CN117491648A - Human body autoantibody detection material and preparation method thereof - Google Patents
Human body autoantibody detection material and preparation method thereof Download PDFInfo
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Abstract
The invention provides a human body autoantibody detection material and a preparation method thereof, belonging to the technical field of medical detection. The detection material is formed by mixing coded microspheres with a multi-layer composite structure and a composite detection with a specific recognition function. Wherein the encoded microsphere comprises a magnetic core, a spacing layer, a nanocrystal fluorescent layer and a biological recognition layer, and realizes stable fluorescent encoding. The composite detector contains a fluorescent dye providing a second code. The combination of the two kinds of fluorescence realizes hybrid coding and enlarges the coding space. Meanwhile, a method for preparing the detection material is provided, and the detectable effect of the codes is ensured by controlling the fluorescent layer of the coded microspheres. Finally, by using the detection material, the accurate high-flux detection of the human autoantibody can be effectively realized.
Description
Technical Field
The invention belongs to the technical field of medical detection, and particularly relates to a human body autoantibody detection material and a preparation method thereof.
Background
Autoantibodies are an important class of biomarkers whose abnormal levels often indicate the presence of an imbalance in the immune system or a particular disease in the body. In order to discover autoimmune diseases early, development of a rapid and efficient autoantibody detection kit is not easy. The traditional immunological detection reagent and the analysis method have various defects such as complex operation, low detection flux and the like, and the accurate high-flux detection of the autoantibody is difficult to realize. Therefore, the development of the novel autoantibody detection kit has important clinical application value.
The encoding microsphere technology brings new opportunities for biological detection. The specific biological recognition elements on the surfaces of different coded microspheres are utilized to generate specific combination with target biological elements, different targets are distinguished according to microsphere codes, and high-flux parallel detection of multiple targets is realized. The encoded microspheres may carry different types of encoded information, such as fluorescent encoding, shape encoding, magnetic encoding, and the like. However, the conventional coding microsphere technology also has certain limitations, such as poor coding stability, limited coding combination number and the like.
Referring to the published related document, the technical scheme with the publication number of CN112147324A provides a kit with a specific carrier, and the specific carrier is combined with cytokinin-3 in a sample, so that the detection of the target antibody is realized; the technical proposal with publication number US10794906B2 proposes an assay for the presence or level of neutralizing and non-neutralizing autoantibodies carried in biological agents, useful for monitoring the level of formation of neutralizing and/or non-neutralizing anti-drug antibodies over time when a subject is subjected to biological therapy; the technical proposal of the publication No. EP4119575A1 proposes a monoclonal antibody or antigen binding fragment thereof for detecting SARS-COV-2 antibody, which realizes the rapid and high-accuracy detection of the antibody.
The technical schemes above all provide a plurality of technical schemes for detecting antibodies in human bodies, but the schemes for detecting high-flux antibodies are less proposed at present, and a larger lifting space is provided.
The foregoing discussion of the background art is intended to facilitate an understanding of the present invention only. This discussion is not an admission or admission that any of the material referred to was common general knowledge.
Disclosure of Invention
The invention aims to provide a human autoantibody detection material and a preparation method thereof, belonging to the technical field of medical detection. The detection material is formed by mixing coded microspheres with a multi-layer composite structure and a composite detection with a specific recognition function. Wherein the encoded microsphere comprises a magnetic core, a spacing layer, a nanocrystal fluorescent layer and a biological recognition layer, and realizes stable fluorescent encoding. The composite detector contains a fluorescent dye providing a second code. The combination of the two kinds of fluorescence realizes hybrid coding and enlarges the coding space. Meanwhile, a method for preparing the detection material is provided, and the detectable effect of the codes is ensured by controlling the fluorescent layer of the coded microspheres. Finally, by using the detection material, the accurate high-flux detection of the human autoantibody can be effectively realized.
The invention adopts the following technical scheme:
a human autoantibody detection material, the detection material comprising:
a set of one or more combinatorial agents; each set of said combinatorial agents comprises: encoding the microsphere and the composite detector;
the structure of the coding microsphere is from inside to outside, and the coding microsphere comprises:
the magnetic core comprises high-molecular polymer microspheres and magnetic nano particles attached to the surfaces of the high-molecular polymer microspheres;
a spacer layer formed alternately of one or more pairs of polyelectrolyte layers having positive and negative charges;
a nanocrystal layer composed of nanocrystals having a specified fluorescent color;
a biological capture molecule coated on the outermost layer of the encoded microsphere for specifically binding to a designated antibody protein molecule;
the complex detector has a fluorescent dye and a second biological capture molecule bound to the fluorescent dye; the second biological capture molecule is the same as the biological capture molecule possessed by the encoded microsphere;
wherein the absorption spectrum of the fluorescent dye of the composite detector is consistent with the absorption spectrum of the nanocrystal layer of the encoded microsphere; the emission spectrum of the fluorescent dye of the composite detector is inconsistent with the emission spectrum of the nanocrystal layer of the encoded microsphere;
preferably, the magnetic nano-particles are superparamagnetic nano-particles, and the size of the magnetic nano-particles is 10-50 nm;
preferably, the preparation of the magnetic nanoparticles comprises mixing long-chain fatty acid, medium-short-chain fatty acid and original magnetic nanoparticles and heating the mixture together to obtain the molded magnetic nanoparticles;
preferably, the nanocrystals employed in the nanocrystal layer are semiconductor nanocrystals;
preferably, the diameter of the encoded microsphere is 10-200 μm;
preferably, the negatively charged polyelectrolyte layer is composed of a polyacid and/or a polyacrylate; the positively charged polyelectrolyte layer is composed of a polybasic and/or polybasic salt;
furthermore, a preparation method of the human body autoantibody detection material is provided; the preparation method is used for preparing the human body autoantibody detection material; the preparation method comprises two parts of preparation of the coded microsphere and preparation of the composite detector;
preferably, the preparation of the encoded microsphere comprises the following steps:
s100: mixing long-chain fatty acid, medium-chain fatty acid and short-chain fatty acid with original magnetic nano particles, and heating to obtain molded magnetic nano particles;
s200: dissolving the formed magnetic nano particles and high molecular polymer microspheres in a solvent to prepare a first mixed solution; dehydrating and cooling the first mixed solution to obtain a magnetic core;
s300: alternately coating a pair of polyelectrolyte layers with positive charges and more than one polyelectrolyte layer with negative charges on the surface of the magnetic core in sequence to form a spacing layer on the surface of the magnetic core;
s400: attaching the microsphere prepared in the step S300 to the nanocrystal to form a nanocrystal layer;
s500: coating biological capture molecules on the surfaces of the microspheres with the nanocrystal layers;
preferably, the preparation of the composite detector comprises the following steps:
e100: selecting a proper biological recognition molecule, and preparing a fluorescent dye solution by using an organic fluorescent dye with emission wavelength different from that of fluorescent nanocrystals of the encoded microspheres;
e200: mixing a second biological capture molecule with the fluorescent dye solution to prepare a mixed solution;
e300: adding a cross-linking agent into the mixed solution obtained in the step E200, connecting a second biological capture molecule and a fluorescent dye in a covalent cross-linking mode, and reacting to generate a fluorescent-labeled biological recognition molecule compound;
e400: centrifuging and washing to remove unreacted fluorescent dye and cross-linking agent, thus obtaining a composite detector;
preferably, in step S400, the volume V of the desired nanocrystals is calculated by the following calculation formula;
wherein h is the ideal coating thickness of the nanocrystals on the surface of the encoded microsphere, which is set by the relevant technicians according to the needs; n is the statistical quantity of the coded microspheres prepared at this time, S is the statistical surface area of the microspheres, phi is the volume fraction of the nanocrystals, and rho is the material density of the nanocrystals; n and S are obtained from factory data provided by the manufacturer of the microspheres; phi is obtained by sampling and detecting the nanocrystals; k is the redundancy factor, which is determined and set by the relevant technician by using the current nanocrystal to perform the post-experiment coating effect.
The beneficial effects obtained by the invention are as follows:
1. the detection material of the technical scheme has high specificity for antibody recognition, and can realize high affinity and specificity combination with the corresponding targeting antibody through the specific biological capture molecules connected with the surface of the coded microsphere, so that the detection accuracy is ensured;
2. the detection material of the technical scheme has large coding space and can form rich coding combinations; the combination of fluorescent nanocrystal codes on the surfaces of the coded microspheres and fluorescent dye codes in the composite detector realizes multi-dimensional mixed codes and greatly expands possible combinations of the codes;
3. the detection material of the technical scheme can realize the single or combined detection of various antibodies by matching with the biological capture molecules suitable for various antibodies, and can be widely used for producing mass production products suitable for various antibodies.
Drawings
The invention will be further understood from the following description taken in conjunction with the accompanying drawings. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the embodiments. Like reference numerals designate corresponding parts throughout the different views.
Reference numerals illustrate: 10-encoded microspheres; 12-a magnetic core; 14-a positively charged polyelectrolyte layer; 16-a negatively charged polyelectrolyte layer; 18-a nanocrystal layer; 20-a biological capture molecule; 22-fluorescent dye; 24-a second biological capture molecule;
FIG. 1 is a schematic diagram of the structure of a coded microsphere according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of the surface layer structure of the encoded microsphere according to an embodiment of the present invention;
FIG. 3 is a schematic diagram showing a combination structure of a fluorescent dye and a second biological capture molecule according to an embodiment of the invention.
Detailed Description
In order to make the technical scheme and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the following examples thereof; it should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. Other systems, methods, and/or features of the present embodiments will be or become apparent to one with skill in the art upon examination of the following detailed description. It is intended that all such additional systems, methods, features and advantages be included within this description. Included within the scope of the invention and protected by the accompanying claims. Additional features of the disclosed embodiments are described in, and will be apparent from, the following detailed description.
The same or similar reference numbers in the drawings of embodiments of the invention correspond to the same or similar components; in the description of the present invention, it should be understood that, if any, the terms "upper," "lower," "left," "right," and the like indicate an orientation or a positional relationship based on the orientation or the positional relationship shown in the drawings, this is for convenience of description and simplification of the description, and does not indicate or imply that the apparatus or component to be referred to must have a specific orientation. The terms describing the positional relationship in the drawings are merely for illustrative purposes and are not to be construed as limiting the present patent, and specific meanings of the terms are understood by those of ordinary skill in the art according to specific circumstances.
Embodiment one: illustratively, a human autoantibody detection material is presented, the detection material comprising:
a set of one or more combinatorial agents; each set of said combinatorial agents comprises: encoding the microsphere and the composite detector;
the structure of the coding microsphere is from inside to outside, and the coding microsphere comprises:
the magnetic core comprises high-molecular polymer microspheres and magnetic nano particles attached to the surfaces of the high-molecular polymer microspheres;
a spacer layer formed alternately of one or more pairs of polyelectrolyte layers having positive and negative charges;
a nanocrystal layer composed of nanocrystals having a specified fluorescent color;
a biological capture molecule coated on the outermost layer of the encoded microsphere for specifically binding to a designated antibody protein molecule;
the complex detector has a fluorescent dye and a second biological capture molecule bound to the fluorescent dye; the second biological capture molecule is the same as the biological capture molecule possessed by the encoded microsphere;
wherein the absorption spectrum of the fluorescent dye of the composite detector is consistent with the absorption spectrum of the nanocrystal layer of the encoded microsphere; the emission spectrum of the fluorescent dye of the composite detector is inconsistent with the emission spectrum of the nanocrystal layer of the encoded microsphere;
preferably, the magnetic nano-particles are superparamagnetic nano-particles, and the size of the magnetic nano-particles is 10-50 nm;
preferably, the preparation of the magnetic nanoparticles comprises mixing long-chain fatty acid, medium-short-chain fatty acid and original magnetic nanoparticles and heating the mixture together to obtain the molded magnetic nanoparticles;
preferably, the nanocrystals employed in the nanocrystal layer are semiconductor nanocrystals;
preferably, the diameter of the encoded microsphere is 10-200 μm;
preferably, the negatively charged polyelectrolyte layer is composed of a polyacid and/or a polyacrylate; the positively charged polyelectrolyte layer is composed of a polybasic and/or polybasic salt;
furthermore, a preparation method of the human body autoantibody detection material is provided; the preparation method is used for preparing the human body autoantibody detection material; the preparation method comprises two parts of preparation of the coded microsphere and preparation of the composite detector;
preferably, the preparation of the encoded microsphere comprises the following steps:
s100: mixing long-chain fatty acid, medium-chain fatty acid and short-chain fatty acid with original magnetic nano particles, and heating to obtain molded magnetic nano particles;
s200: dissolving the formed magnetic nano particles and high molecular polymer microspheres in a solvent to prepare a first mixed solution; dehydrating and cooling the first mixed solution to obtain a magnetic core;
s300: alternately coating a pair of polyelectrolyte layers with positive charges and more than one polyelectrolyte layer with negative charges on the surface of the magnetic core in sequence to form a spacing layer on the surface of the magnetic core;
s400: attaching the microsphere prepared in the step S300 to the nanocrystal to form a nanocrystal layer;
s500: coating biological capture molecules on the surfaces of the microspheres with the nanocrystal layers;
preferably, the preparation of the composite detector comprises the following steps:
e100: selecting a proper biological recognition molecule, and preparing a fluorescent dye solution by using an organic fluorescent dye with emission wavelength different from that of fluorescent nanocrystals of the encoded microspheres;
e200: mixing a second biological capture molecule with the fluorescent dye solution to prepare a mixed solution;
e300: adding a cross-linking agent into the mixed solution obtained in the step E200, connecting a second biological capture molecule and a fluorescent dye in a covalent cross-linking mode, and reacting to generate a fluorescent-labeled biological recognition molecule compound;
e400: centrifuging and washing to remove unreacted fluorescent dye and cross-linking agent, thus obtaining a composite detector;
preferably, in step S400, the volume V of the desired nanocrystals is calculated by the following calculation formula;
wherein h is the ideal coating thickness of the nanocrystals on the surface of the encoded microsphere, which is set by the relevant technicians according to the needs; n is the statistical quantity of the coded microspheres prepared at this time, S is the statistical surface area of the microspheres, phi is the volume fraction of the nanocrystals, and rho is the material density of the nanocrystals; n and S are obtained from factory data provided by the manufacturer of the microspheres; phi is obtained by sampling and detecting the nanocrystals; k is a redundancy coefficient, and the coating effect after the experiment is carried out by using the current nanocrystal is measured and set by relevant technicians;
as shown in fig. 1 and 2, one structural combination of the fluorescent-encoded microspheres 10 is schematically illustrated; comprising
A magnetic core 12, a positively charged polyelectrolyte layer 14, a negatively charged polyelectrolyte layer 16, a nanocrystal layer 18, and a biological capture molecule 20;
as shown in fig. 3, a second biological capture molecule 24 associated with the fluorescent dye 22 in the composite detector is schematically illustrated;
in an exemplary embodiment, the high molecular polymer microsphere may be one or more of a polystyrene microsphere, a polymethyl methacrylate microsphere, an agarose microsphere, a polystyrene-divinylbenzene microsphere, and a methyl methacrylate-glycerol methacrylate microsphere;
in an exemplary embodiment, the nanocrystals are a mixture of one or more of the following: pbS, cdS, znS, cuInS 2 、ZnS、Ag 2 S, S; the crystals of the materials have the semiconductor quantum dot materials with fluorescence luminescence characteristics; the fluorescence luminescence of the quantum dots is derived from the energy level transition process, and belongs to the inherent characteristics of the materials; therefore, the quantum dot can be directly used for constructing a fluorescent coding layer of the coding microsphere without additional processing to obtain a fluorescent effect;
in an exemplary embodiment, the fluorescent dye in the composite detector may be selected from a plurality of organic fluorescent dyes with different luminescence ranges, such as fluorescein isothiocyanate, pyrene derivative fluorescent dye, rhodamine fluorescent dye, luminol fluorescent dye, etc.;
specifically, in the technical scheme, fluorescent nanocrystals are adopted as main fluorescent coding substances on fluorescent coding microspheres, and meanwhile, a composite detector containing fluorescent dye is introduced into a detection material, wherein the fluorescent emission ranges of the fluorescent coding substances are different; wherein the fluorescent nanocrystal layer provides a primary fluorescent code for the encoded microsphere; the fluorescent signal generated by the fluorescent dye in the composite detector can be regarded as another fluorescent code of the encoded microsphere; the combination of the two fluorescence coding signals realizes the expansion of coding space;
in actual detection, after the fluorescent nanocrystal layer on the surface of the coded microsphere is excited by the selected excitation wavelength, a unique fluorescent emission wavelength is generated and is used as a main coded identification signal; meanwhile, the fluorescent dye on the composite detector has the same absorption spectrum as that of the fluorescent nanocrystal, so that the fluorescent dye can generate a fluorescent signal different from the wavelength excited by the fluorescent nanocrystal; thus, the intensity combination of two independent fluorescent signals in the material is detected, and mixed coding with more dimensions can be realized; by combining the advantages of two fluorescent materials, the stable and uniform coding effect of the fluorescent nanocrystals is realized, and the coding space is enlarged by introducing another fluorescent source; the two are used as orthogonal fluorescent sources, so that the number of coding combinations can be greatly increased, and high-flux accurate detection of more various targets can be realized;
further, the adoption of the magnetically encoded microspheres can have the following advantages:
(1) The magnetic separation is convenient, and the coded microspheres have magnetic cores, so that the coded microspheres can be adsorbed, aggregated and separated conveniently under the action of an external magnetic field, and the magnetic separation method can be used for rapidly separating the unbound coded microspheres;
(2) Preventing the encoded microsphere from settling, wherein the magnetic nano particles in the encoded microsphere have a certain density, and if the encoded microsphere is not magnetic, the encoded microsphere gradually settles in the storage and reaction process. The superparamagnetic nano particles can prevent the sedimentation of the encoding microspheres under the action of an external magnetic field;
(3) The recombination of the microsphere in the medium is facilitated, and the coded microsphere can be recombined in the solution by the aid of an externally applied rotating magnetic field, so that the detection sensitivity is improved;
(4) The uniform dispersion of the coded microspheres is ensured, and the coded microspheres in the reaction system can be ensured to be uniformly dispersed by proper amount of magnetism and magnetic field stirring, so that the detection sensitivity is improved;
further, to ensure a detectable fluorescence brightness, the volume V of the nanocrystals used needs to be calculated;
the volume V is calculated to accurately set the nano crystal amount on the surface of each encoding microsphere, so that the thickness of the coating and the fluorescence intensity are ensured to meet the encoding requirement; if the amount of the nanocrystals is too small, the fluorescence signals of the encoded microspheres are insufficient, and the detection sensitivity is affected; if excessive, the resources are wasted, and the cost is increased;
further, in an exemplary embodiment, the analyte capture molecules may be selected in the following manner:
(1) Firstly, the specificity of the human autoantibody to be detected needs to be determined, for example, a certain autoantibody detection kit is used for detecting an anti-heparin antibody, an anti-thyroglobulin antibody or the like;
(2) Then selecting as analyte capture molecules the corresponding antigens that bind with high affinity and specificity to these specific autoantibody types; for example, detection of anti-heparin antibodies, heparin-BSA conjugates can be selected as capture molecules; detection of anti-thyroglobulin antibodies thyroglobulin may be selected as a capture molecule;
(3) Through connecting the corresponding antigens on the surfaces of the coded microspheres, when the corresponding autoantibodies exist in the sample, the corresponding autoantibodies can be specifically combined with the sample, so that the capture and detection of the autoantibodies are realized; different types of autoantibodies employ different functionalized encoded microspheres, each of which binds to its corresponding autoantibody to which it is linked.
Embodiment two: this embodiment should be understood to include at least all of the features of any one of the preceding embodiments, and be further modified based thereon;
in step S200 of the preparation method, the magnetic nanoparticles are preferably dissolved in a solvent, such as methyl chloride, methylene chloride, chloroform, carbon tetrachloride, dichloroethane; thereafter, the high molecular polymer microspheres are dissolved in a solvent such as ethanol, methanol, isopropanol, ethanol, ethylene glycol, propionic acid alcohol, n-octanol, undecanol, isobutanol or n-propanol; mixing the two mixed solutions, and stirring to obtain a first mixed solution;
in step S400, the microsphere prepared in step S300 is attached to a nanocrystal to form a nanocrystal layer; the method for attaching nanocrystals includes:
self-assembly, i.e., utilizing the affinity between the nanocrystal surface modifying molecule (e.g., thiol) and the microsphere surface material to spontaneously form a self-assembled monolayer;
an electrostatic adsorption method, in which the surface charges of the nanocrystals and the microspheres are adjusted, and the nanocrystals are attached to the surfaces of the microspheres by using electrostatic attraction;
covalent bonding method, coupling functional groups on the surfaces of microsphere and nanocrystal in advance, and connecting the microsphere and nanocrystal through covalent bonds;
embedding the nano crystal and the microsphere together in sol gel or polymer, curing to form a film, and releasing the microsphere on the surface;
the above-presented attachment modes are only examples, and the specific attachment mode may be selected according to actual production conditions.
Embodiment III: this embodiment should be understood to include at least all of the features of any one of the preceding embodiments, and be further modified based thereon;
furthermore, signals are read out through fluorescence detection and other modes, so that multiple detection of various autoantibodies can be realized; among the detection methods that can be employed are:
fluorescence detection, distinguishing various codes by using nanocrystal layers with different luminous wavelengths on the coded microspheres; detecting the bound analyte using the fluorescent label of the composite detector;
detecting by flow cytometry, wherein coded microspheres with different sizes and fluorescence can be distinguished in a flow cytometer, and meanwhile, fluorescent signals of composite detection objects on the microspheres can be detected;
magnetic detection, namely detecting magnetic response by utilizing a magnetic core on the encoded microsphere, and also carrying out magnetic separation;
microscopic image detection, namely directly observing fluorescent signals of the coded microspheres through a fluorescent microscope, and distinguishing the coded microspheres with different luminous wavelengths;
further, the mixed solution mixed with the sample to be analyzed and the detection material in the technical scheme is put into a magnetic frame, and the buffer solution is replaced by acetic acid buffer solution with the pH value of 4.8; in this buffer, the amphiphilic polyelectrolyte shell encoding the microsphere surface will degrade, thereby releasing the detected target analytes and corresponding detection components from the microsphere surface into solution, which is then pipetted for subsequent further molecular biological studies.
In the foregoing embodiments, the descriptions of the embodiments are emphasized, and in part, not described or illustrated in any particular embodiment, reference is made to the related descriptions of other embodiments.
While the invention has been described above with reference to various embodiments, it should be understood that many changes and modifications can be made without departing from the scope of the invention. That is, the methods, systems and devices discussed above are examples. Various configurations may omit, replace, or add various procedures or components as appropriate. For example, in alternative configurations, the methods may be performed in a different order than described, and/or various components may be added, omitted, and/or combined. Moreover, features described with respect to certain configurations may be combined in various other configurations, such as different aspects and elements of the configurations may be combined in a similar manner. Furthermore, as the technology evolves, elements therein may be updated, i.e., many of the elements are examples, and do not limit the scope of the disclosure or the claims.
Specific details are given in the description to provide a thorough understanding of exemplary configurations involving implementations. However, configurations may be practiced without these specific details, e.g., well-known circuits, processes, algorithms, structures, and techniques have been shown without unnecessary detail in order to avoid obscuring configurations. This description provides only an example configuration and does not limit the scope, applicability, or configuration of the claims. Rather, the foregoing description of the configuration will provide those skilled in the art with an enabling description for implementing the described techniques. Various changes may be made in the function and arrangement of elements without departing from the spirit or scope of the disclosure.
It is intended that the foregoing detailed description be regarded as illustrative rather than limiting, and that it be understood that it is intended that it be regarded as illustrative rather than limiting. Various changes and modifications to the present invention may be made by one skilled in the art after reading the teachings herein, and such equivalent changes and modifications are intended to fall within the scope of the invention as defined in the appended claims.
Claims (9)
1. A human autoantibody detection material, the detection material comprising:
a set of one or more combinatorial agents; each set of said combinatorial agents comprises: encoding the microsphere and the composite detector;
the structure of the coding microsphere is from inside to outside, and the coding microsphere comprises:
the magnetic core comprises high-molecular polymer microspheres and magnetic nano particles attached to the surfaces of the high-molecular polymer microspheres;
a spacer layer formed alternately of one or more pairs of polyelectrolyte layers having positive and negative charges;
a nanocrystal layer composed of nanocrystals having a specified fluorescent color;
a biological capture molecule coated on the outermost layer of the encoded microsphere for specifically binding to a designated antibody protein molecule;
the complex detector has a fluorescent dye and a second biological capture molecule bound to the fluorescent dye; the second biological capture molecule is the same as the biological capture molecule possessed by the encoded microsphere;
wherein the absorption spectrum of the fluorescent dye of the composite detector is consistent with the absorption spectrum of the nanocrystal layer of the encoded microsphere; the emission spectrum of the fluorescent dye of the composite detector is not consistent with the emission spectrum of the nanocrystal layer of the encoded microsphere.
2. The detection material according to claim 1, wherein the magnetic nanoparticles are superparamagnetic nanoparticles, and the size of the magnetic nanoparticles is 10-50 nm.
3. The detecting material of claim 2, wherein the preparation of the magnetic nanoparticles comprises mixing long-chain fatty acids, medium-short-chain fatty acids and the original magnetic nanoparticles and heating the mixture together to obtain the shaped magnetic nanoparticles.
4. A test material according to claim 3, wherein the nanocrystals employed in the nanocrystal layer are semiconductor nanocrystals.
5. The detection material according to claim 4, wherein the encoded microspheres have a diameter of 10 to 200. Mu.m.
6. The detection material according to claim 5, wherein the negatively charged polyelectrolyte layer is composed of a polyacid and/or a polyacrylate; the positively charged polyelectrolyte layer is composed of a polybasic and/or polybasic salt.
7. A method for preparing a human autoantibody detection material, wherein the method is used for preparing a human autoantibody detection material according to claim 6; the preparation method comprises two parts of preparation of the coded microsphere and preparation of the composite detector;
wherein, the preparation of the encoded microsphere comprises the following steps:
s100: mixing long-chain fatty acid, medium-chain fatty acid and short-chain fatty acid with original magnetic nano particles, and heating to obtain molded magnetic nano particles;
s200: dissolving the formed magnetic nano particles and high molecular polymer microspheres in a solvent to prepare a first mixed solution; dehydrating and cooling the first mixed solution to obtain a magnetic core;
s300: alternately coating a pair of polyelectrolyte layers with positive charges and more than one polyelectrolyte layer with negative charges on the surface of the magnetic core in sequence to form a spacing layer on the surface of the magnetic core;
s400: attaching the microsphere prepared in the step S300 to the nanocrystal to form a nanocrystal layer;
s500: the surface of the microsphere with the nanocrystal layer is coated with biological capture molecules.
8. The method of preparing of claim 7, wherein the preparation of the composite detector comprises the steps of:
e100: selecting a proper biological recognition molecule, and preparing a fluorescent dye solution by using an organic fluorescent dye with emission wavelength different from that of fluorescent nanocrystals of the encoded microspheres;
e200: mixing a second biological capture molecule with the fluorescent dye solution to prepare a mixed solution;
e300: adding a cross-linking agent into the mixed solution obtained in the step E200, connecting a second biological capture molecule and a fluorescent dye in a covalent cross-linking mode, and reacting to generate a fluorescent-labeled biological recognition molecule compound;
e400: and (3) centrifugally washing to remove unreacted fluorescent dye and cross-linking agent, thereby obtaining the composite detector.
9. The method of claim 8, wherein in step S400, the volume void volume V of the desired nanocrystal is calculated by the following equation;
wherein h is the ideal coating thickness of the nanocrystals on the surface of the encoded microsphere, which is set by the relevant technicians according to the needs; n is the statistical quantity of the coded microspheres prepared at this time, S is the statistical surface area of the microspheres, phi is the volume fraction of the nanocrystals, and rho is the material density of the nanocrystals; n and S are obtained from factory data provided by the manufacturer of the microspheres; phi is obtained by sampling and detecting the nanocrystals; k is the redundancy factor, which is determined and set by the relevant technician by using the current nanocrystal to perform the post-experiment coating effect.
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