CN111485016A

CN111485016A - Method for detecting nucleic acid and micro-fluidic chip

Info

Publication number: CN111485016A
Application number: CN202010236204.0A
Authority: CN
Inventors: 颜菁; 申炳阳; 刘文佳
Original assignee: Shanghai Bangxian Medical Technology Co ltd
Current assignee: Shanghai Bangxian Medical Technology Co ltd
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2020-08-04

Abstract

The invention discloses a simple, rapid and effective method for detecting nucleic acid and a microfluidic chip. The method comprises the following steps: A. mixing fluorescent microparticles and a nucleic acid amplification product and hybridizing, wherein a capture object capable of being specifically bound with a target nucleic acid molecule is coupled on the surface of the fluorescent microparticles; B. washing to remove nucleic acid molecules that do not specifically bind to the fluorescent microparticles; C. c, adding the liquid containing the fluorescent microparticles treated in the step C into a microfluidic chip, and intercepting the microparticles into the microfluidic chip; and D, reading the fluorescence intensity of the label on each fluorescent microparticle and accumulating to obtain the fluorescence intensity of the target nucleic acid molecule.

Description

Method for detecting nucleic acid and micro-fluidic chip

Technical Field

The invention belongs to the technical field of biological detection, and relates to a method for detecting nucleic acid and a micro-fluidic chip.

Background

The gene chip is mainly characterized in that the fluid is controlled in a micron-scale space, basic operation units such as sample biochemical reaction, separation, detection and the like can be integrated in a chip system, and a network is formed by microchannels so that the controllable fluid can penetrate through the whole system, thereby realizing various functions of a conventional laboratory. The gene chip is widely used in the field of biochemical analysis, such as nucleic acid detection fields of bacteria, viruses, protein detection and the like, due to the characteristics of easy integration, automation, controllable fluid and small required sample amount. However, in the existing gene chip detection process, a sample applicator, a hybridization instrument and the like are used, and a plurality of instruments are used in a matching way, so that the operation of professional personnel is required, the steps are complicated, the detection cost is high, and the detection requirements of site, rapidness and portability are difficult to meet.

Disclosure of Invention

The invention aims to provide a simple, quick and effective method for detecting nucleic acid and a microfluidic chip.

In order to achieve the purpose, the invention adopts a technical scheme that:

a method for nucleic acid detection comprising the steps of:

A. mixing fluorescent microparticles and a nucleic acid amplification product and hybridizing, wherein a capture object capable of being specifically bound with a target nucleic acid molecule is coupled on the surface of the fluorescent microparticles;

B. washing to remove nucleic acid molecules that do not specifically bind to the fluorescent microparticles;

C. c, adding the liquid containing the fluorescent microparticles treated in the step C into a microfluidic chip, and intercepting the microparticles into the microfluidic chip; and

D. reading the fluorescence intensity of the label on each fluorescent microparticle and accumulating to obtain the fluorescence intensity of the target nucleic acid molecule.

Preferably, the fluorescent microparticles have a plurality of sizes, and each size of fluorescent microparticle is coupled with a capture substance corresponding to one target nucleic acid molecule, and the capture substances coupled with different sizes of fluorescent microparticles are different. The arrangement and combination of microparticles with different particle sizes, colors and shapes improve the detection flux of the gene chip.

In one embodiment, any two sizes of fluorescent microparticles differ in at least one of particle size, color, shape.

In a preferred embodiment, the fluorescent microparticles have a particle size of 10 to 500 μm.

In a preferred embodiment, the color of the fluorescent microparticles is selected from at least one of green, yellow, red, blue and purple.

In a preferred embodiment, the shape of the fluorescent microparticles is selected from at least one of spheres, cubes and triangles.

In one embodiment, in the step C, the microfluidic chip traps fluorescent microparticles with different particle size ranges in the corresponding chambers.

In one embodiment, in the step D, the fluorescence intensity of the label on each fluorescent microparticle is read, and the fluorescence intensities of the fluorescent microparticles with the same specification are respectively added to obtain the fluorescence intensity of each target nucleic acid molecule.

In particular, the capture objects may be oligonucleotide probes.

In one embodiment, in the step a, the fluorescent microparticles and the nucleic acid amplification product are mixed and hybridized in a container, and the mixed solution is poured out after the hybridization is finished. In particular, the container may be a centrifuge tube.

In a preferred embodiment, the fluorescent microparticles have magnetism, and after the fluorescent microparticles are adsorbed by disposing a magnetic substance on the wall of the container, the mixed solution in the centrifugal tube is poured out.

In a preferred embodiment, the contents of the container are centrifuged and the mixture is decanted by removing the supernatant.

In a preferred embodiment, in the step B, a washing solution is added to the container to wash away the nucleic acid molecules not bound to the fluorescent microparticles, and the washing solution of the last washing is retained in the container; and in the step C, adding the liquid in the container into the microfluidic chip.

In one embodiment, in the step C, the microparticles are trapped in the trapping chamber of the microfluidic chip by the trapping part of the microfluidic chip.

More preferably, the trapping part includes a plurality of trapping columns, one trapping chamber is formed between the sample port and the trapping column on the frontmost side and between any two adjacent trapping columns, the liquid enters from the sample port on the front side of the microfluidic chip and passes through each trapping column in sequence, and microparticles with different particle sizes are trapped in different trapping chambers respectively.

More preferably, the volume of the entrapment chamber on the front side is greater than the volume of the entrapment chamber on the rear side thereof.

More preferably, the rear side of the microfluidic chip is provided with an air vent.

The other technical scheme adopted by the invention is as follows:

the microfluidic chip for detecting nucleic acid comprises a hollow shell, wherein a sample injection port is formed in the front end of the shell, and the microfluidic chip further comprises an interception part which is arranged in the shell and used for intercepting microparticles.

In one embodiment, the trapping part includes a first trapping column, a first trapping chamber is formed between the first trapping column and the sample addition port, the first trapping column includes a plurality of first columnar protrusions, a first trapping channel is formed between adjacent first columnar protrusions, and the first trapping channel is used for trapping microparticles with a particle size greater than or equal to an inner diameter of the first trapping channel in the first trapping chamber.

In a more preferred embodiment, the trapping part further includes a second trapping column, a second trapping chamber is formed between the second trapping column and the first trapping column, the second trapping column includes a plurality of second cylindrical protrusions, a second trapping channel is formed between adjacent second cylindrical protrusions, and the second trapping channel is used for trapping micro-particles with a particle size larger than or equal to an inner diameter of the second trapping channel in the second trapping chamber.

More preferably, the inner diameter of the second trapping channel is smaller than the inner diameter of the first trapping channel.

More preferably, the volume of the second entrapment chamber is less than the volume of the first entrapment chamber.

In a further preferred embodiment, the microfluidic chip further includes a third trapping column, a third trapping chamber is formed between the third trapping column and the second trapping column, the third trapping column includes a plurality of third columnar protrusions, a third trapping channel is formed between adjacent third columnar protrusions, and the third trapping channel is configured to trap microparticles having a particle size greater than or equal to an inner diameter of the third trapping channel in the third trapping chamber.

More preferably, the third trapping channel has an inner diameter smaller than that of the second trapping channel.

More preferably, the volume of the third entrapment chamber is less than the volume of the second entrapment chamber.

In one embodiment, the housing includes a base plate and a cover plate, the trap portion is formed on the base plate, and the cover plate and the base plate are fixedly connected and cover the trap portion.

More preferably, the sample addition port is opened on the front side of the cover plate.

More preferably, the rear side of the cover plate is provided with an air vent.

Compared with the prior art, the invention has the following advantages by adopting the scheme:

in the invention, the specific fluorescent microparticles coupled with the capture object are hybridized with the fluorescence-labeled nucleic acid amplification product, so that a spotting instrument and a hybridization instrument used in the existing gene chip detection process are avoided, the specific fluorescent microparticles combined with target nucleic acid molecules are intercepted and separated through a microfluidic chip, and the fluorescence intensity of the surfaces of the microparticles is read on the microfluidic chip, so that the detection is convenient, the detection cost is reduced, and the nucleic acid molecules can be simply, quickly and effectively detected.

Drawings

In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

Fig. 1 is an exploded schematic view of a microfluidic chip according to an embodiment of the present invention;

FIG. 2 is a schematic diagram of the structure of the microfluidic chip shown in FIG. 1;

FIG. 3 is a cross-sectional view taken along line A-A of FIG. 2;

FIG. 4 is a sectional view taken along line G-G of FIG. 2;

FIG. 5 is a schematic diagram of the entrapment of example 1;

FIG. 6 is a schematic diagram of the entrapment of example 2;

FIG. 7 is a schematic diagram of the entrapment of example 3.

In the above-described figures of the drawings,

1. a housing; 11. a base plate; 12. a cover plate; 2a, a first trapping column; 21a, a first columnar projection; 22a, a first trapped channel; 2b, a second trapping column; 21b, second columnar projections; 22b, a second trapping channel; 2c, a third trapping column; 21c, a third columnar projection; 22c, a third trapping channel; 3a, a first entrapment chamber; 3b, a second entrapment chamber; 3c, a second entrapment chamber; 4. a sample addition port; 5. and (4) a vent.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings so that the advantages and features of the invention may be more readily understood by those skilled in the art. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto.

A method for detecting nucleic acid, which adopts a micro-fluidic chip coupled with fluorescent microparticles of a capture substance (such as an oligonucleotide probe) capable of specifically binding target nucleic acid molecules and trapped fluorescent microparticles, reduces the cost of nucleic acid detection and improves the detection flux.

The method is implemented as follows:

the method comprises the following steps: selecting fluorescent microparticles with certain particle size, color and shape, wherein the particle size selection range is 10-500 μm, preferably 20-200 μm, the color is mainly green, yellow and red, and can also be other colors such as purple, blue and the like, and the shape is an entity with a regular structure such as a sphere, a cube, a triangle and the like; coupling a capture substance (a specific oligonucleotide probe) capable of being specifically combined with a target nucleic acid molecule on the surface of the fluorescent microparticle, mixing a nucleic acid amplification product with a fluorescent label and the fluorescent microparticle in a centrifugal tube, then placing the mixture in a constant-temperature oven for hybridization for 1-4 h to combine complementary paired DNA single strands, and pouring out the mixed solution after the hybridization is finished; the fluorescent microparticles can have magnetism at the same time, and the mixed solution can be poured out by attaching a magnet to the wall of the centrifugal tube to control the magnetic beads; or if the fluorescent micro-particles are nonmagnetic, pouring the mixed solution in a mode of centrifuging to remove the supernatant;

step two: adding a cleaning solution into a centrifuge tube containing fluorescent microparticles, and washing away nucleic acid molecules which are not specifically combined with the probe, wherein the cleaning solution is removed in the manner described in the step one; cleaning for 2 times totally, wherein the cleaning solution is not poured in the last cleaning for standby;

step three: adding a solution containing fluorescent microparticles into a microfluidic chip, and controlling microparticles with different sizes in corresponding chambers under the action of a specific-size retention part on the chip;

step four: placing the microfluidic chip on a chip scanner, and respectively reading the fluorescence intensity marked on the single fluorescent microparticles; the fluorescence intensities on the fluorescent microparticles which are completely the same (the particle size, the color and the shape are all the same) are added up to obtain the actual fluorescence intensity for detecting each target nucleic acid.

Preferably, the fluorescent microparticles used have a plurality of sizes, and each size of fluorescent microparticle is conjugated with a capture substance corresponding to one target nucleic acid molecule, and the capture substances conjugated to different sizes of fluorescent microparticles are different. The particle size of at least one different fluorescent microparticle in the particle sizes, colors and shapes of any two specifications of fluorescent microparticles is 10-500 mu m; the color of the fluorescent microparticles is selected from at least one of green, yellow, red, blue and purple; the shape of the fluorescent microparticles is selected from at least one of spheres, cubes and triangles. In the step C, the micro-fluidic chip intercepts fluorescent micro-particles with different particle size ranges in corresponding chambers; in step D, reading the fluorescence intensity of the label on each fluorescent microparticle, and accumulating the fluorescence intensities of the fluorescent microparticles with the same specification to obtain the fluorescence intensity of each target nucleic acid molecule. Thereby realizing the purpose of simultaneously detecting various nucleic acid molecules and improving the detection flux.

The microfluidic chip for nucleic acid detection described above is shown in fig. 1 to 4. Referring to fig. 1 to 4, the microfluidic chip for nucleic acid detection includes a hollow housing 1, a sample addition port 4 is opened on a front end portion of the housing 1, and an air vent 5 is opened on a rear end portion of the housing 1. The micro-fluidic chip also comprises a retention part arranged in the shell 1, and the retention part is positioned between the sample adding port 4 and the vent 5. The interception part is used for intercepting microparticles with the particle size larger than or equal to a preset value, and the interception part is provided with an interception channel for liquid and microparticles with the particle size smaller than the preset value to pass through.

The intercepting part comprises an intercepting column, the intercepting column comprises a plurality of columnar protrusions, the plurality of columnar protrusions are arranged at intervals to provide intercepting channels for liquid and fluorescent microparticles with particle sizes smaller than the intervals to pass through, and the intercepting channels are formed between the columnar protrusions of the intercepting part. And a trapping chamber is formed between the trapping columns or between the trapping columns and the sample adding port 4.

Preferably, the trapping part comprises a plurality of trapping columns arranged in sequence from front to back, and the interval of any trapping column is larger than that of the trapping column at the back side thereof and/or smaller than that of the trapping column at the front side thereof. The interval between two adjacent columnar projections of the retaining column on the foremost side is the largest, the interval of the retaining column positioned in the middle is the next to the interval, and the interval of the retaining column on the rearmost side is the smallest.

An entrapment chamber is formed between the sample addition port 4 and the foremost entrapment column and between two adjacent entrapment columns respectively. The distance between two adjacent trapping columns is decreased from front to back, so that the volume of any one trapping chamber on the front side is larger than that of the trapping chamber on the back side. A sample addition region is formed at the sample addition port 4. A waste liquid zone is formed between the last trap column and the vent 5. The liquid carrying the fluorescent microparticles enters the sample adding area from the sample adding port 4, then flows through each interception column in sequence so as to intercept the fluorescent microparticles with different sizes in the corresponding interception cavities, and finally enters the waste liquid area.

Specifically, in the present embodiment, as shown in fig. 1 and fig. 2, the number of the trapping columns is three, and the trapping columns are a first trapping column 2a, a second trapping column 2b, and a third trapping column 2c, which are sequentially arranged from front to back at intervals. The first trap column 2a includes a plurality of first columnar projections 21a, a first trap channel 22a is formed between two connected first columnar projections 21a, and a first trap chamber 3a is formed between the first trap column 2a and the sample addition port. The second trapping column 2b includes a plurality of second columnar projections 21b, a second trapping passage 22b is formed between adjacent two second columnar projections 21b, and a second trapping chamber 3b is formed between the second trapping column 2b and the first trapping column 2 a. The third trapping column 2c includes a plurality of third columnar projections 21c, a third trapping channel 22c is formed between adjacent two of the third columnar projections 21c, and a third trapping chamber 3c is formed between the third trapping column 2c and the second trapping column 2 b.

The first trapping channel 22a traps microparticles having a particle size larger than or equal to its inner diameter in the first trapping chamber 3a, the second trapping channel 22b traps microparticles having a particle size larger than or equal to its inner diameter in the second trapping chamber 3b, and the third trapping channel 22c traps microparticles having a particle size larger than or equal to its inner diameter in the third trapping chamber 3 c. The first trapping passage 22a, the second trapping passage 22b, and the third trapping passage 22c have successively smaller inner diameters, and the first trapping chamber 3a, the second trapping chamber 3b, and the third trapping chamber 3c have successively smaller volumes. The three interception chambers are respectively used for intercepting the fluorescent microparticles with three particle size ranges of high, medium and low. Fluorescent microparticles with the particle size of 150-500 micrometers are trapped by the first trapping column 2a and stored in the first trapping chamber 3 a; fluorescent microparticles with the particle size of 80-150 micrometers are intercepted by the second intercepting column 2b and stored in the second intercepting chamber 3 b; fluorescent microparticles having a particle size of 10 to 80 μm are trapped by the third trapping column 2c and stored in the third trapping chamber 3 c. Preferably, the first trapping column 2a traps fluorescent microparticles having a particle size of 200 microns, the second trapping column 2b traps fluorescent microparticles having a particle size of 100 microns, and the third trapping column 2c traps fluorescent microparticles having a particle size of 50 microns.

The whole occupied space of each interception column is equal, and the number of the columnar protrusions contained in the interception column on the front side is smaller than that of the columnar protrusions contained in the interception column on the rear side. The front trapping column has the largest and smallest number of columnar projections, the middle trapping column is next to the middle trapping column, and the last trapping column has the smallest and largest number of columnar projections. The columnar bulges of each interception column are distributed in an array mode, and any four adjacent columnar bulges are arranged in a square mode. For example, the number of the first columnar projections 21a is smaller than the number of the second columnar projections 21b, and the number of the second columnar projections 21b is smaller than the number of the third columnar projections 21 c; the first columnar projection 21a has a volume larger than that of the second columnar projection 21b, and the second columnar projection 21b has a volume larger than that of the third columnar projection 21 c.

The housing 1 is made of a material such as a high polymer (e.g., PS, PMMA, PDMA, etc.), glass, or metal, and can be manufactured by a method including, but not limited to, photolithography, injection molding, machining, and laser cutting. The housing 1 specifically includes a bottom plate 11 and a cover plate 12, the trapping column and the trapping chamber are formed on the bottom plate 11, and the cover plate 12 is fixedly connected to the bottom plate 11 to cover the trapping column and the trapping chamber on the bottom plate 11. The cover plate 12 may be fixedly connected to the base plate 11 by thermal bonding, anodic bonding, low temperature bonding, or the like. The sample addition port 4 and the vent 5 are provided on the front and rear sides of the cover plate 12, respectively. The cylindrical projection of the trapping column extends upward from the bottom plate 11 to the lower surface of the cover plate 12.

Furtherly, hold back post integrated into one piece on bottom plate 11, be formed with the whole recess that is the rectangle on bottom plate 11, have in the recess to the multiunit column arch (being first column arch 21a, second column arch 21b and third column arch 21c respectively) that extend upward and the interval set up, every group column arch forms one and holds back the post, and the bellied degree of depth that highly equals the recess of column. The groove is divided into a plurality of chambers by the columnar bulges, and the chambers comprise a sample adding area, a trapping chamber and a waste liquid area. The shape of the columnar bump includes, but is not limited to, a cylinder, a triangular prism, and a quadrangular prism.

The length of bottom plate 11 and apron 12 is equal, and the width is equal, is 10 ~ 100 millimeters respectively. The thickness of the whole micro-fluidic chip is 500-1000 microns, wherein the thickness of the bottom plate 11 is 100-500 microns.

The fluid driving mode on the micro-fluidic chip mainly comprises two modes, one mode is capillary force, and the fluid is driven to move in the micro-channel by surface tension; the other is gas pressure provided by an external injection pump, which is used for adjusting internal compressed air pressure, so that fluid in the micro-channel can move in a certain direction. The use mode of the microfluidic chip is as follows:

the method comprises the steps of horizontally placing a bonded microfluidic chip, adding a 500-plus-5000 mu L sample into a sample adding area of the microfluidic chip by using a pipette, wherein the interior of a chamber and a microchannel both have capillary forces, so that the sample flows forwards along the microchannel under the capillary forces.

The method comprises the following steps of placing a bonded microfluidic chip in a horizontal position, adding a 500-.

The invention can be used for reducing the detection cost of the gene chip and improving the detection flux, the use of the fluorescent microparticles replaces a spotting instrument and a hybridization instrument in the detection process of the gene chip, the detection cost is greatly reduced, and the detection flux of the gene chip is improved by the arrangement and combination of the microparticles with different particle sizes, colors and shapes, so that the invention can simply, quickly and effectively detect and provides a new thought for the popularization of the detection of the gene chip.

The process of the present invention is described in detail below by means of specific examples.

Example 1

The method comprises the following steps: 30 red fluorescent microspheres with the particle sizes of 50 microns, 100 microns and 200 microns are taken respectively, corresponding oligonucleotide probes are coupled to the surface of each microsphere with the particle size, and the oligonucleotide probes coupled to the three microspheres are different from each other. Mixing the nucleic acid amplification product with the fluorescent label and the microspheres in a centrifuge tube, then putting the mixture in a constant-temperature oven for hybridization for 2 hours to combine complementary paired DNA single strands, and pouring out the mixed solution after the hybridization is finished. The fluorescent microspheres have magnetism, and the mixed liquid can be poured out by attaching a magnet to the wall of the centrifugal tube to control the magnetic beads.

Step two: and (3) adding a cleaning solution into the centrifuge tube containing the fluorescent microspheres, and washing away the nucleic acid molecules which are not specifically bound with the probes, wherein the cleaning solution is removed in the manner as described in the step one. The cleaning is carried out for 2 times totally, and the cleaning solution is not poured out in the last cleaning for standby.

Step three: the microsphere solution is added into the microfluidic chip, and the red fluorescent microspheres P of 200, 100 and 50 micrometers are respectively trapped in the first trapping chamber 3a, the second trapping chamber 3b and the third trapping chamber 3c through the action of the first trapping column 2a, the second trapping column 2b and the third trapping column 2c with specific sizes on the chip, as shown in fig. 5.

Step four: and (3) placing the microfluidic chip on a chip scanner, identifying the particle size of each fluorescent microsphere, and reading the fluorescence intensity of the label bound on the surface of the fluorescent microsphere. The fluorescence intensities on the fluorescent microspheres with the same particle size are accumulated to obtain the actual fluorescence intensity for detecting each target nucleic acid.

Example 2

The method comprises the following steps: 10 red fluorescent microspheres with the particle sizes of 50 micrometers, 100 micrometers and 200 micrometers respectively, 10 green fluorescent microspheres with the particle sizes of 50 micrometers, 100 micrometers and 200 micrometers respectively and 10 yellow fluorescent microspheres with the particle sizes of 50 micrometers, 100 micrometers and 200 micrometers respectively are taken. The surface of each microsphere is coupled with a corresponding oligonucleotide probe, and the oligonucleotide probes coupled on the nine microspheres are different from each other. Mixing the nucleic acid amplification product with the fluorescent label and the microspheres in a centrifuge tube, then putting the mixture in a constant-temperature oven for hybridization for 2 hours to combine complementary paired DNA single strands, and pouring out the mixed solution after the hybridization is finished. The fluorescent microspheres have magnetism, and the mixed liquid can be poured out by attaching a magnet to the wall of the centrifugal tube to control the magnetic beads.

Step three: the microsphere solution is added into the microfluidic chip, and the fluorescent microspheres P of 200, 100 and 50 micrometers are respectively trapped in the first trapping chamber 3a, the second trapping chamber 3b and the third trapping chamber 3c by the action of the first trapping column 2a, the second trapping column 2b and the third trapping column 2c with specific sizes on the chip, as shown in fig. 6.

Step four: and (3) placing the microfluidic chip on a chip scanner, identifying the particle size and the color of each fluorescent microsphere, and reading the fluorescence intensity of the label bound on the surface of the fluorescent microsphere. The fluorescence intensities on the fluorescent microspheres with the same particle size and color are accumulated to obtain the actual fluorescence intensity for detecting each target nucleic acid.

Example 3

The method comprises the following steps: 10 red fluorescent microparticles with the particle sizes of 50 micrometers, 100 micrometers and 200 micrometers are taken, wherein each microparticle with the particle size is 5 in a sphere and a triangle respectively; similarly, 10 green fluorescent microparticles each having a particle size of 50, 100, or 200 micrometers, 10 yellow fluorescent microparticles each having a particle size of 50, 100, or 200 micrometers, and 5 spheres and triangles of each of the fluorescent microparticles having the same color were taken. The surface of each micro particle is coupled with a corresponding oligonucleotide probe, and the oligonucleotide probes coupled on the eighteen micro particles are different from each other. Mixing the nucleic acid amplification product with the fluorescent label with the microparticles in a centrifuge tube, then placing the mixture in a constant-temperature oven for hybridization for 3h to combine complementary paired DNA single strands, and pouring out the mixed solution after the hybridization is finished. Because the fluorescent microparticles are nonmagnetic, the mixed solution is poured out by a mode of centrifuging to remove the supernatant.

Step two: and (3) adding a cleaning solution into the centrifuge tube containing the fluorescent microparticles, and washing away the nucleic acid molecules which are not specifically bound with the probe, wherein the cleaning solution is removed in the manner as described in the step one. The cleaning is carried out for 2 times totally, and the cleaning solution is not poured out in the last cleaning for standby.

Step three: the micro-particle solution is added into the micro-fluidic chip, and the fluorescent micro-particles P of 200, 100 and 50 micrometers are respectively trapped in the first trapping chamber 3a, the second trapping chamber 3b and the third trapping chamber 3c by the action of the first trapping column 2a, the second trapping column 2b and the third trapping column 2c with specific sizes on the chip, as shown in FIG. 7.

Step four: and (3) placing the microfluidic chip on a chip scanner, identifying the particle size, color and shape of each fluorescent microparticle, and reading the fluorescence intensity of the label bound on the surface of the fluorescent microparticle. The fluorescence intensities on the fluorescent microspheres with the same specification (the particle size, the color and the shape are all the same) are added up to obtain the actual fluorescence intensity for detecting each target nucleic acid.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and are preferred embodiments, which are intended to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the scope of the present invention. All equivalent changes or modifications made according to the principles of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for nucleic acid detection, comprising the steps of:

2. The method of claim 1, wherein: the fluorescent microparticles have a plurality of specifications, a capture object corresponding to one target nucleic acid molecule is coupled on each specification of fluorescent microparticles, and the capture objects coupled on the fluorescent microparticles with different specifications are different.

3. The method of claim 2, wherein: at least one of the particle size, the color and the shape of any two specifications of fluorescent microparticles are different.

4. The method of claim 2, wherein: the particle size of the fluorescent microparticles is 10-500 mu m; the color of the fluorescent microparticles is selected from at least one of green, yellow, red, blue and purple; the shape of the fluorescent microparticles is selected from at least one of spheres, cubes and triangles.

5. The method of claim 2, wherein: in the step C, the micro-fluidic chip intercepts fluorescent micro-particles with different particle size ranges in corresponding chambers; and/or in the step D, reading the fluorescence intensity of the label on each fluorescent microparticle, and respectively accumulating the fluorescence intensities of the fluorescent microparticles with the same specification to obtain the fluorescence intensity of each target nucleic acid molecule.

6. The method of claim 1, wherein: the capture object is an oligonucleotide probe.

7. The method of claim 1, wherein: in the step A, the fluorescent microparticles and the nucleic acid amplification product are mixed and hybridized in a container, and the mixed solution is poured out after the hybridization is finished; the fluorescent microparticles are magnetic, and after a magnetic substance is arranged on the wall of the container to adsorb the fluorescent microparticles, the mixed solution in the centrifugal tube is poured out; alternatively, the contents of the vessel are centrifuged and the mixture is decanted by removing the supernatant.

8. The method of claim 7, wherein: in the step B, adding a cleaning solution into the container to clean and wash away the nucleic acid molecules which are not bonded to the fluorescent microparticles, wherein the cleaning solution of the last cleaning is remained in the container; and in the step C, adding the liquid in the container into the microfluidic chip.

9. The method of claim 1, wherein: and in the step C, the micro-particles are intercepted into the interception cavity of the micro-fluidic chip through the interception part of the micro-fluidic chip.

10. The method of claim 9, wherein: the interception part comprises a plurality of interception columns, one interception chamber is formed between the sample adding port and the interception column at the foremost side and between two adjacent interception columns respectively, the liquid enters from the sample adding port at the front side of the microfluidic chip and sequentially passes through the interception columns, and microparticles with different particle sizes are intercepted in different interception chambers respectively.

11. The utility model provides a micro-fluidic chip for nucleic acid detects, includes hollow casing, seted up the sample injection port on the front end of casing, its characterized in that: the micro-fluidic chip also comprises an interception part which is arranged in the shell and used for intercepting micro-particles.

12. The microfluidic chip of claim 11, wherein: the trapping part comprises a first trapping column, a first trapping chamber is formed between the first trapping column and the sample adding port, the first trapping column comprises a plurality of first columnar protrusions, a first trapping channel is formed between every two adjacent first columnar protrusions, and the first trapping channel is used for trapping micro-particles with the particle size larger than or equal to the inner diameter of the first trapping channel in the first trapping chamber.

13. The microfluidic chip of claim 12, wherein: the interception part further comprises a second interception column, a second interception chamber is formed between the second interception column and the first interception column, the second interception column comprises a plurality of second cylindrical protrusions, a second interception channel is formed between every two adjacent second cylindrical protrusions, and the second interception channel is used for intercepting micro-particles with the particle size larger than or equal to the inner diameter of the second interception channel into the second interception chamber.

14. The microfluidic chip of claim 13, wherein: the second trapping channel has an inner diameter smaller than that of the first trapping channel.

15. The microfluidic chip of claim 13, wherein: the volume of the second entrapment chamber is less than the volume of the first entrapment chamber.

16. The microfluidic chip of claim 13, wherein: the microfluidic chip further comprises a third interception column, a third interception chamber is formed between the third interception column and the second interception column, the third interception column comprises a plurality of third columnar bulges, a third interception channel is formed between every two adjacent third columnar bulges, and the third interception channel is used for intercepting micro-particles with the particle size larger than or equal to the inner diameter of the third interception channel in the third interception chamber.

17. The microfluidic chip of claim 16, wherein: the third trapping channel has an inner diameter smaller than that of the second trapping channel.

18. The microfluidic chip of claim 16, wherein: the volume of the third entrapment chamber is less than the volume of the second entrapment chamber.

19. The microfluidic chip of claim 11, wherein: the shell comprises a bottom plate and a cover plate, the interception part is formed on the bottom plate, and the cover plate is fixedly connected with the bottom plate and covers the interception part.

20. The microfluidic chip of claim 19, wherein: the sample adding port is formed in the front side of the cover plate; and/or the rear side of the cover plate is provided with an air vent.