阅读说明：本技术 一种基于磁珠混匀的微流控芯片检测方法 (Micro-fluidic chip detection method based on magnetic bead uniform mixing ) 是由 许行尚 杰弗瑞·陈 刘祥 于 2021-07-29 设计创作，主要内容包括：本发明公开了一种基于磁珠混匀的微流控芯片检测方法,包括以下步骤：步骤1,微流控芯片内的固态试剂预置；步骤2,微流控芯片组装；步骤3,进样；步骤4,免疫反应；步骤5,吹除多余液体；步骤6,光学检测。本发明免除使用清洗液清洗步骤,使操作步骤更加简化,进一步缩短了检测时间,提高了检测效率。相应外部配套液路装置及芯片相应清洗流道相关的设计结构也得到简化,且有效避免了清洗液对免疫反应后复合物的影响,提高了检测结果的稳定性和准确性。该检测方法采用上下磁力交互吸附的混匀反应方式,不仅提高了免疫复合物的特异性结合效率,利于与其他组分相分离,还免除了复合物的清洗步骤,使外部配套检测设备结构大大简化、降低了生产成本。(The invention discloses a micro-fluidic chip detection method based on magnetic bead uniform mixing, which comprises the following steps: step 1, presetting a solid reagent in a microfluidic chip; step 2, assembling the microfluidic chip; step 3, sample introduction; step 4, immune reaction; step 5, blowing off redundant liquid; and 6, optically detecting. The invention avoids the cleaning step by using the cleaning solution, simplifies the operation steps, further shortens the detection time and improves the detection efficiency. The corresponding external matched liquid path device and the design structure related to the corresponding cleaning flow channel of the chip are also simplified, the influence of the cleaning liquid on the compound after immune reaction is effectively avoided, and the stability and the accuracy of the detection result are improved. The detection method adopts a mixing reaction mode of upper and lower magnetic force interactive adsorption, not only improves the specific binding efficiency of the immune complex and is beneficial to separating from other components, but also avoids the cleaning step of the complex, greatly simplifies the structure of external matching detection equipment and reduces the production cost.)

1. A micro-fluidic chip detection method based on magnetic bead uniform mixing is characterized by comprising the following steps:

step 1, presetting a solid reagent in a microfluidic chip: the solid reagent is preset in a quantitative-reaction cavity of the microfluidic chip and is used for specifically combining with a sample to form an immunoreaction compound;

step 2, assembling the microfluidic chip: bonding the microfluidic chip into a whole;

step 3, sample injection of the microfluidic chip: adding a sample to be detected, allowing the sample to be detected to enter, and conveying the sample to be detected into the quantitative-reaction cavity;

step 4, mixing and immunoreaction in the microfluidic chip: performing up-and-down magnetic force interactive adsorption on the upper part and the lower part of a quantitative-reaction cavity of the microfluidic chip, so that the sample to be detected in the quantitative-reaction cavity and the solid reagent are fully and uniformly mixed, and performing immunoreaction;

step 5, blowing off excess liquid in the quantitative-reaction cavity: after the immunoreaction is finished, fixing immunoreaction compound formed by the immunoreaction in the quantitative-reaction cavity in a magnetic adsorption mode, and blowing off redundant liquid through the gas path device;

and 6, optically detecting the microfluidic chip: and reading the fluorescence intensity in the quantitative-reaction cavity of the microfluidic chip by using an optical detection component, and calculating and giving a result.

2. The microfluidic chip detection method based on magnetic bead mixing according to claim 1, wherein in step 1, the solid reagent comprises immunomagnetic beads and fluorescent microsphere labeled antibodies.

3. The microfluidic chip detection method based on uniform mixing of magnetic beads according to claim 1, wherein in step 1, the solid reagent comprises immunomagnetic beads and a fluorophore-labeled antibody.

4. The microfluidic chip detection method based on uniform mixing of magnetic beads according to claim 1, wherein in step 2, the microfluidic chip comprises an upper chip, a middle chip and a lower chip, and the solid reagent is packaged inside the microfluidic chip through chip bonding.

5. The microfluidic chip detection method based on uniform mixing of magnetic beads according to claim 1, wherein in step 4, the upper and lower magnetic force interactive adsorption is realized by carrying out interactive energization on electromagnets.

6. The microfluidic chip detection method based on uniform mixing of magnetic beads according to claim 1, wherein in step 4, the upper and lower magnetic force interaction adsorption is realized by adjusting the distance between a magnet and the microfluidic chip.

7. The microfluidic chip detection method based on uniform mixing of magnetic beads according to claim 1, wherein the inside of a quantification-reaction chamber of the microfluidic chip is subjected to hydrophobic treatment.

8. The microfluidic chip detection method based on uniform mixing of magnetic beads according to claim 1, wherein in step 6, the detection result is displayed through an instrument screen.

9. The microfluidic chip detection method based on uniform mixing of magnetic beads according to claim 1, wherein in step 6, the detection result can be printed or data transmitted.

10. The microfluidic chip detection method based on uniform mixing of magnetic beads according to claim 1, wherein the microfluidic chip comprises a sample injection cavity, a quantitative-reaction cavity and a waste liquid cavity.

Technical Field

The invention relates to the field of microfluidic detection, in particular to a microfluidic chip detection method based on uniform mixing of magnetic beads.

Background

The superparamagnetic polymer microsphere is used as a novel solid phase carrier reagent, specific chemical groups on the surface of the superparamagnetic polymer microsphere can be combined with specific biomolecules, the obtained immunomagnetic beads can be specifically combined with corresponding target substances to form a new compound, and the compound can be retained and separated from other components when passing through a magnetic field. The immunomagnetic beads have the characteristics of superparamagnetism and nano particles in a magnetic field. The superparamagnetism of the method enables the solid-liquid separation operation to be simpler and more convenient, and omits the complicated traditional operations such as centrifugal filtration and the like; and the nano-sized immunomagnetic beads have small particles, large specific surface area, large coupling capacity and good suspension stability, are beneficial to the smooth implementation of specific reaction, and are widely applied in the analysis fields of biomedicine such as cell separation, immunoreaction, DNA extraction and the like.

Conventional magnetic bead technology has mature designs and methods, has wide application in biological sample detection, and many designs are commercialized. However, the conventional immunomagnetic bead technology has the following defects to be improved:

1) in the conventional magnetic bead technology, a conventional vessel is used for containing a sample solution, and operations such as cleaning are performed by using a pipettor and the like, so that exogenous pollutants are easily introduced.

2) The conventional magnetic bead technology needs manual operation and control of experimental steps, such as sampling, cleaning and the like, and the experimental failure is caused by the fact that manual operation error factors are easily introduced.

3) The conventional magnetic bead technology experiment platform is huge, needs larger instrument and equipment, is difficult to integrate and miniaturize, and is difficult to realize portability.

The micro-fluidic chip technology takes a micro-pipeline network as a structural characteristic, and the micro-pipeline network and other functional units are etched on a chip with the size of a few square centimeters by adopting a micro-processing technology, so that a rapid, high-efficiency and low-consumption micro analysis device which integrates sample introduction, reaction, separation and detection is prepared. The micro-fluidic chip has the characteristics of less reagent consumption, short reaction time and high automation degree in the detection platform. In the current research, the miniaturization of the chip brings many difficulties to the separation and matching integration of the early sample, and simultaneously, the chip also has many problems in the aspects of sample fixation, elution and the like.

The magnetic bead technology is combined with the microfluidic technology to realize a micro-separation chip system, the characteristic of high-efficiency separation of magnetic beads and the microfluidic control liquid flow are utilized to carry out biological reaction, and the manufactured micro-magnetic component and the microfluidic device can be used for constructing a high-integration immune detection micro-system. Compared with the conventional magnetic bead technology, the method has the following advantages:

1) the device is tiny and has no manual operation, and the reagent consumption is little.

2) The mixing reaction of different reagents in the same chip is realized by designing different micro flow paths, so that the complicated biological experiment operation is reduced, and the time required by detection is shortened.

3) The microfluidic chip can be connected with a circuit to realize automatic control.

4) The micro-fluidic chip and the matched detection equipment have small volume and are easy to carry.

However, in the detection technology combining the existing magnetic bead technology with the microfluidic chip, although the detection system has made great improvements in the above-mentioned advantages, the following disadvantages still exist: the cleaning step of the cleaning solution after the immune reaction makes the operation still complicated and the detection time correspondingly longer; the implementation of the cleaning step also makes the design of the chip structure relatively complex; and the remaining of the cleaning solution may cause inaccuracy of the detection result. In addition, ultrasonic mixing is generally adopted in the immunoreaction, an ultrasonic generator needs to be correspondingly equipped, so that external matched detection equipment is relatively complex, and the outer wall of the bottom surface of the reaction cavity is easily damaged by the traditional ultrasonic mixing mode, so that the acquisition of subsequent fluorescent signals is influenced.

Disclosure of Invention

Therefore, the invention provides a micro-fluidic chip detection method based on magnetic bead uniform mixing, which adopts a uniform mixing reaction mode of upper and lower magnetic force interactive adsorption in the immune reaction process, and avoids the cleaning step of cleaning solution after immune reaction. The invention not only simplifies the operation steps, but also greatly simplifies the structure of the chip and the external matched detection equipment, shortens the detection time, reduces the production cost and ensures that the detection result is more accurate and stable.

In order to achieve the purpose, the invention mainly adopts the following technical scheme:

a micro-fluidic chip detection method based on magnetic bead uniform mixing comprises the following steps:

step 1, presetting a solid reagent in a microfluidic chip: the solid reagent is preset in a quantitative-reaction cavity of the microfluidic chip and is used for specifically combining with a sample to form an immunoreaction compound;

step 2, assembling the microfluidic chip: bonding the microfluidic chip into a whole;

step 3, sample injection of the microfluidic chip: adding a sample to be detected, allowing the sample to be detected to enter, and conveying the sample to be detected into the quantitative-reaction cavity;

step 4, mixing and immunoreaction in the microfluidic chip: performing up-and-down magnetic force interactive adsorption on the upper part and the lower part of a quantitative-reaction cavity of the microfluidic chip, so that the sample to be detected in the quantitative-reaction cavity and the solid reagent are fully and uniformly mixed, and performing immunoreaction;

step 5, blowing off excess liquid in the quantitative-reaction cavity: after the immunoreaction is finished, fixing immunoreaction compound formed by the immunoreaction in the quantitative-reaction cavity in a magnetic adsorption mode, and blowing off redundant liquid through the gas path device;

and 6, optically detecting the microfluidic chip: and reading the fluorescence intensity in the quantitative-reaction cavity of the microfluidic chip by using an optical detection component, and calculating and giving a result.

Preferably, in step 1, the solid reagent comprises immunomagnetic beads and fluorescent microsphere labeled antibodies.

Preferably, in step 1, the solid reagent comprises immunomagnetic beads and fluorophore-labeled antibodies.

Preferably, in step 2, the microfluidic chip includes an upper chip, a middle chip and a lower chip, and the solid reagent is packaged inside the microfluidic chip by chip bonding.

Preferably, in step 4, the upper and lower magnetic force mutual adsorption is realized by alternately electrifying the electromagnet.

Preferably, in step 4, the upper and lower magnetic force mutual adsorption is realized by adjusting the distance between the magnet and the microfluidic chip.

Preferably, the inside of the quantitative-reaction chamber of the microfluidic chip is subjected to hydrophobic treatment.

Preferably, in step 6, the detection result is displayed on an instrument screen.

Preferably, in step 6, the detection result can be printed or transmitted.

Preferably, the microfluidic chip comprises a sample injection cavity, a quantitative-reaction cavity and a waste liquid cavity.

Compared with the prior art, the invention has the beneficial effects that: 1) the detection method avoids a cleaning step, simplifies the operation steps and shortens the detection time; 2) the influence of the residual cleaning liquid on the light path detection is effectively avoided, and the accuracy and the stability of the detection result are improved; 3) the structure of the micro-fluidic chip can not comprise structures related to the cleaning steps, such as a necessary cleaning solution micro-channel and the like, so that the structure of the chip is simplified; 4) the detection method adopts a mixing reaction mode of upper and lower magnetic force interactive adsorption, not only improves the specific binding efficiency of the immunoreaction compound, but also does not damage the outer wall of the bottom surface of the quantitative-reaction cavity, further does not influence the acquisition of subsequent fluorescence signals, greatly simplifies the structure of external matched detection equipment and reduces the production cost.

Drawings

Fig. 1 is a schematic front view of a layer chip in a first microfluidic chip according to an embodiment of the present disclosure;

FIG. 2 is a schematic diagram of a back side structure of a layer chip in a first microfluidic chip according to an embodiment of the present disclosure;

FIG. 3 is a schematic front view of a lower chip of a first microfluidic chip according to an embodiment of the present disclosure;

FIG. 4 is a schematic front view of a layer chip in a second microfluidic chip according to an embodiment of the present disclosure;

FIG. 5 is a schematic diagram of a back side structure of a layer chip in a second microfluidic chip according to an embodiment of the present disclosure;

FIG. 6 is a schematic front view of a lower chip of a second microfluidic chip according to an embodiment of the present disclosure;

FIG. 7 is an exploded view of a second microfluidic chip according to an embodiment of the present invention;

fig. 8 is a schematic structural diagram of a second microfluidic chip during detection according to an embodiment of the present invention.

The reference numbers illustrate:

2. a middle layer chip; 2-1, sample injection part; 2-1-1, air holes; 2-5, a cleaning liquid interface; 2-6. color developing liquid interface; 2-8, a through middle layer through hole; 2-9, a middle layer groove; 2-10, a reaction tank cover plate; 3. a lower chip; 3-1, a whole blood filter tank; 3-2, a quantitative-reaction tank; 3-3, a waste liquid pool; 3-4, a cleaning liquid conveying branch; 3-5, a color developing liquid conveying branch; 6. a valve means; 9-a. a first backflow prevention device; 9-b. a second backflow prevention device;

an upper chip 1'; a middle layer chip 2'; a lower chip 3'; a sample injection part 2-1'; 2-1-1' of an air hole; a through middle layer through hole 2-8'; middle layer groove 2-9'; 2-10' of a cover plate of the reaction tank; a whole blood filtering pool 3-1'; a quantitative-reaction tank 3-2'; 3-3' of a waste liquid pool; a first backflow prevention device 9-a'; 100. an electromagnet; 200. a second microfluidic chip.

Detailed Description

The following describes in more detail embodiments of the present invention with reference to the schematic drawings. The advantages and features of the present invention will become more apparent from the following description. It is to be noted that the drawings are in a very simplified form and are not to precise scale, which is merely for the purpose of facilitating and distinctly claiming the embodiments of the present invention.

The invention provides a detection method of a micro-fluidic chip based on uniform mixing of magnetic beads, which comprises the steps of detecting by means of matched equipment such as a chip carrier and a chip contact device, placing the micro-fluidic chip on the chip carrier, and detecting the chip by the chip contact device, wherein the chip contact device comprises a gas circuit device, a valve pressure point with a conductive rubber probe and an adjustable and controllable strong magnet.

In one embodiment, the microfluidic chip used in the detection of the present invention may adopt a chip structure in the prior art, that is, a chip structure in the chinese patent application No. 201710531301.0, which is referred to as a first microfluidic chip in this example. As shown in fig. 1, 2 and 3, the microfluidic chip is provided with a light path scanning window, a sample injection cavity, a quantitative-reaction cavity and a waste liquid cavity, and the microfluidic chip is of a three-piece structure, and an upper chip, a middle chip 2 and a lower chip 3 are sequentially stacked from top to bottom, and are stacked in a manner that every two chips are connected in a matching manner through a positioning column and a positioning hole. The upper chip comprises a sample injection part through hole, a light path scanning window, a cleaning fluid interface through hole, a color development liquid interface through hole and an upper layer vent hole. The middle layer chip 2 comprises a sample introduction part 2-1 corresponding to the through hole of the upper layer sample introduction part, a reaction tank cover plate 2-10, a middle layer cover plate, a cleaning solution interface 2-5 and a color development solution interface 2-6, wherein the sample introduction part 2-1 is used for butting a whole blood filter tank 3-1 of the lower layer chip and is provided with an annular groove and an annular flange, an air source interface is arranged in the annular groove, air holes 2-1-1 are formed in the end face of the annular flange and a conical transition surface close to the inner wall of the annular flange, when the air hole part 2-1 is not communicated, air can continuously push a sample to advance through the air holes 2-1-1, and the sample introduction part 2-1 is combined with the sample introduction part through hole of the upper layer chip and the whole blood filter tank 3-1 of the lower layer chip 3 to form a sample introduction cavity; the reaction tank cover plate 2-10 is positioned on the back surface of the middle chip 2 and is used for butting the quantitative-reaction tank 3-2 on the lower chip 3 so as to seal the tank port to form a quantitative-reaction cavity; the middle layer cover plate is arranged corresponding to the waste liquid pool 3-3 of the lower layer chip 3 to seal the pool mouth of the waste liquid pool to form a waste liquid cavity, and the middle layer cover plate comprises a middle layer through hole 2-8 and a middle layer groove 2-9 which are communicated. The lower chip 3 is provided with a whole blood filtering pool 3-1, a quantitative-reaction pool 3-2, a waste liquid pool 3-3, a cleaning liquid conveying branch 3-4 and a color developing liquid conveying branch 3-5, a liquid outlet of the whole blood filtering pool 3-1 is communicated with a liquid inlet of the quantitative-reaction pool 3-2 through a first anti-backflow device 9-a, a liquid outlet of the cleaning liquid conveying branch 3-4 is converged with a liquid outlet of the color developing liquid conveying branch 3-5 and then communicated with a liquid inlet of the quantitative-reaction pool 3-2 through a second anti-backflow device 9-b, a valve device 6 is arranged on a microfluidic flow channel between the quantitative-reaction pool 3-2 and the waste liquid pool 3-3, and the valve device 6 comprises a detection mechanism for detecting whether a fluid flows through the installation position of the valve device or not, the valve device 6 is normally open, and can be automatically closed when the value fed back by the detection mechanism indicates that the fluid flows through the installation position of the valve device 6, but the valve device 6 in the cut-off state can be opened at regular time, so that the fluid can circulate under the action of the airflow. The whole blood filtering pool 3-1 is arranged corresponding to the sample injection part 2-1 and can be communicated with an external gas circuit, whole blood sample filtering paper is paved in the pool to realize sample filtering, and different filtering settings can be carried out according to requirements, such as filtering into plasma or serum; the quantitative-reaction tank 3-2 is equally divided into three separate cavities, wherein a marked antibody is placed in the separate cavity in the middle, coated antibodies (immunomagnetic beads) are placed in the separate cavities on two sides, a reaction tank cover plate 2-10 corresponding to the quantitative-reaction tank 3-2 is equally divided into two cover plate split bodies, and each cover plate split body is filled with the coated antibodies (immunomagnetic beads); the waste liquid pool 3-3 is provided with two communicated waste liquid pool split bodies, the waste liquid pool split body a corresponds to a through middle layer through hole 2-8 of the middle layer chip 2, the waste liquid pool split body b corresponds to a middle layer groove 2-9 of the middle layer chip 2, absorbent paper is placed in the waste liquid pool 3-3, an airflow channel can be formed between the inner wall of the waste liquid cavity split body a and the absorbent paper, and the waste liquid cavity split body b is provided with a ventilation port.

The first microfluidic chip can be used in the detection method, namely, the existing chip and the existing resource can be fully utilized for detection under the condition that the microfluidic chip is not required to be redesigned, and the detection method has good applicability. Because the detection method of the invention avoids the cleaning step of the cleaning solution, when the first microfluidic chip is used in combination with the detection method of the invention, the cleaning solution delivery branch, the color development solution delivery branch, the corresponding through hole and the corresponding interface in the chip are not used.

In another embodiment, the microfluidic chip used in the detection may also be a new microfluidic chip, as shown in fig. 4, fig. 5, fig. 6 and fig. 7, the microfluidic chip only includes a sample injection cavity, a quantitative-reaction cavity, a waste liquid cavity and related channels therebetween, that is, the chip has a structure substantially similar to that of the first microfluidic chip except that interfaces, channels and corresponding backflow prevention devices related to the cleaning solution and the color developing solution are not required, compared with the chip in the prior art, the chip structure is simplified, and the design and processing costs are indirectly reduced, and in this embodiment, the chip structure is referred to as the second microfluidic chip 200. In this embodiment, the chip is divided into three layers, which are stacked from top to bottom into an upper chip 1 ', a middle chip 2 ' and a lower chip 3 '. The upper chip 1' comprises a sample injection part through hole and an upper layer vent hole. The middle layer chip 2 'comprises a sample injection part 2-1' corresponding to the through hole of the upper layer sample injection part, an air vent 2-1-1 ', a reaction tank cover plate 2-10', a through middle layer through hole 2-8 'and a middle layer groove 2-9'. The lower chip 3 ' is provided with a whole blood filtering pool 3-1 ', a quantitative-reaction pool 3-2 ' and a waste liquid pool 3-3 ', and the liquid outlet of the whole blood filtering pool 3-1 ' is communicated with the liquid inlet of the quantitative-reaction pool 3-2 ' through a first anti-backflow device 9-a '. The quantitative-reaction tank 3-2 'and the reaction tank cover plate 2-10' form a quantitative-reaction cavity, the waste liquid tank 3-3 ', the through middle layer through hole 2-8' and the upper layer chip form a waste liquid cavity at a corresponding position, and a conductive rubber valve device is arranged on a microfluidic flow channel between the quantitative-reaction cavity and the waste liquid cavity.

The process of implementing the detection method will now be specifically described using the second microfluidic chip 200 as an example, as shown in fig. 8, including the following steps:

step 1: the solid reagents in the microfluidic chip 200 are preset.

Before the sample injection of the chip 200, a solid reagent is required to be pre-placed in a quantitative-reaction cavity of the microfluidic chip 200, wherein the solid reagent comprises immunomagnetic beads and fluorescent microsphere labeled antibodies. In the embodiment, the presetting of the reagent is realized by coating and drying the sealing liquid, the sealing liquid does not participate in the reaction, and can be used for placing the fluorescent marker and the immunomagnetic beads, and in the drying process, the sealing liquid can still keep the biological activity and avoid the non-specific combination with the micro-control chip. After the sealing liquid is dried, adding immunomagnetic beads into a reaction pool cover plate 2-10 ' on the middle chip 2 ', and then drying to fix the immunomagnetic beads and the middle chip 2 '; and adding the fluorescent microsphere labeled antibody into a quantitative-reaction tank 3-2 ' on the lower chip 3 ', and drying to fix the fluorescent microsphere labeled antibody and the lower chip 3 '. In other embodiments, the immunomagnetic beads and the fluorescent microsphere labeled antibodies can be prepared as freeze-dried beads, and the solid reagents can be placed in the quantitative-reaction cavity in this way.

The solid reagent can also adopt immunomagnetic beads and fluorescent groups to mark antibodies, the difference is that fluorescent microspheres are cancelled, fluorescent groups are connected with the antibodies instead, the fluorescent microspheres have larger volume, when the solid reagent reacts with sample antigens, the capture reaction with the sample antigens is influenced due to larger steric hindrance, the reaction is insufficient, the detection result is inaccurate, the reaction efficiency with the sample antigens can be enlarged by directly connecting the fluorescent groups with the antibodies, and the detection result can be more fully reacted, so that the accuracy of the detection result is improved.

Step 2: the microfluidic chip 200 is assembled.

Sequentially assembling and combining an upper chip 1 ', a middle chip 2 ' fixed with immunomagnetic beads and a lower chip 3 ' fixed with fluorescent labeling antibodies in a chip ultrasonic bonding mode.

And step 3: and injecting a sample into the microfluidic chip 200.

And transferring the quantitative whole blood sample to the sample injection part of the microfluidic chip 200, and then putting the microfluidic chip 200 after sample injection into external matched detection equipment. Then, an external gas circuit device in external matching detection equipment is connected with a chip sample injection cavity, pressurized air pushes a whole blood sample to be changed into plasma/serum under the filtration of a whole blood filtering membrane, the plasma/serum enters a whole blood filtering pool 3-1 ', continuous air pressure pushes the plasma/serum to continuously flow forwards along a micro-channel and flows into a conductive rubber valve device after passing through a first backflow prevention device 9-a' and a quantitative-reaction cavity, capacitance change causes the valve device to be closed, namely the micro-channel entering a waste liquid cavity is closed, meanwhile, the external gas circuit is closed, and pressurization is stopped.

Step 4; and (3) uniformly mixing and carrying out immune reaction on the micro-fluidic chip 200.

The immune magnetic beads and the fluorescence labeling antibody are redissolved and are uniformly mixed with the blood plasma/blood serum in the quantitative-reaction cavity for reaction for a certain time, and the control time of the embodiment is 5-10 minutes to form an immune reaction compound.

The mixing method of the invention is that an adjustable strong magnet is arranged in external matching detection equipment and is positioned at the upper part and the lower part of a quantitative-reaction cavity of a chip 200, the adjustable strong magnet uses an electromagnet 100 in the embodiment, reaction liquid in the quantitative-reaction cavity is fully mixed and reacted under the action of the interaction and the adsorption of the upper and lower magnetic forces by the up-and-down interactive electrification of fixed frequency, the adjustable strong magnet can be the electromagnet 100 and can also be replaced by a common magnetic material, for example, a common magnet, the mixing reaction of the reaction liquid in the quantitative-reaction cavity is controlled by adjusting the distance between the common magnet and the chip, the farther the magnet is away from the chip, the smaller the magnetic force is, otherwise, the larger the magnetic force is, and the arrangement position of the adjustable strong magnet relative to the chip can be adjusted according to the requirements. The mixing method enables the sample to be specifically combined with the target substance under the action of the vertical magnetic force in the reaction process of the immunomagnetic beads and the fluorescence labeling antibodies to form an immunoreaction compound, has high reaction efficiency, and can not damage the bottom surface of the outer wall of the quantitative-reaction cavity to cause scratches and further not influence the acquisition of subsequent fluorescence signals compared with the traditional ultrasonic mixing method, thereby being beneficial to improving the accuracy of detection results.

And 5: the gas circuit device blows off redundant liquid.

After the immunoreaction is finished, an immunoreaction compound formed by the sample magnetic bead labeled antibody in the quantitative-reaction cavity is adsorbed by the electromagnet, the valve device can be opened at regular time, namely, in a circulating state, the external gas circuit device is started, continuous gas pressure pushes redundant liquid in the microfluidic chip 200 to flow to the waste liquid cavity until the liquid in the microfluidic channel is dried, at the moment, the capacitance value sensed by the capacitance probe in the valve device is restored to an initial value, and the external gas circuit device is closed and removes the adsorption force of the electromagnet.

According to the invention, the external gas circuit device is started for purging, so that an immunoreaction compound formed by the sample magnetic bead labeled antibody can be directly and effectively separated from the reaction liquid, and repeated cleaning and blow-drying are not needed to be carried out in cooperation with a cleaning solution, so that the operation steps are simplified, the time is saved, the design structure of external matched detection equipment and a chip is simplified, corresponding liquid flow channels in an external liquid circuit and the chip are omitted, the production cost is reduced, meanwhile, the influence of cleaning solution residue on light path detection is avoided, and the accuracy and the stability of the detection result are also improved.

In addition, in order to better complete step 5, the chamber of the quantitative-reaction chamber in the microfluidic chip 200 may be subjected to hydrophobic treatment before the reagent is preset, that is, before step 1, so that the waste liquid is blown out more simply and completely.

Step 6: the microfluidic chip 200 performs optical detection.

Starting an optical detection part of external matching detection equipment, reading the fluorescence intensity in the quantitative-reaction cavity on the microfluidic chip 200 through the outer wall of the bottom surface of the quantitative-reaction cavity, and calculating and giving a result report. And after operation, the detection result can be displayed on an instrument screen, and the detection result can be printed or subjected to data transmission.

The first microfluidic chip is also applicable to the detection method of the present invention, and when the first microfluidic chip is used in combination with the detection method of the present invention, the cleaning solution delivery branch, the developing solution delivery branch, the corresponding through hole, and the interface in the chip are not used.

According to the steps of the method disclosed by the invention, the method is applied to actual clinical detection, and a corresponding detection result is obtained through a specific detection sample. In the clinical acute myocardial infarction disease detection, cardiac troponin I (cTnI) is an early sensitive index for judging whether a thrombolytic therapy blood vessel is recanalized, and serum cTnI has clinical application value in the judgment of myocardial inflammation and slight injury and is also a sensitive index in the judgment of myocardial injury in CABG perioperative period. Therefore, in the invention, for the detection of cTnI as an example, the antigen is set to 9 concentrations (ng/mL), each concentration value is set to 3 parallel, the antigens are added into a microfluidic chip, the reaction is carried out for 10min according to the steps, and the fluorescence signal value after the reaction is detected:

1) when the solid-state reagent is an antibody marked by immunomagnetic beads and fluorescent microspheres, the detection data are as follows:

2) when the solid reagent is an immunomagnetic bead and a fluorophore-labeled antibody, the detection data are as follows:

the data in the table show that the fluorescence signal values of the two solid reagents are positively correlated with the increase of the antigen concentration in use, and the detected CV value is within 15 percent, so that the experiment stability is better, and the product development requirements are met.

In conclusion, the invention discloses a detection method of a micro-fluidic chip based on uniform mixing of magnetic beads, which not only simplifies the operation steps, but also greatly simplifies the chip structure and the structure of external matched detection equipment. The cleaning step is omitted, so that the operation steps are simplified, the detection time is further shortened, and the detection efficiency is improved. Because no cleaning step exists, the design structures related to corresponding external matched liquid path devices and corresponding cleaning flow channels of the chip can be simplified, the influence of cleaning liquid on the compound after immune reaction is effectively avoided, and the stability and the accuracy of the detection result are also improved. The detection method adopts a uniform mixing reaction mode of upper and lower magnetic force interactive adsorption, not only improves the specific binding efficiency of the immunoreaction compound, is beneficial to separating with other components, but also avoids the cleaning step of the compound, greatly simplifies the structure of external matched detection equipment and reduces the production cost.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and those skilled in the art can make various changes, modifications, substitutions and alterations without departing from the principle and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.