CN114907834B - Magnetic microcapsule for measuring vibrio parahaemolyticus and preparation method thereof - Google Patents

Magnetic microcapsule for measuring vibrio parahaemolyticus and preparation method thereof Download PDF

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CN114907834B
CN114907834B CN202210692355.6A CN202210692355A CN114907834B CN 114907834 B CN114907834 B CN 114907834B CN 202210692355 A CN202210692355 A CN 202210692355A CN 114907834 B CN114907834 B CN 114907834B
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李新章
戴任祥
张陈成
陶婷
丁超
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Nantong Kinghunt Biotechnology Development Co ltd
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Abstract

The invention belongs to the technical field of microcapsule preparation, and in particular relates to a magnetic microcapsule for measuring vibrio parahaemolyticus, which comprises porous ZIF-8 particles, magnetic CdS quantum dots dispersed in pore channels of the ZIF-8 particles, and a microcapsule coated on the magnetic CdS quantum dots/ZIF-8 nanoparticles; the preparation method of the magnetic microcapsule comprises the steps of firstly preparing magnetic CdS quantum dots, then preparing magnetic CdS quantum dots/ZIF-8 nano particles, and finally coating the obtained magnetic CdS quantum dots/ZIF-8 nano particles with the microcapsule to obtain the final magnetic microcapsule. The magnetic microcapsule obtained by the invention is nontoxic and harmless, is simple and easy to obtain in preparation, has quick detection aging, and improves the detection efficiency of vibrio parahaemolyticus.

Description

Magnetic microcapsule for measuring vibrio parahaemolyticus and preparation method thereof
Technical Field
The invention belongs to the technical field of microcapsule preparation, and particularly relates to a magnetic microcapsule for measuring vibrio parahaemolyticus and a preparation method thereof.
Background
Vibrio parahaemolyticus is gram-negative polymorphic bacillus or Vibrio parahaemolyticus, and Vibrio parahaemolyticus food poisoning is also called halophilic bacteria food poisoning, is caused by eating food containing the bacteria, clinically takes acute onset, abdominal pain, vomit, diarrhea and watery stool as main symptoms, and mainly causes pathological changes such as slight erosion of jejunum and ileum, gastric mucositis, viscera (liver, spleen, lung) congestion and the like. At present, the detection method of vibrio parahaemolyticus has the defects of molecular biological detection and immunological detection, but the molecular biological detection has the defects of complicated detection, long test period, unfavorable large-scale screening and the like, and the immunological detection mainly depends on imported detection products, has higher detection cost, is unfavorable for large-scale and rapid detection, and limits the application and development of the vibrio parahaemolyticus detection to a certain extent.
In order to meet market demands, the invention creatively designs the magnetic nano particles, combines the magnetic CdS quantum dots with the ZIF-8 nano particles, and coats the microcapsule to increase the specific surface area and magnetic adsorption capacity of the particles, and then uses the obtained magnetic microcapsule in combination with a PCR technology for measuring the vibrio parahaemolyticus, thereby simplifying the detection steps, remarkably improving the detection rate and accuracy, having low use cost and greatly meeting the requirements of quick detection rate, high detection precision and wide detection range of the vibrio parahaemolyticus required in the current market.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a magnetic microcapsule for measuring vibrio parahaemolyticus, which is convenient to apply, economical, practical, efficient and environment-friendly. In order to achieve the purpose of the invention, the following technical scheme is adopted:
a magnetic microcapsule for measuring vibrio parahaemolyticus comprises porous ZIF-8 particles, magnetic CdS quantum dots dispersed in pore channels of the ZIF-8 particles, and a microcapsule coated on the magnetic CdS quantum dots/ZIF-8 nanoparticles.
In order to achieve the above object, the present invention also provides a method for preparing a magnetic microcapsule for measuring vibrio parahaemolyticus, comprising the steps of:
(1) Preparing magnetic CdS quantum dots: dropwise adding thioglycollic acid into a cadmium chloride aqueous solution at a rotating speed of 40-80r/min, regulating the acidity of the solution to be pH1, dropwise adding sodium hydroxide until the acidity of the solution is pH2, then adding a sodium sulfide solution, continuously stirring for 1-2h, centrifugally collecting precipitate, washing 3-5 times with ethanol, and vacuum freeze-drying at the temperature of- (20-40) ℃ for 5-8h to prepare the magnetic CdS quantum dots for later use;
(2) Preparing magnetic CdS quantum dot/ZIF-8 nano particles: dispersing zinc acetate and the magnetic CdS quantum dots prepared in the step (1) in 2-methylimidazole aqueous solution in an ultrasonic manner, adding phosphate buffer aqueous solution containing polyimide with the concentration of 1-2% wt%, and stirring for 1-2h to prepare magnetic CdS quantum dots/ZIF-8 nano particles;
(3) Preparing magnetic CdS quantum dot/ZIF-8 nano particles coated with microcapsules: stirring chitosan, distilled water and glacial acetic acid at 30-60 ℃ for 0.5-1.5-h, regulating the acidity of the solution to be pH3, adding the magnetic CdS quantum dots/ZIF-8 nano particles obtained in the step (2), continuously stirring, adding a cross-linking agent, stirring for 1-3 h under ice bath to prepare microcapsule solution, and freeze-drying the microcapsule solution for 72-96h to prepare the magnetic CdS quantum dots/ZIF-8 nano particles coated with the microcapsule, thus obtaining the final magnetic microcapsule.
Preferably, in the step (1), the pH1 is 2-3, and the pH2 is 6-7; the pH3 in the step (3) is 6.8-7.5.
Preferably, the concentration of the cadmium chloride aqueous solution in the step (1) is 0.01-0.05mol/L, and the concentration of the sodium hydroxide is 1-2mol/L.
Preferably, the concentration of the sodium sulfide solution in the step (1) is 0.005-0.01 mol/L.
Preferably, the molar ratio of sodium sulfide to cadmium chloride in step (1) is 1: (1.3-1.8).
Preferably, the concentration of the 2-methylimidazole aqueous solution in the step (2) is 2-5 mol/L.
Preferably, in the step (2), the mass ratio of the zinc acetate to the magnetic CdS quantum dots is 1: (0.5-1).
Preferably, the cross-linking agent in step (3) is glutaraldehyde.
Compared with the prior art, the invention has the beneficial effects that: the invention carries out microcapsule coating on the magnetic CdS quantum dot/ZIF-8 nano particles, is used for extracting vibrio parahaemolyticus DNA, and combines the PCR combined technology to screen and determine the vibrio parahaemolyticus of aquatic products, and the technology is nontoxic and harmless, is simple and convenient to operate, has quick detection aging, and improves the detection efficiency of the vibrio parahaemolyticus; the design of the magnetic microcapsule fills the defects of complex, time-consuming and labor-consuming and expensive operation of the existing nucleic acid extraction, and the like, and finally the magnetic microcapsule which is high-efficiency, ecological and environment-friendly and applied to the determination of vibrio parahaemolyticus is obtained, is convenient for large-scale detection, and is beneficial to popularization, application and development.
Drawings
FIG. 1 is a schematic flow chart of a method for preparing a magnetic microcapsule for measuring Vibrio parahaemolyticus according to the invention;
FIG. 2 is an SEM image of a magnetic microcapsule for determination of Vibrio parahaemolyticus prepared according to the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The invention provides a magnetic microcapsule for measuring vibrio parahaemolyticus, which comprises porous ZIF-8 particles, magnetic CdS quantum dots dispersed in pore channels of the ZIF-8 particles, and a microcapsule coated on the magnetic CdS quantum dots/ZIF-8 nanoparticles.
The magnetic microcapsule of this embodiment is prepared by the following preparation method, and fig. 1 is a schematic flow chart of a preparation method of a magnetic microcapsule for measuring vibrio parahaemolyticus according to the invention, and it can be seen that the preparation method specifically comprises the following steps:
step S1: preparing magnetic CdS quantum dots: and (3) dropwise adding thioglycollic acid into a cadmium chloride aqueous solution at a rotating speed of 40r/min, regulating the pH value of the solution to 2, dropwise adding sodium hydroxide until the pH value of the solution is 6, adding a sodium sulfide solution, continuously stirring for 1h, centrifugally collecting precipitate, washing with ethanol for 3 times, and performing vacuum freeze-drying at-20 ℃ for 5h to obtain the magnetic CdS quantum dot for later use. In this step, the concentration of the aqueous solution of cadmium chloride was 0.01mol/L, the concentration of sodium hydroxide was 1mol/L, the concentration of the sodium sulfide solution was 0.005 mol/L, and the molar ratio of sodium sulfide to cadmium chloride was 1:1.3.
step S2: preparing magnetic CdS quantum dot/ZIF-8 nano particles: zinc acetate and the magnetic CdS quantum dots prepared in the step S1 are mixed according to the mass ratio of 1:0.5 of the magnetic CdS quantum dot/ZIF-8 nano particles are dispersed in 2mol/L of 2-methylimidazole aqueous solution by ultrasonic, phosphate buffer aqueous solution containing 1wt% of polyimide is added, and the mixture is stirred for 1h, so that the magnetic CdS quantum dot/ZIF-8 nano particles are prepared.
Step S3: preparing magnetic CdS quantum dot/ZIF-8 nano particles coated with microcapsules: stirring chitosan, distilled water and glacial acetic acid at 30 ℃ for 1.5 and h, regulating the pH of the solution to 6.8, adding the magnetic CdS quantum dots/ZIF-8 nano particles obtained in the step S2, continuously stirring, adding 4 drops of cross-linking agent, namely glutaraldehyde, stirring for 1h in an ice bath to prepare microcapsule solution, and freeze-drying the microcapsule solution at-20 ℃ for 72 hours to prepare the magnetic CdS quantum dots/ZIF-8 nano particles coated with the microcapsule, thus obtaining the final magnetic microcapsule.
When the method is applied, the prepared magnetic microcapsule is combined with a PCR technology to be used for measuring the vibrio parahaemolyticus, and the method specifically comprises the following steps of:
firstly, respectively taking 150 mu L of phosphate buffer aqueous solution and 300 mu L of isopropanol in a 2mL centrifuge tube, adding 0.5g of magnetic microcapsule powder, uniformly oscillating, and then adjusting the pH value to 9.0 to respectively prepare two groups of reagents; then adding the purified vibrio parahaemolyticus DNA solution in the aquatic sample into a centrifuge tube, placing the centrifuge tube into a magnetic rack, standing for 1 min, and removing the liquid in the centrifuge tube after the solution is clear; next, 600 μl of rinse solution and protein solution were added, respectively, and the liquid in the tube was removed one by one; finally, 100 mu L of eluent is added, evenly mixed, incubated for 5min at 65 ℃, and supernatant fluid is removed, so that vibrio parahaemolyticus DNA adsorbed by the magnetic microcapsule is obtained, and the DNA is measured by a fluorescence analyzer.
Example 2
The invention provides a magnetic microcapsule for measuring vibrio parahaemolyticus, which comprises porous ZIF-8 particles, magnetic CdS quantum dots dispersed in pore channels of the ZIF-8 particles, and a microcapsule coated on the magnetic CdS quantum dots/ZIF-8 nanoparticles.
The magnetic microcapsule of this embodiment is prepared by the following preparation method, and fig. 1 is a schematic flow chart of a preparation method of a magnetic microcapsule for measuring vibrio parahaemolyticus according to the invention, and it can be seen that the preparation method specifically comprises the following steps:
step S1: preparing magnetic CdS quantum dots: and (3) dropwise adding thioglycollic acid into a cadmium chloride aqueous solution at a rotating speed of 80r/min, regulating the pH value of the solution to 3, dropwise adding sodium hydroxide until the pH value of the solution is 7, adding a sodium sulfide solution, continuously stirring for 2 hours, centrifugally collecting precipitate, washing with ethanol for 5 times, and performing vacuum freeze-drying at-40 ℃ for 8 hours to obtain the magnetic CdS quantum dot for later use. In this step, the concentration of the aqueous solution of cadmium chloride was 0.05mol/L, the concentration of sodium hydroxide was 2mol/L, the concentration of the sodium sulfide solution was 0.01mol/L, and the molar ratio of sodium sulfide to cadmium chloride was 1:1.8.
step S2: preparing magnetic CdS quantum dot/ZIF-8 nano particles: zinc acetate and the magnetic CdS quantum dots prepared in the step S1 are mixed according to the mass ratio of 1:1, dispersing the magnetic CdS quantum dot/ZIF-8 nano particles in a 5 mol/L2-methylimidazole aqueous solution by ultrasonic, adding a phosphate buffer aqueous solution containing 2. 2 wt% of polyimide, and stirring for 2 hours.
Step S3: preparing magnetic CdS quantum dot/ZIF-8 nano particles coated with microcapsules: stirring chitosan, distilled water and glacial acetic acid at 60 ℃ for 0.5 and h, regulating the pH of the solution to 7.5, adding the magnetic CdS quantum dots/ZIF-8 nano particles obtained in the step S2, continuously stirring, adding 6 drops of cross-linking agent, namely glutaraldehyde, stirring for 3 h in an ice bath to prepare a microcapsule solution, and freeze-drying the microcapsule solution at-40 ℃ for 96 hours to prepare the magnetic CdS quantum dots/ZIF-8 nano particles coated with the microcapsule, thus obtaining the final magnetic microcapsule.
When the method is applied, the prepared magnetic microcapsule is combined with a PCR technology to be used for measuring the vibrio parahaemolyticus, and the method specifically comprises the following steps of:
firstly, respectively taking 150 mu L of phosphate buffer aqueous solution and 300 mu L of isopropanol in a 2mL centrifuge tube, adding 0.5g of magnetic microcapsule powder, uniformly oscillating, and then adjusting the pH value to 9.0 to respectively prepare two groups of reagents; then adding the vibrio parahaemolyticus DNA solution in the purified aquatic sample into a centrifuge tube, placing the centrifuge tube into a magnetic rack, standing for 2 min, and removing the liquid in the centrifuge tube after the solution is clear; next, 600 μl of rinse solution and protein solution were added, respectively, and the liquid in the tube was removed one by one; finally, 100 mu L of eluent is added, evenly mixed, incubated for 5min at 65 ℃, and supernatant fluid is removed, so that vibrio parahaemolyticus DNA adsorbed by the magnetic microcapsule is obtained, and the DNA is measured by a fluorescence analyzer.
Example 3
The invention provides a magnetic microcapsule for measuring vibrio parahaemolyticus, which comprises porous ZIF-8 particles, magnetic CdS quantum dots dispersed in pore channels of the ZIF-8 particles, and a microcapsule coated on the magnetic CdS quantum dots/ZIF-8 nanoparticles.
The magnetic microcapsule of this embodiment is prepared by the following preparation method, and fig. 1 is a schematic flow chart of a preparation method of a magnetic microcapsule for measuring vibrio parahaemolyticus according to the invention, and it can be seen that the preparation method specifically comprises the following steps:
step S1: preparing magnetic CdS quantum dots: and (3) dropwise adding thioglycollic acid into a cadmium chloride aqueous solution at a rotating speed of 60r/min, regulating the pH value of the solution to 2.5, dropwise adding sodium hydroxide until the pH value of the solution is 6.5, then adding a sodium sulfide solution, continuously stirring for 1h, centrifugally collecting precipitate, washing with ethanol for 4 times, and performing vacuum freeze-drying at-30 ℃ for 6h to obtain the magnetic CdS quantum dots for later use. In this step, the concentration of the aqueous solution of cadmium chloride was 0.03mol/L, the concentration of sodium hydroxide was 1mol/L, the concentration of the sodium sulfide solution was 0.005 mol/L, and the molar ratio of sodium sulfide to cadmium chloride was 1:1.5.
step S2: preparing magnetic CdS quantum dot/ZIF-8 nano particles: zinc acetate and the magnetic CdS quantum dots prepared in the step S1 are mixed according to the mass ratio of 1:0.8 of the magnetic CdS quantum dot/ZIF-8 nano particles are dispersed in 3mol/L of 2-methylimidazole aqueous solution by ultrasonic, phosphate buffer aqueous solution containing 2 wt% of polyimide is added, and the mixture is stirred for 2 hours to prepare the magnetic CdS quantum dot/ZIF-8 nano particles.
Step S3: preparing magnetic CdS quantum dot/ZIF-8 nano particles coated with microcapsules: stirring chitosan, distilled water and glacial acetic acid at 40 ℃ for 1h, regulating the pH of the solution to 7.4, adding the magnetic CdS quantum dots/ZIF-8 nano particles obtained in the step S2, continuously stirring, adding 5 drops of cross-linking agent, namely glutaraldehyde, stirring for 2h under ice bath to prepare microcapsule solution, and freeze-drying the microcapsule solution at-30 ℃ for 82 hours to prepare the magnetic CdS quantum dots/ZIF-8 nano particles coated with the microcapsule, thus obtaining the final magnetic microcapsule.
When the method is applied, the prepared magnetic microcapsule is combined with a PCR technology to be used for measuring the vibrio parahaemolyticus, and the method specifically comprises the following steps of:
firstly, respectively taking 150 mu L of phosphate buffer aqueous solution and 300 mu L of isopropanol in a 2mL centrifuge tube, adding 0.5g of magnetic microcapsule powder, uniformly oscillating, and then adjusting the pH value to 9.0 to respectively prepare two groups of reagents; then adding the vibrio parahaemolyticus DNA solution in the purified aquatic sample into a centrifuge tube, placing the centrifuge tube into a magnetic rack, standing for 2 min, and removing the liquid in the centrifuge tube after the solution is clear; next, 600 μl of rinse solution and protein solution were added, respectively, and the liquid in the tube was removed one by one; finally, 100 mu L of eluent is added, evenly mixed, incubated for 5min at 65 ℃, and supernatant fluid is removed, so that vibrio parahaemolyticus DNA adsorbed by the magnetic microcapsule is obtained, and the DNA is measured by a fluorescence analyzer.
The detection results of the prepared magnetic CdS quantum dot/ZIF-8 nanoparticle coated with the microcapsule in vibrio parahaemolyticus in different aquatic samples are shown in Table 1:
table 1 shows the results of detection of Vibrio parahaemolyticus in different aquatic samples
Sample species Number of samples Number of detected Detection rate% Time consuming h
Prawn (prawn) 40 6 15.0 5.9
Oyster shell 40 11 27.5 6.5
Mussel 40 13 32.5 6.2
According to the embodiment of the invention, the magnetic CdS quantum dot/ZIF-8 nanoparticle coated by the microcapsule is prepared, the particle size is uniform, the dispersion is good (as shown in figure 2), the magnetic microcapsule is combined with the PCR combined technology in application, the detection rate of different aquatic products is good, the operation is simple and convenient, the detection aging is quick, and the detection efficiency of the vibrio parahaemolyticus is improved. The invention fills the defects of complex, time-consuming and labor-consuming and expensive operation of the existing nucleic acid extraction, and finally obtains the magnetic microcapsule which is high-efficiency, ecological and environment-friendly and is applied to the determination of vibrio parahaemolyticus.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any modifications and substitutions made by those skilled in the art are deemed to fall within the scope of the present invention, which is defined by the appended claims.

Claims (9)

1. A magnetic microcapsule for use in the determination of vibrio parahaemolyticus, characterized in that: comprises porous ZIF-8 particles, magnetic CdS quantum dots dispersed in pores of the ZIF-8 particles, and microcapsules coated on the magnetic CdS quantum dots/ZIF-8 nanoparticles.
2. A preparation method of a magnetic microcapsule for measuring vibrio parahaemolyticus is characterized by comprising the following steps of: the method comprises the following steps:
(1) Preparing magnetic CdS quantum dots: dropwise adding thioglycollic acid into a cadmium chloride aqueous solution at a rotating speed of 40-80r/min, regulating the acidity of the solution to be pH1, dropwise adding sodium hydroxide until the acidity of the solution is pH2, then adding a sodium sulfide solution, continuously stirring for 1-2h, centrifugally collecting precipitate, washing 3-5 times with ethanol, and vacuum freeze-drying at the temperature of- (20-40) ℃ for 5-8h to prepare the magnetic CdS quantum dots for later use;
(2) Preparing magnetic CdS quantum dot/ZIF-8 nano particles: dispersing zinc acetate and the magnetic CdS quantum dots prepared in the step (1) in 2-methylimidazole aqueous solution in an ultrasonic manner, adding phosphate buffer aqueous solution containing polyimide with the concentration of 1-2% wt%, and stirring for 1-2h to prepare magnetic CdS quantum dots/ZIF-8 nano particles;
(3) Preparing magnetic CdS quantum dot/ZIF-8 nano particles coated with microcapsules: stirring chitosan, distilled water and glacial acetic acid at 30-60 ℃ for 0.5-1.5-h, regulating the acidity of the solution to be pH3, adding the magnetic CdS quantum dots/ZIF-8 nano particles obtained in the step (2), continuously stirring, adding a cross-linking agent, stirring for 1-3 h under ice bath to prepare microcapsule solution, and freeze-drying the microcapsule solution for 72-96h to prepare the magnetic CdS quantum dots/ZIF-8 nano particles coated with the microcapsule, thus obtaining the final magnetic microcapsule.
3. The method for preparing a magnetic microcapsule for vibrio parahaemolyticus assay according to claim 2, wherein: the pH1 in the step (1) is 2-3, and the pH2 is 6-7; the pH3 in the step (3) is 6.8-7.5.
4. A method for preparing a magnetic microcapsule for vibrio parahaemolyticus assay according to claim 2 or 3, characterized in that: the concentration of the cadmium chloride aqueous solution in the step (1) is 0.01-0.05mol/L, and the concentration of sodium hydroxide is 1-2mol/L.
5. The method for preparing a magnetic microcapsule for vibrio parahaemolyticus assay according to claim 4, wherein: the concentration of the sodium sulfide solution in the step (1) is 0.005-0.01 mol/L.
6. The method for preparing a magnetic microcapsule for vibrio parahaemolyticus assay according to claim 5, wherein: the molar ratio of the sodium sulfide to the cadmium chloride in the step (1) is 1: (1.3-1.8).
7. The method for preparing a magnetic microcapsule for vibrio parahaemolyticus assay according to claim 2, wherein: the concentration of the 2-methylimidazole aqueous solution in the step (2) is 2-5 mol/L.
8. The method for producing a magnetic microcapsule for vibrio parahaemolyticus assay according to claim 2 or 7, characterized in that: the mass ratio of the zinc acetate to the magnetic CdS quantum dots in the step (2) is 1: (0.5-1).
9. The method for preparing a magnetic microcapsule for vibrio parahaemolyticus assay according to claim 2, wherein: the cross-linking agent in the step (3) is glutaraldehyde.
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