CN108344866B - Micro-fluidic chip detection system and method for detecting sample based on same - Google Patents
Micro-fluidic chip detection system and method for detecting sample based on same Download PDFInfo
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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
Technical Field
The invention relates to the fields of life science and medicine, in particular to a micro-fluidic chip detection system and a method for detecting a sample based on the system.
Background
Biomarkers are effective means of detecting and assessing the pharmacological response of conventional biological processes, pathogenesis, or therapeutic interventions, and therefore, convenient and accurate detection and quantification of biomarkers is a central goal of modern biotechnology. For clinical applications, sandwich immunoassays are the most widely used means of detection of target biomarkers. Generally, these methods require some type of label (enzymatic or fluorescent, etc.) to selectively and sensitively detect the target analyte. While also requiring special instrumentation (e.g., plate readers) and numerous washing steps. Recently, the concept of instant diagnosis (also called bedside detection) has become a reality due to the rapid development of rapid, accurate, portable, and marker-free quantitative detection methods for biomarkers. 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 formed label by immobilizing a specific receptor on the sensor surface. Another promising label-free detection method is based on bio-functionalized 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. Colorimetric methods or particle size analyzers have been used for the detection of biomarkers by analyzing the aggregation state of these particles. Nanoparticles can be made to form aggregates by specific protein interactions (e.g., antigen-antibody) between receptors immobilized on the particle surface and the target analyte in solution. Generally, the higher the biomarker concentration, the more aggregates are produced, and the larger.
However, the exact relationship between nanoparticle aggregation and biomarker concentration has not been fully revealed because of the lack of compatible methods or tools to accurately quantify the aggregation state (including information such as the number of nanoparticles within each aggregate). In addition to nanoparticles, antibodies have been modified on the surface of microparticles, and an impedance sensor is used to detect the aggregation state between the microparticles modified with specific antibodies, thereby detecting antigens. Although the impedance sensor can directly read the number of aggregates, this method has problems of low flux, easy clogging, and low resolution. In addition, developing an impedance analysis system requires specialized fluid transport devices and expensive data acquisition systems to achieve fast and low noise electrical signal processing. This has prevented the development of such electrical impedance systems into portable detection devices.
Therefore, there is a need for a detection device to overcome the problems of the prior art, such as low throughput, easy blockage, and low resolution, which results in difficult detection.
Disclosure of Invention
In view of this, the present application provides a microfluidic chip detection system and a method for performing sample detection based on the same, so as to overcome the problems of difficult detection due to low flux, easy blockage and low resolution in the detection in the prior art. High-flux, non-blocking and high-resolution detection is realized.
The application provides a micro-fluidic chip detecting system, includes:
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.
From the above, the flow rate is controlled by the microfluidic operation device, the sample after the microsphere reaction is dispersed and deposited in the micro-well of the microfluidic detection chip by combining the structure, and further information in the micro-well is collected by the portable image collection device (such as a mobile phone) for analysis, so that the defects of low flux, easy blockage and low resolution caused by non-dispersion and superposition of the sample in the prior art are overcome, and the high-flux, non-blockage, portable and high-resolution detection of the sample to be detected is realized.
Preferably, the microfluidic operating 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.
Therefore, the sample which is not deposited in the micro-well is washed, and the interference on the detection of the sample in the micro-well is favorably reduced. During the rinsing process, the dimer in the micro-well is not washed away due to the existence of the micro-well.
Preferably, the system further comprises:
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 is also used as the sample reaction chamber.
By last, above-mentioned sample reaction chamber can be test tube, centrifuging tube, pipe, microcavity body etc. that set up alone, also can directly regard as sample reaction chamber with sample introduction pipe for equipment is succinct more easily 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; sequentially adjacent microfluidic detection chips are sequentially communicated in series through connecting pipelines; the micro-well arrays in each micro-fluidic detection chip have the same size; the sizes of the micro wells of different microfluidic detection chips are sequentially increased along the flow direction of the sample.
Therefore, when a single microfluidic detection chip is arranged, the micro-well array is arranged in the microfluidic detection chip, inflowing samples are not easy to block, the flux is high, and the samples to be detected fall into each micro-well in a dispersed manner, so that the detection is clearer.
When more than two microfluidic detection chips (for example, 3) are arranged, the microspheres can be classified and captured stage by stage: the first stage micro fluid cavity is used for capturing single microspheres in a microsphere solution after reaction, the second stage micro fluid cavity is mainly used for capturing a dimer consisting of two microspheres, and the third stage micro fluid cavity is used for capturing a polymer consisting of three or more microspheres. The grading treatment can reduce the difficulty of detection and counting of the microspheres, and further reduce detection errors.
Preferably, the connecting pipeline between the sequentially adjacent microfluidic detection chips is also provided with a controllable valve.
Therefore, the capture of the microspheres with different polymerization degrees can be better realized in a grading manner by arranging the valve. 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 microfluidic cavity of the first stage and the microfluidic cavity of the second stage is closed; when a buffer solution is introduced to wash a sample which is not deposited in the microwell of the first-stage microfluidic cavity, opening a pipeline between the first-stage microfluidic cavity and the second-stage microfluidic cavity; simultaneously closing the conduit between the second and third stage microfluidic chambers; resuspending and washing the sample which is not deposited into the microwell of the first stage microfluidic cavity into the second stage microfluidic cavity, so that the dimer consisting of two microspheres in the sample is captured into the microwell; when a buffer solution is introduced to wash the sample which is not deposited in the microwell of the microfluid cavity of the second stage, a pipeline between the microfluid cavity of the second stage and the microfluid cavity of the third stage is opened; and re-suspending and flushing the sample which is not deposited into the microwell of the microfluidic cavity of the second stage into the microfluidic cavity of the third stage, so that the aggregate consisting of three or more microspheres in the microwell is captured. The grading treatment can reduce the difficulty of detection and counting of the microspheres, and further reduce detection errors.
Preferably, the microfluidic operating device comprises: a pneumatic fluid pump or a syringe pump.
Preferably, the microspheres have a diameter in the range of: 1 micron to 20 microns;
the material of the microspheres includes at least one of the following: 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 but is not limited to one of the following: DNA, RNA, protein molecules, biotin molecules;
wherein the impedance molecules include at least one of, but are not limited to: bovine serum albumin BSA, polyethylene glycol PEG.
From the above, the microspheres may range directly from 1 to 100 microns, preferably from 1 to 10 microns, and may be selected as desired.
Preferably, the diameter of the microwell is 3 to 10 times 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.
From above, the microwell diameter is preferably 3-5 times the diameter of the individual microspheres; the microwell depth is preferably 2-3 times the diameter of an individual microsphere. The height of the microfluidic cavity is preferably 2-10 times the diameter of a single microsphere.
Preferably, the materials for fabricating the microfluidic cavity and the microwell include at least one of the following materials: transparent organic polymers, glass, silica.
From the above, the material for making the microfluidic cavity and the microwell can be any transparent material. To facilitate subsequent image acquisition.
Preferably, the material for making the leading-in conduit and the leading-out conduit at least comprises but is not limited to one of the following materials: plastic tubes, Teflon tubes, glass tubes, metal tubes.
The application also provides a method for detecting a sample based on the microfluidic chip detection system, which comprises the following steps:
A. placing a sample to be detected in a sample reaction chamber containing microspheres, and reacting target molecules to be detected in the sample to be detected with the microspheres to form a dimer;
B. the reacted sample enters a micro-fluid cavity of a micro-fluid control detection chip through a sample introducing conduit by a micro-fluid operation device, and the dimer is deposited into the micro-well by controlling the flow rate;
C. passing a buffer solution through the sample introduction conduit into the microfluidic chamber via the microfluidic operating device, and washing the sample that is not deposited into the microwell by controlling the flow rate of the buffer solution to flow out through the sample introduction conduit;
D. collecting information of the dimer deposited in the microwell by an image collecting device;
E. and analyzing the information of the dimer collected by the image collection device through a data analysis device to obtain the concentration of the molecule to be detected in the sample to be detected.
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; sequentially adjacent microfluidic detection chips are sequentially communicated in series through connecting pipelines; the micro-well arrays in each micro-fluidic detection chip have the same size; the sizes of the micro wells of different microfluidic detection chips 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 microfluidic detection chips.
Therefore, the capture of the microspheres with different polymerization degrees can be better realized in a grading manner by arranging the valve. 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 microfluidic cavity of the first stage and the microfluidic cavity of the second stage is closed; when a buffer solution is introduced to wash a sample which is not deposited in the microwell of the first-stage microfluidic cavity, opening a pipeline between the first-stage microfluidic cavity and the second-stage microfluidic cavity; simultaneously closing the conduit between the second and third stage microfluidic chambers; resuspending and washing the sample which is not deposited into the microwell of the first stage microfluidic cavity into the second stage microfluidic cavity, so that the dimer consisting of two microspheres in the sample is captured into the microwell; when a buffer solution is introduced to wash the sample which is not deposited in the microwell of the microfluid cavity of the second stage, a pipeline between the microfluid cavity of the second stage and the microfluid cavity of the third stage is opened; and re-suspending and flushing the sample which is not deposited into the microwell of the microfluidic cavity of the second stage into the microfluidic cavity of the third stage, so that the aggregate consisting of three or more microspheres in the microwell is captured. The grading treatment can reduce the difficulty of detection and counting of the microspheres, and further reduce detection errors.
To sum up, this application through with the sample dispersion deposit after the microballon reaction to in the microwell of micro-fluidic detection chip to further gather the information in the microwell through portable image acquisition device (for example cell-phone) and carry out the analysis, overcome among the prior art because the sample does not disperse, the stack leads to the flux low, easily block up to and the low defect of resolution ratio, realized to the sample high flux that awaits measuring, unblock, portable, the detection of high resolution ratio. And this application can also realize catching the grading of the polymer of different types to reduce the degree of difficulty that detects with the count, and then reduced the detection error.
Drawings
Fig. 1 is a top view of a microfluidic detection chip provided in an embodiment of the present application;
fig. 2 is a schematic three-dimensional view of a microfluidic detection chip provided in an embodiment of the present application;
FIG. 3 is a schematic diagram illustrating a reaction between microspheres in a sample reaction chamber and target molecules to be detected in a sample according to an embodiment of the present disclosure;
fig. 4 is a schematic view of the overall structure and the working principle of the microfluidic chip detection system provided in the embodiment of the present application;
FIG. 5 is a schematic view of a sample reaction chamber as provided in an embodiment of the present application when the sample introduction conduit is a sample reaction chamber;
FIG. 6 is a schematic diagram of the sample reaction chamber when the microfluidic cavity is provided in an embodiment of the present application;
FIG. 7 is a schematic diagram of a microfluidic detection chip provided in an embodiment of the present application when there are more than two microfluidic detection chips (3 microfluidic detection chips in this embodiment, which are connected through a conduit);
FIG. 8 is a schematic diagram of microspheres of different sizes modified with different molecular probes and mixed with a sample to be tested to form different dimers;
FIG. 9 is a schematic diagram of the structure of integrating the microfluidic detection chip into a portable black box;
FIG. 10 is a schematic view of an image acquired by an image acquisition device;
FIG. 11 is a graph showing the results of detecting prostate cancer antibody (PSA) according to one embodiment of the present disclosure;
fig. 12 is a schematic flow chart of a method for sample detection based on a microfluidic chip detection system according to an embodiment of the present application.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
The application provides a micro-fluidic chip detection system and a method for detecting a sample based on the system, which are beneficial to realizing the detection of a sample to be detected, wherein the sample to be detected has high flux, no blockage, portability and low detection limit.
As shown in fig. 1-4, the present application provides a microfluidic chip detection system comprising:
a microfluidic detection chip comprising:
a microfluidic chamber 100;
a microwell array 101 disposed on a bottom surface within the microfluidic cavity;
a sample introduction conduit 102, a sample discharge conduit 103; which are respectively connected with and arranged at two sides of the microfluid cavity;
a microfluidic operation device 200 for allowing the sample reacted with the microspheres to enter the microfluidic cavity through a sample introduction conduit and depositing the sample reacted with the microspheres into the microwells by controlling a flow rate; wherein the microfluidic operating 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. The sample that is not deposited in the microwell is advantageously flushed, and the interference on the detection of the sample in the microwell is advantageously reduced. Wherein the microfluidic operating device comprises: pneumatic fluid pumps or syringe pumps or other principles such as electrically driven fluid pumps or the like.
And an image acquisition device 300 disposed above the microwell array for acquiring information of the sample deposited in the microwell.
And the data analysis device is used for analyzing the sample information acquired by the image acquisition device and acquiring an analysis result. And processing the pictures by using picture processing technologies, such as Matlab programs and deep learning, so as to extract the shape, size, quantity and proportion information of the microsphere polymer, and further obtain the concentration of the target molecules to be detected.
Wherein, the system still includes:
a sample reaction chamber 400 connected to the microfluidic manipulation device; in the sample reaction chamber, the microspheres react with target molecules to be detected in the sample to form dimers. The specific principle is shown in fig. 3. The surface of the microsphere 31 is modified with a molecular probe 33 which specifically adsorbs a target molecule 32 in a sample. In addition, the sample 34 to be measured also contains other molecules 35 and 36, etc. Using the specific binding of the molecular probes 33 to the target molecules 32, a microsphere aggregate 37 is formed.
Wherein the sample reaction chamber is a separately arranged chamber; alternatively, the sample is introduced into a conduit or microfluidic chamber as a sample reaction chamber. Wherein, above-mentioned sample reaction chamber can be test tube, centrifuging tube, pipe, little cavity etc. that set up alone, also can directly be with sample introduction pipe or microfluid cavity conduct sample reaction chamber for equipment is succinct more easily to carry.
Wherein the microfluidic cavity is a single cavity; or
More than two cavities which are communicated in parallel are connected through connecting pipelines; wherein, each cavity is provided with a micro-well array; wherein the microwells in each cavity are the same in size, and the microwells in different cavities are sequentially increased in size along the direction of the sample introduction conduit, the microfluidic cavity and the sample discharge conduit.
By last, single cavity sets up the microwell array in it, and the sample that sets up the inflow is difficult for blockking up, and flux is high, and the sample dispersion that awaits measuring falls into each microwell for detect more clearly.
The setting of cavity more than two can realize catching classifying step by step of microballon: the first stage micro fluid cavity is used for capturing single microspheres in a microsphere solution after reaction, the second stage micro fluid cavity is mainly used for capturing a dimer consisting of two microspheres, and the third stage micro fluid cavity is used for capturing a polymer consisting of three or more microspheres. The grading treatment can reduce the difficulty of detection and counting of the microspheres, and further reduce detection errors.
Wherein, the diameter range of the microspheres combined with the target molecules to be detected in the sample is as follows: 1 micron to 20 microns; wherein the microspheres may range directly from 1 to 100 microns, preferably from 1 to 10 microns, or from 1 micron to 20 microns; can be selected according to specific requirements.
The material of the microspheres includes at least one of the following: 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 but is not limited to one of the following: (deoxyribonucleic acid) DNA, (ribonucleic acid) RNA, protein molecules, biotin molecules;
wherein, the impedance molecule at least comprises but not limited to one of (bovine serum albumin) BSA, (oligo-ethylene glycol-poly L lysine) P LL-OEG.
Preferably, the diameter of the microwell is 3 to 10 times the diameter of a single microsphere; preferably 3-5 times the diameter of the individual microspheres.
The depth of the micro-well is 1-5 times of the diameter of a single microsphere; the depth is preferably 2-3 times the diameter of an individual 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: various topologies are possible, such as circular, elliptical, or polygonal (diamond, trapezoid, triangle, square, rectangle, parallelogram, etc.).
Wherein, the materials for manufacturing the micro-fluid cavity and the micro-well at least comprise but are not limited to one of the following materials: transparent organic polymers, glass, silica, plastics, and the like. From the above, the material for making the microfluidic cavity and the microwell can be any transparent material. To facilitate subsequent image acquisition. The microfluidic cavity and the microwell are combined together by bonding or mechanical clamping. The main methods for processing the micro-fluid cavity and the micro-well with the micron scale include wet etching, dry etching, laser etching, mechanical milling and the like.
Wherein, the leading-in conduit and the leading-out conduit are made of materials at least comprising but not limited to one of the following materials: plastic tubes, Teflon tubes, glass tubes, metal tubes.
For a more clear description of the technical solution of the present application, the working principle of the microfluidic chip detection system of the present application under various schemes is described as follows:
firstly, 1, as shown in fig. 1-4, placing a sample to be detected in a sample reaction chamber 400 provided with microspheres, so that molecules to be detected in the sample to be detected react with molecular probes 33 on the surfaces of the microspheres 31 to form dimers 37;
2. the reacted sample is introduced into the microfluidic cavity through the sample introduction conduit 102 by the microfluidic handling device 200 (pneumatic fluid pump or syringe pump or other principle such as electrically driven fluid pump) and the dimer is deposited into the microwell 101 by controlling the flow rate;
3. a buffer is introduced into the microfluidic chamber (single) through the sample introduction conduit by the microfluidic operation device, and a sample not deposited in the microwell is washed by controlling the flow rate of the buffer to flow out through the sample introduction conduit 103. Due to the presence of the microwells, dimers therein are not washed away.
4. Collecting information of the dimers 37 deposited in the microwells 101 by an image collecting device 300; the microfluidic detection chip is fixed on a portable microscope (image acquisition device 300), and a mobile phone is arranged on the microscope, and a camera of the mobile phone acquires images of the microfluidic detection chip through an eyepiece of the microscope. The acquired image is shown in fig. 10.
5. And analyzing the dimer information acquired by the image acquisition device through a data analysis device, and processing the picture by using picture processing technologies, such as Matlab program and deep learning to extract the shape, size, quantity and proportion information of the microsphere polymer, thereby further acquiring the concentration of the target molecule to be detected.
Results of the experiment
FIG. 11 shows the results of the detection of PSA protein molecules in serum, according to one embodiment of the present invention. In serum experiments, PSA serum solutions at concentrations of 0, 3.67pM,36.7pM, and 367pM were combined with 2 x 10 concentrations6The method comprises the steps of (1)/m L, reacting a polystyrene microsphere solution with the diameter of 5.1 microns, (modifying an anti-PSA molecular probe for specifically capturing PSA) on the surface of the polystyrene microsphere, (reacting the microsphere solution and a serum sample in a 1m L centrifugal tube for 30 minutes after the microsphere solution and the serum sample are fully mixed, introducing the centrifugal tube into a microwell microfluidic chip for detection, and taking the quantity and the proportion of microsphere dimers as detection indexes, wherein the detection results are shown in figure 11, and the experimental results shown in figure 11 not only verify the feasibility of a microsphere polymerization method, but also verify the practicability of the embodiment.
Second, as shown in fig. 5, when the sample reaction chamber is a sample introduction conduit, the difference from the principle in the first embodiment is that 1 is replaced with: the microspheres are mixed with a sample solution containing the molecules to be detected and other molecules, and introduced into the sample introduction tube 102. The rest is consistent with 2-5 in the first scheme.
Thirdly, as shown in fig. 6, when the sample reaction chamber is a microfluidic cavity, the difference from the first principle is that the following steps 1 and 2 are replaced by: the microspheres are fully mixed with a solution of a sample to be tested containing a molecule to be tested and other molecules, and then introduced into the microfluidic cavity through the sample introduction conduit 102 to react therein. The rest is consistent with 2-5 in the first scheme.
Fourthly, as shown in fig. 7, different from the first to third methods, the microfluidic detection chip shown in fig. 7 is composed of two or more microfluidic cavities. After the reaction between the microspheres and the sample solution to be measured, the reaction solution is introduced into the microfluidic cavity through the sample introduction conduit 102. The sample to be measured sequentially passes through the primary microfluid cavity 71, the secondary microfluid cavity 72 and the tertiary microfluid cavity 73. The multi-stage microfluidic chambers communicate through flow channels 74. The microwells in the microwell arrays 711, 721, 731 in the multi-stage microfluidic cavity increase in size in sequence. For example, microwell array 711 is one to two times the size of the microspheres used to capture a single microsphere; microwell array 721 is two to four times the size of the microspheres used to capture the microsphere aggregates formed by the two microspheres; microwell array 731 is four to ten times the size of the microspheres used to capture the microsphere aggregates formed by the multiple microspheres. The inlet and the outlet are respectively communicated with the conduit for leading in and leading out the sample solution. By utilizing the hierarchical micro-well chip in the embodiment, the microspheres can be classified and captured stage by stage: the first stage micro fluid cavity is used for capturing single microspheres in a microsphere solution after reaction, the second stage micro fluid cavity is mainly used for capturing a dimer consisting of two microspheres, and the third stage micro fluid cavity is used for capturing a polymer consisting of three or more microspheres. Wherein, the valve 741 is arranged on the flow channel 74, so that microspheres with different polymerization degrees can be captured in a better grading manner. 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 microfluidic cavity of the first stage and the microfluidic cavity of the second stage is closed; when a buffer solution is introduced to wash a sample which is not deposited in the microwell of the first-stage microfluidic cavity, opening a pipeline between the first-stage microfluidic cavity and the second-stage microfluidic cavity; simultaneously closing the conduit between the second and third stage microfluidic chambers; resuspending and washing the sample which is not deposited into the microwell of the first stage microfluidic cavity into the second stage microfluidic cavity, so that the dimer consisting of two microspheres in the sample is captured into the microwell; when a buffer solution is introduced to wash the sample which is not deposited in the microwell of the microfluid cavity of the second stage, a pipeline between the microfluid cavity of the second stage and the microfluid cavity of the third stage is opened; and re-suspending and flushing the sample which is not deposited into the microwell of the microfluidic cavity of the second stage into the microfluidic cavity of the third stage, so that the aggregate consisting of three or more microspheres in the microwell is captured. The grading treatment can reduce the difficulty of detection and counting of the microspheres, and further reduce detection errors.
And fifthly, as shown in fig. 8, different from the first to fourth, microspheres 84, 85, 86 with different sizes modified with different molecular probes are simultaneously mixed with a sample 87 to be detected to form dimers 88, 89, 810 respectively, wherein target objects 81, 82, 83 are simultaneously detected. And (3) after the microspheres react with the detected sample solution, introducing the reaction solution into the microfluidic detection chip. And subsequent operations are consistent. It should be noted that the method for detecting different target detection objects by using microspheres with various sizes provided in this embodiment can be combined with the scheme provided in fig. 8. The specific mode is that the function design of the multi-stage microfluid cavity is as follows: the primary microfluid cavity is used for capturing microspheres with the minimum size, and then the size of the captured microspheres is gradually increased in the downstream microfluid cavity, so that the classified capture of the microspheres with different sizes is realized, and the spatial differential detection of detection results of different target detection objects is realized.
Sixthly, as shown in fig. 9, unlike the first to the fourth, the present embodiment integrates the microfluidic detection chip into a portable black box 94, which includes: the loading platform 96 for fixing the microfluidic detection chip 97 can focus on the observation objective 98 in the microfluidic detection chip 97. In practical operation, the microfluidic control device 91 is connected to the microfluidic detection chip 97 in the portable black box 94 through the introduction conduit 92 and the extraction conduit 95, so that the introduction and capture of the microsphere sample can be performed. Finally, the mobile phone 93 (or other image capturing devices) is placed on the observation objective 98 of the black box 94 for image extraction.
To sum up, the micro-fluidic chip detection system of the application disperses and deposits the sample after the microsphere reaction into the micro-well of the micro-fluidic detection chip, and further collects the information in the micro-well through a portable image collection device (such as a mobile phone) for analysis, overcomes the defects of low flux, easy blockage and low resolution caused by non-dispersion and superposition of the sample in the prior art, and realizes the detection of high flux, no blockage, portability and high resolution of the sample to be detected. And the height, the width of the microfluid cavity, the diameter and the height of the microwell set a proper range, so that the blockage problem is effectively eliminated.
And this application can also realize catching the grading of the polymer of different types to reduce the degree of difficulty that detects with the count, and then reduced the detection error.
Compared with the traditional detection system based on the optical and Culecounter principle, the device realized by the invention does not need complex optical and electrical detection equipment. The micro-fluidic chip used in the invention has small volume and very low cost, and the cost of each chip can be controlled within dozens of RMB and can be repeatedly used. The subsequent detection system is an image detection method based on mobile phone photographing and mobile phone APP realization, and one mobile phone can solve the task to be realized by a complex detection module in the traditional technology. The microscopic magnification device is realized by a portable microscope or a small objective lens. The microfluid introducing system can be realized by a syringe pump. Compared with the prior detection technology, the technical scheme provided by the invention greatly reduces the threshold of the detection device, and each module can be conveniently assembled by adopting a portable device and can be operated in hospitals, homes and the field. In microsphere detection, microspheres with surface modified with specific molecular probes are combined with target molecules, so that two adjacent microspheres can form a sandwich structure, and further a dimer or even a polymer is formed. The number or concentration of these microsphere polymers is related to the concentration of the target molecule. According to the method, two microspheres can be connected through a single target molecule to form a microsphere dimer, so that the microsphere polymerization method can theoretically detect extremely low molecular concentration. The micro-fluidic chip detection system can reduce the influence of the detection means on the microsphere polymer at the minimum. As shown in fig. 11, by detecting PSA, the lowest detection limit is 3.67pM, i.e., the present application can detect samples with extremely low concentration, and can sufficiently meet the detection requirement of clinical interest in the 30pM to 300 pM PSA diagnostic concentration range.
Example two
As shown in fig. 12, the present application also provides a detection method based on the above-mentioned microfluidic chip detection system, which includes:
s121, placing a sample to be detected in the sample reaction chamber provided with the microspheres, and enabling molecules to be detected in the sample to be detected to react with the microspheres to form a dimer;
s122, passing the reacted sample through the sample introduction conduit into the microfluidic cavity via the microfluidic operation device, and controlling the flow rate to deposit the dimer into the microwell;
s123, enabling a buffer solution to enter the microfluidic cavity through the sample introduction conduit by the microfluidic operation device, and flushing the sample which is not deposited in the microwell by controlling the flow rate of the buffer solution so as to enable the sample to flow out through the sample introduction conduit;
s124, collecting the information of the dimer deposited in the microwell through an image collecting device;
and S125, analyzing the dimer information acquired by the image acquisition device through a data analysis device to acquire the concentration of the molecules to be detected in the sample to be detected.
To sum up, the micro-fluidic chip detection system of the application deposits the sample after reacting with the microsphere in the micro-well, and further collects the information in the micro-well through the image acquisition device, and analyzes the information, so that the detection of the sample to be detected, which has high flux, no blockage, portability and low detection limit, is realized.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
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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| CN109406813A (en) * | 2018-12-14 | 2019-03-01 | 贵州大学 | A kind of microfluidic control device for high-throughput droplet array microfluid point sample |
| CN110095316A (en) * | 2019-04-04 | 2019-08-06 | 天津大学 | A kind of particle acquisition device, detection system and detection method |
| CN110068528A (en) * | 2019-04-23 | 2019-07-30 | 中国石油大学(华东) | Particle detection technique in detection device and suspension |
| CN110395688A (en) * | 2019-06-24 | 2019-11-01 | 金华职业技术学院 | A kind of micro-nano particle precipitation equipment |
| CN112387316B (en) * | 2019-08-19 | 2021-09-21 | 中国科学院亚热带农业生态研究所 | 3D micro-fluidic chip model, 3D micro-fluidic chip, and preparation method and application thereof |
| CN112268886A (en) * | 2020-09-04 | 2021-01-26 | 武汉光谷航天三江激光产业技术研究院有限公司 | Laser rapid detection and disinfection integrated device and method for virus and bacteria |
| CN113834808A (en) * | 2021-08-10 | 2021-12-24 | 南京师范大学 | Three-dimensional photonic crystal microsphere for detecting Escherichia coli O157H 7, detection platform and non-labeling detection method thereof |
| CN116338159B (en) * | 2022-12-13 | 2024-02-09 | 西交利物浦大学 | Full-automatic paper-based micro-fluidic system based on smart phone local detection |
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