic nanoparticles is: dissolving ferric chloride and trisodium citrate in ethylene glycol, then adding sodium acetate under stirring, stirring the mixture and sealing it in a polytetrafluoroethylene-lined hydrothermal reactor for heating, and then cooling it; washing the black product with ethanol and deionized water and drying it to obtain magnetic nanoparticles. 5.如权利要求2所述一种纳米探针的制备方法，其特征在于：表面修饰有多粘菌素B的绿色长余辉纳米粒子的制备方法包括以下步骤：5. A method for preparing a nanoprobe as claimed in claim 2, characterized in that: the method for preparing green long-lasting nanoparticles with surface modified with polymyxin B comprises the following steps: 将绿色长余辉纳米粒子分散在氢氧化钠溶液中搅拌，去离子水洗涤，干燥后得到羟基化的绿色长余辉纳米粒子，将羟基化的绿色长余辉纳米粒子溶解在N,N-二甲基甲酰胺，电磁搅拌下加入3-氨丙基三乙氧基硅烷，置于油浴，电磁搅拌后离心分离，用N,N-二甲基甲酰胺和无水乙醇洗涤，干燥后得到氨基化的绿色长余辉纳米粒子，将氨基化的绿色长余辉纳米粒子溶解在N,N-二甲基甲酰胺，搅拌下依次加入丁二酸酐的N,N-二甲基甲酰胺溶液、4-二甲基氨基吡啶的N,N-二甲基甲酰胺溶液，继续搅拌后离心分离，无水乙醇洗涤干燥后得到羧基化的绿色长余辉纳米粒子，将羧基化的绿色长余辉纳米粒子溶解在PBS缓冲液中，超声，加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺、N-羟基琥珀酰亚胺磺酸钠盐孵育，随后加入多粘菌素B，用碳酸氢钠调节pH，孵育后离心去除未结合的多粘菌素B，加入甲氧基聚乙二醇胺基封闭，得到多粘菌素B修饰的绿色长余辉纳米粒子。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.如权利要求5所述一种纳米探针的制备方法，其特征在于：绿色长余辉纳米粒子的制备方法为：将氧化锗粉末溶解在氢氧化钠溶液中，得到锗酸钠溶液，然后将硝酸锌、氯化锰和硝酸镨的水溶液在磁力搅拌下混合，加入硝酸溶液，得到前体物混合溶液；随后，在持续搅拌的状态下向前体物混合溶液中逐滴加入锗酸钠，将混合溶液的pH调节至碱性，进行超声，再置于磁力搅拌器上搅拌，将所得溶液置于高压反应釜中水热反应，冷却离心后将沉淀用乙醇洗涤，干燥后即得绿色长余辉纳米粒子。6. A method for preparing a nanoprobe as described in claim 5, characterized in that: the method for preparing green long afterglow nanoparticles is: dissolving germanium oxide powder in sodium hydroxide solution to obtain sodium germanate solution, then mixing aqueous solutions of zinc nitrate, manganese chloride and praseodymium nitrate under magnetic stirring, adding nitric acid solution to obtain a precursor mixed solution; subsequently, adding sodium germanate dropwise to the precursor mixed solution under continuous stirring, adjusting the pH of the mixed solution to alkaline, performing ultrasound, and then placing it on a magnetic stirrer for stirring, placing the resulting solution in a high-pressure reactor for hydrothermal reaction, cooling and centrifuging, washing the precipitate with ethanol, and drying to obtain green long afterglow nanoparticles. 7.如权利要求2所述一种纳米探针的制备方法，其特征在于：表面修饰有万古霉素的红色长余辉纳米粒子的制备方法包括：将红色长余辉纳米粒子分散在氢氧化钠溶液中搅拌，去离子水洗涤干燥后得到羟基化的红色长余辉纳米粒子，将羟基化的红色长余辉纳米粒子溶解在N,N-二甲基甲酰胺，电磁搅拌下加入3-氨丙基三乙氧基硅烷，置于油浴，电磁搅拌后离心分离，用N,N-二甲基甲酰胺和无水乙醇洗涤，干燥后得到氨基化的红色长余辉纳米粒子，将万古霉素溶于含有EDC和NHS的2-(N-吗啉基)乙磺酸缓冲液中；然后将氨基化的红色长余辉纳米粒子加入上述混合物中并再搅拌，将获得的纳米粒子离心清洗，加入甲氧基聚乙二醇胺基封闭，得到万古霉素修饰的红色长余辉纳米粒子。7. A method for preparing a nanoprobe as claimed in claim 2, characterized in that: the method for preparing 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, and 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 groups to seal to obtain vancomycin modified red long afterglow nanoparticles. 8.如权利要求7所述一种纳米探针的制备方法，其特征在于：红色长余辉纳米粒子的制备方法为：将硝酸锌、硝酸镓、硝酸铬混合在一起搅拌；加入浓缩氢氧化铵溶液调整pH，形成白色沉淀，再继续搅拌，将混合物置于反应釜中高温高压反应，然后自然冷却到室温，离心后得到的白色沉淀物分散在盐酸中，形成透明溶液，然后用异丙醇混合洗涤，干燥后即得红色长余辉纳米粒子。8. A method for preparing a nanoprobe as described in claim 7, characterized in that: the method for preparing red long afterglow nanoparticles is: mix zinc nitrate, gallium nitrate, and chromium nitrate together and stir; add concentrated ammonium hydroxide solution to adjust the pH to form a white precipitate, and continue stirring, place the mixture in a reactor for high temperature and high pressure reaction, and then naturally cool to room temperature, disperse the white precipitate obtained after centrifugation in hydrochloric acid to form a transparent solution, and then mix and wash with isopropanol, and dry to obtain red long afterglow nanoparticles. 9.如权利要求1所述一种纳米探针或如权利要求2-8任一项所述一种纳米探针的制备方法在鉴定革兰阴性阳性细菌中的应用。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.如权利要求9所述应用，其特征在于：鉴定革兰阴性阳性细菌的方法包括：将革兰阴性细菌和革兰阳性细菌混合在一起，与刀豆蛋白A修饰的磁性纳米粒子、万古霉素修饰的红色长余辉纳米粒子、多粘菌素B修饰的绿色长余辉纳米粒子混合反应后，通过磁分离，用酶标仪检测其磁吸沉淀组与磁吸上清组的磷光强度，并计算强度比值，利用绿色余辉与红色余辉发射波段的差异，对革兰阳性细菌和阴性细菌进行鉴定与定量检测；10. The use as claimed in claim 9, characterized in that: the method for identifying Gram-negative and Gram-positive bacteria comprises: 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, and 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; 将革兰阴性细菌和革兰阳性细菌的混合细菌稀释到不同浓度，与刀豆蛋白A修饰的磁性纳米粒子、万古霉素修饰的红色长余辉纳米粒子、多粘菌素B修饰的绿色长余辉纳米粒子混合反应后，通过磁分离，用酶标仪检测其磁吸沉淀组与磁吸上清组的磷光强度，并计算强度比值，建立强度比值与目标分析物浓度的标准曲线；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. 将采集样本与刀豆蛋白A修饰的磁性纳米粒子、万古霉素修饰的红色长余辉纳米粒子、多粘菌素B修饰的绿色长余辉纳米粒子混合反应，通过磁分离，用酶标仪检测其磁吸沉淀组与磁吸上清组的磷光强度，计算出强度比值，与标准曲线进行对比得到样本中目标检测物的浓度。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 an enzyme marker, and 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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