CN116539869B - Structural color coded magnetic microcarrier, preparation method thereof and gibberellin detection method - Google Patents
Structural color coded magnetic microcarrier, preparation method thereof and gibberellin detection method Download PDFInfo
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- 229930191978 Gibberellin Natural products 0.000 title claims abstract description 136
- 239000003448 gibberellin Substances 0.000 title claims abstract description 136
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- 238000001514 detection method Methods 0.000 title claims abstract description 33
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- 235000019441 ethanol Nutrition 0.000 description 4
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 4
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
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- -1 diterpenoid compound Chemical class 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
- G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/20—Magnetic particle immunoreagent carriers the magnetic material being present in the particle core
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to the field of biological materials, and discloses a structural color coding magnetic microcarrier, a preparation method thereof and a gibberellin detection method. The structural color coded magnetic microcarrier comprises a photonic crystal microsphere and gibberellin antibody grafted on the surface of the photonic crystal microsphere, wherein the photonic crystal microsphere is formed by self-assembly of core-shell structure nano particles, and the core-shell structure nano particles comprise Fe 3 O 4 Inner core formed by magnetic nano particles and SiO wrapped around outer periphery of inner core 2 A nano layer; the preparation method comprises the following steps: fe is added to 3 O 4 SiO is wrapped around the periphery of the magnetic nano particles 2 The nano layer is used for obtaining core-shell structure nano particles, and the core-shell structure nano particles are self-assembled to form photonic crystal microspheres; and (3) after the photonic crystal microsphere is activated, mixing the photonic crystal microsphere with gibberellin antibody to perform reaction I, and mixing the photonic crystal microsphere with a blocking agent to perform reaction II, so that gibberellin antibody is grafted on the surface of the photonic crystal microsphere. The microcarrier can specifically recognize gibberellin, can enhance the sensitivity and accuracy of gibberellin detection, and has high detection efficiency.
Description
Technical Field
The invention relates to a biological material technology, in particular to a structural color coding magnetic microcarrier, a preparation method thereof and a gibberellin detection method.
Background
Gibberellin (GA) is a diterpenoid compound that is widely distributed among angiosperms, gymnosperms, bacteria, and various fungi. There are also many kinds of gibberellins, and up to now, hundreds of gibberellins have been isolated and extracted from plants, fungi and bacteria. Although the structures of these gibberellins are quite similar, only a few gibberellins can exhibit biological activity in plants. In the plant body, gibberellin is used as a plant growth regulator for regulating the processes of plant growth, germination, flowering, fruiting and the like, the content of gibberellin in the plant body is very important, and the plant growth and development can be promoted only when the gibberellin concentration is proper; when the gibberellin concentration is too high, plant bakanae disease is caused, so that plants grow crazy, roots and stems become slender, the plants are difficult to bear the weight of the plants, and the plants are lodged and difficult to fruiting. In addition, when the human body ingests a lot of gibberellin remained on vegetables and fruits, the endocrine system of the human body is disturbed, and chronic organ poisoning and even cancer are caused. Therefore, a GA detection method with high sensitivity and specificity is required to monitor plant growth environment and food safety problems.
To date, researchers have established a variety of GA detection strategies including high performance liquid chromatography, GAs chromatography-mass spectrometry, liquid chromatography-electrospray tandem mass spectrometry, and the like; however, these methods require complex and time-consuming extraction and separation of the sample, involving expensive detection equipment. Therefore, there is a need to develop a novel rapid, efficient and accurate-result GA detection platform.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a structural color coding magnetic microcarrier, a preparation method thereof and a method for detecting gibberellin, wherein the microcarrier can specifically identify gibberellin, can enhance the sensitivity and accuracy of gibberellin detection, and has high detection efficiency.
In order to achieve the above object, a first aspect of the present invention provides a structural color-coded magnetic microcarrier comprising a photonic crystal microsphere and gibberellin antibody grafted on the surface of the photonic crystal microsphere, wherein the photonic crystal microsphere is formed by self-assembly of core-shell nanoparticles, and the core-shell nanoparticles comprise Fe 3 O 4 Inner core formed by magnetic nano particlesSiO wrapping the periphery of the inner core 2 A nano layer.
Preferably, the Fe 3 O 4 The magnetic nano particles are Fe modified by poly (4-styrenesulfonic acid-co-maleic acid) sodium salt 3 O 4 And (3) nanoparticles.
Preferably, the Fe 3 O 4 Magnetic nanoparticles and SiO 2 The weight ratio of the nano layer is 1:5-10, wherein the weight ratio of the photonic crystal microsphere to the gibberellin antibody is 2-4:1.
the second aspect of the present invention provides a method for preparing a structural color-coded magnetic microcarrier, comprising the steps of:
(1) Fe is added to 3 O 4 SiO is wrapped around the periphery of the magnetic nano particles 2 The nano layer is used for obtaining core-shell structure nano particles, and the core-shell structure nano particles are self-assembled to form photonic crystal microspheres;
(2) And (3) after the photonic crystal microsphere is activated, mixing the photonic crystal microsphere with gibberellin antibody to perform reaction I, and mixing the photonic crystal microsphere with a blocking agent to perform reaction II, so that gibberellin antibody is grafted on the surface of the photonic crystal microsphere.
Preferably, fe is added in step (1) 3 O 4 SiO is wrapped around the periphery of the magnetic nano particles 2 The process of the nano layer comprises the following steps: by Stober method in the Fe 3 O 4 SiO formation on the periphery of magnetic nanoparticles 2 A nano layer.
Preferably, the Stober method forms SiO 2 The conditions of the nanolayer include: the temperature is 45-55 ℃.
Preferably, the Fe 3 O 4 The magnetic nano particles are Fe modified by poly (4-styrenesulfonic acid-co-maleic acid) sodium salt 3 O 4 A nanoparticle; the Fe is 3 O 4 Magnetic nanoparticles and SiO 2 The weight ratio of the nano layer is 1:5-10.
Preferably, the self-assembly process in step (1) comprises: forming microfluidic liquid drops by taking a solution containing the core-shell structure nano particles as an internal phase and n-hexadecane as an external phase, solidifying the microfluidic liquid drops to form primary microspheres, and calcining the primary microspheres.
Preferably, the microfluidic droplet is formed using a microfluidic device.
Preferably, the flow rate of the inner phase in the microfluidic device is 0.3-0.5mL/h and the flow rate of the outer phase is 2-5mL/h.
Preferably, the solution containing the core-shell structure nano particles adopts ultrapure water as a solvent, and the concentration of the core-shell structure nano particles in the solution is 200-300mg/mL.
Preferably, the curing conditions include: the temperature is 70-85 ℃ and the time is 8-15h.
Preferably, the calcination process comprises: washing and drying the primary microspheres by normal hexane, and calcining at the temperature of 750-850 ℃.
Preferably, the activating in step (2) comprises: and mixing and activating the photonic crystal microsphere and 3-aminopropyl triethoxysilane.
The conditions of the mixed activation include: the temperature is 5-40deg.C, and the time is 25-35min.
Preferably, the gibberellin antibody is a GA3 antibody conjugated with N-hydroxysuccinimide and (N-ethyl-N' -3-dimethylaminopropyl) carbodiimide hydrochloride.
Preferably, the weight ratio of the photonic crystal microsphere, the 3-aminopropyl triethoxysilane and the gibberellin antibody is 2-4:2-4:1.
preferably, the conditions of reaction I include: the temperature is 5-40deg.C, and the time is 50-70min.
Preferably, the blocker in step (2) is bovine serum albumin and the weight ratio of gibberellin antibody to blocker is 1:1.5-2.5.
Preferably, the conditions of reaction II include: the temperature is 5-40deg.C, and the time is 50-70min.
The third aspect of the invention provides the magnetic microcarrier and the application of the magnetic microcarrier prepared by the method in gibberellin detection.
In a fourth aspect, the present invention provides a method for detecting gibberellin, the method comprising the steps of:
s1, respectively mixing gibberellin standard solutions with different concentrations with the magnetic microcarrier according to claim 1 or 2 and/or the magnetic microcarrier prepared by the method according to any one of claims 3 to 8 for reaction III, and detecting reflection peak offset values of the magnetic microcarrier before and after the reaction III;
s2, establishing a relation curve equation between the reflection peak offset value and the concentration of the gibberellin standard solution;
s3, mixing the solution to be detected with the magnetic microcarrier to carry out a reaction III, detecting reflection peak offset values of the magnetic microcarrier before and after the reaction III, and then calculating the content of gibberellin in the solution to be detected according to the relation curve equation.
Preferably, the conditions of reaction III include: the temperature is 0-40 ℃ and the time is 1.5-2.5h.
Preferably, the relation equation is y= -7.83 x+78.90, where y is the offset value of the reflection peak, and x is the negative logarithmic value of the gibberellin standard solution concentration.
Through the technical scheme, the invention has the beneficial effects that:
according to the structural color coding magnetic microcarrier provided by the invention, based on photonic crystal microspheres formed by core-shell structure nano-particles, an antigen-antibody combination method is introduced, gibberellin antibody is grafted on the microspheres, so that the constructed microcarrier can specifically identify gibberellin, has higher specific surface area and porosity, can provide more abundant molecular binding sites, and enhances the specificity and accuracy of gibberellin detection; furthermore, the structural color coding magnetic microcarrier provided by the invention is constructed into a movable enriched liquid phase chip to capture gibberellin molecules and carry out content measurement, so that the structural color coding magnetic microcarrier can be fully contacted with gibberellin in a solution to be detected, the reaction time of gibberellin antibody and gibberellin is greatly shortened, and the detection efficiency of gibberellin is effectively improved.
Compared with the traditional gibberellin detection method, the gibberellin detection method provided by the invention has the advantages that the flow is simple, the change of structural color and characteristic reflection peak is utilized, the gibberellin can be observed by naked eyes, and expensive instruments are not needed; further combining with the offset value of the reflection peak, the quantitative determination of gibberellin can be realized, and the detection process is rapid and efficient.
Drawings
FIG. 1 is a schematic diagram of a method for preparing a structural color-coded magnetic microcarrier and a flow chart for detecting gibberellin;
FIG. 2 shows the structural color of the aqueous dispersion of core-shell nanoparticles of example 1 and example 2 by magnetic attraction, wherein a is the nanoparticle of 270nm in example 1 and b is the nanoparticle of 300nm in example 1;
FIG. 3 is an electron microscope image of the photonic crystal microsphere of example 1, wherein a is an external structure and b is an internal structure;
FIG. 4 is a photomicrograph of the photonic crystal microsphere, the structural color-coded magnetic microcarrier and the magnetic microcarrier after gibberellin molecule adsorption in example 1, wherein a is the photonic crystal microsphere, b is the structural color-coded magnetic microcarrier, and c is the magnetic microcarrier after gibberellin molecule adsorption;
FIG. 5 is a standard curve of gibberellin concentration detection for the structural color-coded magnetic microcarrier obtained in example 1.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In a first aspect, the present invention provides a structural color-coded magnetic microcarrier, which comprises a photonic crystal microsphere and gibberellin antibody grafted on the surface of the photonic crystal microsphere, wherein the photonic crystal microsphere is formed by self-assembly of core-shell structure nanoparticles, and the core-shell structure nanoparticles comprise Fe 3 O 4 Inner core formed by magnetic nano particles and wrapping the periphery of the inner coreSiO 2 A nano layer.
The structural color coding magnetic microcarrier provided by the invention is based on photonic crystal microspheres (belonging to positive structural photonic crystal microspheres) formed by core-shell structural nanoparticles, and an antigen-antibody combination method is introduced, gibberellin antibody is grafted on the microspheres, so that the constructed microcarrier can specifically identify gibberellin, has higher specific surface area and porosity, can provide richer molecular binding sites, and enhances the specificity and accuracy of gibberellin detection; furthermore, the structural color coding magnetic microcarrier provided by the invention is constructed into a movable enriched liquid phase chip to capture gibberellin molecules and carry out content measurement, so that the structural color coding magnetic microcarrier can be fully contacted with gibberellin in a solution to be detected, the reaction time of gibberellin antibody and gibberellin is greatly shortened, and the detection efficiency of gibberellin is effectively improved.
According to the present invention, preferably, the Fe 3 O 4 The magnetic nano particles are Fe modified by poly (4-styrenesulfonic acid-co-maleic acid) sodium salt (PSSMA for short) 3 O 4 And (3) nanoparticles. The inventors found that under this preferred embodiment, fe 3 O 4 The particles of the magnetic nano particles are more uniform, the specific surface area and the porosity of the photonic crystal microsphere are improved, the stability of the structural color coding magnetic microcarrier is further improved, and the better color development effect is achieved.
In the invention, fe modified by sodium salt (PSSMA) of poly (4-styrenesulfonic acid-co-maleic acid) 3 O 4 Nanoparticles are commercially available or can be prepared by themselves according to existing preparation methods, for example, chemical high temperature hydrothermal methods can be used.
Exemplary PSSMA modified Fe 3 O 4 The preparation process of the nano particle comprises the following steps: adding 0.2-0.3g ferric chloride, 1-1.5g sodium acetate, 0.3-0.5g poly (4-styrenesulfonic acid-co-maleic acid) sodium (PSSMA), 20-70 mu L water and 0.004-0.0048g vitamin C into 10-20mL of ethylene glycol, and stirring until the solution is transparent yellow; adding 0.2-0.3g sodium hydroxide, stirring until the solution is black transparent solution, transferring into autoclave, placing into muffle furnace, calcining for 8-10 hr, and adding wineFine: the water=1:1 mixed solution and pure water are respectively washed for three times to obtain PSSMA modified Fe 3 O 4 The nanoparticle was dissolved in 12mL of pure water and sealed.
According to the present invention, preferably, the Fe 3 O 4 Magnetic nanoparticles and SiO 2 The weight ratio of the nano layer is 1:5-10, wherein the weight ratio of the photonic crystal microsphere to the gibberellin antibody is 2-4:1. the inventors found that in this preferred embodiment, it is advantageous to further improve the stability of the structural color-coded magnetic microcarrier and the detection efficiency of gibberellin.
In the invention, the process of forming the photonic crystal microsphere by self-assembly of the core-shell structure nano-particles preferably adopts a microfluidic technology, and the process specifically comprises the following steps: forming microfluidic liquid drops by taking a solution containing the core-shell structure nano particles as an internal phase and n-hexadecane as an external phase, solidifying the microfluidic liquid drops to form primary microspheres, and calcining the primary microspheres. The inventor finds that under the preferred embodiment, the core-shell structure nano particles can be quickly and efficiently formed into the photonic crystal microsphere with the positive structure by adopting a microfluidic technology.
The second aspect of the present invention provides a method for preparing a structural color-coded magnetic microcarrier, comprising the steps of:
(1) Fe is added to 3 O 4 SiO is wrapped around the periphery of the magnetic nano particles 2 The nano layer is used for obtaining core-shell structure nano particles, and the core-shell structure nano particles are self-assembled to form photonic crystal microspheres;
(2) And (3) after the photonic crystal microsphere is activated, mixing the photonic crystal microsphere with gibberellin antibody to perform reaction I, and mixing the photonic crystal microsphere with a blocking agent to perform reaction II, so that gibberellin antibody is grafted on the surface of the photonic crystal microsphere.
According to the present invention, preferably, fe is added in the step (1) 3 O 4 SiO is wrapped around the periphery of the magnetic nano particles 2 The process of the nano layer comprises the following steps: by Stober method in the Fe 3 O 4 SiO formation on the periphery of magnetic nanoparticles 2 A nano layer.
According to the invention, preference is given toThe Stober method forms SiO 2 The conditions of the nanolayer include: the temperature is 45-55 ℃. The inventors found that in this preferred embodiment, siO is favored 2 Nanolayer pair Fe 3 O 4 The magnetic nano particles are better wrapped, and the core-shell structure nano particles have better structural stability, so that the detection stability and the sensitivity of the structural color coding magnetic microcarrier are improved.
In the present invention, the Stober method is a physicochemical method for synthesizing monodisperse silicon particles, and generally refers to a method for generating nano silicon dioxide particles by adding Tetraethoxysilane (TEOS) to ethanol and aqueous ammonia. Illustratively, the core-shell nanoparticle may be prepared by: taking Fe 3 O 4 Adding magnetic nanoparticles into 30-50mL of absolute ethanol, adding 1-3mL of ammonia water (NH) 3 The concentration is 20-25wt%) ultrasonic for 4-6min, then transferring into a three-neck flask, water-bathing at 45-55deg.C, stirring for 8-12min, then adding 150-250 μl tetraethyl orthosilicate (TEOS) every 15-25min, adding three times, stirring for 0.8-1.2 hr to obtain reaction solution; and (3) centrifuging the reaction solution to obtain solid particles, respectively cleaning the solid particles with absolute ethyl alcohol and pure water for three times, and drying to obtain the core-shell structure nano particles.
According to the present invention, preferably, the Fe 3 O 4 The magnetic nano particles are Fe modified by poly (4-styrenesulfonic acid-co-maleic acid) sodium salt 3 O 4 A nanoparticle; the Fe is 3 O 4 Magnetic nanoparticles and SiO 2 The weight ratio of the nano layer is 1:5-10.
According to the present invention, preferably, the self-assembly process in step (1) includes: forming microfluidic liquid drops by taking a solution containing the core-shell structure nano particles as an internal phase and n-hexadecane as an external phase, solidifying the microfluidic liquid drops to form primary microspheres, and calcining the primary microspheres.
According to the invention, preferably, the microfluidic droplets are formed using conventional microfluidic devices. Further preferably, the flow rate of the inner phase in the microfluidic device is 0.3-0.5mL/h and the flow rate of the outer phase is 2-5mL/h. The inventors have found that in this preferred embodiment, it is advantageous to promote the formation of microfluidic droplets from n-hexadecane-encapsulated core-shell structured nanoparticles.
According to the present invention, preferably, the solution containing the core-shell structured nanoparticles adopts ultrapure water as a solvent, and the concentration of the core-shell structured nanoparticles in the solution is 200-300mg/mL.
According to the present invention, preferably, the curing conditions include: the temperature is 70-85 ℃ and the time is 8-15h. The inventors have found that in this preferred embodiment, not only is it advantageous to increase the efficiency of solidification of the microfluidic droplets, but the morphology of the primary microspheres can be effectively controlled.
According to the present invention, preferably, the calcination process includes: washing and drying the primary microspheres by normal hexane, and calcining at the temperature of 750-850 ℃. The inventor finds that under the preferred embodiment, the photonic crystal microsphere with higher specific surface area and porosity is favorable for forming, so that the specificity and accuracy of the structural color coding magnetic microcarrier on gibberellin detection are enhanced.
According to the present invention, preferably, the activating in step (2) includes: the photonic crystal microsphere is mixed and activated with 3-aminopropyl triethoxysilane (APTES). The inventor finds that under the preferred embodiment, the gibberellin antibody grafted on the surface of the photonic crystal microsphere is facilitated, and the structural stability of the photonic crystal microsphere after grafting is improved.
According to the present invention, preferably, the conditions of the mixed activation include: the temperature is 5-40deg.C, and the time is 25-35min. The inventors have found that, in this preferred embodiment, it is advantageous to increase the activation efficiency of the photonic crystal microsphere,
according to the present invention, preferably, the gibberellin antibody is an N-hydroxysuccinimide (abbreviated as NHS) and (N-ethyl-N' -3-dimethylaminopropyl) carbodiimide hydrochloride (abbreviated as EDC) coupled GA3 antibody, wherein the GA3 antibody is commercially available, and the NHS and EDC coupled GA3 antibody is self-prepared by the methods in the prior art. The inventors found that in this preferred embodiment, the antibody can be immobilized to improve the coupling effect of the antibody and the photonic crystal microsphere.
According to the present invention, preferably, the weight ratio of the photonic crystal microsphere, the 3-aminopropyl triethoxysilane, and the gibberellin antibody is 2 to 4:2-4:1.
according to the present invention, preferably, the conditions of reaction I include: the temperature is 5-40deg.C, and the time is 50-70min. The inventors have found that in this preferred embodiment, not only is the temperature readily set (typically at room temperature), but the efficiency of grafting gibberellin antibodies to the surface of photonic crystal microspheres can be increased.
According to the present invention, preferably, the blocking agent in step (2) is bovine serum albumin, and the weight ratio of the gibberellin antibody to the blocking agent is 1:1.5-2.5. The inventors have found that under this preferred embodiment, bovine serum albumin is able to efficiently block the unwanted gibberellin binding sites on photonic crystal microspheres, terminating the progress of reaction I.
According to the present invention, preferably, the conditions of reaction II include: the temperature is 5-40deg.C, and the time is 50-70min. The inventors have found that in this preferred embodiment, it is advantageous to increase the efficiency of blocking unwanted gibberellin-binding sites on photonic crystal microspheres.
According to a particularly preferred embodiment of the present invention, the method for preparing a structural color-coded magnetic microcarrier comprises the following steps (scheme shown in FIG. 1):
(1) At 45-55deg.C under the condition of Stober method 3 O 4 SiO formation on the periphery of magnetic nanoparticles 2 Nano-layer to obtain core-shell structure nano-particle (Fe 3 O 4 @SiO 2 Wherein Fe is 3 O 4 Magnetic nanoparticles and SiO 2 The weight ratio of the nano layer is 1: 5-10), mixing the core-shell structure nano particles with ultrapure water to form a core-shell structure nano particle solution with the concentration of 200-300mg/mL, forming microfluidic liquid drops by a microfluidic device by taking the core-shell structure nano particle solution as an internal phase (the flow rate is 0.3-0.5 mL/h) and n-hexadecane as an external phase (the flow rate is 2-5 mL/h), solidifying the microfluidic liquid drops for 8-15h at the temperature of 70-85 ℃ to form primary microspheres, and carrying out the steps ofWashing and drying the primary microspheres by normal hexane, and calcining at 750-850 ℃ to form photonic crystal microspheres with positive structures;
(2) Mixing and activating the photonic crystal microsphere and 3-aminopropyl triethoxysilane at the temperature of 5-40 ℃ for 25-35min, mixing with N-hydroxysuccinimide and (N-ethyl-N' -3-dimethylaminopropyl) carbodiimide hydrochloride-coupled GA3 antibody, reacting for 50-70min at the temperature of 5-40 ℃, mixing with bovine serum albumin, and reacting for 50-70min at the temperature of 5-40 ℃ to enable gibberellin antibody to be grafted on the surface of the photonic crystal microsphere.
The structural color coded magnetic microcarrier provided by the invention can generate structural color and a reflection spectrum with a specific reflection peak, captures gibberellin molecules and performs content measurement by constructing a movable enriched liquid chip in the microcarrier, can be fully contacted with gibberellin in a solution to be detected, greatly shortens the reaction time of gibberellin antibody and gibberellin, and effectively improves the detection efficiency of gibberellin. Based on this, a third aspect of the present invention provides the use of the magnetic microcarrier described above, the magnetic microcarrier produced by the method described above, in the detection of gibberellin.
In a fourth aspect, the present invention provides a method for detecting gibberellin, the method comprising the steps of:
s1, respectively mixing gibberellin standard solutions with different concentrations with the magnetic microcarrier according to claim 1 or 2 and/or the magnetic microcarrier prepared by the method according to any one of claims 3 to 8 for reaction III, and detecting reflection peak offset values of the magnetic microcarrier before and after the reaction III;
s2, establishing a relation curve equation between the reflection peak offset value and the concentration of gibberellin standard solution to represent the relation between the reflection peak offset value and the concentration of gibberellin of the structural color coded magnetic microcarrier before and after the reaction III;
s3, mixing the solution to be detected with the magnetic microcarrier to carry out a reaction III, detecting reflection peak offset values of the magnetic microcarrier before and after the reaction III, and then calculating the content of gibberellin in the solution to be detected according to the relation curve equation.
The method for detecting gibberellin provided by the invention is characterized in that a solution to be detected is mixed with a structural color coding magnetic microcarrier to quantitatively detect gibberellin, and the detection principle is as follows: gibberellin solutions with different concentration gradients are respectively added into the structural color coding magnetic microcarrier, the combination of gibberellin and gibberellin antibody causes the refractive index of the magnetic microcarrier to fluctuate, the structural color and the characteristic reflection peak of the magnetic microcarrier to correspondingly change, and the concentration of gibberellin can be quantified through measuring the characteristic reflection peak of the magnetic microcarrier (the flow is shown in figure 1).
The structural color coded magnetic microcarrier provided by the invention can generate various structural colors (before gibberellin is adsorbed), and the refractive index changes after gibberellin is adsorbed, so that the reflection peak wavelength of the structural color coded magnetic microcarrier generates red shift; the reflection peak offset value is the difference between the reflection peak of the structural color-coded magnetic microcarrier before reaction III and the reflection peak of the structural color-coded magnetic microcarrier after reaction III. The reflection peak wavelength of the structural color coded magnetic microcarrier before and after the reaction III can be obtained by detection by adopting an optical fiber spectrometer.
According to the present invention, preferably, the conditions of reaction III include: the temperature is 0-40 ℃ and the time is 1.5-2.5h.
Preferably, according to the present invention, the relational equation is y= -7.83 x+78.90, where y is the offset value of the reflection peak, and x is the negative logarithmic value of the gibberellin standard solution concentration.
The present invention will be described in detail by examples.
In the following examples, a scanning electron microscope was purchased from Japanese electronics Co., ltd (JEOL), model JSM-5610LV, the structural color of the particles was obtained by an optical microscope (purchased from Olympic Bas, model BX53 front microscope), a microfluidic device was purchased from Pond, model LSP02-1B, and a fiber-optic spectrometer was purchased from Shanghai-Kabushiki optical Co., model PG2000-Pro-EX. GA3 antibody is purchased from Shenzhen Anti Biotechnology Co., ltd, product number is AT01GAAb, and the rest reagents and raw materials are conventional commercial products without special description.
In the examples which follow, room temperature refers to 25.+ -. 5 ℃ without specific description.
The preparation process of the GA3 antibody coupled with N-hydroxysuccinimide and (N-ethyl-N' -3-dimethylaminopropyl) carbodiimide hydrochloride comprises the following steps: 2g of GA3 antibody was added to 50mL of an as-prepared 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC solution (EDC concentration 4.2 mg/mL), followed by stabilization by 50mL of N-hydroxysuccinimide NHS solution (NHS concentration 5 mg/mL) and incubation at room temperature for 60min.
Example 1
(1) 16mL of ethylene glycol was taken, 0.26g of ferric chloride, 1.2g of sodium acetate, 0.4g of sodium poly (4-styrenesulfonic acid-co-maleic acid) (PSSMA), 50. Mu.L of water, 0.0045g of vitamin C were added, and the solution was stirred until it became clear yellow; then adding 0.24g of sodium hydroxide, continuously stirring until the solution is black transparent solution, transferring into an autoclave, putting into a muffle furnace, calcining for 9h, and after calcining, using alcohol: the water=1:1 mixed solution and pure water are respectively washed for three times to obtain PSSMA modified Fe 3 O 4 Nanoparticle, dissolving the particles in 12mL of pure water, and sealing;
(2) Taking 6mL of the aqueous solution obtained in the step (1), adding the aqueous solution into 40mL of absolute ethyl alcohol, and adding 2mL of ammonia water (NH) into the solution system 3 Concentration 25 wt%) and sonicated for 5min; then transferring into a three-neck flask, carrying out water bath at 50 ℃ with stirring for 10min, then adding 200 mu L of tetraethyl orthosilicate (TEOS) every 20min, adding the TEOS for three times, and stirring for 1h to obtain a reaction solution; the solid particles obtained after the reaction solution is centrifuged are respectively washed three times by absolute ethyl alcohol and pure water to obtain core-shell structure nano particles (Fe) with the average particle diameter of 270nm 3 O 4 @SiO 2 Nanoparticles, fe 3 O 4 Magnetic nanoparticles and SiO 2 The weight ratio of the nano layer is 1: 5) The method comprises the steps of carrying out a first treatment on the surface of the The structural color of the water dispersion liquid of the core-shell structure nano-particles under the action of magnet adsorption is shown as a in figure 2 a, and the core-shell structure nano-particles can be observed to have obvious structural color at the moment;
(3) Mixing the core-shell structure nano particles obtained in the step (2) with ultrapure water to form a core-shell structure nano particle solution with the concentration of 250mg/mL, taking the core-shell structure nano particle solution as an internal phase (the flow rate is 0.4 mL/h), taking n-hexadecane as an external phase (the flow rate is 4 mL/h), generating microfluidic liquid drops through a microfluidic device, solidifying the microfluidic liquid drops for 10 hours at the temperature of 75 ℃ to form preliminary microspheres, pouring the n-hexadecane as clean as possible, replacing the preliminary microspheres with excessive n-hexane, soaking the preliminary microspheres for 2 hours, continuously blowing and washing the preliminary microspheres with the n-hexane when the preliminary microspheres can float freely, repeating the step for 4 times, naturally air-drying the preliminary microspheres, and calcining the preliminary microspheres at 800 ℃ in a muffle furnace to form photonic crystal microspheres with positive structures; the photonic crystal microsphere is characterized by using a scanning electron microscope, as shown in fig. 3, the microsphere sphericity is good, and the internal magnetic nano particles are orderly arranged; the initial structural color of the photonic crystal microsphere is shown as a in fig. 4;
(4) Mixing and activating 6g of photonic crystal microspheres obtained in the step (3) and 6g of 3-aminopropyl triethoxysilane (APTES) at room temperature, and incubating for 30min to activate the photonic crystal microspheres; then mixing with 2g of N-hydroxysuccinimide (NHS) and (N-ethyl-N' -3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) coupled GA3 antibody, incubating for 60min at room temperature to obtain an incubation liquid, mixing the incubation liquid with 4g of Bovine Serum Albumin (BSA), and reacting for 60min at room temperature to obtain photonic crystal microspheres with gibberellin antibody grafted on the surface as a structural color coded magnetic microcarrier.
The structural color-coded magnetic microcarrier obtained in example 1 was photographed using a frontal microscope instrument (manufacturer Olinbas model BX 53) to obtain the structural color as shown in FIG. 4 b.
Example 2
(1) 10mL of ethylene glycol is taken, 0.2g of ferric chloride, 1g of sodium acetate, 0.3g of PSSMA, 30 mu L of water and 0.004g of vitamin C are added, and the mixture is stirred until the solution is transparent yellow; then adding 0.2g of sodium hydroxide, continuously stirring until the solution is black transparent solution, transferring into an autoclave, placing into a muffle furnace, calcining for 8h, and after calcining, using alcohol: the water=1:1 mixed solution and pure water are respectively washed for three times to obtain PSSMA modified Fe 3 O 4 Nanoparticle, dissolving the particles in 12mL of pure water, and sealing;
(2) Taking 4mL of the aqueous solution obtained in the step (1), adding the aqueous solution into 30mL of absolute ethyl alcohol, and adding 1mL of ammonia water (NH) into the solution system 3 Concentration 20 wt%) and ultrasonic treatment for 4min; then transferring into a three-neck flask, carrying out water bath at 45 ℃ with stirring for 8min, then adding 150 mu L of tetraethyl orthosilicate (TEOS) every 15min, adding the TEOS for three times and stirring for 45min to obtain a reaction solution; the solid particles obtained after the reaction solution is centrifugated are respectively washed three times by absolute ethyl alcohol and pure water, and are dried to obtain the core-shell structure nano particles (Fe) with the average particle diameter of 300nm 3 O 4 Magnetic nanoparticles and SiO 2 The weight ratio of the nano layer is 1: 8) The method comprises the steps of carrying out a first treatment on the surface of the The structural color of the water dispersion liquid of the core-shell structure nano-particles under the action of magnet adsorption is shown as b in fig. 2, and the core-shell structure nano-particles can be observed to have obvious structural color at the moment;
(3) Mixing the core-shell structure nano particles obtained in the step (2) with ultrapure water to form a core-shell structure nano particle solution with the concentration of 200mg/mL, taking the core-shell structure nano particle solution as an internal phase (the flow rate is 0.3 mL/h) and n-hexadecane as an external phase (the flow rate is 2 mL/h), generating microfluidic liquid drops through a microfluidic device, solidifying the microfluidic liquid drops for 15h at the temperature of 70 ℃ to form preliminary microspheres, pouring the n-hexadecane as clean as possible, replacing the preliminary microspheres with excessive n-hexane, soaking the preliminary microspheres for 2.5h, continuously blowing and washing the preliminary microspheres with the n-hexane when the preliminary microspheres can freely float, repeating the step for 3 times, naturally air-drying the preliminary microspheres, and calcining the preliminary microspheres in a muffle furnace at the temperature of 750 ℃ to form photonic crystal microspheres with the positive structure;
(4) Mixing and activating 4g of the photonic crystal microsphere obtained in the step (3) and 4g of 3-aminopropyl triethoxysilane (APTES) at room temperature, and incubating for 25min to activate the photonic crystal microsphere; then mixing with 2g of NHS and EDC coupled GA3 antibody, incubating for 50min at room temperature to obtain an incubation liquid, mixing the incubation liquid with 3g of Bovine Serum Albumin (BSA), and reacting for 50min at room temperature to obtain photonic crystal microsphere with gibberellin antibody grafted on the surface as a structural color coding magnetic microcarrier.
Example 3
(1) 20mL of ethylene glycol is taken, 0.3g of ferric chloride, 1.5g of sodium acetate, 0.5g of PSSMA, 70 mu L of water and 0.0048g of vitamin C are added, and the mixture is stirred until the solution is transparent yellow; then adding 0.3g of sodium hydroxide, continuously stirring until the solution is black transparent solution, transferring into an autoclave, placing into a muffle furnace, calcining for 10h, and after calcining, using alcohol: the water=1:1 mixed solution and pure water are respectively washed for three times to obtain PSSMA modified Fe 3 O 4 Nanoparticle, dissolving the particles in 12mL of pure water, and sealing;
(2) Taking 3mL of the aqueous solution obtained in the step (1), adding the aqueous solution into 50mL of absolute ethyl alcohol, and adding 3mL of ammonia water (NH) into the solution system 3 Concentration is 20wt%) and ultrasonic treatment is carried out for 6min; then transferring into a three-neck flask, carrying out water bath at 55 ℃ with stirring for 12min, then adding 250 mu L of tetraethyl orthosilicate (TEOS) every 25min, adding the TEOS for three times and stirring for 75min to obtain a reaction solution; the solid particles obtained after the reaction solution is centrifugated are respectively washed three times by absolute ethyl alcohol and pure water, and are dried to obtain core-shell structure nano particles (Fe) with the average particle diameter of 350nm 3 O 4 Magnetic nanoparticles and SiO 2 The weight ratio of the nano layer is 1:10 A) is provided;
(3) Mixing the core-shell structure nano particles obtained in the step (2) with ultrapure water to form a core-shell structure nano particle solution with the concentration of 300mg/mL, taking the core-shell structure nano particle solution as an internal phase (the flow rate is 0.5 mL/h), taking n-hexadecane as an external phase (the flow rate is 5 mL/h), generating microfluidic liquid drops through a microfluidic device, solidifying the microfluidic liquid drops for 8 hours at the temperature of 85 ℃ to form preliminary microspheres, pouring the n-hexadecane as completely as possible, replacing the preliminary microspheres with excessive n-hexane, soaking the preliminary microspheres for 2.5 hours, continuously blowing and washing the preliminary microspheres with the n-hexane when the preliminary microspheres can freely float, repeating the step for 3 times, naturally air-drying the preliminary microspheres, and calcining the preliminary microspheres in a muffle furnace at 850 ℃ to form photonic crystal microspheres with the positive structure;
(4) Mixing 8g of photonic crystal microspheres obtained in the step (3) with 8g of 3-aminopropyl triethoxysilane (APTES) at room temperature for activation, and incubating for 35min for activation; then mixing with 2g of N-hydroxysuccinimide and (N-ethyl-N' -3-dimethylaminopropyl) carbodiimide hydrochloride-coupled GA3 antibody, incubating for 70min at room temperature to obtain an incubation liquid, mixing the incubation liquid with 5g of Bovine Serum Albumin (BSA), and reacting for 70min at room temperature to obtain photonic crystal microspheres with gibberellin antibody grafted on the surfaces, wherein the photonic crystal microspheres are used as a structural color coded magnetic microcarrier.
Example 4
A structural color-coded magnetic microcarrier was prepared as in example 3, except PSSMA modified Fe 3 O 4 Substitution of nanoparticles with Fe 3 O 4 A nanoparticle;
the step (1) is replaced by:
(1) 2.16g FeCl at normal temperature 3 ·6H 2 O is dissolved in 100mL deionized water, and Na is added under the protection of nitrogen 2 SO 3 Stirring the solution by magnetic force for 15min, quickly adding 5mL of concentrated ammonia water with mass fraction of 25%, quickly blackening the solution, reacting in an oil bath at 60 ℃ for 30min, then dropwise adding 0.3g of citric acid, adjusting the temperature to 80 ℃, cooling to room temperature after reacting for 1h, settling the magnetic ball, and cleaning the magnetic ball with acetone and deionized water for 3 times to obtain Fe 3 O 4 Nanoparticle of Fe 3 O 4 The nanoparticles were dissolved in 20mL of purified water and sealed.
Test example 1
(1) Preparing a standard working solution: weighing gibberellin standard (product number is AT01GAAg, purchased from Shenzhen Anti Biotechnology Co., ltd.) 0.35mg in a 100mL volumetric flask, dissolving with PBS, fixing volume to scale line, and performing ultrasonic treatment for 10min to obtain standard solution; sucking the 10mL standard solution into a 100mL volumetric flask, diluting with PBS to scale to obtain mixed intermediate solution with concentration gradient of 10 -5 、10 -6 、10 -7 、10 -8 、10 -9 Gibberellin standard working solution with mol/L;
(2) Measuring the reflection spectrum of the structural color coding magnetic microcarrier prepared in the example 1 by adopting an optical fiber spectrometer, recording the characteristic peak position of 498nm before reaction, respectively adding 100mg of the structural color coding magnetic microcarrier prepared in the example 1 into 2mL of gibberellin standard working solutions with different concentration gradients obtained in the step (1), and reacting for 2h at room temperature; then, separating out the magnetic microcarrier by using a magnet (the structural color of the magnetic microcarrier after gibberellin adsorption is shown as c in figure 4), measuring the reflection spectrum of the magnetic microcarrier after reaction by using an optical fiber spectrometer, and recording the position of a characteristic peak after reaction; drawing a relation between the detected reflection peak offset value (difference value of characteristic peak positions before and after reaction) and the concentration of the gibberellin solution into a standard curve, wherein a formula for obtaining the standard curve is y= -7.83 x+78.90, wherein y is the reflection peak offset value, and x is a negative logarithmic value of the concentration of the gibberellin standard solution as shown in fig. 5;
(3) 100mg of the structure color-coded magnetic microcarrier prepared in example 1 was added to 2mL of the solution I to be tested (using a concentration of 3.73X10) -5 Fully mixing the gibberellin standard working solution with mol/L as the solution I) to be tested, and stirring the mixture at room temperature for reaction for 2 hours; then separating out the magnetic microcarrier by using a magnet, measuring the reflection spectrum of the magnetic microcarrier after reaction, recording the characteristic peak position as 537nm, calculating the reflection peak offset value of the solution to be measured, substituting the reflection peak offset value into the standard curve obtained in the step (2), and finally calculating to obtain the concentration of gibberellin in the solution to be measured as 3.7X10 -5 M。
The detection limit of gibberellin of the structural color-coded magnetic microcarrier obtained in example 1 is 10 -10 M。
As can be seen from FIG. 4, the positive-structure photonic crystal microsphere, the structural color-coded magnetic microcarrier and the magnetic microcarrier after gibberellin molecule adsorption obtained in example 1 have different structural colors and gradually red-shifted phenomena.
Test example 2
(1) Preparing gibberellin standard working solution according to step (1) in test example 1;
(2) Measuring the reflection spectrum of the structural color coding magnetic microcarrier prepared in the example 2 by adopting an optical fiber spectrometer, recording the characteristic peak position to be 512nm before the reaction, respectively adding 100mg of the structural color coding magnetic microcarrier prepared in the example 2 into 2mL of gibberellin standard working solutions with different concentration gradients obtained in the step (1), and reacting for 1.5h at room temperature; then separating out the magnetic microcarrier by using a magnet, measuring the reflection spectrum of the magnetic microcarrier after reaction by using an optical fiber spectrometer, and recording the position of a characteristic peak after reaction; drawing a relation between the detected reflection peak offset value (difference value of characteristic peak positions before and after reaction) and the concentration of the gibberellin solution into a standard curve, and obtaining a formula of the standard curve as y= -6.18 x+69.27, wherein y is the reflection peak offset value, and x is a negative logarithmic value of the concentration of the gibberellin standard solution;
(3) 100mg of the structure color-coded magnetic microcarrier prepared in example 2 was added to 2mL of solution II to be tested (using a concentration of 6.87X 10) -8 Fully mixing the gibberellin standard working solution with mol/L as the solution II) to be tested, and stirring the mixture at room temperature for reaction for 1.5 hours; then separating out the magnetic microcarrier by using a magnet, measuring the reflection spectrum of the magnetic microcarrier after reaction, recording the characteristic peak position as 532nm, calculating the reflection peak offset value of the solution to be measured, substituting the reflection peak offset value into the standard curve obtained in the step (2), and finally calculating to obtain the concentration of gibberellin in the solution to be measured as 7.13 multiplied by 10 -8 M。
The detection limit of gibberellin of the structural color-coded magnetic microcarrier obtained in example 2 is 10 -11 M。
Test example 3
(1) Preparing gibberellin standard working solution according to step (1) in test example 1;
(2) Measuring the reflection spectrum of the structural color coding magnetic microcarrier prepared in the embodiment 3 by adopting an optical fiber spectrometer, recording the characteristic peak position to be 582nm before the reaction, respectively adding 100mg of the structural color coding magnetic microcarrier prepared in the embodiment 2 into 2mL of gibberellin standard working solutions with different concentration gradients obtained in the step (1), and reacting for 2.5 hours at room temperature; then separating out the magnetic microcarrier by using a magnet, measuring the reflection spectrum of the magnetic microcarrier after reaction by using an optical fiber spectrometer, and recording the position of a characteristic peak after reaction; drawing a relation between the detected reflection peak offset value (difference value of characteristic peak positions before and after reaction) and the concentration of the gibberellin solution into a standard curve, and obtaining a formula of the standard curve as y= -6.89 x+70.97, wherein y is the reflection peak offset value, and x is a negative logarithmic value of the concentration of the gibberellin standard solution;
(3) 100mg of the structural color-coded magnetic microcarrier prepared in example 3 was added to 2mL solution III to be measured (with a concentration of 4.45X10) -7 Fully mixing the gibberellin standard working solution with mol/L as a solution III) to be tested, and stirring the mixture at room temperature for 2.5 hours; then separating out the magnetic microcarrier by using a magnet, measuring the reflection spectrum of the magnetic microcarrier after reaction, recording the characteristic peak position as 605nm, calculating the reflection peak offset value of the solution to be measured, substituting the reflection peak offset value into the standard curve obtained in the step (2), and finally calculating to obtain the concentration of gibberellin in the solution to be measured as 4.97X10 -7 M。
The detection limit of gibberellin of the structural color-coded magnetic microcarrier obtained in example 3 is 10 -10 M。
Comparative example 1
Preparing gibberellin standard working solution according to the step (1) in the test example 1, detecting the characteristic peak area of a gibberellin standard substance by utilizing HPLC-MS-MS, and drawing a standard curve of the characteristic peak area and gibberellin concentration; detecting the solution I to be detected by HPLC-MS-MS to obtain the characteristic peak area of gibberellin, and substituting the characteristic peak area into a standard curve to obtain the gibberellin content of 3.4X10 in the solution to be detected - 5 M。
Compared with comparative example 1, the structural color-coded magnetic microcarrier provided in example 1 has better detection specificity, sensitivity and accuracy.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. The structural color coding magnetic microcarrier is characterized by being applied to detection of gibberellin, and comprises photonic crystal microspheres and gibberellin antibodies grafted on the surfaces of the photonic crystal microspheres, wherein the photonic crystal microspheres are formed by self-assembly of core-shell structure nanoparticles, and the core-shell structure nanoparticles comprise Fe 3 O 4 Inner core formed by magnetic nano particles and SiO wrapping periphery of inner core 2 A nano layer;
the preparation method of the structural color coding magnetic microcarrier comprises the following steps:
(1) Fe is added to 3 O 4 SiO is wrapped around the periphery of the magnetic nano particles 2 The nano layer is used for obtaining core-shell structure nano particles, and the core-shell structure nano particles are self-assembled to form photonic crystal microspheres;
(2) After the photonic crystal microsphere is activated, mixing the photonic crystal microsphere with gibberellin antibody to perform reaction I, and then mixing the photonic crystal microsphere with a blocking agent to perform reaction II, so that gibberellin antibody is grafted on the surface of the photonic crystal microsphere;
the self-assembly process in step (1) comprises: forming microfluidic liquid drops by taking a solution containing the core-shell structure nano particles as an internal phase and n-hexadecane as an external phase, solidifying the microfluidic liquid drops to form primary microspheres, and calcining the primary microspheres.
2. The structural color-coded magnetic microcarrier of claim 1, wherein Fe is added in step (1) 3 O 4 SiO is wrapped around the periphery of the magnetic nano particles 2 The process of the nano layer comprises the following steps: by Stober method in the Fe 3 O 4 SiO formation on the periphery of magnetic nanoparticles 2 A nano layer.
3. The structural color-coded magnetic microcarrier of claim 2, wherein said Stober process forms SiO 2 The conditions of the nanolayer include: the temperature is 45-55 ℃;
the Fe is 3 O 4 The magnetic nano particles are Fe modified by poly (4-styrenesulfonic acid-co-maleic acid) sodium salt 3 O 4 A nanoparticle; the Fe is 3 O 4 Magnetic nanoparticles and SiO 2 The weight ratio of the nano layer is 1:5-10.
4. A structural colour coded magnetic microcarrier according to any one of claims 1 to 3, characterized in that the microfluidic droplet is formed using a microfluidic device.
5. The structural color-coded magnetic microcarrier of claim 4, wherein the flow rate of the inner phase in the microfluidic device is 0.3-0.5mL/h and the flow rate of the outer phase is 2-5mL/h;
the solution containing the core-shell structure nano particles adopts ultrapure water as a solvent, and the concentration of the core-shell structure nano particles in the solution is 200-300mg/mL.
6. The structural color-coded magnetic microcarrier of claim 1, wherein the conditions for curing comprise: the temperature is 70-85 ℃ and the time is 8-15h;
the calcination process comprises the following steps: washing and drying the primary microspheres by normal hexane, and calcining at the temperature of 750-850 ℃.
7. A structural color-coded magnetic microcarrier according to any one of claims 1-3, characterized in that the activation process in step (2) comprises: mixing and activating the photonic crystal microsphere and 3-aminopropyl triethoxysilane;
the conditions of the mixed activation include: the temperature is 5-40deg.C, and the time is 25-35min;
the gibberellin antibody is a GA3 antibody coupled with N-hydroxysuccinimide and (N-ethyl-N' -3-dimethylaminopropyl) carbodiimide hydrochloride;
the weight ratio of the photonic crystal microsphere to the 3-aminopropyl triethoxysilane to the gibberellin antibody is 2-4:2-4:1, a step of;
the conditions for reaction I include: the temperature is 5-40deg.C, and the time is 50-70min.
8. A structural color-coded magnetic microcarrier according to any one of claims 1-3, wherein the blocker in step (2) is bovine serum albumin and the weight ratio of gibberellin antibody to blocker is 1:1.5-2.5;
the conditions for reaction II include: the temperature is 5-40deg.C, and the time is 50-70min.
9. A method for detecting gibberellin, the method comprising the steps of:
s1, respectively mixing gibberellin standard solutions with different concentrations with the structural color-coded magnetic microcarrier according to any one of claims 1 to 8 to perform a reaction III, and detecting reflection peak offset values of the structural color-coded magnetic microcarrier before and after the reaction III;
s2, establishing a relation curve equation between the reflection peak offset value and the concentration of the gibberellin standard solution;
s3, mixing the solution to be detected with the structural color coding magnetic microcarrier to carry out a reaction III, detecting reflection peak offset values of the structural color coding magnetic microcarrier before and after the reaction III, and then calculating the content of gibberellin in the solution to be detected according to the relation curve equation.
10. The method of claim 9, wherein the conditions of reaction III comprise: the temperature is 0-40 ℃ and the time is 1.5-2.5h;
the relation curve equation is y= -7.83 x+78.90, wherein y is a reflection peak offset value, and x is a negative logarithmic value of the concentration of the gibberellin standard solution.
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