CN112858670B - Multiple digital ELISA detection method and microfluidic chip - Google Patents
Multiple digital ELISA detection method and microfluidic chip Download PDFInfo
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Abstract
The invention discloses a multiple digital ELISA detection method and a microfluidic chip, wherein the microfluidic chip comprises a top chip, a microsphere partition arrangement chip and size and color double-coding microspheres, wherein the microsphere partition arrangement chip and the size and color double-coding microspheres are respectively used in combination with the top chip. The microsphere partition arrangement chip is provided with microcolumn arrays which are arranged in parallel and have different radius size intervals, the microcolumn array of each size is a capture area of microspheres with corresponding size, and the microsphere surface is a detected solid phase surface. The size and color double-coded microspheres are mixed and superposed with different radii and sizes and different proportions of two colors to construct high-flux double-classified coded microspheres. The microsphere surface is a detected solid phase surface, and is obtained by double coding of size and color, and the characteristics of color are obtained by mixing different colors in different proportions. Each microsphere corresponds to one target, so that large-scale simultaneous detection of multiple targets is realized, and the problems of large sample and labor consumption, high time and high economic cost in single target detection are solved.
Description
Technical Field
The invention relates to the field of rapid detection of medical instruments, in particular to a multiple digital ELISA detection method and a microfluidic chip.
Background
The on-site rapid inspection is also called instant inspection, is immediate inspection and analysis carried out at the patient bedside or the sampling site, does not depend on clinical inspectors and complex laboratory processes, and can meet the requirements of remote areas with poor medical conditions, emergent public health events or disease screening and the like on-site rapid inspection.
The diagnosis of diseases and the judgment of curative effect need to carry out combined detection and analysis on a plurality of targets (including genes or proteins and the like), which provides a new challenge for the rapidity and comprehensiveness of the traditional detection field.
The single target detection consumes more samples, consumes long time, has high labor and reagent cost, and cannot meet the requirements of large-scale screening and diagnosis of clinical diseases, and the existing multiple detection method has only a few or more detection fluxes, so that the detection efficiency can be obviously improved and the real-time rapid monitoring of the diseases can be realized if the detection fluxes can be greatly improved to carry out parallel multi-path detection.
Disclosure of Invention
In order to solve the above problems, the present invention provides a multiplex digital ELISA detection method to achieve the purposes of parallel detection of high-throughput targets, less sample consumption, low time and labor cost, high sensitivity and specificity; a microfluidic chip is also provided.
The purpose of the invention is realized by the following technical scheme:
the invention provides a multiple digital ELISA detection method which is applied to a microfluidic chip and comprises the following steps:
step S1, respectively combining the double-classification coded microspheres with different radius sizes and color settings with corresponding detection antibodies;
step S2, filling the microspheres obtained in the step S1 into a microsphere partition arrangement chip of the microfluidic chip, so that the microspheres are arranged in corresponding areas in the microsphere partition arrangement chip according to the size under the action of self fluid;
step S3, pouring a sample with target molecules and a binding antibody which corresponds to the detection antibody and contains fluorescent molecules into the microfluidic chip, wherein the target molecules in the sample are firstly bound with the detection antibody on the microspheres, and the binding antibody is then bound with the target molecules in the sample, so that the microspheres carry the fluorescent molecules with the content corresponding to the content of the target molecules;
and step S4, collecting fluorescence signals of the microfluidic chip, distinguishing the types of target molecules according to the areas of the microspheres in the microsphere partition arrangement chip and the autofluorescence of the microspheres, and analyzing the concentration of the target molecules in the sample according to the fluorescence intensity of the bound antibodies on the microspheres.
Further, in step S2, the microsphere partition arrangement chip is provided with a microsphere arrangement region, the microsphere arrangement region includes a plurality of microsphere capture arrays, and the plurality of microsphere capture arrays have microsphere placement positions at different intervals.
Furthermore, each microsphere capture array consists of a plurality of microcolumns with the same interval size; the micro-fluidic chip is provided with micro-column barriers which have openings with different sizes and are arranged in parallel and used for screening the microspheres with different sizes.
Further, in step S1, two colors mixed in different proportions are set on the microspheres, and the two colors and the different radius sizes of the microspheres form a three-dimensional encoding mode.
Further, the micro-fluidic chip is prepared by a micro-nano technology or a 3D printing technology.
Furthermore, from the inlet to the outlet of the microfluidic chip, the spacing size of the microcolumns in the microsphere capture array is reduced from large to small in sequence.
Further, the microcolumn includes a square column, a circular column, a triangular column, a diamond column, an octagonal column, a petal-shaped column, and a star-shaped column.
A micro-fluidic chip comprises a bottom chip, a micro-sphere partition arrangement chip arranged on the bottom chip and micro-spheres with different radiuses, sizes and colors and double codes, wherein the micro-sphere partition arrangement chip is provided with a micro-sphere arrangement area, the micro-sphere arrangement area comprises a plurality of micro-sphere capture arrays, the micro-sphere capture arrays are provided with micro-sphere placement positions with different intervals, and each micro-sphere capture array consists of a plurality of micro-columns with the same interval size; the micro-fluidic chip is provided with micro-column barriers which have openings with different sizes and are arranged in parallel and used for screening the microspheres with different sizes.
Further, the microcolumn includes a square column, a circular column, a triangular column, a diamond column, an octagonal column, a petal-shaped column, and a star-shaped column.
Further, the bottom chip comprises an upper shell and a lower shell which are connected, and an outlet and an inlet are arranged at the front end and the rear end of the bottom chip; from the inlet to the outlet of the microfluidic chip, the spacing size of the microcolumns in the microsphere capture array is reduced from large to small in sequence.
In the invention, microspheres with different radius sizes and color dependence are respectively combined with corresponding detection antibodies; then, pouring the microspheres into a microsphere partition arrangement chip of the microfluidic chip, so that the microspheres are arranged in corresponding areas in the microsphere partition arrangement chip according to the size; pouring a sample with target molecules and a corresponding binding antibody into the microfluidic chip, wherein the binding antibody is bound with the target molecules in the sample, and the target molecules in the sample are bound with the detection antibody in the microspheres, so that the microspheres carry fluorescent molecules with the content corresponding to the content of the target molecules; finally, the micro-fluidic chip is subjected to fluorescent signal acquisition, the types of targets are distinguished according to the areas of the microspheres in the microsphere partition arrangement chip and the fluorescent light with different classification colors, and the concentration of target molecules in a sample is analyzed according to the fluorescent intensity of the binding antibody on the microspheres; so as to achieve the purposes of high-throughput target parallel detection, less sample consumption, low time and labor cost, high sensitivity and specificity.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings:
FIG. 1 is a block diagram of the steps of the multiplex digital ELISA detection method of the present invention;
FIG. 2 is a schematic plan view of a microsphere chip of the present invention;
FIG. 3 is a schematic perspective view of a microsphere chip according to the present invention;
FIG. 4 is a schematic illustration of a microsphere of the present invention;
FIG. 5 is a schematic diagram of detection of the microsphere partition arrangement chip according to the present invention;
wherein the reference numerals are: 1-bottom chip, 2-micro-column capture array, 3-micro-sphere partition arrangement chip, 4-inlet, 5-outlet.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in fig. 1 to 5, the multiplex digital ELISA detection method of the present invention, applied to a microfluidic chip, comprises the following steps:
step S1, respectively combining the double-classification coded microspheres with different radius sizes and color settings with corresponding detection antibodies;
step S2, filling the microspheres obtained in the step S1 into a microsphere partition arrangement chip of the microfluidic chip, so that the microspheres are arranged in corresponding areas in the microsphere partition arrangement chip according to the size under the action of self fluid;
step S3, pouring a sample with target molecules and a binding antibody which corresponds to the detection antibody and contains fluorescent molecules into the microfluidic chip, wherein the target molecules in the sample are firstly bound with the detection antibody on the microspheres, and the binding antibody is then bound with the target molecules in the sample, so that the microspheres carry the fluorescent molecules with the content corresponding to the content of the target molecules;
and step S4, collecting fluorescence signals of the microfluidic chip, distinguishing the types of target molecules according to the areas of the microspheres in the microsphere partition arrangement chip and the autofluorescence of the microspheres, and analyzing the concentration of the target molecules in the sample according to the fluorescence intensity of the bound antibodies on the microspheres.
In the invention, microspheres with different radius sizes and color dependence are respectively combined with corresponding detection antibodies; then, pouring the microspheres into a microsphere partition arrangement chip of the microfluidic chip, so that the microspheres are arranged in corresponding areas in the microsphere partition arrangement chip according to the size; pouring a sample with target molecules and a corresponding binding antibody into the microfluidic chip, wherein the binding antibody is bound with the target molecules in the sample, and the target molecules in the sample are bound with the detection antibody in the microspheres, so that the microspheres carry fluorescent molecules with the content corresponding to the content of the target molecules; finally, the micro-fluidic chip is subjected to fluorescent signal acquisition, the types of targets are distinguished according to the areas of the microspheres in the microsphere partition arrangement chip and the fluorescent light with different classification colors, and the concentration of target molecules in a sample is analyzed according to the fluorescent intensity of the binding antibody on the microspheres; so as to achieve the purposes of high-throughput target parallel detection, less sample consumption, low time and labor cost, high sensitivity and specificity.
Specifically, the method is applied to the simultaneous detection of multiple targets, and comprises the following steps:
s1, respectively combining the double-classification coded microspheres with different radius sizes, different radius sizes and set colors with corresponding detection antibodies;
s2, pouring the microspheres obtained in the step S1 into a microfluidic chip containing micro-column barriers with different sizes, so that the microspheres are arranged in corresponding barrier areas in the chip according to the sizes of the micro-column barrier openings under the action of a fluid;
step S3, pouring a sample with target molecules and a binding antibody which corresponds to the detection antibody and carries fluorescent molecules into the microfluidic chip, firstly, combining the target molecules in the sample with the detection antibody on the microspheres, and then combining the binding antibody with the target molecules in the sample to enable the microspheres to carry the fluorescent molecules with the content corresponding to the content of the target molecules;
and step S4, collecting fluorescent signals of the microfluidic chip, distinguishing the types of target molecules according to the barrier area of the microspheres in the microfluidic chip and the autofluorescence of the microspheres, and analyzing the concentration of the target molecules in the sample according to the fluorescence intensity of the bound antibodies on the microspheres.
In step S2, the microfluidic chip is provided with microcolumn barriers having openings of different sizes and arranged in parallel for screening microspheres of different sizes.
Wherein, each micro-column barrier is composed of a series of continuously repeated upright columns with the same interval, and the opening size of each micro-column barrier corresponds to the size of the microsphere.
Wherein, the microcolumn comprises a cubic column, a cylinder, a triangular column, a rhombic column and an eight-sided column.
Wherein, from the inlet to the outlet of the micro-fluidic chip, the openings of the micro-column barrier are reduced from large to small in sequence.
In step S1, the mixed colors with different proportions are set on the microspheres, and the colors and the different radius sizes of the microspheres form a dual classification coding method.
Wherein, the color characteristics of the microspheres are obtained by mixing different colors in different proportions.
The method for mixing different colors of the microspheres comprises the steps of adding fluorescent dye, compounding quantum dots and the microspheres, doping the microspheres with multicolor luminous up-conversion nanoparticles, fluorescent life coding microspheres, surface enhanced Raman scattering spectrum coding microspheres and structural color coding microspheres.
In the invention, microspheres with different radius sizes and color dependence are respectively combined with corresponding detection antibodies; then pouring the microspheres into the microcolumn barriers with different sizes of the microfluidic chip, so that the microspheres are arranged in corresponding barrier areas in the chip according to the sizes; pouring a sample with target molecules and a corresponding kind of binding antibody into the microfluidic chip, wherein the binding antibody is bound with the target molecules in the sample, and the target molecules in the sample are bound with the detection antibody in the microspheres, so that the microspheres carry fluorescent molecules; finally, the micro-fluidic chip is subjected to fluorescent signal acquisition, the types of targets are distinguished according to the barrier area of the microspheres in the micro-fluidic chip and the fluorescence of different classification colors, and the concentration of target molecules in a sample is analyzed according to the fluorescence intensity of the binding antibody on the microspheres; so as to achieve the purposes of high-throughput target parallel detection, less sample consumption, low time and labor cost, high sensitivity and specificity.
In step S1, different colors or sizes are set on the microspheres, and the settable colors of the microspheres with different radius sizes are obtained by mixing different colors in different proportions. Specifically, if two dyes red and blue are selected, the addition of nine parts of red with one part of blue will result in a microsphere color that is different from the color of a microsphere with nine parts of blue with one part of red, and so on, assuming there are a dyes, each dye has B parts, and there are BA/2 of the types of microsphere colors that can be obtained. And assuming that the size of the microsphere has C types, and the microspheres coded by double codes with different radius sizes and colors have BA/2 XC types. The microspheres with double-coded sizes and colors are used as solid phase surfaces for detection, BA/2 xC detection antibodies are loaded, and BA/2 xC targets are detected, namely, one type of microspheres corresponds to one detection antibody, wherein, one type of microspheres refers to microspheres with the same diameter size, color type and ratio, and any element (diameter size, color type and color ratio) is different, namely, microspheres with different types are represented.
The type of microspheres, in addition to being different in size, also includes differences in color. The color is characterized by being obtained by mixing different colors in different proportions. If two dyes red and blue are chosen, the addition of nine parts of red with one part of blue will give microspheres with a different colour than the addition of nine parts of blue with one part of red, and so on, assuming that there are a dyes, each with B parts, there will be BA/2 of the available microsphere colour types.
In step S2, the microfluidic chip is provided with microcolumn barriers with different openings and arranged in parallel for screening microspheres with different sizes. Each microcolumn barrier is composed of a series of continuously repeated upright columns with the same interval, each microcolumn comprises a cubic column, a cylinder, a triangular column, a rhombic column and an octagonal column, and the size of an opening of each microcolumn barrier corresponds to the size of the microsphere. From the inlet to the outlet of the microfluidic chip, the openings of the micro-column barriers are sequentially reduced from large to small, and the microspheres with the radius size between the sizes of the openings of the two micro-column barriers are arranged between the two micro-column barriers.
The method for mixing different colors of the microspheres comprises the steps of adding fluorescent dye, compounding quantum dots and the microspheres, doping the microspheres with multicolor luminous up-conversion nanoparticles, fluorescent life-span coding microspheres, surface-enhanced Raman scattering spectrum coding microspheres and structural color coding microspheres.
In step S2, the microsphere partition arrangement chip is provided with a microsphere arrangement region, the microsphere arrangement region contains microsphere capture arrays arranged at different intervals, the microsphere capture arrays are composed of microcolumns with different intervals, the interval sizes of the microcolumns in the microsphere capture arrays are sequentially reduced from large to small from the inlet to the outlet of the microfluidic chip, and the microcolumns include square pillars, circular pillars, triangular pillars, rhombic pillars, octagonal pillars, petal-shaped pillars, and star-shaped pillars; specifically, the bottom chip 1 is provided with a microcolumn capture array 2 with different radius size intervals, a microsphere partition arrangement chip 3, an inlet 4 and an outlet 5, as shown in fig. 3, the microsphere partition arrangement chip 3 is provided with the microcolumn capture arrays 2 which are arranged in parallel and have different radius size intervals, the interval sizes of the microcolumns from the inlet 4 to the outlet 5 are reduced from large to small, and microspheres with diameters between the two microcolumn interval sizes are distributed between the two microcolumn arrays.
The invention designs a micro-fluidic chip containing micro-column arrays with different sizes, so that microspheres with different sizes are arranged at corresponding positions in the chip in a partitioning manner. Microspheres are fixed in a chip without external means, and microspheres with different sizes can be separated by size limitation.
In the invention, the microcolumn of the microsphere partition arrangement chip can be obtained by micro-nano processing technology or 3D printing. The micro-fluidic chip designed by the invention is prepared by a micro-nano technology or a 3D printing technology, and the microspheres are fixed on the chip interface after being limited by the opening size of the micro-column barrier, so that an external complex equipment means is not needed. The surface of the chip can be modified differently to allow better adhesion of the microspheres to it. In addition, the chip has an integrated function, and can realize integration of multiple functions such as microsphere fixation, antibody incubation, signal detection and the like. On the other hand, the micro-fluidic chip is not open, provides a relatively closed space for detection, reduces sample consumption and external pollution, and simultaneously can improve detection sensitivity due to reaction with smaller volume.
In the invention, microspheres with different radius sizes and color dependence are respectively combined with corresponding detection antibodies, the microspheres are filled into the microsphere partition arrangement chip, the microspheres are arranged in corresponding areas according to the sizes, then a sample and corresponding binding antibodies are filled, when the sample and the binding antibodies flow through different microsphere arrangement areas, target molecules in the sample are combined with the microspheres through the detection antibodies on the microspheres, the binding antibodies are combined with the target molecules to carry fluorescent molecules corresponding to the content of the target molecules, finally, the microfluidic chip is placed in a fluorescence microscope or a fluorescence scanner for detection, the types of the targets are distinguished according to the areas where the microspheres are located and the fluorescence of different classification colors, and the concentration of the target in the sample is analyzed according to the fluorescence intensity of the binding antibodies on the microspheres.
In the invention, the surface of the microsphere can be provided with carboxyl/amino/benzenesulfonyl groups, the microsphere is combined with a combined antibody or antigen with amino/carboxyl groups, a target molecule in a sample is detected by a double-antibody sandwich method or a competition method, the detected antibody (antigen) is provided with a fluorescence molecule, the molecule of the sample to be detected carries fluorescence after being combined with the sample to be detected, the visualization of a detection result is realized, and the fluorescence signal intensity of the detection result is in positive correlation or negative correlation with the content of the target molecule in the sample.
In the invention, the detection antibody is modified on the surface of the microsphere, the detection antibody can be rapidly combined with a target molecule in a sample, the whole immunoreaction (a double antibody sandwich method or a competition method) is carried out in a uniform liquid phase system, the speed is high, the reaction uniformity is good, in addition, the microsphere can be subjected to color coding, different colors can correspond to different detection items, and the detection flux is further increased.
Within the present invention, the fluorescent molecule carried by the detection antibody bound to the target molecule causes the target molecule to fluoresce, and the concentration of the sample is inferred by measuring the intensity of the fluorescence.
According to the invention, the size classification code is added on the basis of the color classification code of the microsphere, so that the detection flux is improved, and meanwhile, all information of the detection target can be obtained only by acquiring and analyzing the fluorescence signal, so that the rapid response requirement of multi-target detection can be met.
In the invention, different detection antibodies are modified on the surface of the microsphere, when a sample flows through the microsphere, a target molecule in the sample is combined with the detection antibody on the microsphere, the target molecule which is not combined is discharged out of the microfluidic chip along with fluid, then the combined antibody containing fluorescent molecules is added, and when the sample flows through a reagent channel of the microfluidic chip, the combined antibody is combined with the target molecule on the microsphere again, so that the target molecule carries a fluorescent signal, and the fluorescent intensity of the target molecule on the microsphere is related to the concentration of the target molecule in the sample, therefore, the corresponding concentration of the target molecule in the sample to be detected can be deduced according to a standard curve of the target molecule, thereby achieving the purpose of rapid detection.
In addition, the microfluidic chip adopts the sealing agent to seal the non-detection areas on the surface of the channel and the surface of the microsphere of the chip, prevents target molecules in a sample from generating non-specific adsorption, improves the detection specificity of the microfluidic chip, and promotes the sample and the microsphere to fully contact and react by utilizing the reciprocating motor, thereby improving the detection accuracy and sensitivity.
Within the present invention, the mixing of the microspheres with the sample and the bound antibody may be performed in various ways, for example, the mixing may include a split mixing and a simultaneous mixing; the separated mixing mode is as follows: fully mixing the microspheres containing the detection antibodies with the sample in advance, incubating and then cleaning, adding the binding antibodies, enabling the detection antibodies on the microspheres to be firstly bound with target molecules of the sample, incubating and then cleaning; the synchronous mixing mode is as follows: the sample, the microspheres and the bound antibody are mixed, incubated and washed.
In the split mixing method, two cases are divided, one of which is: pouring the microspheres into a microfluidic chip in advance for capture, adding a sample into the microfluidic chip, fully mixing, incubating and cleaning, adding a binding antibody into the microfluidic chip, incubating and cleaning, and collecting fluorescence signals of the microfluidic chip, wherein the second step is as follows: fully mixing the microspheres and a sample outside the microfluidic chip, incubating and cleaning, adding the mixed mixture into the microfluidic chip for capturing the microspheres, finally adding the binding antibody into the microfluidic chip for fully mixing, incubating and cleaning, and collecting and analyzing fluorescent signals of the microfluidic chip.
In the synchronous hybrid system, there are two cases, one of which is: mixing a sample, microspheres and a binding antibody outside the microfluidic chip, incubating and cleaning, filling the mixture into the microfluidic chip for capturing the microspheres, and collecting fluorescent signals of the microfluidic chip; the second step is as follows: and adding the sample, the microspheres and the combined detection antibody into the microfluidic chip, fully mixing, incubating, cleaning, and collecting the fluorescent signal of the microfluidic chip.
It may specifically include the following 4 cases:
1) and (3) pouring the microspheres into a microfluidic chip in advance for capture, adding a sample into the microfluidic chip, fully mixing, incubating and then cleaning, adding a binding antibody into the microfluidic chip, incubating and then cleaning, and finally detecting a fluorescent signal of the microfluidic chip.
2) And fully mixing the microspheres and the sample outside the microfluidic chip, incubating, cleaning, adding the mixture into the microfluidic chip to capture the microspheres, finally adding the binding antibody to fully mix, incubating, cleaning, and detecting a fluorescent signal.
3) Mixing the sample, the microspheres and the combined antibody outside the microfluidic chip, incubating, cleaning, filling the mixture into the microfluidic chip for capturing the microspheres, and finally detecting the fluorescent signal.
4) And adding the sample, the microspheres and the combined antibody into the microfluidic chip, fully mixing, incubating, cleaning and finally detecting the fluorescent signal.
For example, polystyrene microspheres of various sizes are mixed and then poured into the micro-fluidic chip from an inlet of the micro-fluidic chip, the micro-fluidic chip is erected and placed on a reciprocating motor to perform circulating oscillation for several minutes, and due to the gravity effect and the limiting effect of openings of different micro-column barriers on the sizes of the microspheres, the microspheres with different radius sizes are distinguished according to the difference of the openings of the micro-column barriers and are arranged in corresponding barrier areas; after entering through an inlet 5 of the micro-fluidic chip, a sample to be detected carrying target molecules is placed on the reciprocating motor for full oscillation reaction, the chip is washed for 3 times by using buffer salt solution, then the binding antibody is added for full contact and reaction, the chip is washed for 3 times by using the buffer salt solution, and finally, the micro-fluidic chip is subjected to acquisition and analysis of fluorescence signals.
In the invention, the microfluidic chip comprises an upper layer and a lower layer which are connected, wherein the lower layer is sealed with the upper layer by glue or hot pressing or plasma welding or ultrasonic welding; the corresponding materials of the upper layer and the lower layer of the microfluidic chip can be polymethyl methacrylate, polydimethylsiloxane, acrylonitrile-butadiene-styrene copolymer or cyclic olefin copolymer, the upper layer and the lower layer can be bonded by glue or double faced adhesive tape, or hot pressing, ultrasonic welding, bonding process and the like.
In the invention, the front end and the rear end of the microfluidic chip are provided with an outlet 5 and an inlet 4. Sealing agents are coated on all channels in the bottom chip 1, and the outlet 5 and the inlet 4 are designed by threads, so that the air tightness of the chip is improved, the operation is simple and convenient, and the sample adding and waste liquid discharging can be carried out quickly; the channels in the chip are modified with a blocking agent for preventing proteins from generating nonspecific adsorption, so that false positive signals are avoided, and the specificity of detection is enhanced.
The microcolumn barrier comprises 2 or more than 2 microcolumn barriers with different opening sizes, and is used for capturing 2 or more than 2 microspheres with different particle sizes respectively, wherein the particle size of the microspheres is in a nanometer, micron or millimeter level; the microspheres in the microfluidic chip comprise polystyrene microspheres, polystyrene maleic anhydride microspheres, silicon dioxide microspheres, hydrogel microspheres, photonic crystals, magnetic iron oxide microspheres or noble metal nanoparticles; the coupling group on the surface of the microsphere comprises at least one of amino, carboxyl, hydroxyl, tosyl, sulfydryl, chloromethyl, aldehyde group, sulfydryl, hydrazide, epoxy, silicon hydroxyl and succinimide ester; the bioactive molecules coupled to the surface of the microsphere comprise antibodies, antigens, nucleic acids, haptens, hormone receptors, nucleic acids, oligonucleotides, enzymes and aptamers; the immune complex formed on the surface of the microsphere can be analyzed by a chemiluminescence immunoassay method, an electrochemiluminescence immunoassay method, an enzyme-linked immunosorbent assay method, an enzymatic immunoassay method, a biotin-avidin system assay method, a radioimmunoassay method and an immunofluorescence assay method.
The sealant used for sealing the channels and the non-detection areas on the microspheres in the microfluidic chip comprises: calf serum protein, casein, tween20, and the like.
The present invention provides three embodiments:
example 1:
the flow and detection results for detecting various allergen antibodies based on the microfluidic chip provided in example 1 are as follows:
microfluidic chip assembly
Establishing a micro-fluidic chip model by using modeling software, manufacturing the micro-fluidic chip by using a polymethyl methacrylate material, sealing the micro-fluidic chip by using acrylic glue, wherein the sizes of openings of cubic column barriers in the micro-fluidic chip are respectively 500 micrometers, 450 micrometers, 400 micrometers, 350 micrometers and 300 micrometers, corresponding to the sizes of the different diameters in the step S1, the polystyrene microspheres with the five sizes are taken, the peanut agglutinin, the beta-lactoglobulin, the apple rMal d4, the hazelnut rCor a1 and the anemone pollen allergen are respectively modified and respectively used for detecting corresponding IgE antibodies in human serum (a sample with target molecules), before the microspheres are filled into the microfluidic chip, the channel of the microfluidic chip is sealed by 0.5 percent Tween-20 and 1 percent BSA in advance to prevent the generation of non-specific adsorption, the polystyrene microspheres are filled into the microfluidic chip, and the reciprocating motor is started to enable the microspheres to be uniformly mixed and arranged in the area corresponding to the sizes of the microspheres.
(II) sample detection
Injecting sample serum and a fluorescence-labeled peanut agglutinin-resistant IgE antibody, a beta-lactoglobulin IgE antibody, an apple-resistant rMal d4IgE antibody, a hazelnut rCor a1IgE antibody and a bellflower pollen-resistant IgE antibody through an inlet of a microfluidic chip, incubating at 37 ℃, cleaning for 20 minutes by using a buffer salt solution, discharging redundant samples and secondary antibodies, detecting specific allergen IgE in the sample serum by using a double-antigen sandwich method, placing the microfluidic chip on a fluorescence microscope after reaction is finished, collecting fluorescence signal intensity, wherein the fluorescence signal intensity is in positive correlation with the content of target molecules in the samples, the total detection time is 10 minutes, measuring 3 times by using 3 microfluidic chips respectively for each sample, averaging, drawing a standard curve, and obtaining the peanut agglutinin IgE antibody in the sample according to the standard curve, wherein the higher the content of the IgE in the sample is the stronger the luminescence signal, The respective concentrations of beta-lactoglobulin IgE, apple rMal d4IgE, hazelnut rCor a1IgE and bellflower pollen IgE are as follows: 3.2nM, 1.7nM, 4.1nM, 1.5nM, 2.6nM, the results show that the correlation coefficient R is not less than 0.99, the repeatability is better, and the reference can be provided for the diagnosis of allergic diseases.
Example 2:
Microfluidic chip assembly
Establishing a micro-fluidic chip model by using modeling software, manufacturing the micro-fluidic chip by using a polymethyl methacrylate material, sealing the micro-fluidic chip by using acrylic glue, wherein the openings of micro-column barriers in the micro-fluidic chip are respectively 60 micrometers, 40 micrometers and 20 micrometers, respectively modifying alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and carbohydrate chain antigen 199(CA125) antibodies by using photonic crystal microspheres with the three sizes, filling the micro-fluidic chip with the microspheres, wherein before filling the microspheres into the micro-fluidic chip, the channels of the micro-fluidic chip are sealed by using a sealant in advance to prevent the generation of non-specific adsorption, and filling the microspheres into the micro-fluidic chip to uniformly mix the microspheres and arrange the microspheres in an area corresponding to the sizes of the microspheres.
(II) sample detection
Injecting patient sample serum and a fluorescence-labeled secondary antibody through a chip inlet, incubating at 37 ℃, cleaning for 20 minutes by using a buffer salt solution, discharging redundant samples and antigen secondary antibodies, placing the chip on a fluorescence microscope after the reaction is finished, collecting fluorescence signal intensity, wherein the total detection time is 20 minutes, measuring each sample by using 3 micro-fluidic chips for 3 times, taking an average value, drawing a standard curve, and obtaining respective concentrations of AFP, CEA and CA125 in the sample according to the standard curve as follows: 72ng/mL, 3.42ng/mL, 9.72 ng/mL. The result shows that the correlation coefficient R is more than or equal to 0.99, the repeatability is better, and the reference can be provided for cancer diagnosis.
Example 3:
example 3 hepatitis B Virus serum marker detection based on microfluidic chip
Microfluidic chip assembly
Creating a micro-fluidic chip model by using SU8 photoresist photoetching technology, manufacturing the micro-fluidic chip by using polydimethylsiloxane material, sealing the micro-fluidic chip with organic glass by using plasma, wherein the openings of a micro-column barrier in the micro-fluidic chip are respectively 4.9,3.0 and 1.0 mu m, and respectively modifying the six microspheres with three sizes and two colors with hepatitis B virus surface antigen (HBSAg), hepatitis B virus surface antibody (HBSAb), hepatitis B virus core antigen (HBcAg), hepatitis B virus core antibody (HBcAb), hepatitis B virus e antigen (HBeAg) and hepatitis B virus e antibody (HBeAb). Before the microspheres are filled into the microfluidic chip, the channels of the microfluidic chip are sealed by a sealant in advance to prevent the generation of nonspecific adsorption, and the microspheres are filled into the microfluidic chip to be uniformly mixed and arranged in an area corresponding to the size of the microspheres.
(II) sample detection
Injecting patient sample serum, fluorescence-labeled IgG and fluorescence-labeled HBSAg, HBcAg and HBeAg through an inlet of the microfluidic chip, incubating at 37 ℃, cleaning for 20 minutes by using a buffer salt solution, discharging redundant samples and antigen antibodies, placing the chip on a fluorescence microscope after reaction is finished, collecting fluorescence signal intensity, measuring each sample for 3 times by using 3 microfluidic chips respectively, averaging, and drawing a standard curve; the respective concentrations of hepatitis b virus surface antigen (HBSAg), hepatitis b virus surface antibody (HBSAb), hepatitis b virus core antigen (HBcAg), hepatitis b virus core antibody (HBcAb), hepatitis b virus e antigen (HBeAg), and hepatitis b virus e antibody (HBeAb) in the sample obtained according to the standard curve are: 457IU/mL, 0.15IU/mL, 0.06IU/mL, 0.01IU/mL, 0.02IU/mL and 0.01IU/mL, and the result shows that the correlation coefficient R is more than or equal to 0.99, has better repeatability and can provide reference for the diagnosis of hepatitis B virus infection.
From the results of the above test, it can be concluded that:
(1) the microfluidic chip multiple immunodetection method is suitable for the detection of various different targets.
(2) The detection test detects all antigens to be detected when different antigens are detected, and the linear correlation test, the repeatability, the minimum detection limit and the accuracy can achieve the detection effect of single detection.
(3) The multiple immunodetection method of the microfluidic chip disclosed by the invention proves that large-scale high-flux detection can be rapidly, simply and conveniently carried out.
The size and color double-coding micro-fluidic chip provided by the invention is a high-flux biochip processed by a micro-nano technology, can be used for large-scale gene or protein detection, and has the following advantages compared with the prior art:
1. the microsphere based on size and color dual coding greatly improves the detection flux;
2. the microspheres are limited in corresponding micro-column barrier areas on the chip according to size difference for continuous detection, and detection results can be quickly analyzed only by acquiring fluorescence information;
3. target molecules in the sample are fully mixed with various detection antibodies for detection, so that the sample consumption is reduced, and the reagent cost is saved;
4. the traditional solid phase reaction is converted into a microsphere suspension array, so that the reaction area is enlarged, and the reaction efficiency is improved.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A multiple digital ELISA detection method is applied to a microfluidic chip and is characterized by comprising the following steps:
step S1, respectively combining the double-classification coded microspheres with different radius sizes and color settings with corresponding detection antibodies;
step S2, filling the microspheres obtained in the step S1 into a microsphere partition arrangement chip of the microfluidic chip, so that the microspheres are arranged in corresponding areas in the microsphere partition arrangement chip according to the size under the action of self fluid;
step S3, pouring a sample with target molecules and a binding antibody which corresponds to the detection antibody and contains fluorescent molecules into the microfluidic chip, wherein the target molecules in the sample are firstly bound with the detection antibody on the microspheres, and the binding antibody is then bound with the target molecules in the sample, so that the microspheres carry the fluorescent molecules with the content corresponding to the content of the target molecules;
and step S4, collecting fluorescence signals of the microfluidic chip, distinguishing the types of target molecules according to the areas of the microspheres in the microsphere partition arrangement chip and the autofluorescence of the microspheres, and analyzing the concentration of the target molecules in the sample according to the fluorescence intensity of the bound antibodies on the microspheres.
2. The method according to claim 1, wherein in step S2, the bead partition chip is provided with a bead arrangement region, and the bead arrangement region comprises a plurality of bead capture arrays, and the plurality of bead capture arrays have bead placement positions with different intervals.
3. The multiplexed digital ELISA detection method of claim 2 wherein each of the bead capture arrays is composed of a plurality of microcolumns spaced at equal intervals; the micro-fluidic chip is provided with micro-column barriers which are provided with openings with different sizes and are arranged in parallel and used for screening micro-spheres with different sizes.
4. The method of claim 1, wherein in step S1, two colors mixed in different ratios are set on the microspheres, and the two colors and the different radius sizes of the microspheres form a three-dimensional encoding mode.
5. The multiplex digital ELISA detection method according to claim 4, wherein the microfluidic chip is prepared by micro-nano technology or 3D printing technology.
6. The method according to claim 3, wherein the interval size of the micro-pillars in the microsphere capture array decreases from large to small from the inlet to the outlet of the microfluidic chip.
7. The method according to claim 3, wherein the micro-column comprises a square column, a circular column, a triangular column, a diamond column, an octagonal column, a petal-shaped column, or a star-shaped column.
8. A micro-fluidic chip is characterized by comprising a bottom chip, microsphere partition arrangement chips arranged on the bottom chip and microspheres with different radii and colors and double codes, wherein the microsphere partition arrangement chips are provided with microsphere arrangement areas, the microsphere arrangement areas contain a plurality of microsphere capture arrays, the microsphere capture arrays are provided with microsphere placement positions with different intervals, and each microsphere capture array consists of a plurality of microcolumns with the same interval size; the micro-fluidic chip is provided with micro-column barriers which are provided with openings with different sizes and are arranged in parallel and used for screening micro-spheres with different sizes.
9. The microfluidic chip according to claim 8, wherein the micro-pillars comprise square pillars, circular pillars, triangular pillars, diamond-shaped pillars, octagonal pillars, petal-shaped pillars, and star-shaped pillars.
10. The microfluidic chip according to claim 8, wherein the bottom chip comprises an upper shell and a lower shell which are connected, and the front end and the rear end of the bottom chip are provided with an outlet and an inlet; from the inlet to the outlet of the microfluidic chip, the spacing sizes of the microcolumns in the microsphere capture array are reduced from large to small in sequence.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2078958A1 (en) * | 2007-10-25 | 2009-07-15 | Koninklijke Philips Electronics N.V. | A reconfigurable microfluidic filter based on eletric fields |
| CN203133082U (en) * | 2010-07-23 | 2013-08-14 | 中国检验检疫科学研究院 | Protein suspension array system for detecting various antibodies in serum sample synchronously |
| CN103267749A (en) * | 2013-04-27 | 2013-08-28 | 东南大学 | Photon encoding microsphere-based multielement biomarker assay kit-type chip |
| CN105838801A (en) * | 2016-05-06 | 2016-08-10 | 中国科学院上海微系统与信息技术研究所 | Emulsion droplet digital PCR quantitative method based on microspheres and microcolumn array chips |
| CN107828867A (en) * | 2017-11-22 | 2018-03-23 | 陕西易阳科技有限公司 | A kind of preparation method of micro-pillar array chip |
| CN109762728A (en) * | 2019-01-07 | 2019-05-17 | 东南大学 | A kind of space transcript profile detection chip and method |
| CN110093247A (en) * | 2019-05-07 | 2019-08-06 | 西安交通大学 | A kind of micro-fluidic chip of enrichment capture different specification size target cell |
-
2021
- 2021-01-26 CN CN202110103868.4A patent/CN112858670B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2078958A1 (en) * | 2007-10-25 | 2009-07-15 | Koninklijke Philips Electronics N.V. | A reconfigurable microfluidic filter based on eletric fields |
| CN203133082U (en) * | 2010-07-23 | 2013-08-14 | 中国检验检疫科学研究院 | Protein suspension array system for detecting various antibodies in serum sample synchronously |
| CN103267749A (en) * | 2013-04-27 | 2013-08-28 | 东南大学 | Photon encoding microsphere-based multielement biomarker assay kit-type chip |
| CN105838801A (en) * | 2016-05-06 | 2016-08-10 | 中国科学院上海微系统与信息技术研究所 | Emulsion droplet digital PCR quantitative method based on microspheres and microcolumn array chips |
| CN107828867A (en) * | 2017-11-22 | 2018-03-23 | 陕西易阳科技有限公司 | A kind of preparation method of micro-pillar array chip |
| CN109762728A (en) * | 2019-01-07 | 2019-05-17 | 东南大学 | A kind of space transcript profile detection chip and method |
| CN110093247A (en) * | 2019-05-07 | 2019-08-06 | 西安交通大学 | A kind of micro-fluidic chip of enrichment capture different specification size target cell |
Non-Patent Citations (2)
| Title |
|---|
| Quantitative Single Cell Analysis and Sorting;Paul K. Horan;《SCIENCE》;20150524;全文 * |
| Recent developments in emerging microimmunoassays;Christine F Woolley et al;《Bioanalysis》;20131231;全文 * |
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