CN111060683A - Multiplex immune molecule detection method and kit - Google Patents
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Abstract
The invention discloses a multiplex immune molecule detection method, which comprises the following steps: obtaining coding microspheres with capture molecules connected to the surfaces; capturing target immune molecules through the capture molecules, adding an enzyme-labeled reagent, and forming an enzyme-labeled immune sandwich compound on the surface of the coding microsphere; driving the coded microspheres with the immune sandwich compound modified on the surfaces into micropores of a microporous plate and sealing; and after the preset time, the microporous plate is excited by light to detect the microporous plate. The invention enables the protein detection to reach the femto-gram level (fg/ml), and the sensitivity is 1000 times higher than that of the traditional ELISA method. Especially has great advantages for single molecule detection, and has wide application prospect in the aspects of life science, in vitro diagnosis, companion diagnosis, blood screening, drug research and development and the like.
Description
Technical Field
The invention belongs to the field of immune molecule detection, and particularly relates to a multiple immune molecule detection method and a kit.
Background
The traditional ELISA reaction system is relatively large (100 mu l), millions of molecules are needed to generate tens of thousands of fluorophore signals to be detected by the microplate reader, so that the traditional ELISA method can only detect signals in a picomolar range (10pg/ml) and adopts an analog algorithm. The method has reached the limit in modern science and is far from meeting the 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 the traditional ELISA cannot detect the molecules, and cannot achieve sensitive monitoring. ELISA only can detect one index in one sample experiment. However, in the case of acute and severe diagnosis or when there are few biological samples (such as blood of infants and children, cerebrospinal fluid, etc.), the immunoassay is clinically required to be capable of detecting multiple protein indexes simultaneously in one sample, i.e., multiplex detection.
In order to improve the sensitivity of immunodiagnosis, in vitro diagnostics manufacturers have developed and commercialized magnetic particle chemiluminescence and magnetic particle electrochemiluminescence based on traditional enzyme-linked immunosorbent assay (ELISA). The two methods adopt a detection methodology identical to that of an enzyme-linked immune sandwich compound, namely, a stationary phase loaded with a capture antibody is used for capturing molecules of a substance to be detected, and then a detection antibody for identifying the molecules of the substance to be detected is added to form the immune sandwich compound. The detection antibody and the capture antibody recognize different epitopes on the molecules of the substance to be detected, and the detection antibody can be marked by enzyme capable of chemically amplifying signals or micromolecular catalyst capable of electrochemically amplifying signals. And finally, positively correlating the detected amplified signal with the molecular concentration of the substance to be detected. And testing the standard substance of the object to be tested with different concentrations, performing linear fitting on the signal value and the concentration value of the object to be tested to draw a calibration curve, testing the unknown sample by using the same method, and substituting the obtained signal value into the standard curve by using an interpolation method to obtain the concentration of the object to be tested in the unknown sample. Compared with enzyme-linked immunosorbent assay, the magnetic particle chemiluminescence assay and the magnetic particle electrochemiluminescence assay adopt magnetic particles as stationary phases, and can capture molecules to be detected more quickly and efficiently. Compared with enzyme-linked immunosorbent assay, the enzyme-linked immunosorbent assay has the advantage that the signal-to-noise ratio of chemiluminescence and electrochemiluminescence signals is improved by about ten times. In practical application, the two new methods have 10-100 times higher sensitivity than the traditional ELISA, and the sensitivity can reach 1pg/ml at most. At this sensitivity, about 200-300 clinically significant protein markers in blood can be effectively detected, but thousands of other or potentially clinically significant blood protein markers cannot be effectively detected due to their low concentrations.
The main factor limiting the sensitivity of the magnetic particle chemiluminescence/electrochemiluminescence method is the method of detection. The magnetic beads with the enzyme-labeled sandwich complex are collectively detected and give a continuous signal in a macroscopic manner. When the concentration of the antigen is 10fg/ml to 10pg/ml, the result of macroscopic concentration observation is not much different from the background (0 enzyme molecules are labeled on a single magnetic bead) when the number of the enzyme molecules labeled on a single magnetic bead is less than 10-100.
In order to realize multiplex detection, in vitro diagnostics manufacturers have developed and commercialized flow-type fluorescence and microporous electrochemiluminescence methods using detection methodologies of immune sandwich complexes. The flow-type fluorescence method adopts microspheres as a stationary phase, uses different fluorescence codes to load microspheres with different capture antibodies, and uses a flow cytometer to read signals of the fluorescence codes of the microspheres and fluorescent markers of the immune sandwich compound on the microspheres. The micropore electrochemiluminescence method is used for micro-printing different capture antibodies to different positions in a micropore, and simultaneously reading position information and an electrochemical signal of the immune sandwich complex through an imaging method. These two methods are capable of achieving multiplex detection. However, fluorescently encoded microspheres, flow cytometry, and microprinting in well plates have dramatically increased the cost of detection. And the sensitivity of the two methods is reduced compared with the magnetic particle chemical/electrochemical luminescence method.
The highest detection sensitivity is single-molecule detection, and each detection molecule is digitalized under the detection condition of the traditional analog signal ELISA, so that the protein detection reaches the Feike level (fg/ml), and the sensitivity is 1000 times higher than that of the traditional ELISA method. And a sample detection mode of a fluorescence coding liquid chip is adopted, and at least 15 marker proteins can be detected, so that the method has extremely high application value for saving the sample amount and improving the efficiency in the aspect of detection research. Has wide application prospect in the aspects of life science, in-vitro diagnosis, companion diagnosis, blood screening, drug research and development and the like.
Disclosure of Invention
The invention aims to solve the problems of low oxygen reduction electrocatalytic activity and low electrochemical stability of the existing silver-based nano catalyst.
Based on the above, the invention provides a multiple immune molecule detection method, which comprises the following steps:
obtaining coding microspheres with capture molecules connected to the surfaces;
capturing target immune molecules through the capture molecules, adding an enzyme-labeled reagent, and forming an enzyme-labeled immune sandwich compound on the surface of the coding microsphere;
driving the coded microspheres with the immune sandwich compound modified on the surfaces into micropores of a microporous plate and sealing;
and after the preset time, the microporous plate is excited by light to detect the microporous plate.
In some embodiments, the encoded microspheres include a body of polymeric material and an encoding material distributed in the body of polymeric material.
In some embodiments, the encoded microspheres further comprise magnetic nanoparticles that are orderly distributed in the polymeric material body.
Preferably, the particle size of the coding microsphere is 0.5-50 μm, and the particle size of the magnetic nanoparticle is 1-100 nm.
In some embodiments, the encoded microspheres comprise at least two luminescent encoding materials;
preferably, the luminescent coding material is an organic fluorescent material or an inorganic fluorescent material;
preferably, the luminescent coding material is at least one of an organic dye and a quantum dot.
In some embodiments, the method of preparing the encoded microspheres comprises the steps of:
mixing at least two luminescent materials and microspheres in a high polymer material, dispersing a high polymer solution in a water phase to form uniform liquid drops through a multiple-coupling physical field, and then wrapping the luminescent materials and magnetic nanoparticles in the liquid drops through a cross-linking polymerization reaction to obtain the coding microspheres.
In some embodiments, one target immune molecule is captured on the surface of each of the encoded microspheres.
In some embodiments, 1 of the encoded microspheres is contained in each of the microwells.
In some embodiments, the encoded microspheres with the immune sandwich complex modified on the surface are driven into the microwells of the microwell plate by an electric field and sealed.
The invention also provides a kit for immune molecule detection, which comprises a coding microsphere, detection molecules and hybridization buffer solution, wherein the surface of the coding microsphere is connected with the capture molecules, the coding microsphere comprises a polymer material main body, magnetic nanoparticles and a coding material, and the magnetic nanoparticles and the coding material are distributed in the polymer material main body.
In some embodiments, the kit further comprises a microplate chip;
preferably, the volume of a single micropore on the micropore plate chip is (20-100) multiplied by 10-15L.
According to the invention, each detection molecule is digitalized under the detection condition of the traditional analog signal ELISA, so that the protein detection reaches the femto-gram level (fg/ml), and the sensitivity is 1000 times higher than that of the traditional ELISA method. And a sample detection mode of a fluorescence coding liquid chip is adopted, and at least 15 marker proteins can be detected, so that the method has extremely high application value for saving the sample amount and improving the efficiency in the aspect of detection research. The invention has great advantages especially for single molecule detection, and has wide application prospects in the aspects of life science, in-vitro diagnosis, companion diagnosis, blood screening, drug research and development and the like.
Drawings
FIG. 1 is a signal of a labeled enzyme (galactosidase) detected by a front scattering imaging module after fluorescent-encoded microspheres modified with a sandwich complex are introduced into a microplate and oil-sealed when samples of IL-2 and IL-6 cytokines with unknown concentrations are measured in example 1.
FIG. 2 is a signal of a labeled enzyme (galactosidase) detected by a front scattering imaging module after oil sealing of fluorescent encoded microspheres modified with a sandwich complex introduced into a microplate when samples of IL-10 and IFNr cytokines with unknown concentrations are measured in example 2.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
It is to be noted that, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention provides a method for giving a macroscopic continuous signal by intensively detecting all magnetic particles of an enzyme-labeled immune sandwich compound during detection, which is fundamentally abandoned, but each magnetic particle is sealed in a femto-liter-grade volume for independent detection, and simultaneously, the detection is carried out>10,000 magnetic particles. Chemical signals generated by individual enzyme molecules can be detected at this volume. When the concentration of the molecules to be detected is in the fg/ml level, most of the magnetic particles are not labeled by enzyme molecules, and no signal is generated. The remaining magnetic particles are predominantly labeled with one enzyme molecule and a small portion labeled with two or more enzyme molecules, thereby generating a chemical signal that can be detected by the system. The ratio of the total number of magnetic beads to which chemical signals were detected (fon) to the total number of labeled Enzyme molecules and the total number of magnetic beads (AEB, Average Enzyme per Bead) followed a Poisson distribution (AEB ═ ln (1-fon)). When the concentration of the analyte reaches pg/ml grade, the majority of the magnetic particles after sample treatment carry 1-10 labeled enzyme molecules (AEB is 1-10), the AEB is still positively correlated with the concentration of the analyte, and the AEB is Ibead/Ienzyme，IbeadIs the average of the signals generated by all beads in a single test, IenzymeIs the average of the signals generated by the individual enzyme molecules, Ienzyme＝fon*Ibead/AEB, at this time IbeadMeans all messages generated in a single testThe average signal value of magnetic beads No. AEB ═ ln (1-fon), calculated using assay concentration on the fg/ml scale, fon<Data for samples at 0.5, where the calculated AEB is more accurate and the majority of signal-producing magnetic particles carry only one tagged enzyme molecule, thus ensuring the calculated IenzymeAccuracy of). And (3) the AEB and the concentration of the molecules of the object to be detected form positive correlation, the standard substance of the object to be detected with different concentrations is tested, the AEB value and the concentration value of the object to be detected are subjected to linear fitting to draw a calibration curve, the unknown sample is tested by using the same method, and the obtained signal value is introduced into the standard curve by using an interpolation method, so that the concentration of the object to be detected in the unknown sample is measured. By adopting the method of independently detecting each magnetic particle after being sealed, the detection sensitivity can be improved from pg/ml magnitude to fg/ml level by about 1000 times.
In some preferred embodiments, multiple protein indicators are detected simultaneously in order to achieve a single assay. Different capture antibodies are coupled on the magnetic particles with different fluorescence codes, and the same sample is detected after equal proportion mixing.
Two or more than two fluorescent materials (organic and inorganic fluorescent molecules, including quantum dots and the like), magnetic nanoparticles and other functional nanoparticles are mixed in a plurality of high polymer materials (with different molecular weights and different functional groups) to be synthesized, a high polymer solution is dispersed in a water phase through a multi-coupling physical field to form uniform liquid drops/micro reaction groups, then the functional materials are wrapped and buried in the high polymer microspheres through crosslinking and polymerization reactions, and the oligomeric polymers with reactivity are exposed on the surfaces of the microspheres through micro phase separation. The exposed oligomeric polymer or other surface polymers of the microsphere can be subjected to further surface chemical reaction, so that the surface has weak nonspecific affinity to nucleic acid molecules, strong specific coupling reaction activity and high density, the microsphere can be tolerant under different reaction conditions (organic phase and high temperature), and finally the controllable capture antibody for specifically recognizing target molecules coupled on the microsphere surface can be realized.
For a particular analyte molecule, the capture antibody and the detection antibody each recognize different epitopes with high affinity specificity. Affinity and specificity were characterized and tested using methods such as surface plasmon resonance and protein chips. The antibody with higher affinity is used as a capture antibody, and the antibody with relatively low affinity is used as a detection antibody. The detection antibody needs to be labeled with corresponding enzyme, such as biotin, digoxigenin, etc. The magnetic particle surface has epoxy, carboxyl and other functional groups, and after the capture antibody is covalently immobilized on the magnetic bead surface in a suitable catalyst (EDC) and pH, the magnetic bead surface needs to be blocked by a suitable blocking agent such as whey protein, bovine blood albumin and the like to reduce non-specific adsorption on the magnetic particle surface.
Enzyme-labeled immune sandwich complex formation can be performed in a three-step, two-step, or one-step process. The three-step method is to add magnetic particles with capture antibodies to the unknown sample. Incubate 30 minutes to 3 hours at 25C with shaking and wash. The detection antibody is added and incubated at 25C with shaking for 30 minutes to 3 hours before washing. The enzyme-labeled reagent is added, incubated at 25 ℃ with shaking for 30 minutes to 3 hours, and then washed. The two-step method is that magnetic particles with capture antibody and detection antibody are added to unknown sample, and washed after incubation for 30 minutes to 3 hours at 25C with shaking. The enzyme-labeled reagent is added, incubated at 25 ℃ with shaking for 30 minutes to 3 hours, and then washed. The one-step method is that magnetic particles with capture antibodies, detection antibodies and enzyme labeling reagents are added into unknown samples, and the unknown samples are washed after being incubated for 30 minutes to 3 hours at 25 ℃ with shaking. The enzyme-labeled reagent needs to be capable of amplifying chemical signals, such as horseradish peroxidase, galactosidase and the like, and also needs to identify detection antibodies, such as streptavidin, biotin on the detection antibody, rabbit anti-mouse antibody, mouse-derived detection antibody and the like.
The composite structure of 'microsphere-capture antibody-target molecule-detection antibody-marker enzyme' is successfully formed, and a plurality of orders of magnitude of reaction substrate molecules are generated by catalysis of one catalyst molecule through the reaction of a marker enzyme molecule catalytic solution carried at the tail end in a reaction substrate, so that the effective amplification of signals is realized.
Based on the single molecule detection of the microsphere composite structure and the microporous plate chip: because a traditional chemiluminescence catalysis mode is used, in order to achieve unimolecular sensitivity without amplification, the concentration of a catalyst needs to be amplified to the maximum extent, so that each microsphere composite structure is placed in a micropore in a micro-reactor mode, the volume of the micropore is about 10e-15 liters, and then each micropore (oil phase or polymer film and the like) is sealed and locked, so that each microsphere composite structure is in a single small volume, the concentration of only one catalyst molecule on the composite structure is also improved by multiple orders of magnitude, and the single micropore light reflection is catalyzed sufficiently.
The density of the microwells on the microplate chip determines the amount of data collected and the dynamic range of detection. The densely packed microwells need to be clearly resolved by optical detection and ensure that there is only one or no microsphere in each microwell. The disposable microporous plate is produced by injection molding. Non-disposable microwells can be fabricated by the chip MEMS method.
Assembling the microsphere composite structure and the micropores: in order to ensure efficient and rapid assembly of microspheres and micropores in a one-to-one pairing manner, several methods can be adopted: 1. by utilizing the characteristic that the density of the microspheres is greater than that of water, the microspheres can naturally sink into the micropores by gravity; 2. because the microspheres can wrap the magnetic material, the microspheres can be manipulated into the micropores by using magnetic force; 3. because the dielectric constant of the microsphere is greatly different from that of the solution, the microsphere can be pushed into the micropore by applying a non-uniform-strength alternating electric field to generate dielectrophoresis force, and after the reaction detection is finished, the direction of the force can be changed by adjusting the frequency of the electric field to push the microsphere out of the micropore, so that the repeated use of the micropore is realized.
If the single molecule detection with extremely low concentration needs to be carried out, the proportion of the microspheres to the target molecules needs to be adjusted according to the Poisson distribution optimization, so that only one or no target molecules are captured on one microsphere.
Microsphere-based sample processing and microplate-based detection device
Sample treatment: the sample processing part of the device is integrated with modules of liquid transfer, mixing, magnetic attraction, liquid prestoring, liquid path cleaning and the like, and after the sample is correspondingly diluted by an online dilution system integrated in the device, the sample is mixed and washed with reagents such as magnetic beads loaded with detection antibodies, enzyme labeling reagents, washing liquor and the like. Finally obtaining pure magnetic particle solution with the enzyme-labeled immune sandwich compound.
And (3) detection: the detection part of the device integrates modules such as multicolor fluorescence excitation, front scattering imaging, fluorescence emission filtering, magnetic attraction, alternating electric field control and the like, microspheres with multiple fluorescence codes are manipulated into a microporous plate in the aforementioned mode, the surface of the microporous plate is sealed by using an oil phase through automatic control of a liquid path, after catalytic reaction occurs, fluorescence with different wavelengths is used for near-field excitation, images under different filtering wavelengths are obtained by using front scattering light imaging for photographing, and then the microspheres are manipulated out of holes and then the liquid path is cleaned.
Software and automatic control: all modules are automatically controlled by a bottom layer program, the software is integrated with an image automatic identification and analysis module, a system automatically identifies the brightness in each micropore and obtains the distribution of the micropore brightness, whether a reaction occurs in each micropore is automatically judged, and the number of the microsphere corresponding to each reaction is judged. And obtaining the dynamic detection range of the analog signal through brightness analysis, and taking the number of the reaction occurrences of the microspheres with the same number as the dynamic detection range of the digital signal.
The following is further illustrated with reference to specific examples.
Example 1
High sensitivity detection for simultaneous detection of IL-2 and IL6 cytokines
1. Two fluorescent coding microspheres were synthesized: styrene monomer, polymethyl methacrylate, initiator, cross-linking agent and acrylic oligomer are mixed in chloroform to form polymer solution. Taking two parts of 4.5mL of macromolecular solution, marking as a reaction solution 1 and a reaction solution 2, adding 0.5mL of rhodamine, 0.5mL of fluorescein and 90mg of nano magnetic particles into the reaction solution 1, and adding 0.5mL of rhodamine, 0.5mL of fluorescein and 90mg of nano magnetic particles into the reaction solution 2. Then placing the reaction solution 1 and the reaction solution 2 in two reactors with 300mL of deionized water and a surfactant respectively, uniformly dispersing the reaction solution 1 and the reaction solution 2 into microemulsion droplets with the size of about 10 microns under the conditions of stirring, ultrasound and surface force adjustment, adding an initiator into the solution, heating for polymerization and crosslinking reaction, slowly dissolving chloroform in each droplet in water and volatilizing after 24 hours, polymerizing and crosslinking monomers, and finally forming the fluorescent coding microspheres 1 and the fluorescent coding microspheres 2. The surfaces of the two polymer fluorescent coding microspheres are provided with carboxyl functional groups.
2. Microspheres 1 and 2 were coupled to capture antibodies to IL-2 and IL-6, respectively, and using 1 as an example, microspheres 1mg were dispersed in 1ml PBS buffer, 5mg EDC and 5mg Sulfo-NHS were added, mixed well and stirred for 10 minutes, washed with 1ml PBS, and 50ug IL-2 capture antibody was added. Incubate with shaking at room temperature for 30 minutes to 3 hours. After addition of blocking agents such as BSA, etc., incubation was performed at room temperature for 30 minutes with shaking, microspheres 1 were washed by magnetic separation and finally dispersed in PBS. Mixing the loaded magnetic beads 1 and 2 in equal volume, wherein the concentration of each magnetic bead is 0.5mg/ml
3. The IL2 and IL6 standards were spiked into 10% bovine serum at concentrations of (0, 0.001, 0.005, 0.01, 0.3, 1.0, and 10pg/ml), respectively. 100ul of each standard was taken, 50ul of the 1-medium magnetic bead mixed solution was added, and after incubation for 3 hours at 25C with shaking, the mixture was washed three times with 5 XPBS + 0.1% Tween. Dispersed into 100ul PBS, added 50ul 1ug/ml IL2 detection antibody and 50ul 1ug/ml IL6 detection antibody, after 25C shaking incubation for half an hour, washed three times with 5 XPBS + 0.1% Tween. Dispersed in 100ul PBS, and added with streptavidin-beta-galactosidase conjugate enzyme labeling reagent 50 ul. Wash four times with 5X PBS + 0.1% Tween. Dispersed in 100ul enzyme substrate solution. Samples containing unknown concentrations of analyte were processed in the same manner.
4. Adding the microsphere composite structure into a reactor with a microporous plate chip through microfluid, adding 10MHz alternating current, pushing microspheres into micropores, sealing the surfaces of the micropores with silicone oil, reacting for 2 minutes, exciting with 488nm wavelength light, photographing with an optical filter 1, photographing with an optical filter 2, exciting with 532 nm wavelength light, photographing with an optical filter 3, flowing ethanol, then flowing cleaning liquid, changing the alternating current frequency to 10kHz, then flowing cleaning liquid, and cleaning the reactor.
5. Data processing and concentration determination. The image processing software first identifies all the beads and classifies the beads according to the intensity of the fluorescence under filter 1 and filter 2 (bead 1 with IL-2 capture antibody and bead 2 with IL-6 capture antibody). The ratio of the total number of labeled Enzyme molecules to the total number of magnetic beads was calculated for each concentration of each magnetic bead (AEB, Average Enzyme perBead). When the concentration of the substance to be detected is low, the magnetic beads generating chemical signals are detected to account for the total magnetismRatio of beads (fon)<0.5), AEB ═ ln (1-fon)). When fon>0.5 AEB ═ Ibead/Ienzyme, Ibead is the mean value of the signals generated by all beads in a single test, Ienzyme is the mean value of the signals generated by individual enzyme molecules, IenzymeI.e. fon Ibead/AEB, AEB-ln (1-fon), when calculated IenzymeWith all fons<Data for 0.5 sample. And after linearly fitting the AEB value and the concentration value of the object to be detected to draw a calibration curve, testing the unknown sample by using the same method, and substituting the obtained AEB value into a standard curve by using an interpolation method to obtain the concentration of the object to be detected in the unknown sample. IL-2 concentration of 1.1pg/ml and IL-6 concentration of 0.8pg/ml were determined as described above.
6. Determination of detection Limit
And (3) parallelly measuring the sample with the concentration of 0 for 10 times, and bringing the average value of the measured AEB value plus the standard deviation of 3 times into a standard curve of 4 to obtain the solubility of the substance to be measured, namely the detection limit of the method. The detection limit of IL-2 was 0.069pg/ml, and that of IL-6 was 0.030 pg/ml.
FIG. 1 is a signal of a labeled enzyme (galactosidase) detected by a front scattering imaging module after fluorescent-encoded microspheres modified with a sandwich complex are introduced into a microplate and oil-sealed when samples of IL-2 and IL-6 cytokines with unknown concentrations are measured in example 1. Each microwell contains 1 or 0 magnetic microsphere. Because of the low concentration of IL-2 and IL-6 cytokines detected, most of the sphere surfaces did not form an intact complex with a galactosidase label and did not produce a fluorescent signal amplified by the galactosidase label. A small fraction of the sphere surface forms one or a few intact complexes with a galactosidase label, which is amplified to produce a fluorescent signal. The proportion of the spheres generating the fluorescence signals in the total microspheres is positively correlated with the concentration of the substance to be detected.
Example 2
High-sensitivity simultaneous detection of IL10 and IFNr cytokines
1. Two fluorescent coding microspheres were synthesized: styrene monomer, polymethyl methacrylate, initiator, cross-linking agent and acrylic oligomer are mixed in chloroform to form polymer solution. Taking two parts of 4.5mL of macromolecular solution as a reaction solution 1 and a reaction solution 2, adding 0.5mL of rhodamine, 0.5mL of fluorescein and 90mg of nano magnetic particles into the reaction solution 1, and adding 0.5mL of rhodamine, 0.5mL of fluorescein and 90mg of nano magnetic particles into the reaction solution 2. Then placing the solution 1 and the solution 2 in two reactors with 300mL of deionized water and a surfactant respectively, taking 1 as an example, uniformly dispersing the solution 1 into droplets of 10 micrometers under the conditions of stirring, ultrasound and surface force adjustment, adding an initiator into the solution and heating for polymerization crosslinking reaction, after 24 hours, slowly dissolving chloroform in each droplet in water and volatilizing, polymerizing and crosslinking monomers, and finally forming the polymer microspheres 1, wherein the process of 2 is the same. The surfaces of the two polymer microspheres are provided with carboxyl functional groups.
2. Microspheres 1 and 2 were coupled to capture antibodies for IL-10 and IFNr, respectively, and using 1 as an example, microspheres 1mg were dispersed in 1ml PBS buffer, 5mg EDC and 5mg Sulfo-NHS were added, mixed and stirred for 10 minutes, washed with 1ml PBS, and 50ug IL-2 capture antibody was added. Incubate with shaking at room temperature for 30 minutes to 3 hours. After addition of blocking agents such as BSA, etc., incubation was performed at room temperature for 30 minutes with shaking, microspheres 1 were washed by magnetic separation and finally dispersed in PBS. Mixing the loaded magnetic beads 1 and 2 in equal volume, wherein the concentration of each magnetic bead is 0.5mg/ml
3. IL10 and INFr standards were spiked into 10% bovine serum at concentrations of (0, 0.001, 0.005, 0.01, 0.3, 1.0, and 10pg/ml), respectively. 100ul of each standard was taken, 50ul of the 1-medium magnetic bead mixed solution was added, and after incubation for 3 hours at 25C with shaking, the mixture was washed three times with 5 XPBS + 0.1% Tween. Dispersed into 100ul PBS, added 50ul 1ug/ml IL-10 detection antibody and 50ul 1ug/ml IFNr detection antibody, in 25C oscillation incubation for half an hour, with 5X PBS + 0.1% Tween washing three times. Dispersed in 100ul PBS, and added with streptavidin-beta-galactosidase conjugate enzyme labeling reagent 50 ul. Wash four times with 5X PBS + 0.1% Tween. Dispersed in 100ul enzyme substrate solution. Samples containing unknown concentrations of analyte were processed in the same manner.
4. Adding the microsphere composite structure into a reactor with a microporous plate chip through microfluid, adding 10MHz alternating current, pushing microspheres into micropores, sealing the surfaces of the micropores with silicone oil, reacting for 2 minutes, exciting with 488nm wavelength light, photographing with an optical filter 1, photographing with an optical filter 2, exciting with 532 nm wavelength light, photographing with an optical filter 3, flowing ethanol, then flowing cleaning liquid, changing the alternating current frequency to 10kHz, then flowing cleaning liquid, and cleaning the reactor.
5. Data processing and concentration determination. The image processing software first identifies all the beads and classifies the beads according to the intensity of the fluorescence under filter 1 and filter 2 (bead 1 with IL-2 capture antibody and bead 2 with IL-6 capture antibody). The ratio of the total number of labeled Enzyme molecules to the total number of magnetic beads was calculated for each concentration of each magnetic bead (AEB, Average Enzyme perBead). When the concentration of the analyte is low, the ratio of the magnetic beads generating the chemical signal to the total magnetic beads is detected (fon <0.5), and AEB ═ ln (1-fon)). When fon >0.5, AEB ═ Ibead/Ienzyme, Ibead is the average of the signals generated by all beads in a single test, Ienzyme is the average of the signals generated by a single enzyme molecule, Ienzyme ═ fon ═ Ibead/AEB, AEB ═ ln (1-fon), Ienzyme used the data for all fon <0.5 samples when calculated. And after linearly fitting the AEB value and the concentration value of the object to be detected to draw a calibration curve, testing the unknown sample by using the same method, and substituting the obtained AEB value into a standard curve by using an interpolation method to obtain the concentration of the object to be detected in the unknown sample. IL-10 was measured at a concentration of 10pg/ml and INFr at a concentration of 0.3 pg/ml.
6. Determination of detection Limit
And (3) parallelly measuring the sample with the concentration of 0 for 10 times, and bringing the average value of the measured AEB value plus the standard deviation of 3 times into a standard curve of 4 to obtain the solubility of the substance to be measured, namely the detection limit of the method. The detection limit of IL-10 is 0.027pg/ml, and the detection limit of INFr is 0.024 pg/ml.
FIG. 2 is a signal of a labeled enzyme (galactosidase) detected by a front scattering imaging module after oil sealing of fluorescent encoded microspheres modified with a sandwich complex introduced into a microplate when samples of IL-10 and IFNr cytokines with unknown concentrations are measured in example 2. Each microwell contains 1 or 0 magnetic microsphere. Because of the low concentration of IL-2 and IL-6 cytokines detected, most of the sphere surfaces did not form an intact complex with a galactosidase label and did not produce a fluorescent signal amplified by the galactosidase label. A small fraction of the sphere surface forms one or a few intact complexes with a galactosidase label, which is amplified to produce a fluorescent signal. The proportion of the spheres generating the fluorescence signals in the total microspheres is positively correlated with the concentration of the substance to be detected.
In conclusion, the multiple immunodetection method and the kit provided by the invention can enable the immune molecule detection to reach a femto-gram level (fg/ml), embody ultrahigh sensitivity, are matched with a nucleic acid molecule diagnosis device without amplification, and have the advantages of simple detection steps and accurate and reliable detection results.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A multiplex immune molecule detection method is characterized by comprising the following steps:
obtaining coding microspheres with capture molecules connected to the surfaces;
capturing target immune molecules through the capture molecules, adding an enzyme-labeled reagent, and forming an enzyme-labeled immune sandwich compound on the surface of the coding microsphere;
driving the coded microspheres with the immune sandwich compound modified on the surfaces into micropores of a microporous plate and sealing;
and after the preset time, the microporous plate is excited by light to detect the microporous plate.
2. The multiplex immunoassay molecule of claim 1, wherein the encoded microspheres comprise a polymeric material body and an encoding material disposed in the polymeric material body.
3. The multiplex immuno molecule detection method of claim 2, wherein said encoded microspheres further comprise magnetic nanoparticles distributed in order in said polymeric material body;
preferably, the particle size of the coding microsphere is 0.5-50 μm, and the particle size of the magnetic nanoparticle is 1-100 nm.
4. The multiplex immunoassay molecule of any of claims 1-3, wherein the encoded microspheres comprise at least two luminescent encoding materials;
preferably, the luminescent coding material is an organic fluorescent material or an inorganic fluorescent material;
preferably, the luminescent coding material is at least one of an organic dye and a quantum dot.
5. The multiplex immunoassay molecule detection method of claim 1, wherein the preparation method of the encoded microspheres comprises the steps of:
mixing at least two luminescent materials and microspheres in a high polymer material, dispersing a high polymer solution in a water phase to form uniform liquid drops through a multiple-coupling physical field, and then wrapping the luminescent materials and magnetic nanoparticles in the liquid drops through a cross-linking polymerization reaction to obtain the coding microspheres.
6. The method of claim 1, wherein each of the encoded microspheres captures a target biomolecule on the surface.
7. The multiplex immuno molecule detection method of claim 1 or 6, wherein 1 of said encoded microspheres is contained in each of said microwells.
8. The multiplex immuno-molecular detection method of claim 1 or 6, wherein the encoded microspheres with the surface modified with the immuno-sandwich complexes are driven into the microwells of the microwell plate by an electric field and sealed.
9. The kit for detecting the immune molecules is characterized by comprising a coding microsphere, a detection molecule and a hybridization buffer solution, wherein the surface of the coding microsphere is connected with a capture molecule, the coding microsphere comprises a polymer material main body, magnetic nanoparticles and a coding material, and the magnetic nanoparticles and the coding material are distributed in the polymer main body material.
10. The kit of claim 9, further comprising a microplate chip;
preferably, the volume of a single micropore on the micropore plate chip is (20-100) multiplied by 10-15L。
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