CN108344866B - Micro-fluidic chip detection system and method for detecting sample based on same - Google Patents

Abstract

The invention provides a micro-fluidic chip detection system and a method for detecting a sample based on the system, wherein the method comprises the following steps: a microfluidic detection chip comprising: a microfluidic cavity; a microwell array disposed on a bottom surface within the microfluidic cavity; a sample introduction conduit and a sample discharge conduit; which are respectively connected with and arranged at two sides of the microfluid cavity; a microfluidic operation device for allowing the sample reacted with the microspheres to enter the microfluidic cavity through a sample introduction conduit and controlling a flow rate so that the sample reacted with the microspheres is deposited into the microwells; the image acquisition device is arranged above the microwell array and is used for acquiring information of samples deposited in the microwells; and the data analysis device is used for analyzing the sample information acquired by the image acquisition device and acquiring an analysis result. Therefore, the method is favorable for realizing the detection of the sample to be detected with high flux, no blockage, portability and low detection limit.

Description

A microfluidic chip detection system and a method for sample detection based on the system

technical field

The invention relates to the fields of life science and medicine, and in particular, to a microfluidic chip detection system and a method for sample detection based on the system.

Background technique

Biomarkers are effective means to detect and assess the pharmacological response of routine biological processes, pathogenesis or therapeutic interventions, therefore, convenient and accurate detection and quantification of biomarkers is a core goal of modern biotechnology. For clinical applications, sandwich immunoassays are the most widely used method for detecting biomarkers of interest. Generally, these methods require some type of label (enzymatic or fluorescent, etc.) to selectively and sensitively detect the target analyte. It also requires specific equipment (eg a plate reader) and many washing steps. Recently, the rapid development of rapid, accurate, portable and marker-free biomarker quantification methods has made the concept of point-of-care diagnosis (also known as point-of-care testing) a reality. Among them, biosensors such as impedance spectroscopy, potentiometric sensors, surface-enhanced Raman scattering techniques, and gravimetric sensors have been developed and used for direct quantitative measurement of target biomarkers. These methods do not require any label formation by immobilizing specific receptors on the sensor surface. Another promising label-free detection method is based on biofunctionalized micro/nanoparticles. Molecular probes or carriers formed with such micro/nanoparticles have been widely used to directly capture target proteins or DNA from complex samples. By analyzing the aggregation state of these particles, colorimetric methods or particle size analyzers have been used for biomarker detection. Nanoparticles can be made to form aggregates through specific protein interactions (eg, antigen-antibody) between receptors immobilized on the particle surface and the target analyte in solution. In general, the higher the biomarker concentration, the more aggregates are produced, and the larger they are.

However, due to the lack of compatible methods or tools to accurately quantify the aggregation state (including information such as the number of nanoparticles within each aggregate), the precise relationship between nanoparticle aggregation and biomarker concentration has not been thoroughly revealed. In addition to nanoparticles, people have also begun to modify the surface of microparticles with antibodies, and use impedance sensors to detect the aggregation state between microspheres modified with specific antibodies, thereby realizing the detection of antigens. Although impedance sensors can directly read out the number of aggregates, this method suffers from low throughput, easy clogging, and low resolution. Furthermore, developing an impedance analysis system requires dedicated fluid transport devices and expensive data acquisition systems for fast and low-noise electrical signal processing. This hinders the development of this electrical impedance system into a portable detection device.

Therefore, there is an urgent need for a detection device to overcome the problems of difficult detection caused by low throughput, easy clogging, and low resolution in the prior art.

SUMMARY OF THE INVENTION

In view of this, the present application provides a microfluidic chip detection system and a method for sample detection based on the system, to overcome the difficulty in detection due to low throughput, easy blockage, and low resolution in the prior art. question. High-throughput, non-blocking, high-resolution detection is achieved.

The application provides a microfluidic chip detection system, including:

Microfluidic detection chip, including:

microfluidic chamber;

a microwell array, disposed on the bottom surface of the microfluidic cavity;

The sample introduction conduit and the sample export conduit; they are respectively connected and arranged on both sides of the microfluidic cavity;

A microfluidic operating device, used to make the sample reacted with the microspheres enter the microfluidic cavity through a sample introduction conduit, and control the flow rate to deposit the sample reacted with the microspheres into the microwells;

an image acquisition device, arranged above the micro-well array, for acquiring information of the samples deposited in the micro-well;

A data analysis device, configured to analyze the sample information collected by the image acquisition device and obtain analysis results.

From the above, the present application controls the flow rate through a microfluidic operating device, and in combination with the above-mentioned structure of the present application, the samples after the microsphere reaction are dispersed and deposited into the microwells of the microfluidic detection chip, and further through a portable image acquisition device ( For example, a mobile phone) collects the information in the micro-well for analysis, which overcomes the defects of low throughput, easy clogging and low resolution caused by non-dispersion and superposition of samples in the prior art, and realizes high throughput and no clogging of the sample to be tested. , Portable, high-resolution detection.

Preferably, the microfluidic operating device is further used for: introducing the buffer solution into the microfluidic chamber through the sample introduction conduit, and flushing the sample not deposited in the microwell by controlling the flow rate of the buffer solution, so that the sample can pass through the sample Outflow from the conduit.

From the above, the samples that are not deposited into the micro-wells are washed, which is beneficial to reduce the interference to the detection of the samples in the micro-wells. During the flushing process at this time, due to the existence of the microwell, the dimer in it will not be washed away.

Preferably, the system further includes:

The system of claim 2, further comprising a sample reaction chamber:

The sample reaction chamber is an independent chamber in communication with the microfluidic manipulation device; or

The microfluidic chamber or the sample introduction conduit also serves as the sample reaction chamber connection.

From the above, the above-mentioned sample reaction chamber can be a separately set test tube, centrifuge tube, catheter, micro-cavity, etc., or the sample can be directly introduced into the catheter as the sample reaction chamber, which makes the device more compact and easy to carry.

Preferably, the number of the microfluidic detection chips is 1 or at least 2;

When the number of the microfluidic detection chips is at least 2; successively adjacent microfluidic detection chips are connected in series through connecting pipes; the size of each microwell array in each microfluidic detection chip is the same; The size of the microwells of the detection chip increases sequentially along the flow direction of the sample.

From the above, when a single microfluidic detection chip is set, a microwell array is set in it, the inflowing sample is not easy to be blocked, the throughput is high, and the sample to be tested falls into each microwell dispersedly, making the detection more clear.

When more than two microfluidic detection chips (for example, three) are set up, the microspheres can be classified and captured step by step: the first-stage microfluidic cavity is used to capture a single microsphere in the microsphere solution after the reaction, The second-stage microfluidic cavity is mainly used to capture dimers composed of two or two microspheres, and the third-stage microfluidic cavity is used to capture aggregates composed of three or more microspheres. Such a grading process can reduce the difficulty of detection and counting of microspheres, thereby reducing detection errors.

Preferably, a controllable valve is also provided on the connecting pipes between successively adjacent microfluidic detection chips.

From the above, by setting the above valve, the capture of microspheres with different degrees of polymerization can be better graded. For example, when the microfluidic cavity of the microfluidic detection chip of the first stage captures a single microsphere in the microsphere solution after the reaction, the pipeline between the first stage and the second stage microfluidic cavity is closed; When the buffer rinses the sample not deposited into the microwells of the microfluidic chamber of the first stage, open the pipeline between the first and second stage microfluidic chambers; close the second and third stage microfluidic chambers at the same time. Conduit between fluidic chambers; resuspend the sample not deposited in the microwells of the first-stage microfluidic chamber into the second-stage microfluidic chamber, allowing the dimerization of two microspheres in it The body is captured into the micro-well; when the sample in the micro-well that is not deposited into the micro-fluidic chamber of the second stage is flushed with the buffer solution, the pipeline between the second-stage and the third-stage microfluidic chamber is opened; The samples that were not deposited in the microwells of the second-stage microfluidic chamber were resuspended and rinsed into the third-stage microfluidic chamber, so that the aggregates composed of three or more microspheres were captured in the microfluidic chambers. in the well. Such a grading process can reduce the difficulty of detection and counting of microspheres, thereby reducing detection errors.

Preferably, the microfluidic manipulation device comprises: a pneumatic fluid pump or a syringe pump.

Preferably, the diameter of the microspheres ranges from 1 micron to 20 microns;

The materials of the microspheres include but are not limited to at least one of the following: polystyrene, silicon dioxide, and iron tetroxide;

Wherein, the surface of the microsphere is modified with specific molecular probes and impedance molecules disposed in the gaps of the molecular probes for reducing non-specific adsorption of molecules;

Wherein, the specific molecular probe includes but is not limited to at least one of the following: DNA, RNA, protein molecule, biotin molecule;

Wherein, the impedance molecules include but are not limited to at least one of the following: bovine serum albumin BSA, polyethylene glycol PEG.

From the above, the direct range of the microspheres can be 1-100 microns, preferably 1-10 microns, which can be selected according to needs.

Preferably, the diameter of the microwell is 3-10 times the diameter of a single microsphere;

The depth of the microwell is 1-5 times the diameter of a single microsphere;

Wherein, the height of the microfluidic cavity is 2-10 times the diameter of a single microsphere;

Wherein, the projection of the micro-well in the horizontal direction is: circle, ellipse or polygon.

From the above, the diameter of the microwell is preferably 3-5 times the diameter of the single microsphere; the depth of the microwell is preferably 2-3 times the diameter of the single microsphere. The height of the microfluidic cavity is preferably 2-10 times the diameter of a single microsphere.

Preferably, the fabrication materials of the microfluidic cavity and the microwell include but are not limited to at least one of the following: transparent organic polymers, glass, and silicon dioxide.

From the above, the fabrication materials of the microfluidic cavity and the microwell can be all transparent materials. to facilitate subsequent image acquisition.

Preferably, the materials for making the introduction conduit and the outflow conduit include but are not limited to at least one of the following: a plastic tube, a Teflon tube, a glass conduit, and a metal conduit.

The application also provides a method for sample detection based on a microfluidic chip detection system, including:

A. Place the sample to be tested in a sample reaction chamber containing microspheres, so that the target molecule to be tested in the sample to be tested reacts with the microspheres to form a dimer;

B. The reacted sample is introduced into the microfluidic cavity of the microfluidic detection chip through the sample introduction conduit through the microfluidic operating device, and the dimer is deposited into the microwell by controlling the flow rate;

C. Through the microfluidic operating device, the buffer solution enters the microfluidic chamber through the sample introduction conduit, and the sample not deposited in the microwell is washed by controlling the flow rate of the buffer solution, so that it flows out through the sample export conduit;

D. Collect the information of the dimer deposited in the microwell by an image acquisition device;

E. The information of the dimer collected by the image acquisition device is analyzed by the data analysis device to obtain the concentration of the molecule to be tested in the sample to be tested.

Preferably, the number of the microfluidic detection chips is 1 or at least 2;

When the number of the microfluidic detection chips is at least 2; successively adjacent microfluidic detection chips are connected in series through connecting pipes; the size of each microwell array in each microfluidic detection chip is the same; The size of the microwells of the detection chip increases sequentially along the flow direction of the sample;

Wherein, a controllable valve is also provided on the connecting pipeline between the adjacent microfluidic detection chips.

From the above, by setting the above valve, the capture of microspheres with different degrees of polymerization can be better graded. For example, when the microfluidic cavity of the microfluidic detection chip of the first stage captures a single microsphere in the microsphere solution after the reaction, the pipeline between the first stage and the second stage microfluidic cavity is closed; When the buffer rinses the sample not deposited into the microwells of the microfluidic chamber of the first stage, open the pipeline between the first and second stage microfluidic chambers; close the second and third stage microfluidic chambers at the same time. Conduit between fluidic chambers; resuspend the sample not deposited in the microwells of the first-stage microfluidic chamber into the second-stage microfluidic chamber, allowing the dimerization of two microspheres in it The body is captured into the micro-well; when the sample in the micro-well that is not deposited into the micro-fluidic chamber of the second stage is flushed with the buffer solution, the pipeline between the second-stage and the third-stage microfluidic chamber is opened; The samples that were not deposited in the microwells of the second-stage microfluidic chamber were resuspended and rinsed into the third-stage microfluidic chamber, so that the aggregates composed of three or more microspheres were captured in the microfluidic chambers. in the well. Such a grading process can reduce the difficulty of detection and counting of microspheres, thereby reducing detection errors.

To sum up, the present application overcomes the problem by dispersing and depositing the reacted samples of the microspheres into the microwells of the microfluidic detection chip, and further collecting the information in the microwells through a portable image acquisition device (such as a mobile phone) for analysis. The defects of low throughput, easy blockage, and low resolution caused by non-dispersion and superposition of samples in the prior art are eliminated, and high-throughput, non-blocking, portable, and high-resolution detection of the sample to be tested is realized. In addition, the present application can also realize the hierarchical capture of different types of polymers, thereby reducing the difficulty of detection and counting, thereby reducing detection errors.

Description of drawings

1 is a top view of a microfluidic detection chip provided by an embodiment of the present application;

2 is a three-dimensional schematic diagram of a microfluidic detection chip provided by an embodiment of the present application;

3 is a schematic diagram of the principle of the reaction between the microspheres in the sample reaction chamber and the target molecule to be measured in the sample provided by the embodiment of the present application;

4 is a schematic diagram of the overall structure and working principle of the microfluidic chip detection system provided by the embodiment of the present application;

5 is a schematic diagram when the sample reaction chamber is a sample introduction conduit according to an embodiment of the present application;

FIG. 6 is a schematic diagram when the sample reaction chamber is a microfluidic cavity according to an embodiment of the present application;

FIG. 7 is a schematic diagram when there are more than two microfluidic detection chips (three in this embodiment, which are connected through a conduit) according to an embodiment of the present application;

Fig. 8 is a schematic diagram showing that different sizes of microspheres modified with different molecular probes are simultaneously mixed with samples to be tested to form different put-in dimers;

FIG. 9 is a schematic structural diagram of integrating a microfluidic detection chip into a portable black box;

10 is a schematic diagram of an image collected by an image collection device;

11 is a schematic diagram of the detection result of detecting prostate cancer antibody (PSA) according to an embodiment of the present application;

FIG. 12 is a schematic flowchart of a method for sample detection based on a microfluidic chip detection system according to an embodiment of the present application.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art fall within the protection scope of the present invention.

The present application provides a microfluidic chip detection system and a method for sample detection based on the system, which is conducive to realizing high-throughput, non-blocking, portable, and low detection limit detection of the sample to be tested.

As shown in Figures 1-4, the present application provides a microfluidic chip detection system, including:

Microfluidic detection chip, including:

microfluidic chamber 100;

The microwell array 101 is arranged on the bottom surface of the microfluidic cavity;

The sample introduction conduit 102 and the sample export conduit 103; they are respectively connected to the two sides of the microfluidic cavity;

The microfluidic operating device 200 is used to make the sample reacted with the microspheres enter the microfluidic cavity through a sample introduction conduit, and the sample reacted with the microspheres is deposited into the microwell by controlling the flow rate; wherein, The microfluidic operating device is also used for: introducing the buffer solution into the microfluidic chamber through the sample introduction conduit, and flushing the sample not deposited in the microwell by controlling the flow rate of the buffer solution, so that it flows out through the sample exporting conduit . It is beneficial to flush the samples that are not deposited in the micro-well, and it is beneficial to reduce the interference to the detection of the samples in the micro-well. Wherein, the microfluidic operating device includes: a pneumatic fluid pump or a syringe pump or other principles such as an electrically driven fluid pump.

The image acquisition device 300 is disposed above the micro-well array, and is used for acquiring information of the samples deposited in the micro-wells.

A data analysis device, configured to analyze the sample information collected by the image acquisition device and obtain analysis results. Image processing technology, such as Matlab program and deep learning, is used to process the image to extract the shape, size, quantity and proportion information of the microsphere aggregate, so as to further obtain the concentration of the target molecule to be measured.

Wherein, the system also includes:

The sample reaction chamber 400 is connected to the microfluidic operating device; in the sample reaction chamber, the microspheres react with the target molecules to be detected in the sample to form dimers. The specific principle is shown in Figure 3. The surface of the microsphere 31 is modified with a molecular probe 33 that specifically adsorbs the target molecule 32 in the sample. In addition, the sample 34 to be tested also contains other molecules 35 and 36 and the like. Using the specific binding of the molecular probe 33 to the target molecule 32, the microsphere aggregate 37 is formed.

Wherein, the sample reaction chamber is a separate chamber; or, the sample is introduced into a conduit or a microfluidic cavity as a sample reaction chamber. The above-mentioned sample reaction chamber can be a separately set test tube, centrifuge tube, conduit, micro-cavity, etc., or the sample can be directly introduced into the conduit or microfluidic cavity as the sample reaction chamber, which makes the device more compact and easy to carry.

Wherein, the microfluidic cavity is a single cavity; or

Two or more cavities that communicate with each other are connected in parallel by connecting pipes; wherein, a micro-well array is arranged in each cavity; wherein, the size of the micro-wells in each cavity is the same, and the micro-wells in different cavities have the same size. The size increases sequentially along the direction of the sample introduction conduit—the microfluidic chamber—the sample outflow conduit.

From the above, a single cavity is provided with an array of microwells, the inflowing samples are not easily blocked, the throughput is high, and the samples to be tested fall into each microwell dispersedly, making the detection more clear.

The setting of more than two cavities can realize the step-by-step classification and capture of microspheres: the first-stage microfluidic cavity is used to capture a single microsphere in the microsphere solution after the reaction, and the second-stage microfluidic cavity is mainly used for The third-stage microfluidic chamber is used to capture the polymer composed of three or more microspheres. Such a grading process can reduce the difficulty of detection and counting of microspheres, thereby reducing detection errors.

Wherein, the diameter range of the microspheres to be combined with the target molecule to be detected in the sample is: 1 micrometer-20 micrometers; wherein, the direct range of the microspheres can be 1-100 micrometers, preferably 1-10 micrometers, or 1 micrometer- 20 microns; can be selected according to specific needs.

The materials of the microspheres include but are not limited to at least one of the following: polystyrene, silicon dioxide, and iron tetroxide;

Wherein, the surface of the microsphere is modified with specific molecular probes and impedance molecules disposed in the gaps of the molecular probes for reducing non-specific adsorption of molecules;

Wherein, the specific molecular probes include but are not limited to at least one of the following: (deoxyribonucleic acid) DNA, (ribonucleic acid) RNA, protein molecules, biotin molecules;

Wherein, the impedance molecules include but are not limited to at least one of the following: (bovine serum albumin) BSA, (oligoethylene glycol-poly-L-lysine) PLL-OEG.

Preferably, the diameter of the microwell is 3-10 times the diameter of a single microsphere; preferably, it is 3-5 times the diameter of the single microsphere.

The depth of the microwell is 1-5 times the diameter of a single microsphere; the depth is preferably 2-3 times the diameter of a single microsphere.

Wherein, the height of the microfluidic cavity is 2-10 times the diameter of a single microsphere;

Wherein, the horizontal projection of the microwell can be various topological structures, such as circle, ellipse or polygon (rhombus, trapezoid, triangle, square, rectangle, parallelogram, etc.).

Wherein, the fabrication materials of the microfluidic cavity and the microwell include, but are not limited to, at least one of the following: transparent organic polymers, glass, silicon dioxide, plastic, and the like. From the above, the fabrication materials of the microfluidic cavity and the microwell can be all transparent materials. to facilitate subsequent image acquisition. The microfluidic cavity and the microwell are combined by bonding or mechanical clamping. The main methods of processing micron-scale microfluidic chambers and microwells include wet etching, dry etching, laser etching, and mechanical milling.

Wherein, the materials for making the introduction conduit and the outflow conduit at least include but are not limited to one of the following: a plastic tube, a Teflon tube, a glass conduit, and a metal conduit.

In order to illustrate the technical solutions of the present application more clearly, the working principles of the various solutions of the microfluidic chip detection system of the present application are now described as follows:

1. 1. As shown in Figures 1-4, the sample to be tested is placed in the sample reaction chamber 400 provided with microspheres, so that the molecules to be tested in the sample to be tested and the molecular probes 33 on the surface of the microspheres 31 are connected. The reaction forms dimer 37;

2. Make the reacted sample enter the microfluidic chamber through the sample introduction conduit 102 through the microfluidic operating device 200 (air pressure fluid pump or syringe pump or other principle such as an electrically driven fluid pump), and control the flow rate so that all The dimer is deposited into the microwell 101;

3. Through the microfluidic operating device, the buffer solution enters the microfluidic chamber (single) through the sample introduction conduit, and the sample that is not deposited in the microwell is washed by controlling the flow rate of the buffer solution, so that it is exported through the sample Conduit 103 flows out. Due to the presence of the microwell, the dimer in it will not be washed away.

4. The information of the dimer 37 deposited in the microwell 101 is collected by the image acquisition device 300; wherein, the above-mentioned microfluidic detection chip is fixed on the portable microscope (image acquisition device 300), A mobile phone is arranged on the microscope, and the camera of the mobile phone collects images of the microfluidic detection chip through the eyepiece of the microscope. Figure 10 shows the acquired image.

5. Analyze the information of the dimer collected by the image acquisition device through the data analysis device, and use the image processing technology, such as Matlab program, deep learning to process the image to extract the shape, size, quantity and proportion information of the microsphere aggregate, thereby Further obtain the concentration of the target molecule to be detected according to this.

Experimental results

Figure 11 shows the result of detecting PSA protein molecules in serum using an embodiment of the patent of the present invention. In serum experiments, PSA serum solutions at concentrations of 0, 3.67pM, 36.7pM, and 367pM were reacted with a solution of polystyrene microspheres with a concentration of ² *106/mL and a diameter of 5.1 microns. Anti-PSA molecular probes that specifically capture PSA are modified on the surface of polystyrene microspheres. The microsphere solution and the serum sample were thoroughly mixed, reacted in a 1 mL centrifuge tube for 30 minutes, and then passed into a microwell microfluidic chip for detection. The number and ratio of microsphere dimers were used as detection indicators. The detection results are shown in Figure 11. The experimental results shown in FIG. 11 not only verify the feasibility of the microsphere polymerization method, but also verify the practicability of the embodiments.

2. Among them, as shown in FIG. 5, it is the situation when the sample reaction chamber is the sample introduction conduit. The difference between it and the principle in the above one is that the above 1 is replaced by: Molecules and other molecules in the sample solution to be tested are thoroughly mixed and then introduced into the sample introduction conduit 102 . The rest are consistent with 2-5 in the aforementioned scheme 1.

3. Among them, as shown in Figure 6, it is the case when the sample reaction chamber is a microfluidic cavity. The difference from the above-mentioned principle is that the above 1 and 2 are replaced by: The molecules to be tested and the tested sample solutions of other molecules are thoroughly mixed and then introduced into the microfluidic chamber through the sample introduction conduit 102 to react therein. The rest are consistent with 2-5 in the aforementioned scheme 1.

4. Among them, as shown in FIG. 7 , what is different from the aforementioned 1 to 3 is that the microfluidic detection chip shown in FIG. 7 is composed of two or more microfluidic chambers. After the microspheres react with the sample solution to be tested, the reaction solution is introduced into the microfluidic chamber through the sample introduction conduit 102 . The sample to be tested passes through the first-stage microfluidic cavity 71 , the second-stage microfluidic cavity 72 , and the third-stage microfluidic cavity 73 in sequence. The multi-level microfluidic chambers communicate through the flow channel 74 . The size of the microwells in the microwell arrays 711, 721, 731 in the multi-stage microfluidic chamber increases sequentially. For example, the microwell array 711 is one to two times the size of the microsphere used to capture a single microsphere; the microwell array 721 is two to four times the size of the microsphere used to capture two microspheres. The microsphere aggregates; the microwell array 731 is four to ten times the size of the microspheres used to capture the microsphere aggregates formed by multiple microspheres. The inlet and the outlet are communicated with the conduit for introducing and taking out the sample solution, respectively. Using the hierarchical microwell chip in this embodiment, it is possible to realize the step-by-step classification and capture of the microspheres: the first-stage microfluidic cavity is used to capture a single microsphere in the microsphere solution after the reaction, and the second-stage microfluidic cavity It is mainly used to capture dimers composed of two microspheres, and the third-stage microfluidic cavity is used to capture polymers composed of three or more microspheres. Wherein, by arranging the valve 741 on the flow channel 74, the capture of microspheres with different degrees of polymerization can be achieved in a better classification. For example, when the microfluidic cavity of the microfluidic detection chip of the first stage captures a single microsphere in the microsphere solution after the reaction, the pipeline between the first stage and the second stage microfluidic cavity is closed; When the buffer rinses the sample not deposited into the microwells of the microfluidic chamber of the first stage, open the pipeline between the first and second stage microfluidic chambers; close the second and third stage microfluidic chambers at the same time. Conduit between fluidic chambers; resuspend the sample not deposited in the microwells of the first-stage microfluidic chamber into the second-stage microfluidic chamber, allowing the dimerization of two microspheres in it The body is captured into the micro-well; when the sample in the micro-well that is not deposited into the micro-fluidic chamber of the second stage is flushed with the buffer solution, the pipeline between the second-stage and the third-stage microfluidic chamber is opened; The samples that were not deposited in the microwells of the second-stage microfluidic chamber were resuspended and rinsed into the third-stage microfluidic chamber, so that the aggregates composed of three or more microspheres were captured in the microfluidic chambers. in the well. Such a grading process can reduce the difficulty of detection and counting of microspheres, thereby reducing detection errors.

5. Among them, as shown in FIG. 8 , the difference from the above-mentioned 1 to 4 is that the microspheres 84 , 85 , and 86 of different sizes modified with different molecular probes are mixed with the sample 87 to be tested to form dimers 88 , respectively. , 89, 810, in which the target measured objects 81, 82, 83 are detected at the same time. After the microspheres react with the sample solution to be tested, the reaction solution is introduced into the microfluidic detection chip. Subsequent operations are the same. It should be particularly pointed out that the method for detecting different target detection substances with microspheres of various sizes proposed in this embodiment can be combined with the solution proposed in 4. FIG. 8 . Specifically, the multi-stage microfluidic chambers are functionally designed as follows: the first-stage microfluidic chamber is used to capture the microspheres of the smallest size, and then the size of the captured microspheres is gradually increased in the downstream microfluidic chambers. It realizes the classification and capture of microspheres of different sizes, that is, realizes the spatial difference detection of the detection results of different target detection objects.

6. Among them, as shown in FIG. 9, the difference from the above-mentioned 1 to 4 is that this scheme integrates the microfluidic detection chip into a portable black box 94, including: a loading platform 96 for fixing the microfluidic detection chip 97, which can be The observation objective 98 in the microfluidic detection chip 97 is focused. In actual operation, the microfluidic control device 91 only needs to be connected with the microfluidic detection chip 97 in the portable black box 94 through the introduction conduit 92 and communicated with the extraction conduit 95 to conduct the introduction and capture of the microsphere sample. Finally, the mobile phone 93 (which may also be other image acquisition devices) is placed on the observation objective lens 98 of the black box 94 to perform image extraction.

To sum up, the microfluidic chip detection system of the present application disperses and deposits the reacted samples of the microspheres into the microwells of the microfluidic detection chip, and further collects the microfluidic detection chips through a portable image acquisition device (such as a mobile phone). The information in the well is analyzed, which overcomes the defects of low throughput, easy clogging and low resolution caused by non-dispersion and superposition of samples in the prior art, and realizes high throughput, non-clogging, portable, high-resolution samples to be tested. rate detection. In addition, the height and width of the microfluidic cavity of the present application, and the diameter and height of the microwell are set to a suitable range, which more effectively eliminates the blocking problem.

In addition, the present application can also realize the hierarchical capture of different types of polymers, thereby reducing the difficulty of detection and counting, thereby reducing detection errors.

In addition, the device realized by the present invention does not need complicated optical and electrical detection equipment compared with the traditional detection system based on optics and the principle of the Kühler counter. The microfluidic chip used in the present invention is small in size and very low in cost, and the cost of each chip can be controlled within tens of RMB, and can be reused. The subsequent detection system is an image detection method based on mobile phone photography and mobile phone APP. A mobile phone can solve the tasks to be achieved by the complex detection module in the traditional technology. The microscopic magnification device is realized by a portable microscope or a small objective lens. The microfluidic introduction system can be realized by a syringe pump. Compared with the existing detection technology, the technical solution provided by the present invention greatly reduces the threshold of the detection device, and each module can be easily assembled by using a portable device, and can be operated in hospitals, at home and in the field. In microsphere detection, the surface-modified microspheres with specific molecular probes are combined with target molecules, so that two adjacent microspheres can form a sandwich structure, thereby forming dimers or even multimers. The number or concentration of these microsphere aggregates has a corresponding relationship with the concentration of target molecules. In the present application, two microspheres can be connected to form a microsphere dimer through a single target molecule, so the method of microsphere polymerization can theoretically detect extremely low molecular concentrations. The microfluidic chip detection system of the present invention can minimize the influence of the detection means on the microsphere aggregate itself. As shown in FIG. 11 , through the detection of PSA, the minimum detection limit is 3.67pM, that is, the present application can detect extremely low concentration samples, which can fully meet the detection requirements of the clinically concerned PSA diagnostic concentration range of 30pM to 300pM.

Embodiment 2

As shown in FIG. 12 , based on the microfluidic chip detection system of Embodiment 1, the present application also provides a detection method based on the above-mentioned microfluidic chip detection system, including:

S121, placing the sample to be tested in the sample reaction chamber provided with microspheres, so that the molecules to be tested in the sample to be tested react with the microspheres to form dimers;

S122, using the microfluidic operating device to make the reacted sample enter the microfluidic cavity through the sample introduction conduit, and control the flow rate to deposit the dimer into the microwell;

S123, through the microfluidic operating device, the buffer solution enters the microfluidic chamber through the sample introduction conduit, and by controlling the flow rate of the buffer solution, the sample that is not deposited in the microwell is washed so that it flows out through the sample export conduit;

S124, collecting information of the dimer deposited in the microwell by using an image collecting device;

S125 , analyze the dimer information collected by the image acquisition device by the data analysis device to obtain the concentration of the molecule to be tested in the sample to be tested.

To sum up, the microfluidic chip detection system of the present application realizes the detection method by depositing the sample reacted with the microspheres into the microwell, and further collecting the information in the microwell through the image acquisition device and analyzing it. High-throughput, non-blocking, portable, low detection limit detection of samples.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (7)

1. A microfluidic chip detection system, comprising:

a microfluidic detection chip comprising:

a microfluidic cavity;

a microwell array disposed on a bottom surface within the microfluidic cavity;

a sample introduction conduit and a sample discharge conduit; the micro-fluidic detection chip is respectively connected and arranged at two sides of the micro-fluidic detection chip;

a microfluidic operation device for allowing the sample reacted with the microspheres to enter the microfluidic cavity through a sample introduction conduit and controlling a flow rate so that the sample reacted with the microspheres is deposited into the microwells;

the image acquisition device is arranged above the microwell array and is used for acquiring information of samples deposited in the microwells;

the data analysis device is used for analyzing the sample information collected by the image collection device and acquiring an analysis result;

the microfluidic detection chip is provided with a plurality of stages of microfluidic cavities, at least two microfluidic cavities are sequentially connected in series and communicated through a connecting pipeline to form the microfluidic detection chip, and the microwell arrays on each microfluidic cavity are consistent in size; the sizes of the micro wells of different micro fluid cavities are sequentially increased along the flow direction of the sample;

wherein, a controllable valve is also arranged on the connecting pipeline between the sequentially adjacent microfluid cavities.

2. The system of claim 1, wherein the microfluidic manipulation device is further configured to: a buffer is introduced into the microfluidic cavity through the sample introduction conduit, and a sample that is not deposited into the microwell is washed by controlling the flow rate of the buffer to flow out through the sample introduction conduit.

3. The system of claim 2, further comprising a sample reaction chamber:

the sample reaction chamber is an independently arranged chamber communicated with the microfluid operation device; or

The microfluidic cavity or the sample introduction conduit also serves as the sample reaction chamber.

4. The system of any one of claims 1-3, wherein the microfluidic manipulation device comprises: a pneumatic fluid pump or a syringe pump.

5. The system according to any one of claims 1 to 3,

the diameter range of the microspheres is as follows: 1 micron to 20 microns;

the material of the microsphere comprises at least one of the following materials: polystyrene, silica, ferroferric oxide;

the surface of the microsphere is modified with a specific molecular probe and impedance molecules which are arranged in the gap of the molecular probe and used for reducing the nonspecific adsorption of molecules;

wherein the specific molecular probe at least comprises one of the following: DNA, RNA, protein molecules, biotin molecules;

wherein the impedance molecules comprise at least one of: bovine serum albumin BSA, polyethylene glycol PEG.

6. The system according to any one of claims 1-3, wherein:

the diameter of the micro-well is 3-10 times of the diameter of a single microsphere;

the depth of the micro-well is 1-5 times of the diameter of a single microsphere;

wherein the height of the microfluidic cavity is 2-10 times the diameter of a single microsphere;

wherein, the projection of the micro-well in the horizontal direction is as follows: circular, elliptical, or polygonal.

7. A method for detecting a sample based on the microfluidic chip detection system of any one of claims 1 to 6, comprising:

A. placing a sample to be detected in a sample reaction chamber containing microspheres, and enabling target molecules to be detected in the sample to be detected to react with the microspheres to form a microsphere solution comprising a single microsphere, a dimer consisting of two microspheres and a polymer consisting of three or more microspheres; the single microspheres, dimers and multimers comprise a sample;

B. by controlling the sequential opening of each valve, the microsphere solution reacted by the microfluidic operation device enters the microfluidic detection chip through the sample introduction conduit, then sequentially enters each microfluidic cavity of the multistage microfluidic cavity, and the sequential opening of each valve is controlled to stop flowing when the microsphere solution enters the current stage microfluidic cavity so that the corresponding sample is deposited in the microwell of the microfluidic cavity, and when the next adjacent valve is opened, a buffer solution is introduced to resuspend and flush the sample which is not deposited in the microwell to the next adjacent microfluidic cavity so that the corresponding sample is deposited in the microwell of the next stage microfluidic cavity; the sizes of the micro wells corresponding to the micro fluid cavities of all levels are sequentially increased;

C. after the sample is deposited in the microwells of each stage of microfluidic cavity, enabling the buffer solution to enter the microfluidic cavity through the sample introduction guide pipe by the microfluidic operation device, and flushing the sample which is not deposited in each microwell by controlling the flow rate of the buffer solution so as to enable the sample to flow out through the sample discharge guide pipe;

D. collecting information of each sample deposited in each microwell through an image collecting device;

E. and analyzing the information of each sample acquired by the image acquisition device through a data analysis device to acquire the concentration of the molecules to be detected in the samples to be detected.

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