CN117772082A - Surface-modified biological microsphere, kit and preparation method thereof - Google Patents

Surface-modified biological microsphere, kit and preparation method thereof Download PDF

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CN117772082A
CN117772082A CN202311776637.5A CN202311776637A CN117772082A CN 117772082 A CN117772082 A CN 117772082A CN 202311776637 A CN202311776637 A CN 202311776637A CN 117772082 A CN117772082 A CN 117772082A
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microsphere
carboxyl
ligand
hydroxyl
microspheres
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董伟
夏正龙
赵莉莉
王允军
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Suzhou Xingshuo Nanotech Co Ltd
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Suzhou Xingshuo Nanotech Co Ltd
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Abstract

The application provides a surface-modified biological microsphere, a kit and a preparation method thereof, which have universality in the aspect of reducing the nonspecific adsorption of the microsphere, and the surface structure of the biological microsphere is simple, the cost is low, and the applicability is wide. The preparation method comprises the following steps: s1, activating carboxyl microspheres: mixing carboxyl microspheres with a catalyst to form a first mixed solution, and reacting carboxyl on the surfaces of the microspheres with the catalyst to generate active ester or amine-reactive ester to obtain activated microspheres; s2, mixing the activated microsphere with a hydroxyl ligand and a potential ligand to form a second mixed solution, wherein the hydroxyl ligand and the potential ligand replace the active ester or the amine reactive ester and are connected with the surface of the microsphere to obtain the surface-modified biological microsphere.

Description

Surface-modified biological microsphere, kit and preparation method thereof
Technical Field
The application belongs to the technical field of biological microspheres, and in particular relates to a surface-modified biological microsphere, a kit and a preparation method thereof.
Background
The biological microsphere is formed by wrapping fluorescent materials such as quantum dots and the like in a shell layer, and then modifying carboxyl ligands on the surface of the microsphere. The surface of the biological microsphere contains carboxyl, has water solubility for biological application, and can be used for antibody coupling. The biological microsphere is internally provided with a fluorescent material, the fluorescent material can emit fluorescence, and by means of different fluorescent component types and fluorescent contents, a plurality of lights with different luminous wave bands (colors) can be formed, and the fluorescent material can be used for detecting various detected substances to realize a coding function, so that the fluorescent material is widely applied to the biological medicine fields such as biological markers, disease diagnosis, biological tracers, solid-phase chips, liquid-phase chips, immunochromatography, raman scattering and the like.
Since all proteins possess positively charged amino acid residues and negatively charged amino acid residues randomly distributed on their surface, any positively or negatively charged material will adsorb the protein very easily (charge adsorption), thus causing non-specific adsorption. In the prior art, carboxyl ligands are modified on the surfaces of microspheres, and carboxyl is negatively charged and is easy to be subjected to nonspecific adsorption, so that the detection accuracy is affected, but the carboxyl is easy to be subjected to antibody coupling, and the surfaces of biological microspheres are required to be provided with carboxyl. At the same time, hydrophobic adsorption is also liable to occur, whereby non-specific adsorption occurs. In addition, other groups introduced by the ligand modified on the surface of the microsphere can be converted by enzymes in organisms, and nonspecific adsorption can occur.
Currently, in terms of reducing the nonspecific adsorption of biological microspheres. The PS microsphere is formed by coating a fluorescent material with a polystyrene shell layer, and then PEG ligands such as hydroxyethyl methacrylate, polyethylene glycol dimethacrylate and the like and acrylic acid are modified on the surface of the PS microsphere to form the biological microsphere. The introduction of the PEG ligand reduces the content of acrylic acid on the surface of the PS microsphere, the long chain of the PEG ligand forms steric hindrance, and the strong hydrophilicity of the PEG ligand forms a hydration layer, so that the nonspecific adsorption is reduced. However, in the first aspect, the above-mentioned PEG ligands are expensive (for example, PEG silanes and PEG acrylates are very expensive), and are difficult to be used for industrial mass production. In the second aspect, the molecular weight of the PEG ligand is large, the surface structure of the biological microsphere is complex, and the grafting density is not easy to control; the PEG long-chain steric hindrance also has weak effect on reducing nonspecific adsorption. In the third aspect, the applicability of this solution is narrow, for example, an amphiphilic polymer shell such as PMAO (poly (stearyl maleate)), an inorganic shell such as silica, and a resin shell such as MF (urea-formaldehyde), the PEG ligand cannot be directly modified, and a relatively complex manner is required for connection. How to prepare low-cost and universal biological microspheres with low nonspecific adsorption is a great difficulty for many years, which plagues the biological microsphere industry/the technicians in this field.
In view of the above, the application provides a surface-modified biological microsphere, a kit and a preparation method thereof, which have universality in the aspect of reducing the nonspecific adsorption of the microsphere, and the biological microsphere has the advantages of simple surface structure, low cost and wide applicability.
Disclosure of Invention
The application aims at providing a surface modified biological microsphere, a kit and a preparation method thereof, wherein the surface modified biological microsphere has universality in the aspect of reducing nonspecific adsorption of the microsphere, and the biological microsphere has a simple surface structure, low cost and wide applicability.
In a first aspect of the present application, a method for preparing a surface-modified biological microsphere is provided, comprising the steps of:
s1, activating carboxyl microspheres: mixing carboxyl microspheres with a catalyst to form a first mixed solution, and reacting carboxyl on the surfaces of the microspheres with the catalyst to generate active ester or amine-reactive ester to obtain activated microspheres;
s2, mixing the activated microsphere with a hydroxyl ligand and a potential ligand to form a second mixed solution, wherein the hydroxyl ligand and the potential ligand replace the active ester or the amine reactive ester and are connected with the surface of the microsphere to obtain the surface-modified biological microsphere.
In some embodiments, the PH of the first mixed liquor is between 5 and 8. Preferably, the pH of the first mixed solution is 5-6.
In some embodiments, the concentration of the carboxyl microsphere in the first mixed solution is 2-10mg/ml. Preferably, in the first mixed solution, the concentration of the carboxyl microsphere is 4-6mg/ml.
In some embodiments, the first mixed liquor further comprises: and the dispersion liquid is used for improving the dispersibility of the carboxyl microsphere, and the dispersion liquid is a hydroxyl high-molecular polymer dispersing agent.
Further, in the first mixed solution, the concentration of the dispersion is 0.04 to 0.2% by weight. Preferably, in the first mixture, the concentration of the dispersion is 0.08-0.15% wt.
Further, the carboxyl microsphere is mixed with the dispersion and an acidic pH adjustor, and then a catalyst is added.
In some embodiments, the catalyst is capable of reacting with a carboxyl group at a temperature of 8-50 ℃ to form an active ester or an amine-reactive ester.
Further, the catalyst comprises: a water-soluble carbodiimide salt, or/and an imide salt.
Further, the water-soluble carbodiimide salt includes: 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), or N, N Diisopropylcarbodiimide (DIC); the imide salt includes: n-hydroxysuccinimide (NHS), or N-hydroxysulfosuccinimide (Sulfo-NHS).
Further, in the first mixed solution, the mass ratio of the carboxyl microsphere to the catalyst is 1: (0.1-100). Preferably, in the first mixed solution, the mass ratio of the carboxyl microsphere to the catalyst is 1: (10-40).
In some embodiments, the temperature of the first mixture is adjusted to 8-50 ℃ and maintained for 25-60 minutes, such that the carboxyl microspheres are activated. Preferably, the temperature of the first mixed solution is adjusted to 35-40 ℃ and maintained for 30-40min, so that the carboxyl microsphere is activated.
In some embodiments, the carboxyl microsphere is a microsphere having carboxyl groups on the surface, the microsphere comprising, from inside to outside: fluorescent material, and a shell layer coating the fluorescent material.
Further, the carboxyl is a carboxyl self-contained on the surface of the microsphere, or the carboxyl is a carboxyl modified on the surface of the microsphere.
Further, the fluorescent material includes: at least one of a fluorescent nanoparticle, a fluorescent polymer, and an organic fluorescent dye, the fluorescent nanoparticle comprising at least one of a quantum dot, a metal oxide nanoparticle, a nanorod, or a nanoplatelet.
Further, the shell layer is formed by hydrolyzing and condensing inorganic matters, or is formed by condensing a resin precursor, or is formed by self-assembling amphiphilic polymer coated micro-droplets, or is formed by polymerizing vinyl monomers with carbon-carbon double bonds under the initiation action.
Further, the microsphere also comprises a magnetic material, and the shell layer coats the fluorescent material and the magnetic material.
In some embodiments, the PH of the second mixture is between 5 and 8. Preferably, the pH of the second mixed solution is 7.2-7.8.
Further, the activated microsphere is mixed with an alkaline pH adjustor, followed by the addition of a hydroxyl ligand and a potential ligand.
In some embodiments, the amine group of the hydroxyl ligand replaces the active ester or amine-reactive ester, forming an amide bond to attach to the microsphere surface while providing a hydroxyl group.
Further, the hydroxyl ligand is an alcohol amine compound containing both primary amine or secondary amine and hydroxyl. The hydroxyl ligands include: at least one of ethanolamine, diethanolamine, isopropanolamine, diisopropanolamine, and the like.
In some embodiments, the potentiometric ligand is a carboxamide compound containing two amino groups and one carboxyl group.
Further, one amino group of the potential ligand replaces the active ester or amine-reactive ester, an amide bond is formed and is connected with the surface of the microsphere, and one amino group and one carboxyl group are remained for potential neutralization.
Further, the potential ligand includes: at least one of histidine, arginine and lysine.
In some embodiments, the activated microsphere is mixed with both the hydroxyl ligand and the potential ligand to form a second mixed solution.
Further, adding the activated microsphere, the hydroxyl ligand and the potential ligand into the second mixed solution, wherein the mass ratio of the activated microsphere to the hydroxyl ligand to the potential ligand is 1: (1-20): (0.005-20).
Preferably, in the second mixed solution, the mass ratio of the activated microsphere, the hydroxyl ligand and the potential ligand is 1: (8-12): (0.005-0.02).
Preferably, in the second mixed solution, the mass ratio of the activated microsphere, the hydroxyl ligand and the potential ligand is 1: (8-12): (8-12).
In some embodiments, the second mixed solution is reacted for 2-24 hours at a temperature of 30-50 ℃ under the protection of inert gas to produce the surface modified biological microsphere.
In a second aspect of the present application, there is provided a surface-modified biological microsphere comprising: the microsphere comprises a microsphere body, a hydroxyl ligand and a potential ligand which are connected with the surface of the microsphere body.
In some embodiments, the microsphere comprises, from inside to outside: fluorescent material, and a shell layer coating the fluorescent material.
Further, the fluorescent material includes: at least one of a fluorescent nanoparticle, a fluorescent polymer, and an organic fluorescent dye, the fluorescent nanoparticle comprising at least one of a quantum dot, a metal oxide nanoparticle, a nanorod, or a nanoplatelet.
Further, the shell layer is formed by hydrolyzing and condensing inorganic matters, or is formed by condensing a resin precursor, or is formed by self-assembling amphiphilic polymer coated micro-droplets, or is formed by polymerizing vinyl monomers with carbon-carbon double bonds under the initiation action.
Further, the microsphere also comprises a magnetic material, and the shell layer coats the fluorescent material and the magnetic material.
In some embodiments, the hydroxyl ligand is an alcohol amine compound containing both a primary or secondary amine and a hydroxyl group. The hydroxyl ligands include: at least one of ethanolamine, diethanolamine, isopropanolamine, diisopropanolamine, and the like.
In some embodiments, the potentiometric ligand is a carboxamide compound containing two amino groups and one carboxyl group. The potential ligand comprises: at least one of histidine, arginine and lysine.
In a third aspect of the present application, there is provided a surface-modified biological microsphere obtained by the above preparation method.
In a fourth aspect of the present application, there is provided a kit comprising: the surface modified biological microsphere, the antibody and the blocking agent.
In a fifth aspect of the present application, the use of the surface modified biological microsphere in a biological medicine, the biological medicine comprising: drug loading, biological probes, biological markers, disease diagnosis, biological tracers, solid phase chips, liquid phase chips, immunochromatography, raman scattering.
Compared with the prior art, the biological microsphere and the preparation method thereof have at least the following advantages:
(1) The preparation method of the surface-modified biological microsphere has universality in the aspect of reducing nonspecific adsorption. The method is suitable for the carboxyl microsphere, namely PS (polystyrene) microsphere with carboxyl, or carboxyl microsphere with carboxyl modified later, and the carboxyl on the surface of the microsphere is activated into active ester or amine reactive ester by using the catalyst, so that the active ester or amine reactive ester is easily replaced by other ligands to be combined with the surface of the microsphere, and the method is suitable for all the current carboxyl microspheres.
(2) In the method, on one hand, hydroxyl ligand and potential ligand are added into the activated microsphere, and on the other hand, the hydroxyl ligand replaces part of carboxyl groups on the surface of the microsphere (specifically, the catalyst firstly forms active ester or amine reactive ester with the carboxyl groups on the surface of the microsphere, and the hydroxyl ligand replaces part of active ester or amine reactive ester to form an amide bond to be connected with the surface of the microsphere) so as to provide hydroxyl groups; the hydroxyl forms a hydrogen bond with water to form a hydration layer for protection, so that nonspecific adsorption can be reduced; the hydroxyl group is uncharged, so that the charge is reduced, and the nonspecific adsorption is further reduced; the hydroxyl ligand has low price and low cost; the surface structure of the biological microsphere is simple, and the grafting density is easy to control; and the hydroxyl can improve the acid resistance and the salt resistance of the biological microsphere. In a second aspect, the potential ligands have 2 amino groups and 1 carboxyl group, wherein 1 amino group replaces the carboxyl group of the other part of the microsphere surface (specifically, the catalyst firstly forms active ester or amine-reactive ester with the carboxyl group of the microsphere surface, one amino group of the potential ligand replaces a part of active ester or amine-reactive ester to form an amide bond to be connected with the microsphere surface), each potential ligand also has one amino group (positive charge) and one carboxyl group (negative charge), the potential neutralization and the charge is zero, and the potential neutralized carboxyl group is provided for the rear end coupling antibody, and the nonspecific adsorption is reduced.
(3) The hydroxyl ligand and the potential ligand are added into the activated microsphere at the same time, and the carboxyl content of the surface modified biological microsphere surface is regulated and controlled by controlling the proportion of the added hydroxyl ligand and the potential ligand. Simultaneously adding a hydroxyl ligand and a potential ligand, enabling the activated microsphere to react with the hydroxyl ligand and the potential ligand, and controlling the carboxyl content of the surface of the final biological microsphere by controlling the proportion of the added hydroxyl ligand and the potential ligand through competitive balance between the hydroxyl ligand and the potential ligand.
(4) The carboxyl content and detection performance of the surface of the biological microsphere can be fully regulated by controlling the feeding ratio of the hydroxyl ligand and the potential ligand, and the application requirements of high sensitivity or good linearity can be realized, so that the requirements of being applicable to detection of various rear ends are met, and the applicability is wide.
(5) The biological microsphere solves the problems of charge adsorption and hydrophobic adsorption, and can be used for simple antibody coupling and blocking at the rear end.
Drawings
The foregoing and other features of the present application will be more fully described when read in conjunction with the following drawings. It is appreciated that these drawings depict only several embodiments of the present application and are therefore not to be considered limiting of its scope. The present application will be described more specifically and in detail by using the accompanying drawings.
FIG. 1 is a linear plot of T/C ratio at antigen gradient concentrations for the antibody conjugate of examples 1-4.
FIG. 2 is a linear plot of T/C ratio at antigen gradient concentrations for the antibody coupling validation of examples 5-8.
Detailed Description
The following examples are described to aid in the understanding of the present application and are not, nor should they be construed in any way to limit the scope of the present application.
At least one of the "when preceding or following a list of elements" as for example "is described herein modifies the entire list of elements without modifying individual elements of the list. Unless otherwise defined, all terms (including technical and scientific terms) in the specification can be defined as commonly understood by one of ordinary skill in the art. Terms defined in commonly used dictionaries should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Furthermore, unless expressly stated to the contrary, the words "comprise" and the words "comprising" when used in this specification mean the presence of stated features, regions, integers, steps, operations, elements, and/or components, but does not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof. Accordingly, the above phraseology is to be understood as meaning to include the stated elements, but not to exclude any other elements.
In a first aspect of the present application, a method for preparing a surface-modified biological microsphere is provided, comprising the steps of:
s1, activating carboxyl microspheres: mixing carboxyl microspheres with a catalyst to form a first mixed solution, and reacting carboxyl on the surfaces of the microspheres with the catalyst to generate active ester or amine-reactive ester to obtain activated microspheres;
s2, mixing the activated microsphere with a hydroxyl ligand and a potential ligand to form a second mixed solution, wherein the hydroxyl ligand and the potential ligand replace the active ester or the amine reactive ester and are connected with the surface of the microsphere to obtain the surface-modified biological microsphere.
In some embodiments, the concentration of the carboxyl microsphere in the first mixed solution is 2-10mg/ml. Preferably, in the first mixed solution, the concentration of the carboxyl microsphere is 4-6mg/ml.
In some embodiments, the PH of the first mixed liquor is between 5 and 8. Preferably, the pH of the first mixed solution is 5-6.
Further, the first mixed solution further comprises: an acidic pH adjustor which makes the pH of the first mixed solution 5 to 6.
Acidic PH adjusting agents include: at least one of 2- (N-methyl-4-pyrrolidinyl) ethanesulfonic acid sodium salt (MES) hydrochloride, 4-hydroxyethyl piperazine ethanesulfonate, tris-HCl hydrochloride, phosphate, and bicarbonate hydrochloride.
In some embodiments, the first mixed liquor further comprises: and the dispersion liquid is used for improving the dispersibility of the carboxyl microsphere, and the dispersion liquid is a hydroxyl high-molecular polymer dispersing agent.
Further, the dispersion liquid includes: at least one of Tween 20, tween 60, tween 80, polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP).
Further, in the first mixed solution, the concentration of the dispersion is 0.04 to 0.2% by weight. Preferably, in the first mixture, the concentration of the dispersion is 0.08-0.15% wt.
Further, the carboxyl microsphere is mixed with the dispersion and an acidic pH adjustor, and then a catalyst is added.
The carboxyl microsphere is mixed with the dispersion liquid, so that the carboxyl microsphere is not easy to precipitate due to agglomeration, and the carboxyl microsphere can better resist activation of a high-concentration catalyst, thereby improving the reaction efficiency of the carboxyl microsphere and the catalyst.
In some embodiments, the catalyst is capable of reacting with a carboxyl group at a temperature of 8-50 ℃ to form an active ester or an amine-reactive ester.
Further, the catalyst comprises: a water-soluble carbodiimide salt, or/and an imide salt.
Preferably, the catalyst is a water-soluble carbodiimide salt and an imide salt, and in the first mixed solution, the mass percentage of the imide salt is greater than or equal to the mass percentage of the water-soluble carbodiimide salt. For example, the catalysts are 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), EDC reacting with carboxyl groups to form active esters; NHS reacts with the active ester to form the more active amine-reactive ester.
Further, the water-soluble carbodiimide salt includes: 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), or N, N Diisopropylcarbodiimide (DIC); the imide salt includes: n-hydroxysuccinimide (NHS), or N-hydroxysulfosuccinimide (Sulfo-NHS).
In some embodiments, the mass ratio of the carboxyl microsphere to the catalyst is 1:
(0.1-100)。
preferably, in the first mixed solution, the mass ratio of the carboxyl microsphere to the catalyst is 1: (10-40).
In some embodiments, the temperature of the first mixture is adjusted to 8-50 ℃ and maintained for 25-60 minutes, such that the carboxyl microspheres are activated.
Preferably, the temperature of the first mixed solution is adjusted to 35-40 ℃ and maintained for 30-40min, so that the carboxyl microsphere is activated.
The temperature of the activated carboxyl microsphere is 8-50 ℃, and if the temperature is lower than 8 ℃, the efficiency of the carboxyl on the surface of the carboxyl microsphere to react with a catalyst to generate the active ester is reduced; if the temperature is higher than 50 ℃, part of the carboxyl microsphere is difficult to bear the high temperature, and the fluorescent material is quenched in a fluorescence way or the magnetic material is demagnetized.
In some embodiments, the carboxyl microsphere is a microsphere having carboxyl groups on the surface, the microsphere comprising, from inside to outside: fluorescent material, and a shell layer coating the fluorescent material.
Further, the carboxyl is a carboxyl self-contained on the surface of the microsphere, or the carboxyl is a carboxyl modified on the surface of the microsphere.
Further, the fluorescent material includes: at least one of a fluorescent nanoparticle, a fluorescent polymer, and an organic fluorescent dye, the fluorescent nanoparticle comprising at least one of a quantum dot, a metal oxide nanoparticle, a nanorod, or a nanoplatelet.
The quantum dot comprises at least one of group IIB-VIA, group IIIA-VA, group IVA-VIA, group IVA, group IB-IIIA-VIA, group VIII-VIA, perovskite material and carbon quantum dot. For example, the II-VI compound may include: cdSe, cdTe, znS, znSe, znTe, znO, hgS, hgSe, hgTe, mgSe, mgS, cdSeS, cdSeTe, cdSTe, znSeS, znSeTe, znSTe, hgSeS, hgSeTe, hgSTe, cdZnS, cdZnSe, cdZnTe, cdHgS, cdHgSe, cdHgTe, hgZnS, hgZnSe, hgZnTe, mgZnSe, mgZnS, hgZnTeS, cdZnSeS, cdZnSeTe, cdZnSTe, cdHgSeS, cdHgSeTe, cdHgSTe, hgZnSeS, hgZnSeTe, hgZnSTe or combinations thereof. The III-V compounds may include: gaN, gaP, gaAs, gaSb, alN, alP, alAs, alSb, inN, inP, inAs, inSb, gaNP, gaNAs, gaNSb, gaPAs, gaPSb, alNP, alNAs, alNSb, alPAs, alPSb, inNP, inNAs, inNSb, inPAs, inPSb, inZnP, gaAlNP, gaAlNAs, gaAlNSb, gaAlPAs, gaAlPSb, gaInNP, gaInNAs, gaInNSb, gaInPAs, gaInPSb, inAlNP, inAlNAs, inAlNSb, inAlPAs, inAlPSb or combinations thereof. Quantum dots include nanocrystals having a homogeneous or substantially homogeneous composition, e.g., a core, as well as heterogeneous nanocrystals, such as core/shell quantum dots comprising a core and one or more shells surrounding the core. A shell is defined as a material surrounding a core and may include one or more shell layers. The metal oxide includes: zn, cr, co, dy, er, eu, fe, gd, gd, pr, nd, ni, in, pr, sm, tb, tm, and combinations thereof. The fluorescent polymer has a functional group (such as fluorescein) capable of emitting fluorescence and monomers capable of polymerizing, and the monomers are polymerized or polymerized with other monomers which do not contain fluorescence, so that the fluorescent polymer is prepared. The organic fluorescent dye includes: fluorescein (stilbenes, coumarin, fluoran, benzoxazole, naphthalimide, thiophene dicarboxylic acid amide, polycyclic aromatic hydrocarbon, perylene tetracarboxylic imide, etc.), aromatic condensed ring compounds, intramolecular charge transfer compounds, metal complex fluorescent materials, enzymes, rare earth metal chelates.
Further, the shell layer is formed by hydrolyzing and condensing inorganic matters, or is formed by condensing a resin precursor, or is formed by self-assembling amphiphilic polymer coated micro-droplets, or is formed by polymerizing vinyl monomers with carbon-carbon double bonds under the initiation action.
The shell layer is formed by hydrolyzing and condensing inorganic matters, and the formed inorganic shell layer comprises: a silica shell, a titania shell, or a zirconia shell.
The shell layer is formed by condensing a resin precursor, and the formed organic shell layer comprises: a urea-formaldehyde shell, a urea-paraformaldehyde shell, a melamine-formaldehyde shell, a melamine-paraformaldehyde shell, a urea-melamine-polyoxymethylene shell, or a benzomelamine-formaldehyde shell.
The shell layer is formed by self-assembling amphiphilic polymer coated micro-droplets. The amphiphilic polymer coats the oil-in-water/water-in-oil micro-droplets, and then the internal phase solvent of the micro-droplets is evaporated to self-assemble the amphiphilic polymer so that the amphiphilic polymer physically coats the fluorescent material. The amphiphilic polymer comprises: one of poly (stearyl maleate) (PMAO), poly (cetyl maleate), or poly (tetradecyl maleate). These microspheres have carboxyl groups on their surfaces.
The shell layer is polymerized by vinyl monomers with carbon-carbon double bonds under the initiation action. An organic polymer shell layer formed by polymerization reaction of vinyl monomers with carbon-carbon double bonds under the initiation of an initiator, illumination and the like. The vinyl monomer comprises: vinyl chloride, allyl ether, diethyl diallyl dicarboxylate, diallyl disulfide, methacrylic acid, hydroxyethyl methacrylate, hydroxypropyl methacrylate hydroxyethyl acrylate, hydroxypropyl acrylate, acrylamide, vinylpyrrolidone, acrylonitrile, vinyl acetate, maleic anhydride, itaconic acid, maleic anhydride, and the like styrene sulfonic acid, sodium vinylsulfonate, styrene, methyl styrene, vinyl styrene, methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, hexyl (meth) acrylate, octyl (meth) acrylate, nonyl (meth) acrylate, decyl (meth) acrylate, and undecyl (meth) acrylate, dodecyl (meth) acrylate, cyclohexyl (meth) acrylate, dicyclopentyl (meth) acrylate, lauryl (meth) acrylate, benzyl (meth) acrylate, phenyl (meth) acrylate, dicyclopentanyl (meth) acrylate (HDCPMA), cyclohexyl (meth) acrylate, 4-t-butylcyclohexyl (meth) acrylate, 3, 5-trimethylcyclohexyl (meth) acrylate, isobornyl (meth) acrylate, 1-adamantane (meth) acrylate (AMA), 2-adamantane (meth) acrylate, isooctyl (meth) acrylate, isononyl (meth) acrylate, at least one of 2-ethylhexyl (meth) acrylate, lauric acid (meth) acrylate (LMA), stearic acid (meth) acrylate, ethylene oxide, propylene oxide, butylene oxide, styrene oxide, methyl ethylene oxide, ethylene glycol di-propylene oxide, stilbene oxide, divinylbenzene, trivinyltoluene, divinylbenzene, diallyl diethyl phthalate, diallyl disulfide, cis-butadiene, iso-butadiene, isoprene, tripropylene glycol di (meth) acrylate, tetraethylene glycol dimethacrylate, dodecyl glycol ester, decyl glycol ester, tricyclodecanedimethanol acrylic acid, hexanediol diacrylate, pentaerythritol tri (meth) acrylate, dipentaerythritol tri (meth) acrylate, and trimethyltrimethylolpropane trimethacrylate.
The shell layer coats the fluorescent material and the magnetic material. The magnetic substance includes: ferroferric oxide, ferric oxide, nickel oxide, cobalt oxide, magnetite, ferric oleate, ferric chloride, ferric sulfate, ferric nitrate, ferrous chloride tetrahydrate, ferric chloride hexahydrate, nickel ferrite, aluminum ferrite, manganese ferrite, zinc ferrite, cobalt ferrite, coFe 2 O 4 、NiFe 2 O 4 Or MnFe 2 O 4 At least one of them.
The microsphere surface contains carboxyl, which is convenient for application in the biological field, on one hand, the carboxyl provides water solubility, and on the other hand, the carboxyl is used for coupling antibody. Some microspheres have carboxyl groups on their surface, i.e., the shell of the microsphere contains carboxyl groups (e.g., PMAO microsphere). If the surface of the microspheres is not provided with carboxyl or is provided with a small amount of carboxyl, the surface of the microspheres needs to be modified with carboxyl for making the microspheres water-soluble and convenient for coupling. Carboxyl groups are modified on the surfaces of the microspheres, and the prior art method is adopted.
In some embodiments, the PH of the second mixture is between 5 and 8. Preferably, the pH of the second mixed solution is 7.2-7.8.
Further, the second mixed solution further includes: alkaline pH regulator, alkaline pH regulator makes the pH of the second mixed solution 7.2-7.8.
The alkaline pH adjustor comprises: at least one of borax borate buffer solution, phosphate buffer solution, TRIS, sodium carbonate and sodium bicarbonate.
Further, the activated microsphere is mixed with an alkaline pH adjustor, followed by the addition of a hydroxyl ligand and a potential ligand.
In some embodiments, the amine group of the hydroxyl ligand replaces the active ester or amine-reactive ester, forming an amide bond to attach to the microsphere surface while providing a hydroxyl group.
Further, the hydroxyl ligand is an alcohol amine compound containing both primary amine or secondary amine and hydroxyl. The hydroxyl ligands include: at least one of diisopropanolamine, isopropanolamine, diethanolamine and ethanolamine.
The activated microsphere modifies the hydroxyl ligand so that the carboxyl part on the surface of the carboxyl microsphere is replaced by hydroxyl, on one hand, the hydroxyl can form a hydrogen bond with water molecules to form a hydration layer for protection, and the non-specific adsorption generated by hydrophobic adsorption is overcome, so that the non-specific adsorption is reduced. In the second aspect, the hydroxyl ligand has small molecular weight, and compared with the PEG ligand in the prior art, the biological microsphere has a simple surface structure and easily controlled grafting density. In the third aspect, the hydroxyl ligand of the present application is much cheaper and less expensive (2.5L requires 160 Yuan-ren-zen) than the expensive PEG ligand such as polyethylene glycol dimethacrylate (100 g requires about 1 Yuan-ren-zen). In the fourth aspect, the hydroxyl ligand modifies the surface of the biological microsphere, and can also improve the acid resistance and salt resistance of the biological microsphere. (since the carboxyl group-containing microspheres have relatively good alkali resistance, the carboxyl group-containing microspheres and the biological microspheres of the present application have excellent alkali resistance, and thus comparative comparison of alkali resistance was not performed in examples.)
In some embodiments, the potentiometric ligand is a carboxamide compound containing two amino groups and one carboxyl group.
Further, one amino group of the potential ligand replaces the active ester or amine-reactive ester, an amide bond is formed and is connected with the surface of the microsphere, and one amino group and one carboxyl group are remained for potential neutralization.
Further, the potential ligand includes: at least one of histidine, arginine and lysine.
Since all proteins possess positively charged amino acid residues and negatively charged amino acid residues randomly distributed on their surface, any positively or negatively charged material (e.g., carboxyl microspheres) will adsorb the protein very easily, and thus nonspecific adsorption will occur. The hydroxyl ligand replaces part of carboxyl on the surface of the carboxyl microsphere, and provides hydroxyl, and the hydroxyl is uncharged; and simultaneously, one amino group of the potential ligand replaces the carboxyl group on the surface of the other part of carboxyl microsphere, one amino group (positive charge) and one carboxyl group (negative charge) are remained, the potential is neutralized, the charge is zero, the potential neutralized carboxyl group is provided for the rear end coupling antibody, and the nonspecific adsorption of the biological microsphere is reduced.
In some embodiments, the activated microsphere is mixed with both the hydroxyl ligand and the potential ligand to form a second mixed solution.
Further, adding the activated microsphere, the hydroxyl ligand and the potential ligand into the second mixed solution, wherein the mass ratio of the activated microsphere to the hydroxyl ligand to the potential ligand is 1: (1-20): (0.005-20).
Preferably, in the second mixed solution, the mass ratio of the activated microsphere, the hydroxyl ligand and the potential ligand is 1: (8-12): (0.005-0.02). Under the proportion, the generated surface modified biological microsphere has high sensitivity when applied to antibody coupling.
Preferably, in the second mixed solution, the mass ratio of the activated microsphere, the hydroxyl ligand and the potential ligand is 1: (8-12): (8-12). Under the proportion, the generated surface modified biological microsphere has good linearity when applied to antibody coupling.
And under the condition of simultaneously adding the hydroxyl ligand and the potential ligand, the carboxyl content of the surface modified biological microsphere surface can be regulated by controlling the proportion of the added hydroxyl ligand and the potential ligand. Since the reaction speed of the activated microsphere with the hydroxyl ligand and the potential ligand is fast, for example, if the hydroxyl ligand is added first and then the potential ligand is added, the activated microsphere rapidly reacts with the hydroxyl ligand, and it is difficult to calculate and control the amount of the hydroxyl ligand and the potential ligand bound to the microsphere, respectively. But simultaneously adding the hydroxyl ligand and the potential ligand, and enabling the activated microsphere to react with the hydroxyl ligand and the potential ligand simultaneously, and controlling the carboxyl content on the surface of the final biological microsphere by controlling the proportion of the added hydroxyl ligand and the potential ligand through competitive balance between the hydroxyl ligand and the potential ligand, so as to meet the requirements (such as good sensitivity and good linearity) for various rear-end detection applications.
In some embodiments, the second mixed solution is reacted for 2-24 hours at a temperature of 30-50 ℃ under the protection of inert gas to produce the surface modified biological microsphere.
Preferably, the second mixed solution reacts for 10-18 hours at the temperature of 35-40 ℃ under the protection of inert gas to generate the surface modified biological microsphere.
The inert gas includes: one of nitrogen, helium, neon, argon, krypton, and xenon.
The inert gas such as nitrogen is introduced to prevent the fluorescent material or the magnetic material in the microsphere from being oxidized by oxygen under the heating condition, so that the performance of the microsphere is reduced.
In a second aspect of the present application, there is provided a surface-modified biological microsphere comprising: the microsphere comprises a microsphere body, a hydroxyl ligand and a potential ligand which are connected with the surface of the microsphere body.
In some embodiments, the microsphere comprises, from inside to outside: fluorescent material, and a shell layer coating the fluorescent material.
Further, the fluorescent material includes: at least one of a fluorescent nanoparticle, a fluorescent polymer, and an organic fluorescent dye, the fluorescent nanoparticle comprising at least one of a quantum dot, a metal oxide nanoparticle, a nanorod, or a nanoplatelet.
Further, the shell layer is formed by hydrolyzing and condensing inorganic matters, or is formed by condensing a resin precursor, or is formed by self-assembling amphiphilic polymer coated micro-droplets, or is formed by polymerizing vinyl monomers with carbon-carbon double bonds under the initiation action.
Further, the microsphere also comprises a magnetic material, and the shell layer coats the fluorescent material and the magnetic material.
In some embodiments, the hydroxyl ligand is an alcohol amine compound containing both a primary or secondary amine and a hydroxyl group. The hydroxyl ligands include: at least one of diisopropanolamine, isopropanolamine, diethanolamine and ethanolamine.
In some embodiments, the potentiometric ligand is a carboxamide compound containing two amino groups and one carboxyl group. The potential ligand comprises: at least one of histidine, arginine and lysine.
In a third aspect of the present application, there is provided a surface-modified biological microsphere obtained by the above preparation method.
In a fourth aspect of the present application, there is provided a kit comprising: the surface modified biological microsphere, the antibody and the blocking agent.
In some embodiments, the surface modified biological microspheres of the present application are conjugated to an antibody prior to blocking with a blocking agent. Antibodies are selected according to the antigen to be detected, and blocking agents are common in the prior art.
The surface-modified biological microsphere solves the problems of charge adsorption and hydrophobic adsorption, and can be simply coupled and blocked by an antibody at the rear end. The prior art microsphere is blocked after being connected with an antibody, and a blocking agent is usually macromolecular protein, so that the residual carboxyl on the surface of the microsphere is consumed and the blocking effect is achieved. However, in the first aspect, the reaction efficiency of the blocking agent is low, and only a part of the carboxyl groups on the surface of the microspheres can be consumed, so that the use amount of the coupling agent is increased or the reaction temperature is increased, and the blocking agent has a fatal influence on the antibody. In the second aspect, the blocking agent can reduce the nonspecific adsorption caused by charge adsorption, but the nonspecific adsorption caused by hydrophobic adsorption can only be solved at the synthesis modification end of the biological microsphere. The surface modified biological microsphere has only carboxyl neutralized by potential, the content of the carboxyl is controllable, and the carboxyl with corresponding content ratio is synthesized without influence of hydrophobic adsorption if a small amount of antibody needs to be coupled. After the biological microsphere is coupled with the antibody, only a little carboxyl is remained, and simple blocking is carried out, so that the nonspecific adsorption of a conjugate sample is greatly reduced.
In a fifth aspect of the present application, the use of the surface modified biological microsphere in a biological medicine, the biological medicine comprising: drug loading, biological probes, biological markers, disease diagnosis, biological tracers, solid phase chips, liquid phase chips, immunochromatography, raman scattering.
The present invention will be described in further detail with reference to specific examples and comparative examples, but the present invention is not limited to the following examples, and the implementation conditions adopted in the examples may be further adjusted according to different requirements of specific use, and the conditions not specified are conventional conditions in the industry.
Example 1:
step S1: activation of carboxyl microsphere
50mg of carboxyl microsphere (PMAO shell layer coated CdSe quantum dot microsphere with carboxyl on the surface) is taken and dissolved in 10ml of MES (2- (N-methyl-4-pyrrolidinyl) ethanesulfonic acid sodium salt with tween 20 content of 0.1% prepared in advance and 50mMol concentration) buffer water solution, and the PH is 5-6. 500mg of EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and 1000mg of NHS (N-hydroxysuccinimide) were added, activated for 30min in a water bath at 37℃and centrifuged to obtain activated microspheres.
Step S2: obtaining the surface-modified biological microsphere
Adjusting the pH of the activated microsphere obtained in the step S1 to 7.2-7.8 by adopting borax buffer solution, and then adding 500mg of diethanolamine and 500mg of histidine (1:1) simultaneously; the reaction was carried out under nitrogen atmosphere at 40℃for 14h. And centrifuging after the reaction is finished, washing with deionized water for three times, centrifuging to obtain the surface-modified biological microsphere, and dissolving in deionized water for later use.
Example 2:
step S1: activation of carboxyl microsphere
50mg of carboxyl microsphere (PMAO shell layer coated CdSe quantum dot microsphere with carboxyl on the surface) is taken and dissolved in 10ml of MES (2- (N-methyl-4-pyrrolidinyl) ethanesulfonic acid sodium salt with tween 20 content of 0.1% prepared in advance and 50mMol concentration) buffer water solution, and the PH is 5-6. 500mg of EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and 1000mg of NHS (N-hydroxysuccinimide) were added, activated for 30min in a water bath at 37℃and centrifuged to obtain activated microspheres.
Step S2: obtaining the surface-modified biological microsphere
Adjusting the pH of the activated microsphere obtained in the step S1 to 7.2-7.8 by adopting borax buffer solution, and then adding 500mg of diethanolamine and 50mg of histidine (10:1) simultaneously; the reaction was carried out under nitrogen atmosphere at 40℃for 14h. And centrifuging after the reaction is finished, washing with deionized water for three times, centrifuging to obtain the surface-modified biological microsphere, and dissolving in deionized water for later use.
Example 3:
step S1: activation of carboxyl microsphere
50mg of carboxyl microsphere (PMAO shell layer coated CdSe quantum dot microsphere with carboxyl on the surface) is taken and dissolved in 10ml of MES (2- (N-methyl-4-pyrrolidinyl) ethanesulfonic acid sodium salt with tween 20 content of 0.1% prepared in advance and 50mMol concentration) buffer water solution, and the PH is 5-6. 500mg of EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and 1000mg of NHS (N-hydroxysuccinimide) were added, activated for 30min in a water bath at 37℃and centrifuged to obtain activated microspheres.
Step S2: obtaining the surface-modified biological microsphere
Adjusting the pH of the activated microsphere obtained in the step S1 to 7.2-7.8 by adopting borax buffer solution, and then adding 500mg of diethanolamine and 5mg of histidine (100:1) simultaneously; the reaction was carried out under nitrogen atmosphere at 40℃for 14h. And centrifuging after the reaction is finished, washing with deionized water for three times, centrifuging to obtain the surface-modified biological microsphere, and dissolving in deionized water for later use.
Example 4:
step S1: activation of carboxyl microsphere
50mg of carboxyl microsphere (PMAO shell layer coated CdSe quantum dot microsphere with carboxyl on the surface) is taken and dissolved in 10ml of MES (2- (N-methyl-4-pyrrolidinyl) ethanesulfonic acid sodium salt with tween 20 content of 0.1% prepared in advance and 50mMol concentration) buffer water solution, and the PH is 5-6. 500mg of EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and 1000mg of NHS (N-hydroxysuccinimide) were added, activated for 30min in a water bath at 37℃and centrifuged to obtain activated microspheres.
Step S2: obtaining the surface-modified biological microsphere
Adjusting the pH of the activated microsphere obtained in the step S1 to 7.2-7.8 by adopting borax buffer solution, and then adding 500mg of diethanolamine and 0.5mg of histidine (1000:1) simultaneously; the reaction was carried out under nitrogen atmosphere at 40℃for 14h. And centrifuging after the reaction is finished, washing with deionized water for three times, centrifuging to obtain the surface-modified biological microsphere, and dissolving in deionized water for later use.
Example 5:
step S1: activation of carboxyl microsphere
50mg of carboxyl microsphere (CdSe quantum dot microsphere is coated by a silicon dioxide shell layer, then polyaspartic acid is modified on the surface of the microsphere to enable carboxyl groups to be formed), and the microsphere is dissolved in 10ml of MES (2- (N-methyl-4-pyrrolidinyl) ethanesulfonic acid sodium salt with the tween 20 content of 0.1% which is prepared in advance, and the concentration of 50 mMol) buffer water solution, wherein the PH is 5-6. 500mg of EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and 1000mg of NHS (N-hydroxysuccinimide) were added, activated for 30min in a water bath at 37℃and centrifuged to obtain activated microspheres.
Step S2: obtaining the surface-modified biological microsphere
Adjusting the pH of the activated microsphere obtained in the step S1 to 7.2-7.8 by adopting borax buffer solution, and then adding 500mg of diethanolamine and 500mg of histidine (1:1) simultaneously; the reaction was carried out under nitrogen atmosphere at 40℃for 14h. And centrifuging after the reaction is finished, washing with deionized water for three times, centrifuging to obtain the surface-modified biological microsphere, and dissolving in deionized water for later use.
Example 6:
step S1: activation of carboxyl microsphere
50mg of carboxyl microsphere (CdSe quantum dot microsphere is coated by a silicon dioxide shell layer, then polyaspartic acid is modified on the surface of the microsphere to enable carboxyl groups to be formed), and the microsphere is dissolved in 10ml of MES (2- (N-methyl-4-pyrrolidinyl) ethanesulfonic acid sodium salt with the tween 20 content of 0.1% which is prepared in advance, and the concentration of 50 mMol) buffer water solution, wherein the PH is 5-6. 500mg of EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and 1000mg of NHS (N-hydroxysuccinimide) were added, activated for 30min in a water bath at 37℃and centrifuged to obtain activated microspheres.
Step S2: obtaining the surface-modified biological microsphere
Adjusting the pH of the activated microsphere obtained in the step S1 to 7.2-7.8 by adopting borax buffer solution, and then adding 500mg of diethanolamine and 50mg of histidine (10:1) simultaneously; the reaction was carried out under nitrogen atmosphere at 40℃for 14h. And centrifuging after the reaction is finished, washing with deionized water for three times, centrifuging to obtain the surface-modified biological microsphere, and dissolving in deionized water for later use.
Example 7:
step S1: activation of carboxyl microsphere
50mg of carboxyl microsphere (CdSe quantum dot microsphere is coated by a silicon dioxide shell layer, then polyaspartic acid is modified on the surface of the microsphere to enable carboxyl groups to be formed), and the microsphere is dissolved in 10ml of MES (2- (N-methyl-4-pyrrolidinyl) ethanesulfonic acid sodium salt with the tween 20 content of 0.1% which is prepared in advance, and the concentration of 50 mMol) buffer water solution, wherein the PH is 5-6. 500mg of EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and 1000mg of NHS (N-hydroxysuccinimide) were added, activated for 30min in a water bath at 37℃and centrifuged to obtain activated microspheres.
Step S2: obtaining the surface-modified biological microsphere
Adjusting the pH of the activated microsphere obtained in the step S1 to 7.2-7.8 by adopting borax buffer solution, and then adding 500mg of diethanolamine and 5mg of histidine (100:1) simultaneously; the reaction was carried out under nitrogen atmosphere at 40℃for 14h. And centrifuging after the reaction is finished, washing with deionized water for three times, centrifuging to obtain the surface-modified biological microsphere, and dissolving in deionized water for later use.
Example 8:
step S1: activation of carboxyl microsphere
50mg of carboxyl microsphere (CdSe quantum dot microsphere is coated by a silicon dioxide shell layer, then polyaspartic acid is modified on the surface of the microsphere to enable carboxyl groups to be formed), and the microsphere is dissolved in 10ml of MES (2- (N-methyl-4-pyrrolidinyl) ethanesulfonic acid sodium salt with the tween 20 content of 0.1% which is prepared in advance, and the concentration of 50 mMol) buffer water solution, wherein the PH is 5-6. 500mg of EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and 1000mg of NHS (N-hydroxysuccinimide) were added, activated for 30min in a water bath at 37℃and centrifuged to obtain activated microspheres.
Step S2: obtaining the surface-modified biological microsphere
Adjusting the pH of the activated microsphere obtained in the step S1 to 7.2-7.8 by adopting borax buffer solution, and then adding 500mg of diethanolamine and 0.5mg of histidine (1000:1) simultaneously; the reaction was carried out under nitrogen atmosphere at 40℃for 14h. And centrifuging after the reaction is finished, washing with deionized water for three times, centrifuging to obtain the surface-modified biological microsphere, and dissolving in deionized water for later use.
Comparative example 1:
50mg of carboxyl microsphere (PMAO shell layer coated CdSe quantum dot microsphere, the surface of which is provided with carboxyl) is taken and dissolved in deionized water for standby.
Comparative example 2:
taking 50mg of carboxyl microsphere (a CdSe quantum dot microsphere coated by a silicon dioxide shell layer, and then modifying polyaspartic acid on the surface of the microsphere to enable the microsphere to have carboxyl), and dissolving the microsphere in deionized water for standby.
Acid resistance test:
the standby solutions obtained in examples 1 to 8 and comparative examples 1 to 2 above were centrifuged to obtain surface-modified biological microspheres and carboxyl microspheres, 1mg each, and the biological microspheres and carboxyl microspheres were dissolved in 10ml of phosphate-citric acid buffer solution having a pH of 3, and the toleration of the biological microspheres and carboxyl microspheres was observed, as shown in Table 1.
Salt tolerance test:
the standby solutions obtained in examples 1 to 8 and comparative examples 1 to 2 were centrifuged to obtain surface-modified biological microspheres and carboxyl microspheres, 1mg each, and the biological microspheres and carboxyl microspheres were dissolved in 10ml of saturated saline, and the toleration of the biological microspheres and carboxyl microspheres was observed as shown in Table 1.
And (3) potential detection:
the standby solutions obtained in examples 1 to 8 and comparative examples 1 to 2 were centrifuged to obtain surface-modified biological microspheres and carboxyl microspheres, and the potentials were measured by a Markov particle size meter, and the potential readings are shown in Table 1.
Table 1: test results of acid resistance, salt resistance and potential of example biological microspheres and comparative example carboxyl microspheres
Type(s) Acid resistance Salt tolerance Potential (mV)
Example 1 Does not precipitate Precipitation for 1h -40
Example 2 Does not precipitate 3h precipitation -32
Example 3 Does not precipitate Precipitation for 6 hours -26
Example 4 Does not precipitate Precipitation for 6 hours -24
Comparative example 1 Precipitation for 1min Precipitation by addition -48
Example 5 Does not precipitate Precipitation for 0.5h -44
Example 6 Does not precipitate Precipitation for 2 hours -36
Example 7 Does not precipitate 3h precipitation -30
Example 8 Does not precipitate Precipitation for 4 hours -27
Comparative example 2 Precipitation for 1min Precipitation by addition -52
As can be seen from Table 1, the biological microspheres of examples 1-8 of the present application have significantly improved acid resistance and salt resistance compared to the carboxyl microspheres of the prior art of comparative documents 1-2. The biological microspheres of examples 1-4 have significantly lower negative charges than comparative example 1 and the biological microspheres of examples 5-8 have significantly lower negative charges than comparative example 2, indicating that the biological microspheres of the present application have significantly reduced amounts of charge and thus reduced non-specific adsorption.
Antibody coupling validation:
the standby solutions obtained in examples 1 to 8 and comparative examples 1 to 2 above were centrifuged to obtain surface-modified biological microspheres and carboxyl microspheres, which were subjected to antibody coupling verification using immunochromatographic test paper. The verification method adopts a double antibody sandwich method, the labeled antibody adopts PCT-REAB-G1-016 of the Phpeng biological Co., ltd, and the antigen adopts PCT-Ag1 of the Phpeng biological Co., ltd. The immunochromatographic test paper is prepared, PCT-REAB-G1-015 of the biological Co., ltd. Of the Phpeng is selected as a T line (detection line) synovial membrane antibody, goat Anti-Mouse IgG of the biological Co., ltd. Of the Soilebolo is selected as a C line (quality control line) synovial membrane antibody, and the antibody coupling is completed as a conjugate sample in examples and comparative examples and is added into a conjugate pad to complete the preparation of the immunochromatographic test paper. The specific procedures for antibody coupling in examples and comparative examples are as follows:
S1, taking 0.25mg of each biological microsphere or carboxyl microsphere; then 225uL of MES (2- (N-methyl-4-pyrrolidinyl) ethanesulfonic acid sodium salt, 0.05Mol concentration) was added and the mixture was sonicated at pH 6.0 and 100W for 5min. EDC with the concentration of 2.5uL of 10mg/mL and NHS with the concentration of 5uL of 10mg/mL are added, vortex 10s is fully mixed, then the mixture is put into a vibrating constant temperature metal bath for vibrating for 30min at the temperature of 25 ℃, and the supernatant is removed by centrifugation. 250uL of BB buffer (borate buffer, pH 7.8) at a concentration of 0.05M was added and sonicated at 100W for 5min.
S2, in S1, adding 20ug of antibody (Fei Peng PCT-REAB-G1-016), swirling for 10S, placing into a shaking constant temperature metal bath for shaking at 25 ℃, coupling for 2h, centrifuging, and removing supernatant. 250uL of BB buffer (pH 7.8) at a concentration of 0.05M was added and sonicated at 100W for 5min.
S3, in S2, adding 50uL of 5% casein solution, swirling for 10S, then placing into a vibrating constant temperature metal bath, vibrating at 25 ℃, sealing for 2h, centrifuging to remove supernatant, taking the supernatant as a conjugate sample, and adding the conjugate sample into a conjugate pad to finish the preparation of the immunochromatographic test paper.
Nonspecific adsorption detection:
and (3) adding detection liquid with the concentration of antigen (Fei Peng PCT-Ag 1) of zero into a sample adding port of the immunochromatographic test paper, standing for 15min, and reading a T line by using a dry type fluorescence immunoassay instrument (Guangzhou blue Bo biotechnology Co., ltd.) to obtain a reading result shown in table 2.
Table 2: examples and comparative examples antibody coupling validation of T-line readings at zero antigen concentration
Type(s) 0 concentration antigen T line reading
Example 1 1305
Example 2 168
Example 3 23
Example 4 7
Comparative example 1 2034
Example 5 1568
Example 6 304
Example 7 45
Example 8 6
Comparative example 2 3136
Examples and comparative examples antibody coupling validation was performed, where the antigen concentration of the test solution added at the sample port was zero, i.e., no antigen, and where there was no non-specific adsorption, the reading on the T-line should be zero. At this time, the higher the reading of the T line, the more severe the nonspecific adsorption. As can be seen from Table 2, the biological microspheres of examples 1-8 were subjected to antibody coupling validation, and their nonspecific adsorption was significantly reduced relative to comparative examples 1-2.
And (3) applicability detection:
and respectively adding detection solutions with concentration gradient dilution of antigen (Fei Peng PCT-Ag 1) into the sample adding ports of the immunochromatographic test paper, standing for 15min, adopting a dry type fluorescence immunoassay instrument (Guangzhou blue Bo biotechnology Co., ltd.) to read the T line and the C line, and dividing the T line reading by the C line reading (T/C) to calculate the ratio.
Examples 1-4T/C ratios at antigen concentrations of 0ng/mL, 0.3ng/mL, 0.5ng/mL, 0.7ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 20ng/mL are shown in Table 3, and a linear graph prepared according to Table 3 is shown in FIG. 1.
Examples 5-8T/C ratios at antigen concentrations of 0ng/mL, 0.3ng/mL, 0.5ng/mL, 0.7ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 20ng/mL are shown in Table 4, and a linear graph prepared according to Table 4 is shown in FIG. 2.
Table 3: examples 1-4 antibody coupling validation of T/C ratio results at antigen gradient concentrations
Table 4: examples 5-8 antibody coupling validation of T/C ratio results at antigen gradient concentrations
From FIGS. 1 and 2, as the hydroxyl content of the surface of the biological microsphere is higher (i.e., the feeding ratio of the hydroxyl ligand to the potential ligand is increased, and the ratio of the hydroxyl ligand is increased), the detection sensitivity is higher (the property of the antigen detected at a low concentration), i.e., the sensitivity at the low value end of the antigen concentration is gradually increased. As the hydroxyl ligands of the present application: the lower the charge ratio of the potential ligand (less the ratio of hydroxyl ligand), the greater the correlation coefficient of the curve, the better the linearity, which means that the greater the detection range of the conjugate sample. The technical scheme of the application is verified, the carboxyl content and detection performance of the surface of the biological microsphere can be fully regulated, and the requirements of detection application of various rear ends are met. Some projects pay attention to good linearity in back-end application, and the required detection range is large; some projects focus on sensitivity, requiring that low concentrations of antigen also be sensitive; some items focus on the combination of both, and the present application can be satisfied.
While various aspects and embodiments have been disclosed, other aspects and embodiments will be apparent to those skilled in the art, and many changes and modifications can be made without departing from the spirit of the application, which is intended to be within the scope of the invention. The various aspects and embodiments disclosed herein are for illustration only and are not intended to limit the application, the actual scope of which is subject to the claims.

Claims (10)

1. A method for preparing surface-modified biological microspheres, which is characterized by comprising the following steps:
s1, activating carboxyl microspheres: mixing carboxyl microspheres with a catalyst to form a first mixed solution, and reacting carboxyl on the surfaces of the microspheres with the catalyst to generate active ester or amine-reactive ester to obtain activated microspheres;
s2, mixing the activated microsphere with a hydroxyl ligand and a potential ligand to form a second mixed solution, wherein the hydroxyl ligand and the potential ligand replace the active ester or the amine reactive ester and are connected with the surface of the microsphere to obtain the surface-modified biological microsphere.
2. The method of preparing surface modified biological microspheres according to claim 1, comprising one or more features selected from the group consisting of:
(1) The PH of the first mixed solution is 5-8; in the first mixed solution, the concentration of the carboxyl microsphere is 2-10mg/ml;
(2) The first mixed liquid further comprises: a dispersion liquid, wherein the dispersion liquid is used for improving the dispersibility of carboxyl microspheres, and the dispersion liquid is a hydroxyl high polymer dispersing agent; in the first mixed solution, the concentration of the dispersion liquid is 0.04-0.2%wt;
(3) Mixing carboxyl microspheres with the dispersion liquid and an acidic pH regulator, and then adding a catalyst;
(4) Regulating the temperature of the first mixed solution to 8-50 ℃ and keeping the temperature for 25-60min to activate the carboxyl microsphere;
(5) The carboxyl microsphere is a microsphere with carboxyl on the surface, and the microsphere comprises the following components from inside to outside: a fluorescent material, and a shell layer coating the fluorescent material; the carboxyl is the carboxyl of the microsphere surface, or the carboxyl is the carboxyl of the microsphere surface modification.
3. The method of preparing surface modified biological microspheres according to claim 1, wherein the catalyst is capable of reacting with carboxyl groups to form active esters or amine-reactive esters at 8-50 ℃;
preferably, the catalyst comprises: a water-soluble carbodiimide salt, or/and an imide salt;
Preferably, the water-soluble carbodiimide salt includes: 1-ethyl- (3-dimethylaminopropyl) carbodiimide, or N, N-diisopropylcarbodiimide; the imide salt includes: n-hydroxysuccinimide, or N-hydroxysulfosuccinimide;
preferably, in the first mixed solution, the mass ratio of the carboxyl microsphere to the catalyst is 1: (0.1-100).
4. The method of preparing surface-modified biological microspheres according to claim 1, wherein the amine groups of the hydroxyl ligands replace the active ester or amine-reactive ester to form amide bonds to connect with the microsphere surface while providing hydroxyl groups;
preferably, the hydroxyl ligand is an alcohol amine compound containing both primary amine or secondary amine and hydroxyl;
preferably, the hydroxyl ligand includes: at least one of diisopropanolamine, isopropanolamine, diethanolamine and ethanolamine.
5. The method for preparing the surface-modified biological microsphere according to claim 1, wherein the potential ligand is a carboxamide compound containing two amino groups and one carboxyl group;
preferably, the potential ligand comprises: at least one of histidine, arginine and lysine.
6. The method of preparing surface modified biological microspheres according to claim 1, wherein the activated microspheres are mixed with a hydroxyl ligand and a potential ligand simultaneously to form a second mixed solution;
preferably, in the second mixed solution, the mass ratio of the activated microsphere, the hydroxyl ligand and the potential ligand is 1: (1-20): (0.005-20);
preferably, in the second mixed solution, the mass ratio of the activated microsphere, the hydroxyl ligand and the potential ligand is 1: (8-12): (0.005-0.02); preferably, in the second mixed solution, the mass ratio of the activated microsphere, the hydroxyl ligand and the potential ligand is 1: (8-12): (8-12).
7. A surface-modified biological microsphere, the biological microsphere comprising: the microsphere comprises a microsphere body, a hydroxyl ligand and a potential ligand which are connected with the surface of the microsphere body.
8. The surface modified biological microsphere of claim 7, comprising one or more characteristics selected from the group consisting of:
(1) The microsphere comprises the following components from inside to outside: a fluorescent material, and a shell layer coating the fluorescent material;
(2) The hydroxyl ligand is an alcohol amine compound containing primary amine or secondary amine and hydroxyl; the hydroxyl ligands include: at least one of diisopropanolamine, isopropanolamine, diethanolamine, and ethanolamine;
(3) The potential ligand is a carboxamide compound containing two amino groups and one carboxyl group, and the potential ligand comprises: at least one of histidine, arginine and lysine.
9. A surface-modified biological microsphere, characterized in that it is obtained by the preparation method according to any one of claims 1 to 6.
10. A kit, the kit comprising: surface-modified biological microspheres obtained by the production process according to any one of claims 1 to 6 or surface-modified biological microspheres, antibodies and blocking agents according to any one of claims 7 to 8.
CN202311776637.5A 2023-12-22 2023-12-22 Surface-modified biological microsphere, kit and preparation method thereof Pending CN117772082A (en)

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