WO2021114058A1 - 一种多重免疫分子检测方法及试剂盒 - Google Patents
一种多重免疫分子检测方法及试剂盒 Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Definitions
- the invention belongs to the field of immunomolecular detection, and specifically relates to a multiple immunomolecular detection method and kit.
- the traditional ELISA reaction system is relatively large (100 ⁇ l) and requires millions of molecules to generate tens of thousands of fluorophore signals before it can be detected by the microplate reader. Therefore, the traditional ELISA method can only detect the picomolar range (10pg/ml). )
- the above signals use analog algorithms. This method has reached its limit in modern science and is far from meeting clinical requirements. For example, some low-abundance protein molecules in peripheral blood are of great significance for clinical diagnosis and treatment guidance, but the sensitivity of traditional ELISA cannot detect these molecules, let alone achieve sensitive monitoring. ELISA can only detect one indicator per sample per experiment. However, when there are fewer biological samples (such as infant blood, cerebrospinal fluid, etc.) for acute and severe diagnosis, clinical immunoassays are required to be able to detect multiple protein indicators in one sample at the same time, that is, multiple detection.
- the detection antibody and the capture antibody recognize different epitopes on the molecule to be tested, and the detection antibody can be labeled with an enzyme that can chemically amplify the signal or a small molecule catalyst that can electrochemically amplify the signal.
- the amplified signal detected is positively correlated with the concentration of the analyte molecule.
- magnetic particle chemiluminescence method and magnetic particle electrochemiluminescence method use magnetic particles as a stationary phase, which can capture the molecule to be measured faster and more efficiently.
- the signal-to-noise ratio of chemiluminescence and electrochemiluminescence signals is increased by about ten times.
- these two new methods have increased the sensitivity by 10-100 times compared with the traditional ELISA, and can reach a sensitivity of 1 pg/ml at the highest. With this sensitivity, it can effectively detect about 200-300 clinically significant protein indicators in the blood, but thousands of other blood protein indicators with or potentially clinically significant cannot be effectively detected because of their low concentration.
- the main factor limiting the sensitivity of the magnetic particle chemical/electrochemiluminescence method is the detection method.
- Magnetic beads with enzyme-labeled sandwich complexes focus on detection and give a macroscopic continuous signal.
- concentration of antigen is between 10fg/ml and 10pg/ml, when the number of enzyme molecules labeled on a single magnetic bead is less than 10-100, the macroscopic observation results are similar to the background (0 enzyme molecules are labeled on a single magnetic bead). .
- Flow cytometry uses microspheres as a stationary phase, uses different fluorescent codes to load microspheres with different capture antibodies, and uses a flow cytometer to read the fluorescent codes of the microspheres and the signals of the fluorescent markers of the immunosandwich complex on the microspheres.
- the microporous electrochemiluminescence method microprints different capture antibodies to different positions in the micropores, and simultaneously reads the position information and the electrochemical signal of the immune sandwich complex through the imaging method.
- the highest sensitivity of detection is single-molecule detection.
- This patent digitizes each detection molecule under the detection conditions of traditional analog signal ELISA, so that protein detection reaches the femtogram level (fg/ml), which is 1000 more sensitive than the traditional ELISA method. Times. And it adopts the sample detection method of fluorescent coding liquid phase chip, and can detect at least 15 marker proteins at the same time, which has extremely high application value for saving sample volume and improving the efficiency of detection research. It has broad application prospects in life sciences, in vitro diagnostics, companion diagnostics, blood screening, and drug development.
- the purpose of the present invention is to solve the problems of low oxygen reduction electrocatalytic activity and low electrochemical stability of the current silver-based nanocatalysts.
- the present invention proposes a multiple immune molecule detection method, which includes the following steps:
- the microplate After a predetermined time, the microplate is excited by light to detect the microplate.
- the coded microsphere includes a body of polymer material and a coded material distributed in the body of polymer material.
- the coded microspheres further include magnetic nanoparticles distributed in the polymer material body in an orderly manner.
- the particle size of the coded microspheres is 0.5-50 ⁇ m, and the particle size of the magnetic nanoparticles is 1-100 nm.
- the coded microspheres include at least two light-emitting coding materials
- the luminescent coding material is an organic fluorescent material or an inorganic fluorescent material
- the luminescent coding material is at least one of organic dyes and quantum dots.
- the method for preparing the coded microspheres includes the following steps:
- one target immune molecule is captured on the surface of each of the coded microspheres.
- each of the microwells contains one of the coded microspheres.
- the coded microspheres modified with the immune sandwich complex on the surface are driven into the microwells of the microwell plate and sealed by the electric field.
- the present invention also provides a kit for immunomolecular detection.
- the kit includes coded microspheres with capture molecules attached to the surface, detection molecules, and hybridization buffer.
- the coded microspheres include a polymer material body and a magnetic nanosphere. Particles, coding material, the magnetic nanoparticles and the coding material are distributed in the polymer host material.
- the kit further includes a microplate chip
- the volume of a single microwell on the microplate chip is (20-100) ⁇ 10-15L.
- the invention digitizes each detection molecule under the detection conditions of the traditional analog signal ELISA, so that the protein detection reaches the femtogram level (fg/ml), which is 1000 times more sensitive than the traditional ELISA method. And it adopts the sample detection method of fluorescent coding liquid phase chip, and can detect at least 15 marker proteins at the same time, which has extremely high application value for saving sample volume and improving the efficiency of detection research.
- the invention has great advantages especially for single molecule detection, and has broad application prospects in life sciences, in vitro diagnosis, companion diagnosis, blood screening, drug research and development, and the like.
- Figure 1 shows the labeled enzyme detected by the forward scatter imaging module after oil sealing when the fluorescent coded microspheres modified with the sandwich complex were introduced into the microtiter plate when the IL-2 and IL-6 cytokine samples of unknown concentration were measured in Example 1. (Galactosidase) signal.
- Figure 2 shows the fluorescent coded microspheres modified with sandwich complexes when measuring IL-10 and IFNr cytokine samples at unknown concentrations in Example 2 into the microtiter plate, and the labeled enzyme (half) detected by the forward scatter imaging module after oil sealing. Lactosidase) signal.
- the present invention proposes to fundamentally abandon the method of centralized detection of all magnetic particles with enzyme-labeled immune sandwich complexes during detection to give a macroscopic continuous signal, but to enclose each magnetic particle in a flying volume for independent detection. , And detect >10,000 magnetic particles at the same time. In this volume, the chemical signal generated by a single enzyme molecule can be detected.
- concentration of the molecule to be tested is at the level of fg/ml, most of the magnetic particles are not labeled with enzyme molecules, and no signal is generated. Most of the remaining magnetic particles are labeled with one enzyme molecule, and a small part is labeled with two or more enzyme molecules, thereby generating chemical signals that can be detected by the system.
- the ratio of the magnetic beads that produce chemical signals to the total magnetic beads (fon) and the ratio of the total number of labeled enzyme molecules to the total number of magnetic beads (AEB, Average Enzyme per Bead) follow the Poisson distribution (AEB -ln (1-fon)).
- AEB I bead /I enzyme
- I bead is the average value of all magnetic beads generated signals in a single test
- I enzyme is the average value of signals generated by a single enzyme molecule
- I enzyme fon*I bead /AEB, this
- I bead refers to the average signal value of all magnetic beads that generate signals in a single test
- AEB -ln(1-fon)
- the concentration of the analyte is at the level of fg/ml in the calculation, and the data of the sample when fon ⁇ 0.5
- the calculated AEB is more accurate and most of the signal-generating magnetic particles only carry one labeling enzyme molecule, so as to ensure the accuracy of the calculated I enzyme).
- AEB and the concentration of the analyte molecule are positively correlated.
- Test different concentrations of the analyte standard After linearly fitting the AEB value and the concentration of the analyte to draw a calibration curve, use the same method to test the unknown sample to obtain the signal The value is brought into the standard curve by interpolation to measure the concentration of the analyte in the unknown sample.
- the detection sensitivity can be increased from the pg/ml level by about 1000 times to the fg/ml level.
- multiple protein indicators can be detected at the same time in order to achieve one detection.
- Different capture antibodies are coupled to different fluorescently encoded magnetic particles, and the same sample is tested after mixing in an equal ratio.
- the polymer solution is dispersed in the water phase through multiple coupling physical fields to form uniform droplets/micro-reactive groups, and then the functional materials are wrapped and buried in the polymer microspheres through cross-linking and polymerization reactions, which are reactive
- the oligomers are exposed on the surface of the microspheres through microphase separation.
- the microspheres can undergo further surface chemical reactions, so that the surface can achieve weak non-specific affinity for nucleic acid molecules, strong specific coupling reaction activity and high density, and the microspheres can perform further surface chemical reactions. Tolerate under different reaction conditions (organic phase, high temperature), and finally achieve controllable coupling of capture antibodies that specifically recognize target molecules on the surface of the microspheres.
- the capture antibody and the detection antibody must respectively recognize different epitopes with high affinity and specificity.
- the affinity and specificity are characterized and tested by surface plasmon resonance and protein chip methods.
- Antibodies with higher affinity are used as capture antibodies, and antibodies with relatively low affinity are used as detection antibodies.
- the detection antibody needs to be labeled according to the enzyme, such as biotin, digoxin, etc.
- the surface of the magnetic particles has functional groups such as epoxy and carboxyl.
- EDC catalyst
- the surface of the magnetic beads needs to use a suitable blocking agent such as whey protein and bovine blood albumin. Blocking to reduce non-specific adsorption on the surface of magnetic particles.
- Enzyme-labeled immune sandwich complex can be formed by a three-step method, a two-step method or a one-step method.
- the three-step method involves adding magnetic particles with capture antibodies to an unknown sample. Incubate at 25C with shaking for 30 minutes to 3 hours and then wash. Add detection antibody, incubate at 25C with shaking for 30 minutes to 3 hours and then wash. Add enzyme-labeled reagent, incubate at 25C with shaking for 30 minutes to 3 hours, and then wash.
- the two-step method magnetic particles with capture antibody and detection antibody are added to the unknown sample, incubated at 25C with shaking for 30 minutes to 3 hours, and then washed.
- the one-step method involves adding magnetic particles with capture antibodies, detection antibodies, and enzyme-labeled reagents to the unknown sample, incubating at 25C with shaking for 30 minutes to 3 hours, and then washing.
- Enzyme-labeled reagents need to be able to amplify chemical signals, such as horseradish peroxidase, galactosidase, etc., and also recognize detection antibodies, such as streptavidin to recognize biotin on the detection antibody, and rabbit anti-mouse antibodies to recognize mouse origin The detection antibody and so on.
- microsphere-capture antibody-target molecule-detection antibody-labeled enzyme The composite structure of "microsphere-capture antibody-target molecule-detection antibody-labeled enzyme" is successfully formed.
- the labeled enzyme molecule at the end catalyzes the reaction of the solution, and a catalyst molecule is used to catalyze the reaction of multiple orders of magnitude.
- the density of the micropores on the microplate chip determines the amount of data collected and the dynamic range of detection. Closely arranged micropores need to be clearly distinguished by optical detection, and to ensure that there is only one or no microsphere in each micropore.
- the disposable microplate is produced by injection molding. Non-disposable micro-holes can be processed by chip MEMS method.
- Microsphere composite structure and micropore assembly In order to ensure efficient and quick assembly of microspheres and micropores one by one, the following methods can be used: 1. The density of microspheres is greater than that of water, and gravity can make the microspheres natural Sinking into the micropores; 2. Since the microspheres can wrap the magnetic material, the microspheres can be manipulated into the micropores with magnetic force; 3. Because the dielectric constant of the microspheres is very different from the solution, the application of non-uniform intensity can be used The variable electric field generates dielectrophoresis force to push the microspheres into the micropores. After the reaction detection is completed, the microspheres can be pushed out of the micropores by changing the direction of the force by adjusting the frequency of the electric field to realize the repeated use of the micropores.
- the ratio of the microspheres to the target molecule needs to be adjusted to optimize the Poisson distribution, so that only one or no target molecule can be captured on a microsphere.
- the sample processing part of the device integrates modules such as liquid transfer, mixing, magnetic attraction, liquid pre-storage, and liquid path cleaning. After the sample is diluted correspondingly through the online dilution system integrated in the device, it is loaded with the magnetic beads of the detection antibody , Detection antibody, enzyme-labeled reagent, washing liquid and other reagents are mixed and washed. Finally, a pure magnetic particle solution with an enzyme-labeled immune sandwich complex is obtained.
- the detection part of the device integrates modules such as multi-color fluorescence excitation, forward scattering imaging, fluorescence emission filtering, magnetic attraction, alternating electric field control, etc., and manipulates the microspheres with multiple fluorescence encoding into the microplate through the aforementioned method ,
- the surface of the microwell plate is sealed by the oil phase through automatic control of the liquid path.
- the fluorescence of different wavelengths is used for near-field excitation and forward-scattered light imaging is used to take pictures at different filter wavelengths.
- the fluid path is cleaned.
- All modules are automatically controlled by the underlying program, and the software integrates the image automatic recognition and analysis module.
- the system automatically recognizes the brightness of each micropore and obtains the distribution of micropore brightness, and automatically judges whether there is a reaction in each micropore Occurs, the number of the microsphere corresponding to each reaction.
- the dynamic detection range of the analog signal is obtained through the brightness analysis, and the number of microspheres with the same number is used as the dynamic detection range of the digital signal.
- Synthesis of two kinds of fluorescent coded microspheres mixing styrene monomer, polymethyl methacrylate, initiator, crosslinking agent, and acrylic oligomer in chloroform as a polymer solution. Take two 4.5mL polymer solutions as reaction solution 1 and reaction solution 2. Add 0.5mL rhodamine and 0.5mL fluorescein and 90mg nano-magnetic particles to reaction solution 1, and add 0.5mL rhodamine and 0.5mL fluorescein to reaction solution 2. And 90mg nano magnetic particles.
- reaction solution 1 and reaction solution 2 were respectively placed in two reactors with 300 mL of deionized water and surfactant, and the reaction solution 1 and reaction solution 2 were uniformly dispersed under the conditions of stirring, sonication and adjustment of surface force.
- the initiator is added to the solution and heated for polymerization and crosslinking reaction. After 24 hours, the chloroform in each droplet slowly dissolves in water and volatilizes, the monomer is polymerized and crosslinked, and finally formed Fluorescent coded microsphere 1 and fluorescent coded microsphere 2.
- Two kinds of macromolecule fluorescent coded microspheres have carboxyl functional groups on the surface.
- Microspheres 1 and 2 are respectively coupled with IL-2 and IL-6 capture antibodies. Taking 1 as an example, disperse 1mg of microspheres in 1ml PBS buffer, add 5mg EDC and 5mg Sulfo-NHS, mix and mix well. Keep stirring for 10 minutes, wash with 1ml PBS and add 50ug IL-2 capture antibody. Incubate with shaking at room temperature for 30 minutes to 3 hours. After adding blocking agent such as BSA, incubate with shaking at room temperature for 30 minutes, wash the microspheres 1 by magnetic separation, and finally disperse in PBS. Mix the loaded magnetic beads 1 and 2 in equal volumes, each with a concentration of 0.5mg/ml
- the IL2 and IL6 standards were mixed into 10% bovine serum at concentrations of (0, 0.001, 0.005, 0.01, 0.3, 1.0, and 10 pg/ml). Take 100ul of each standard, add 50ul 1. Medium magnetic beads mixed solution, incubate with shaking at 25C for 3 hours, wash with 5X PBS+0.1% Tween three times. Disperse into 100ul PBS, add 50ul 1ug/ml IL2 detection antibody and 50ul 1ug/ml IL6 detection antibody, incubate with shaking for half an hour at 25C, and wash three times with 5X PBS+0.1% Tween.
- microsphere composite structure to the reactor with microplate chip via microfluid, add 10MHz alternating current, the microspheres are pushed into the micropores, then the surface of the micropores is sealed with silicone oil, after 2 minutes of reaction, use 488nm Wavelength light excitation, filter 1 to take photos, filter 2 to take photos, then use 532 nm wavelength light to excite, filter 3 to take photos, flow into ethanol and then flow into the cleaning solution, change the AC frequency to 10kHz, and then flow into the cleaning solution, cleaning reactor.
- the sample with a concentration of 0 is measured 10 times in parallel, and the average value of the measured AEB value plus 3 times the standard deviation is brought into the standard curve of 4.
- the solubility of the analyte obtained is the detection limit of this method.
- the detection limit of IL-2 is 0.069 pg/ml
- the detection limit of IL-6 is 0.030 pg/ml.
- Figure 1 shows the labeled enzyme detected by the forward scatter imaging module after oil sealing when the fluorescent coded microspheres modified with the sandwich complex were introduced into the microtiter plate when the IL-2 and IL-6 cytokine samples of unknown concentration were measured in Example 1.
- (Galactosidase) signal Each microwell contains 1 or 0 magnetic microspheres. Because of the low concentrations of IL-2 and IL-6 cytokines detected, most of the sphere surfaces did not form a complete complex labeled with galactosidase, and could not produce the fluorescent signal amplified by the labeled galactosidase.
- a small part of the surface of the ball forms one or a few complete complexes labeled with galactosidase to amplify and generate fluorescent signals.
- the ratio of the spheres that produce the fluorescent signal to the total microspheres is positively correlated with the concentration of the analyte.
- Synthesis of two kinds of fluorescent coded microspheres mixing styrene monomer, polymethyl methacrylate, initiator, crosslinking agent, and acrylic oligomer in chloroform as a polymer solution. Take two 4.5mL polymer solutions as reaction solution 1 and reaction solution 2. 1 add 0.5mL rhodamine and 0.5mL fluorescein and 90mg nano-magnetic particles, 2 add 0.5mL rhodamine and 0.5mL fluorescein and 90mg nano-magnetic particles. Then place 1 and 2 in two reactors with 300 mL of deionized water and surfactant. Take 1 as an example.
- the 1 solution Under the conditions of stirring, sonication and adjusting the surface force, the 1 solution is evenly dispersed into a 10 micron liquid. Drop, add initiator to the solution and heat it for polymerization and crosslinking reaction. After 24 hours, the chloroform in each droplet slowly dissolves in water and volatilizes. The monomers are polymerized and crosslinked to finally form polymer microspheres 1, 2 The process is the same. Two polymer microspheres have carboxyl functional groups on the surface.
- Microspheres 1 and 2 are respectively coupled with capture antibodies of IL-10 and IFNr. Take 1 as an example, disperse 1mg of microspheres in 1ml PBS buffer, add 5mg EDC and 5mg Sulfo-NHS, mix and maintain stirring 10 After washing with 1ml PBS, add 50ug IL-2 capture antibody. Incubate with shaking at room temperature for 30 minutes to 3 hours. After adding blocking agent such as BSA, incubate with shaking at room temperature for 30 minutes, wash the microspheres 1 by magnetic separation, and finally disperse in PBS. Mix the loaded magnetic beads 1 and 2 in equal volumes, each with a concentration of 0.5mg/ml
- the IL10 and INFr standards were mixed into 10% bovine serum at concentrations of (0, 0.001, 0.005, 0.01, 0.3, 1.0, and 10 pg/ml). Take 100ul of each standard, add 50ul 1. Medium magnetic beads mixed solution, incubate with shaking at 25C for 3 hours, wash with 5X PBS+0.1% Tween three times. Disperse into 100ul PBS, add 50ul 1ug/ml IL-10 detection antibody and 50ul 1ug/ml IFNr detection antibody, incubate with shaking for half an hour at 25C, and wash three times with 5X PBS+0.1% Tween.
- microsphere composite structure to the reactor with microplate chip via microfluid, add 10MHz alternating current, the microspheres are pushed into the micropores, then the surface of the micropores is sealed with silicone oil, after 2 minutes of reaction, use 488nm Wavelength light excitation, filter 1 to take photos, filter 2 to take photos, then use 532 nm wavelength light to excite, filter 3 to take photos, flow into ethanol and then flow into the cleaning solution, change the AC frequency to 10kHz, and then flow into the cleaning solution, cleaning reactor.
- Ienzyme uses the data of all fon ⁇ 0.5 samples in the calculation. After linearly fitting the AEB value and the concentration value of the analyte to draw a calibration curve, the unknown sample is tested with the same method, and the AEB value obtained is inserted into the standard curve by interpolation to measure the concentration of the analyte in the unknown sample.
- concentration of IL-10 was 10pg/ml
- concentration of INFr was 0.3pg/ml.
- the sample with a concentration of 0 is measured 10 times in parallel, and the average value of the measured AEB value plus 3 times the standard deviation is brought into the standard curve of 4.
- the solubility of the analyte obtained is the detection limit of this method.
- the detection limit of IL-10 was 0.027pg/ml, and the detection limit of INFr was 0.024pg/ml.
- Figure 2 shows the fluorescent coded microspheres modified with sandwich complexes when measuring IL-10 and IFNr cytokine samples at unknown concentrations in Example 2 into the microtiter plate, and the labeled enzyme (half) detected by the forward scatter imaging module after oil sealing. Lactosidase) signal.
- Each microwell contains 1 or 0 magnetic microspheres. Because of the low concentrations of IL-2 and IL-6 cytokines detected, most of the sphere surfaces did not form a complete complex labeled with galactosidase, and could not produce the fluorescent signal amplified by the labeled galactosidase.
- a small part of the surface of the ball forms one or a few complete complexes labeled with galactosidase to amplify and generate fluorescent signals.
- the ratio of the spheres that produce fluorescent signals to the total microspheres is positively correlated with the concentration of the analyte.
- the multiple immunoassay method and kit provided by the present invention can make the immunomolecular detection reach the femtogram level (fg/ml), which embodies ultra-high sensitivity, and cooperates with non-amplified nucleic acid molecular diagnostic equipment, and its detection steps Simple, accurate and reliable test results.
- fg/ml femtogram level
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Claims (10)
- 一种多重免疫分子检测方法,其特征在于,包括如下步骤:获得表面连接有捕获分子的编码微球;通过所述捕获分子捕获目标免疫分子并加入酶标试剂,在所述编码微球表面形成酶标记的免疫夹心复合物;将表面修饰有免疫夹心复合物的编码微球驱动到微孔板的微孔中并密封;预定时间后通过光激发所述微孔板进行检测所述微孔板。
- 根据权利要求1所述的多重免疫分子检测方法,其特征在于,所述编码微球包括聚合物材料主体以及分布在所述聚合物材料主体中的编码材料。
- 根据权利要求2所述的多重免疫分子检测方法,其特征在于,所述编码微球还包括有序分布在所述聚合物材料主体中的磁性纳米颗粒。优选地,所述编码微球的粒径为0.5~50μm,所述磁性纳米颗粒的粒径为1~100nm。
- 根据权利要求1-3中任一项所述的多重免疫分子检测方法,其特征在于,所述编码微球包括至少两种发光编码材料;优选地,所述发光编码材料为有机荧光材料或无机荧光材料;优选地,所述发光编码材料为有机染料、量子点中的至少一种。
- 根据权利要求1所述的多重免疫分子检测方法,其特征在于,所述编码微球的制备方法包括如下步骤:将至少两种发光材料和微球混合在高分子材料中,通过多重耦合的物理场将高分子溶液分散在水相中形成均一的液滴,之后通过交联聚合反应将发光材料与磁性纳米颗粒包裹在所述液滴中得到所述编码微球。
- 根据权利要求1所述的多重免疫分子检测方法,其特征在于,每个所述编码微球表面捕获一个目标免疫分子。
- 根据权利要求1或6所述的多重免疫分子检测方法,其特征在于,每个所述微孔中容纳1个所述编码微球。
- 根据权利要求1或6所述的多重免疫分子检测方法,其特征在于,通过电场将表面修饰有免疫夹心复合物的编码微球驱动到微孔板的微孔中并密封。
- 一种用于免疫分子检测的试剂盒,其特征在于,所述试剂盒包括表面连接有捕获分 子的编码微球、检测分子和杂交缓冲液,所述编码微球包括聚合物材料主体、磁性纳米颗粒、编码材料,所述磁性纳米颗粒和所述编码材料分布在所述聚合物主体材料中。
- 根据权利要求9所述的试剂盒,其特征在于,所述试剂盒还包括微孔板芯片;优选地,所述微孔板芯片上单个微孔的体积为(20~100)×10 -15L。
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