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 PDF

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WO2021114058A1
WO2021114058A1 PCT/CN2019/124142 CN2019124142W WO2021114058A1 WO 2021114058 A1 WO2021114058 A1 WO 2021114058A1 CN 2019124142 W CN2019124142 W CN 2019124142W WO 2021114058 A1 WO2021114058 A1 WO 2021114058A1
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microspheres
detection
coded
enzyme
immune
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PCT/CN2019/124142
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English (en)
French (fr)
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陈忠磊
张敏超
程鹏
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彩科(苏州)生物科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

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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Abstract

一种多重免疫分子检测方法，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。上述方法使蛋白检测达到飞克级别(fg/ml),比传统ELISA方法灵敏度高出1000倍。尤其对单分子检测具有极大的优势，在生命科学、体外诊断、伴随诊断、血液筛查、药物研发等方面有广阔的应用前景。

Description

一种多重免疫分子检测方法及试剂盒 技术领域
本发明属于免疫分子检测领域，具体涉及一种多重免疫分子检测方法及试剂盒。
背景技术
传统的ELISA反应体系相对较大(100μl)，需要数百万分子产生数干万个荧光团信号才可以被酶标仪检测出，因此传统的ELISA方法只能检测到皮摩范围(10pg/ml)以上的信号，采用模拟算法。这种方法在现代科学已经达到了极限，远远不能满足临床要求。例如在外周血中的一些丰度低的蛋白分子对临床诊断及治疗的指导意义重大，但传统ELISA的灵敏度并无法检出这些分子，更无法达到灵敏的监控。ELISA一个样本一次实验只能检测一个指标。但急重症诊断或生物样品较少时(如婴幼儿血液，脑脊液等),临床要求免疫检测能够一个样本同时检测出多个蛋白指标，即多重检测。
为了提高免疫诊断的灵敏度，体外诊断厂家在传统酶联免疫(ELISA)的基础上，开发并商业化了磁微粒化学发光法和磁微粒电化学发光法。这两种方法采用和酶联免疫相同免疫夹心复合物的检测方法学，即用负载了捕获抗体的固定相捕获待测物分子，之后加入识别待测物分子的检测抗体形成免疫夹心复合物。检测抗体和捕获抗体识别待测物分子上不同的表位，而且检测抗体可以被能化学放大信号的酶或者电化学放大信号的小分子催化剂标记。最后检测到的放大信号和待测物分子浓度正相关。测试不同浓度的待测物标准品，将信号值和待测物浓度值进行线性拟合绘制校准曲线后，用相同的方法测试未知样品，得到的信号值用内插法带入标准曲线即测得未知样品中待测物的浓度。磁微粒化学发光法和磁微粒电化学发光法较酶联免疫相比采用磁微粒作为固定相，能更快更高效的捕获待测分子。较酶联免疫采用的酶催化化学显色，化学发光和电化学发光信号的信噪比提高了约十倍。在实际应用中这两种新的方法较传统的ELISA灵敏度提高了10-100倍，最高能到1pg/ml的灵敏度。在这种灵敏度下，可以有效检测血液中约200-300种有临床意义的蛋白指标，但另外上千种有或者潜在有临床意义的血液蛋白指标因为浓度较低，不能被有效检测到。
限制磁微粒化学/电化学发光法灵敏度的主要因素是检测的方法。带有酶标记夹心复合物的磁珠集中检测并给出一个宏观的连续信号。当抗原的浓度在10fg/ml到10pg/ml时，当单个磁珠上标记的酶分子少于10-100个，宏观集中观测的结果和背景(单个磁珠上标记0个酶分子)相差不多。
为了实现多重检测，体外诊断厂家利用免疫夹心复合物的检测方法学，开发并商业化了流式荧光发光法和微孔电化学发光法。流式荧光法采用微球作为固定相，用不同的荧光编码负载不同捕获抗体的微球，用流式细胞仪读取微球荧光编码和微球上免疫夹心复合物荧光标记物的信号。微孔电化学发光法将不同的捕获抗体微印刷到微孔中不同的位置，并通过成像的方法同时读取位置信息和免疫夹心复合物的电化学信号。这两种方法虽然能够实现多重检测。但荧光编码的微球，流式细胞仪，和在孔板中进行微印刷使检测的成本大幅升高。而且这两种方法和磁微粒化学/电化学发光法相比，灵敏度有所下降。
检测的最高灵敏度为单分子的检测，本专利在传统的模拟信号ELISA的检测条件下将每一个检测分子数字化，使蛋白检测达到飞克级别(fg/ml),比传统ELISA方法灵敏度高出1000倍。且采用了荧光编码液相芯片的样品检测方式，同时可检测至少15个标志蛋白，对节省样本量，提高检测研究方面的效率有极高应用价值。在生命科学、体外诊断、伴随诊断、血液筛查、药物研发等方面有广阔的应用前景。
发明内容
本发明的目的是，解决目前银基纳米催化剂的氧还原电催化活性低以及电化学稳定性不高的问题。
基于此，本发明提出一种多重免疫分子检测方法，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。
在一些实施例中，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。
在一些实施例中，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。
在一些实施例中，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、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。
在一些实施例中，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。
在一些实施例中，每个所述编码微球表面捕获一个目标免疫分子。
在一些实施例中，每个所述微孔中容纳1个所述编码微球。
在一些实施例中，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。
本发明还提出一种用于免疫分子检测的试剂盒，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。
在一些实施例中，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。
本发明在传统的模拟信号ELISA的检测条件下将每一个检测分子数字化，使蛋白检测达到飞克级别(fg/ml),比传统ELISA方法灵敏度高出1000倍。且采用了荧光编码液相芯片的样品检测方式，同时可检测至少15个标志蛋白，对节省样本量，提高检测研究方面的效率有极高应用价值。本发明尤其对单分子检测具有极大的优势，在生命科学、体外诊断、伴随诊断、血液筛查、药物研发等方面有广阔的应用前景。
附图说明
图1为实施例1中测未知浓度的IL-2和IL-6细胞因子样品时，修饰有夹心复合物的荧光编码微球导入微孔板，油封后通过前散射成像模块检测到的标记酶(半乳糖苷酶)信号。
图2为实施例2中测未知浓度的IL-10和IFNr细胞因子样品时，修饰有夹心复合物的荧光编码微球导入微孔板，油封后通过前散射成像模块检测到的标记酶(半乳糖苷酶)信号。
具体实施方式
为了便于理解本发明，下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
需要说明的是，除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技 术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本发明提出从根本上放弃检测时带有酶标记的免疫夹心复合物的所有磁微粒集中检测给出一个宏观连续信号的方法，而是将每个磁微粒都封闭到飞升级的体积里单独检测，同时检测>10,000个磁微粒。在该体积下单个酶分子产生的化学信号都可以被检测到。当待测分子的浓度在fg/ml级别时，大部分磁微粒没有酶分子标记，不产生信号。剩余的磁微粒绝大部分被一个酶分子标记，一小部分被两个或两个以上的酶分子标记，从而产生可以被系统检测到的化学信号。检测到产生化学信号的磁珠占总磁珠的比例值(fon)与标记酶分子的总数和磁珠总数的比例值(AEB，Average Enzyme per Bead)之间遵从泊松分布(AEB＝-ln(1-fon))。当待测物的浓度到达pg/ml级别时，样品处理完后的磁微粒绝大多数带有1-10个标记酶分子(AEB＝1-10)，AEB仍然和待测物的浓度呈正向关，且AEB＝I bead/I enzyme，I bead是单次测试中所有磁珠产生信号的平均值，I enzyme是单个酶分子产生信号的平均值，I enzyme＝fon*I bead/AEB,此时I bead指单次测试中所有产生信号的磁珠的平均信号值，AEB＝-ln(1-fon)，计算时用待测物浓度在fg/ml级别，fon<0.5时样品的数据，此时计算的AEB较精确且绝大部分产生信号的磁微粒只带一个标记酶分子，从而保证计算的I enzyme的准确性)。AEB和待测物分子的浓度成正相关，测试不同浓度的待测物标准品，将AEB值和待测物浓度值进行线性拟合绘制校准曲线后，用相同的方法测试未知样品，得到的信号值用内插法带入标准曲线即测得未知样品中待测物的浓度。采用将每个磁微粒封闭后单独检测的方法，可以将检测灵敏度从pg/ml量级提升1000倍左右到fg/ml级别。
在一些优选实施方式中，为了实现一次检测同时检出多个蛋白指标。不同荧光编码的磁微粒上偶联不同的捕获抗体，等比混合后检测同一个样品。
将两种或两种以上的荧光材料(有机、无机荧光分子，包括量子点等)与磁性纳米颗粒及其他功能性纳米颗粒混合在需要合成的多种(不同分子量、不同官能团)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/微反应基团，之后通过交联及聚合反应将功能材料包裹掩埋在高分子微球中，有反应活性的寡聚高分子通过微小相分离暴露在微球表面。通过微球暴露出来的寡聚高分子或其他表面高分子，微球可进行进一步表面化学反应，使表面达到对核酸分子非特异性亲和力弱，特异性偶联反应活性强且密度高，微球可在不同反应条件(有机相、高温)下耐受，最终可实现可控的在微球表面偶连特异性识别目标分子的捕获抗体。
对于特定的待测物分子，捕获抗体和检测抗体分别要高亲和力特异性的识别不同的表位。亲和力和特异性用表面等离子共振和蛋白芯片等方法表征和测试。亲和力较高的抗体作为捕获抗体，亲和力相对低的做检测抗体。检测抗体需要根据酶做相应的标记，如用生物素，地高辛等。磁微粒表面带有环氧，羧基等官能团，捕获抗体在合适的催化剂(EDC)和pH共价固定到磁珠表面后，磁珠表面需要用乳清蛋白，牛血白蛋白等合适的封闭剂封闭以降低磁微粒表面的非特异性吸附。
酶标记的免疫夹心复合物形成可采用三步法、两步法或一步法。三步法为带有捕获抗体的磁微粒加入到未知样品中。在25C振荡孵育30分钟到3个小时后洗涤。加入检测抗体，在25C振荡孵育30分钟到3个小时后洗涤。加入酶标试剂，在25C振荡孵育30分钟到3个小时后洗涤。两步法为带有捕获抗体的磁微粒和检测抗体加入到未知样品中，在25C振荡孵育30分钟到3个小时后洗涤。加入酶标试剂，在25C振荡孵育30分钟到3个小时后洗涤。一步法为带有捕获抗体的磁微粒，检测抗体，及酶标试剂加入到未知样品中，在25C振荡孵育30分钟到3个小时后洗涤。酶标试剂需要能放大化学信号，如辣根过氧化物酶，半乳糖苷酶等，还要识别检测抗体，如链霉亲和素识别检测抗体上的生物素，兔抗鼠抗体识别鼠源的检测抗体等。
成功形成“微球－捕获抗体-目标分子-检测抗体－标记酶”的复合结构在反应底物中通过末端带有的标记酶分子催化溶液的反应，利用一个催化剂分子催化产生多个数量级的反应底物分子，从而实现信号的有效放大。
基于微球复合结构与微孔板芯片的单分子检测：由于使用了传统的化学发光催化方式，为了达到不扩增的单分子灵敏度，需要将催化剂的浓度最大程度的放大，所以本专利采用微反应器的方式将每一个微球复合结构放置在一个微孔中，微孔的体积在10e-15升左右，之后封锁住每一个微孔(油相或高分子薄膜等)，这样每一个微球复合结构都在一个单独的小体积中，即使复合结构上只有一个催化剂分子浓度也提高了多个数量级，足够催化单个微孔反光。
微孔板芯片上微孔的密度决定了所采集的数据量及检测动态范围。密排的微孔需要光学检测能清晰分辨，并保证每一个微孔中只有一个或没有微球。一次性的微孔板采用注塑生产。非一次性的微孔可通过芯片MEMS方法加工。
微球复合结构与微孔的组装：为了保证高效快捷的将微球与微孔一一配对组装，可采用一下几种方法：1.利用微球密度大于水的特性，重力可使微球自然沉入微孔中；2.由于微球可包裹磁性材料，可使用磁力将微球操纵入微孔中；3.由于微球的介电常数与溶液相差极大，可通过施加非匀强交变电场产生介电泳力将微球推入微孔中，在反应检测结束后，可通过调 整电场频率改变力的方向将微球推出微孔从而实现微孔重复使用。
如需进行极低浓度的单分子检测需要调整微球与目标分子的比例依泊松分布优化，使一个微球上只抓取一个或不抓取目标分子。
基于微球的样品处理和基于微孔板的检测设备
样品处理：设备的样品处理部分集成液体转移、混合、磁吸、液体预存、液路清洗等模块，将样品通过集成在设备中的在线稀释系统进行相应的稀释后，和负载检测抗体的磁珠，检测抗体，酶标试剂，洗液等试剂进行混合洗涤等操作。最终得到带有的酶标记的免疫夹心复合物的纯净磁微粒溶液。
检测：设备的检测部分集成多色荧光激发、前散射成像、荧光发射滤光、磁吸、交变电场控制等模块，将多重荧光编码的微球通过之前提到的方式操纵到微孔板中，通过液路自动控制将微孔板表面利用油相封闭，在催化反应发生后，使用不同波长的荧光进行近场激发并利用前散射光成像拍照得到不同滤光波长下的图像，之后将微球操纵出孔后进行液路清洗。
软件与自动控制：所有模块均通过底层程序自动控制，软件集成图像自动识别分析模块，系统自动识别每一个微孔中的亮度并得到微孔亮度的分布，自动判断每个微孔中是否有反应发生，每一个反应对应的微球编号。通过亮度分析得到模拟信号的动态检测范围，通过同一编号微球有反应发生的个数作为数字信号的动态检测范围。
下面结合具体的实施例进一步说明。
实施例1
高灵敏度检测同时检测IL-2和IL6细胞因子
1.两种荧光编码微球合成：将苯乙烯单体、聚甲基丙烯酸甲酯、引发剂、交联剂、丙烯酸寡聚物混合在氯仿中作为高分子溶液。取两份4.5mL高分子溶液记为反应溶液1和反应溶液2，反应溶液1中加入0.5mL罗丹明和0.5mL荧光素和90mg纳米磁颗粒，反应溶液2中加入0.5mL罗丹明和0.5mL荧光素和90mg纳米磁颗粒。之后将反应溶液1和反应溶液2分别置于两个有300mL去离子水和表面活性剂的反应器中，在搅拌、超声与调整表面力的情况下将反应溶液1和反应溶液2均匀分散为约10微米尺寸的微乳液滴，在溶液中加入引发剂并加热进行聚合交联反应，24小时之后，每个液滴中的氯仿缓慢溶于水中并挥发，单体聚合并交联，最终形成荧光编码微球1和荧光编码微球2。两种高分子荧光编码微球表面带有羧基官能团。
2.微球1和2分别偶连IL-2和IL-6的捕获抗体，以1为例，将微球1mg在1ml PBS缓冲液中分散，加入5mg EDC和5mg Sulfo－NHS，混匀并维持搅拌10分钟，用1ml PBS 清洗后加入50ug IL-2捕获抗体。室温振荡孵育30分钟到3小时。加入BSA等封闭剂后室温振荡孵育30分钟，通过磁性分离洗涤微球1，最终分散在PBS中。将负载好的磁珠1和2等体积混合，每个磁珠浓度0.5mg/ml
3.将IL2和IL6标准品掺杂到10％的牛血清中浓度分别为(0，0.001，0.005，0.01，0.3，1.0，和10pg/ml)。取每个标准品100ul，加入50ul 1.中磁珠混合溶液，在25C振荡孵育3个小时后，用5X PBS+0.1％Tween洗涤三遍。分散到100ul PBS中，加入50ul 1ug/ml的IL2检测抗体和50ul 1ug/ml的IL6检测抗体，在25C振荡孵育半个小时后，用5X PBS+0.1％Tween洗涤三遍。分散到100ul PBS中，加入链霉亲和素-beta-半乳糖苷酶共轭酶标试剂50ul。用5X PBS+0.1％Tween洗涤四遍。分散到100ul酶底物溶液中。含有未知浓度待测物的样本按照相同的方式处理。
4.将微球复合结构通过微流体加入有微孔板芯片的反应器中，加10MHz交流电，微球被推入微孔中，之后用硅油将微孔表面封闭，反应2分钟后，使用488nm波长光激发，滤光片1拍照，滤光片2拍照，再使用532纳米波长光激发，滤光片3拍照，流入乙醇后再流入清洗液，改变交流电频率至10kHz，之后流入清洗液，清洗反应器。
5.数据处理和浓度测定。图象处理软件首先识别所有磁珠，根据滤光片1和滤光片2下荧光的强度将磁珠分类(磁珠1，带IL-2捕获抗体和磁珠2,带IL-6捕获抗体)。对于每种磁珠每个浓度计算标记酶分子的总数和磁珠总数的比例值(AEB，Average Enzyme per Bead)。当待测物浓度较低，检测到产生化学信号的磁珠占总磁珠的比例值(fon<0.5)，AEB＝-ln(1-fon))。当fon>0.5,AEB＝Ibead/Ienzyme，Ibead是单次测试中所有磁珠产生信号的平均值，Ienzyme是单个酶分子产生信号的平均值，I enzyme＝fon*Ibead/AEB,AEB＝-ln(1-fon)，计算时I enzyme用所有fon<0.5样品的数据。将AEB值和待测物浓度值进行线性拟合绘制校准曲线后，用相同的方法测试未知样品，得到的AEB值用内插法带入标准曲线即测得未知样品中待测物的浓度。按以上方法测得IL-2浓度为1.1pg/ml,IL-6浓度为0.8pg/ml，所获得的。
6.检出限的测定
将浓度为0的样品平行测10次，测得的AEB值的平均值加3倍的标准偏差带入4.的标准曲线中得到的待测物溶度即为本方法的检出限。IL-2的检出限为0.069pg/ml,IL-6的检出限为0.030pg/ml。
图1为实施例1中测未知浓度的IL-2和IL-6细胞因子样品时，修饰有夹心复合物的荧光编码微球导入微孔板，油封后通过前散射成像模块检测到的标记酶(半乳糖苷酶)信号。 每个微孔中含有1个或者0个磁性微球。因为检测的IL-2和IL-6细胞因子浓度较低，大部分球表面未形成了完整的带有半乳糖苷酶标记的复合物，不能产生由半乳糖苷酶标记放大产生的荧光信号。少部分球表面形成一个或少数几个完整的带有半乳糖苷酶标记的复合物，从而放大产生荧光信号。产生荧光信号的球占总微球的比例和待测物浓度成正相关。
实施例2
高灵敏度同时检测IL10、IFNr两种细胞因子
1.两种荧光编码微球合成：将苯乙烯单体、聚甲基丙烯酸甲酯、引发剂、交联剂、丙烯酸寡聚物混合在氯仿中作为高分子溶液。取两份4.5mL高分子溶液纪为反应溶液1和反应溶液2，1加入0.5mL罗丹明和0.5mL荧光素和90mg纳米磁颗粒，2加入0.5mL罗丹明和0.5mL荧光素和90mg纳米磁颗粒。之后将1和2分别置于两个有300mL去离子水和表面活性剂的反应器中，以1为例，在搅拌、超声与调整表面力的情况下将1溶液均匀分散为10微米的液滴，在溶液中加入引发剂并加热进行聚合交联反应，24小时之后，每个液滴中的氯仿缓慢溶于水中并挥发，单体聚合并交联，最终形成高分子微球1，2过程相同。两种高分子微球表面带有羧基官能团。
2.微球1和2分别偶连IL-10和IFNr的捕获抗体，以1为例，将微球1mg在1mlPBS缓冲液中分散，加入5mg EDC和5mg Sulfo－NHS，混匀并维持搅拌10分钟，用1ml PBS清洗后加入50ug IL-2捕获抗体。室温振荡孵育30分钟到3小时。加入BSA等封闭剂后室温振荡孵育30分钟，通过磁性分离洗涤微球1，最终分散在PBS中。将负载好的磁珠1和2等体积混合，每个磁珠浓度0.5mg/ml
3.将IL10和INFr标准品掺杂到10％的牛血清中浓度分别为(0，0.001，0.005，0.01，0.3，1.0，和10pg/ml)。取每个标准品100ul，加入50ul 1.中磁珠混合溶液，在25C振荡孵育3个小时后，用5X PBS+0.1％Tween洗涤三遍。分散到100ul PBS中，加入50ul 1ug/ml的IL-10检测抗体和50ul 1ug/ml的IFNr检测抗体，在25C振荡孵育半个小时后，用5X PBS+0.1％Tween洗涤三遍。分散到100ul PBS中，加入链霉亲和素-beta-半乳糖苷酶共轭酶标试剂50ul。用5X PBS+0.1％Tween洗涤四遍。分散到100ul酶底物溶液中。含有未知浓度待测物的样本按照相同的方式处理。
4.将微球复合结构通过微流体加入有微孔板芯片的反应器中，加10MHz交流电，微球被推入微孔中，之后用硅油将微孔表面封闭，反应2分钟后，使用488nm波长光激发，滤光片1拍照，滤光片2拍照，再使用532纳米波长光激发，滤光片3拍照，流入乙醇后再流入清洗液，改变交流电频率至10kHz，之后流入清洗液，清洗反应器。
5.数据处理和浓度测定。图象处理软件首先识别所有磁珠，根据滤光片1和滤光片2下荧光的强度将磁珠分类(磁珠1，带IL-2捕获抗体和磁珠2,带IL-6捕获抗体)。对于每种磁珠每个浓度计算标记酶分子的总数和磁珠总数的比例值(AEB，Average Enzyme per Bead)。当待测物浓度较低，检测到产生化学信号的磁珠占总磁珠的比例值(fon<0.5)，AEB＝-ln(1-fon))。当fon>0.5,AEB＝Ibead/Ienzyme，Ibead是单次测试中所有磁珠产生信号的平均值，Ienzyme是单个酶分子产生信号的平均值，Ienzyme＝fon*Ibead/AEB,AEB＝-ln(1-fon)，计算时Ienzyme用所有fon<0.5样品的数据。将AEB值和待测物浓度值进行线性拟合绘制校准曲线后，用相同的方法测试未知样品，得到的AEB值用内插法带入标准曲线即测得未知样品中待测物的浓度。测得IL-10的浓度为10pg/ml,INFr的浓度为0.3pg/ml。
6.检出限的测定
将浓度为0的样品平行测10次，测得的AEB值的平均值加3倍的标准偏差带入4.的标准曲线中得到的待测物溶度即为本方法的检出限。测得IL-10的检出限位0.027pg/ml,INFr检出限为0.024pg/ml。
图2为实施例2中测未知浓度的IL-10和IFNr细胞因子样品时，修饰有夹心复合物的荧光编码微球导入微孔板，油封后通过前散射成像模块检测到的标记酶(半乳糖苷酶)信号。每个微孔中含有1个或者0个磁性微球。因为检测的IL-2和IL-6细胞因子浓度较低，大部分球表面未形成了完整的带有半乳糖苷酶标记的复合物，不能产生由半乳糖苷酶标记放大产生的荧光信号。少部分球表面形成一个或少数几个完整的带有半乳糖苷酶标记的复合物，从而放大产生荧光信号。产生荧光信号的球占总微球的比例和待测物浓度成正相关。
综上所述，本发明提供的多重免疫检测方法和试剂盒，可使免疫分子检测达到飞克级别(fg/ml)，体现了超高灵敏度，配合无扩增核酸分子诊断设备，其检测步骤简单，检测结果准确可靠。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 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。
  2. 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。
  3. 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。
  4. 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。
  5. 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。
  6. 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。
  7. 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。
  8. 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。
  9. 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。
  10. 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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