CN107442186B - Microfluidic chip, analysis device based on microfluidic chip and analysis method - Google Patents
Microfluidic chip, analysis device based on microfluidic chip and analysis method Download PDFInfo
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- CN107442186B CN107442186B CN201610380545.9A CN201610380545A CN107442186B CN 107442186 B CN107442186 B CN 107442186B CN 201610380545 A CN201610380545 A CN 201610380545A CN 107442186 B CN107442186 B CN 107442186B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
Abstract
The invention relates to a micro-fluidic chip which is provided with a micro-fluidic channel, a filter element and a detection area, wherein the filter element is arranged in the micro-fluidic channel and is used for filtering a liquid sample so as to enable liquid containing target particles to flow into the detection area. The invention also relates to an analysis device based on the microfluidic chip, which comprises the microfluidic chip and a detection and analysis unit, wherein the detection and analysis unit is used for detecting and analyzing the detection area to obtain the biological information of the target particles. In addition, the invention relates to a method for analyzing target particles in said liquid sample.
Description
Technical Field
The invention relates to a micro-fluidic chip and application thereof, belonging to the field of biological detection.
Background
Biological samples, such as exosomes, may carry a variety of proteins, mrnas, mirnas, and participate in cellular communication, cell migration, angiogenesis, and tumor cell growth processes. The existing detection method of exosomes mainly comprises enzyme-linked immunosorbent assay (ELISA) and a flow cytometer, and the separation and purification modes of exosomes comprise an ultracentrifugation method, density gradient centrifugation, a magnetic bead immunization method, a PEG-base precipitation method and the flow cytometer.
Enzyme-linked immunosorbent assay has low sensitivity, long time consumption and complex operation, and has certain limitation on the detection of exosomes.
The ultracentrifugation method is the most commonly used method for purifying the exosome at present, the number of the exosomes obtained by simple operation is large, but the process is time-consuming, the recovery rate is unstable, the purity is questioned, and in addition, the repeated centrifugation operation also causes damage to the exosome, thereby reducing the quality of the exosome.
The exosomes separated by the density gradient centrifugation method have high purity, but the preparation work in the previous period is complicated, time-consuming and small in amount, and in addition, the method is very sensitive to the centrifugation time.
The magnetic bead immunization method ensures the completeness of the form of the exosome, has high specificity and simple operation, does not need expensive instruments and equipment, but the non-neutral pH and the non-physiological salt concentration can influence the bioactivity of the exosome.
The principle of PEG-base precipitation is related to competitive binding of free water molecules. There are several problems with precipitation of exosomes with PEG: such as low purity and recovery, more hetero-proteins, non-uniform particle size, resulting in difficult to remove polymers, and physical effects such as mechanical force or chemical additives such as Tween-20 applied to PEG-base will destroy exosomes, etc., and thus are easily questioned.
In the above method, only the flow cytometer is capable of simultaneously detecting and purifying exosomes. However, flow cytometers are commonly used for cell analysis and cell screening. For exosomes of much smaller size than the cell, both the efficiency and sensitivity of the flow cytometer are greatly reduced.
Disclosure of Invention
In view of the above, there is a need for a microfluidic chip, which is provided with a microfluidic channel, and a filter element and a detection area, wherein the filter element is disposed in the microfluidic channel and is used for filtering a liquid sample to make the liquid containing target particles flow into the detection area.
In a preferred embodiment, the filter element is a porous membrane with a pore size of 200-250 nm.
In a preferred embodiment, the microfluidic device further comprises at least one liquid channel connected to the microfluidic channel, wherein the at least one liquid channel is used for introducing a liquid containing at least a label to label the target particles, so that the liquid containing the label target particles flows into the detection region.
In a preferred embodiment, the label includes a fluorescent agent selected from one or more combinations of organic fluorescent molecules, fluorescent liposomes, quantum dots and nanogold, and the label is connected with the target particles to realize the labeling of the target particles.
In a preferred embodiment, the fluid sample is a body fluid of a cancer patient, the target particles are exosomes of cancer cells, and quantum dots containing tumor-specific antibodies are attached to the surfaces of the target particles.
In a preferred embodiment, the at least one liquid channel is further configured to introduce a liquid containing magnetic nanobeads, the liquid containing magnetic nanobeads is configured to identify and capture the target particles, so that the target particles are detected in the detection area, the microfluidic chip further includes a separation unit, and the separation unit includes an electromagnetic control system, and the electromagnetic control system is configured to attract the target particles to be concentrated at a predetermined position of the at least one liquid channel, so as to enrich and separate the target particles.
An analysis device, comprising: the above microfluidic chip; and the detection and analysis unit is used for detecting and analyzing whether the target particles exist in the detection area of the microfluidic chip.
In a preferred embodiment, the detection and analysis unit guides light to the detection area to detect whether the target particles exist, and the detection and analysis unit is further provided with an imaging device for taking a photograph of the detected target particles in real time.
In a preferred embodiment, the analysis device further comprises a separation unit, wherein the separation unit comprises a solenoid control system, and the solenoid control system is used for attracting the detected target particles to gather at a preset position so as to enrich and separate the target particles.
A method of analyzing target particles in a liquid sample, comprising the steps of:
providing a liquid sample and a microfluidic chip, wherein the microfluidic chip is provided with a microfluidic channel, a filter element and a detection area;
filtering the liquid sample using the filter element such that liquid containing target particles flows into the microfluidic channel and into a detection region; and
and detecting and analyzing whether the target particles exist in the detection area by using a detection and analysis unit.
In a preferred embodiment, the method further comprises an enrichment separation step, wherein a liquid containing nano magnetic beads is introduced into the microfluidic chip, and the target particles with the nano magnetic beads connected to the surface are concentrated at a preset position through a magnetic field, so that the target particles are enriched and separated.
Compared with the prior art, the micro-fluidic chip provided by the invention is provided with the micro-fluidic channel, the filter element and the detection area, the liquid sample is filtered by the filter element, the filtered liquid contains target particles and enters the detection area, the target particles in the detection area can be directly detected, and the micro-fluidic chip realizes micro-flow control and can be directly detected.
In addition, the invention provides an analysis device based on the microfluidic chip, and the biological information of the target particles in the microfluidic chip is detected and analyzed by using a detection and analysis unit of the analysis device. The analysis device improves the detection precision and the separation efficiency of the target particles, and realizes the function of integrating the detection and the separation of the target particles.
In the present invention, the target microparticle is an exosome of a cancer cell, and a specific exosome of a cancer cell can be selected by the analyzer. In addition, the analysis device integrates the microfluidic chip and the detection and analysis unit, so that high integration and automation of the analysis device are realized, and the analysis device is small in size and easy and convenient to operate.
Drawings
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below, wherein:
fig. 1 is a schematic structural diagram of a microfluidic chip-based analysis device provided in an embodiment of the present invention;
fig. 2 is a schematic diagram of a sheath flow and a laminar flow of a buffer solution and a liquid containing target particles in a microfluidic chip according to an embodiment of the present invention;
fig. 3 is a flowchart of a method for analyzing target particles in a liquid sample according to an embodiment of the present invention.
Description of the main elements
Detailed Description
For a more clear understanding of the technical features, objects and effects of the present invention, embodiments of the present invention will now be described in detail with reference to the accompanying drawings.
A tumor as used in the present invention is defined as a large number of abnormally growing cells. Tumors useful for the present invention include, but are not limited to, prostate cancer, breast cancer, lung cancer, colon cancer, stomach cancer, endometrial cancer, liver cancer, esophageal cancer, bladder cancer, oral cancer, thyroid cancer, pancreatic cancer, retinal and skin cancer, preferably pancreatic cancer.
"tumor sample" refers to a sample taken before or after removal of a tumor from a patient.
In the present invention "liquid sample" is referred to as "tumor sample" and "liquid sample" may be selected from any tissue, cell extract, saliva, urine, serum, whole blood, plasma concentrate of a patient and any educts isolated from an adhering liquid, such as a sample isolated from a cancer-suffering subject or from a healthy volunteer. The "liquid sample" may also be a cell or cell line created under experimental conditions, rather than being isolated directly from the subject. The subject can be a human, rat, mouse, non-human primate, cat, and the like.
Exosomes were first found in the supernatant of sheep erythrocytes cultured in vitro to be vesicular-like bodies of uniform size, diameter 40-100nm, density 1.10-1.18g/ml actively secreted by the cells. Exosomes can carry various proteins, mRNA and miRNA and participate in processes such as cell communication, cell migration, angiogenesis, tumor cell growth and the like.
Exosomes are secreted by cells under normal and case conditions and contain various membrane proteins and cytoplasmic proteins, and ten major proteins, heat shock protein 8(HSPA8), CD63 antigen (CD63), actin, β (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase 1, α (ENO1), heat shock protein 90AA1(HSP90AA1), CD9 antigen (CD9), CD81 antigen (CD81), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein, zeta polypeptide (yhwa), pyruvate kinase and pyruvate kinase (PKM2) are found in exosomesThe surface membrane protein is selected from CD63 antigen (CD63), CD9 antigen (CD9), CD81 antigen (CD81) and the like. The tetralin is a common exocrine marker, and comprises membrane proteins of CD9, CD63 and CD81, and is used for diagnosis of various tumors and infectious diseases. In particular, CD63+Exosomes are significantly increased in melanoma and other cancer patients, and CD63 fluid is known as a protein marker for cancer. CD81 is a diagnostic marker for hepatitis c virus infection.
Exosomes may be extracted from urine, blood, and serum samples (patient's body fluids) of cancer patients. In addition to serum and urine, other biological fluids may also serve as sources of exosomes, particularly exosomes isolated from saliva that demonstrate diagnostic biomarkers for pancreatic cancer. Each cancer cell has specific exosomes, and in this embodiment, the specific exosomes of the cancer cell generally have membrane proteins common to the cell (e.g., CD81, CD9, CD63, etc.) and all the specific membrane proteins of the parent cancer cell carried by the cell.
In the invention, the surface membrane protein of the exosome, such as CD81, can be specifically modified by 10nm nano magnetic beads, and the nano magnetic beads and the exosome are premixed and combined. The green quantum dot of anti-CD9 is adopted to carry out antibody antigen reaction with surface membrane protein CD9, so that the surface membrane protein of CD9 can be specifically modified. The surface membrane protein of CD63 of an exosome can be specifically modified by adopting red quantum dots of anti-CD 63. The specific receptor protein of the pancreatic cancer is Glypican-1, and the blue quantum dots can specifically modify the Glypican-1 to form an antibody of specific expression of tumors. The Anti-CD63 and Anti-CD9 modified quantum dots with two colors can be detected on each exosome through antibody-antigen reaction, and when an exosome specific to a certain cancer cell appears, the quantum dots with three different colors can be modified on the surface of the exosome of the cancer cell. Only the exosomes of the cancer cells can be connected with quantum dots with three different colors of red, green and blue and 10nm nanometer magnetic beads at the same time, the nonspecific exosomes are connected with anti-CD63 red quantum dots and anti-CD9 green quantum dots at most, and other nonspecific exosomes are connected with single color quantum dots. Based on the above principle, cancer cells can be screened for exosomes from a fluid containing exosomes. In the present embodiment, the exosomes of the cancer cells are target microparticles.
In the present invention, the microfluidic chip product refers to an analysis device for detecting and analyzing a biological or chemical sample, which forms micro-scale micro-channels on a chip by a micro-etching or the like to supply or mix a plurality of sample solutions or reagents, thereby enabling a liquid to react in the channels and perform signal detection in a detection region.
Laminar flow is a fluid flow condition characterized by high momentum diffusion, low momentum convection, and pressure and velocity independent of time. Reynolds number is a dimensionless number that can be used to characterize fluid flow conditions. Where v, ρ, and μ are the flow velocity, density, and viscosity coefficient of the fluid, respectively, and d is the characteristic length. E.g., fluid flowing through a circular pipe, then d is the equivalent diameter of the pipe. The flow of the fluid can be distinguished by the reynolds number as being laminar or turbulent. When the cross-sectional dimension (characteristic length) of the flow channel is small (micron scale), the reynolds number is usually < 1, and laminar flow is formed in the flow channel.
In particular embodiments, the laminar flow is generally achieved using microfluidic techniques. Microfluidic technology refers to devices and systems that manipulate fluid flow and have a characteristic dimension of a microfluidic channel that reaches within 1mm of a test distance. Microfluidic systems have the advantage over conventional systems that they can be operated more quickly and with much less consumption of liquid resources.
Referring to fig. 1, the analysis device 1 based on the microfluidic chip includes a sample introduction unit 10, a microfluidic chip 20, a detection analysis unit 30, a control unit 40, and a separation unit 50. The control unit 40 is respectively connected with the detection and analysis unit 30 and the separation unit 50, and the sample introduction unit 10 is connected with the microfluidic chip 20. The microfluidic chip 20 based analysis device 1 is used for detecting and separating target particles in the liquid sample.
In the present invention, the solution to be introduced by the microfluidic chip 20 includes a buffer, a label, a nano magnetic bead, and a liquid sample. The buffer is a physiological buffer, and comprises Phosphate Buffered Saline (PBS) and 4-hydroxyethylpiperazine ethanesulfonic acid (HEPBS,4- (2-hydroxyethyi) -1-piperazineethanesulfonic acid). The marker is a fluorescent reagent, the fluorescent reagent is selected from one or a combination of several of organic fluorescent molecules, fluorescent liposomes, quantum dots and nanogold, and the marker is connected with the target particles through covalent bonds or non-covalent bonds and realizes the marking of the target particles. The fluorescent reagent comprises red quantum dots connected with anti-CD63, green quantum dots connected with anti-CD9 and blue quantum dots modified by cancer specific antibodies (pancreatic cancer specific Glypican-1 antibodies). The nanometer magnetic beads comprise magnetic bead nanoparticles of 10nm connected with CD81 surface membrane proteins. In this embodiment, magnetic particles, such as magnetic beads with a size of 10nm, are coated on the surface of the exosome, or magnetic nanoparticles are introduced into the cell, so that the magnetic nanoparticles are provided with a magnetic label, and then the exosome with the magnetic label is provided. When the magnetic field flows through the magnetic field area, the target particles are deviated from the original motion orbit by the magnetic force. Because of the difference of different cell sizes, magnetic susceptibility and flow rates, the degree of the cells deviating from the original laminar flow direction is different, so that the target exosome is separated out, and the target exosome is the exosome of the cancer cells. To sum up, the sorting of the magnetic exosomes on the microfluidic chip can realize the sorting of two or more exosomes, and since the exosomes of the cancer cells can be simultaneously connected with three quantum dots modified in different colors and nanoparticles with magnetic beads, when the exosomes of the cancer cells flow through a magnetic field area, the movement direction is changed, so that the exosomes of the cancer cells are separated.
Referring to fig. 1 again, the sample injection unit 10 includes a first sample injection unit 12, a second sample injection unit 14 and a third sample injection unit 16, the first sample injection unit 12 includes a first liquid feeding pump 121 and a first liquid storage tank 122, the second sample injection unit 14 includes a second liquid feeding pump 141 and a second liquid storage tank 142, and the third sample injection unit 16 includes a third liquid feeding pump 161 and a third liquid storage tank 162. The first liquid sending pump 121 is connected to the first liquid storage tank 122, and the first liquid sending pump 121 includes a microfluidic pump, and drives the liquid to flow in the microfluidic chip 20 by a power driving device. The first reservoir 122 includes a device for enclosing liquid by a syringe, and the first liquid sending pump 121 drives the liquid in the first reservoir 122 to flow in the microfluidic chip 20 in a fixed/semi-fixed amount by a certain driving force. In this embodiment, the first liquid sending pump 121 is configured to output a buffer liquid to the microfluidic chip 20. Accurate control of the amount of liquid sample is critical to the separation and detection of the target particles. The flow rate of the liquid is controlled by the first liquid-sending pump 121, and in the present invention, the flow rate of the liquid is set to be less than 1 m/s. The more stable the flow rate of the fluid, the better, if the flow rate is too large, so that the detection and separation process of the target particles becomes difficult.
The second liquid sending pump 141 is connected to the second liquid storage tank 142, the second liquid sending pump 141 includes a microfluidic pump, and drives the liquid to flow in the microfluidic channel by a power driving device, the second liquid storage tank 142 includes a device for packaging the liquid by a syringe, and the second liquid sending pump 141 drives the liquid in the second liquid storage tank 142 to flow in the microfluidic chip 20 in a fixed/semi-fixed amount by a certain driving force. In the present embodiment, the second liquid sending pump 141 is used for outputting the liquid sample to the microfluidic chip 20.
The third liquid sending pump 161 is connected to the third liquid storage tank 162, the third liquid sending pump 225 includes a micro-fluidic pump, and drives the liquid to flow in the micro-fluidic channel by a power driving device, the third liquid storage tank 162 includes a device for packaging the liquid by a syringe, and the third liquid sending pump drives the liquid in the third liquid storage tank to flow in the micro-fluidic chip 20 in a fixed/semi-fixed amount by a certain driving force. In the present embodiment, the third liquid sending pump 161 is configured to output a liquid containing a label containing a fluorescent reagent to the microfluidic chip 20.
Referring to fig. 1 again, the microfluidic chip 20 includes a first sample inlet 22, a second sample inlet 24, a third sample inlet 26, a microchannel 23, a filter element 25, a first sample outlet 28, and a second sample outlet 29. The microchannel 23 includes a first liquid channel 231, a second liquid channel 232, a mixed flow channel 233, and a microfluidic channel 234. The filter element 25 is disposed at an end of the microfluidic channel 234 near an inlet of the microfluidic channel 234, and in the present embodiment, the liquid sample filtered by the filter element 25 contains target particles. The first sample inlet 22 is connected to the first reservoir 122 and the first liquid pump 121, and in this embodiment, the first sample inlet 22 is used for flowing in the buffer solution output by the first liquid pump 121. The second sample inlet 24 is connected to the second reservoir 142 and the second liquid-sending pump 141, and in this embodiment, the second sample inlet 202 is used for flowing in the liquid sample output by the second liquid-sending pump 141. The third sample inlet 25 is connected to the third reservoir 162 and the third liquid sending pump 161, and in this embodiment, the third sample inlet 26 is used for flowing in the liquid containing the marker output by the third liquid sending pump 161. The third reservoir 162 is connected to the third inlet 26 of the microfluidic chip 20 via a tube. The liquid sample containing the target particles and the liquid containing the marker are mixed in the second liquid passage 234 and the marking of the target particles is achieved. The liquid containing the label, the liquid containing the target particles, and the buffer are mixed in the mixed flow channel 233.
The marker comprises a fluorescent reagent, and the fluorescent reagent is selected from one or more of organic fluorescent molecules, fluorescent liposomes, quantum dots and nanogold. In this embodiment, the quantum dots are selected from red quantum dots of anti-CD63, green quantum dots of anti-CD9, and blue quantum dots of tumor-specific antibodies. The tumor specific antibody is Glypican-1 which is used for specifically marking pancreatic cancer. And the second liquid channel also flows into the nano magnetic beads for combining the target particles and realizing the enrichment and separation of the target particles. The 10nm nanometer magnetic bead is used for specifically modifying the exosome. The label is connected with the target particles through ligand molecules in a covalent bond and non-covalent bond mode, and the covalent bond and the non-covalent bond mode comprise a mode of chemical crosslinking, physical crosslinking, molecular specificity action, antigen-antibody reaction and the like. The ligand molecules include one or more combinations including monoclonal antibodies, polyclonal antibodies, haptens and antigens.
The filter element 25 is disposed in the microfluidic chip 20, the filter element 25 includes one or a combination of several of a molecular sieve and a porous membrane, in this embodiment, the filter element 25 is a porous membrane, and the pore size of the porous membrane is 200nm, and is configured to filter out the liquid sample with a pore size smaller than 200nm, including most of exosomes, free genes, and free proteins. The liquid sample after being filtered by the filter element 25 contains target particles, and the liquid containing the target particles flows into the second liquid passage 234 to be mixed with the liquid containing the marker and the marker of the target particles is realized in the second liquid passage 234.
Referring to fig. 2, since the liquid containing the target particles and the liquid containing the marker form a laminar flow after being mixed in the mixed flow channel 233 (as indicated by arrow b in fig. 2), the buffer cannot be mixed with the liquid containing the target particles and the liquid containing the marker after entering the mixed flow channel 233, and a sheath flow liquid can be formed after the buffer enters the mixed flow channel 233 (as indicated by arrow a in fig. 2), and c in fig. 2 refers to an exosome of the target particles, i.e., cancer cells. In this embodiment, the buffer is not mixed with the liquid containing the target particles, and the buffer flows into the mixed flow channel 233 to generate a sheath flow (sheath flow) action, which means that the fluid forms a compressive flow in a shape similar to a sheath under the action of a lateral squeezing effect. And the laminar flow (indicated by arrow b in fig. 2) includes the target particles, if the sheath flow liquid (indicated by arrow a in fig. 2) flows in from both sides of the microchannel 233, the sheath flow liquid isolates the mixed liquid of the target particles labeled with the labeling substance in the same plane (as shown in fig. 2), thereby facilitating the detection and enrichment separation of the target particles. After buffering the sheath flow solution, the specific cancer cell exosomes labeled with the labeling substance may be discharged from the first outlet 28, while most of the non-specific exosomes and the buffer solution are discharged from the second outlet 29.
The detection and analysis unit 30, the control unit 40 and the separation unit 50 are used for detecting and separating the target particles of the liquid sample together with the sample introduction unit 10 and the microfluidic chip 20. The control unit 40 is connected to the detection and analysis unit and the separation unit 50, respectively.
The detection and analysis unit 30 includes a fast ultrahigh resolution fluorescence real-time imaging system and imaging software (not shown), and the lens with ultrahigh resolution may adopt CMOS, CCD, or photodiode and photomultiplier tube according to actual needs. The detection and analysis unit 30 employs a single wavelength excitation light, and it is understood that the single wavelength excitation light is a specific light source. The detection and analysis unit 30 directs the excitation light of the specific light source to the fluid flowing through the mixed flow channel 233 of the microfluidic chip 20. In the present embodiment, the position of the mixed flow channel 233 corresponding to the detection and analysis unit 30 is a detection region 2331, and the detection region 2331 is a part of the mixed flow channel 233. The material of the detection region 2331 is transparent, and the excitation light is transmitted through the surface of the material to irradiate the fluid flowing through the mixed flow channel 233. In addition, the detection region 2331 is positioned downstream of the junction of the two fluids through which the first fluid channel 231 and the second fluid channel 232 flow. The two flows are a buffer, a liquid containing the label after mixing, and a liquid containing the target particles. The target particles can be simultaneously combined with three fluorescent quantum dots (including quantum dots modified by specific antibodies of cancer cells) with different colors and nano magnetic beads, in addition, the flowing liquid also comprises exosomes (common cells) modified by other fluorescent reagents (single-color quantum dots), and the target particles are exosomes of the cancer cells. When a particular single wavelength excitation light is directed to the fluid flowing through detection zone 2331, the presence of the target particle is accounted for by the fact that the target particle, upon receiving excitation from the particular excitation light, emits a different color of light due to the incorporation of the three different color fluorescent quantum dots. When the presence of the target particles in the detection region 2331 is detected by the ultra-high resolution fluorescence imaging system, the imaging software performs real-time fluorescence photographing and recording of the target particles through the fluorescence real-time image, and performs quantitative analysis on the number of the target particles. After quantitative analysis by the imaging software, a detection signal corresponding to the target particle is generated and transmitted to the control unit 40. If the fluid flowing through the detection region 2331 does not contain the target particles, the biological information of the target particles cannot be detected by the ultra-high resolution fluorescence imaging system. In the present embodiment, the detection unit 30 may detect biological information of the target particles, which includes a fluorescence signal.
The control unit 40 is connected to the detection and analysis unit 30 and the separation unit 50, respectively, in this embodiment, the control unit 40 is a rapid analysis computing system, and performs rapid analysis according to the fluorescence real-time image acquired by the control unit 40. In this embodiment, the target particles are labeled with three different colors of fluorescent quantum dots including antibodies specific to pancreatic cancer, and the control unit 40 performs rapid analysis of the fluorescent images and generates control signals according to the fluorescent images of the target particles acquired by the detection analysis unit 30, and transmits the control signals to the separation unit 50.
The separation unit 50 comprises an electromagnetic control system and the separation unit 50 activates the fast-response energizing magnetic field system in response to a control signal generated by the control unit 40. The electromagnetic field system is based on the principle of electromagnetic field, and after the system is electrified, a strong magnetic field is generated to have the function similar to an electromagnetic relay, and the system has strong attraction effect on the particles marked with magnetic labels. In this embodiment, after the electromagnetic control system is activated, the system generates a strong magnetic field, so that the electromagnetic field system can adsorb the exosomes of the cancer cells labeled by the nano magnetic beads and separate the liquid containing the exosomes of the cancer cells from the liquid flowing through the mixed flow channel 233. Since the separation unit 50 is disposed at the left side of the mixed flow channel 233, after the electromagnetic control system is activated, the exosome containing the cancer cells labeled by the nano magnetic beads flows into the left side of the mixed flow channel 233 and flows out from the first sample outlet 28 due to the attraction of the magnetic field, the liquid containing the exosome of the cancer cells flowing out through the first sample outlet 28 realizes the enrichment of the target microparticles, and the liquid containing the exosome of the normal cells (non-target microparticles) and the buffer solution flow out from the other side of the mixed flow channel 233 through the second sample outlet 29, thereby realizing the separation of the target microparticles. In this embodiment, only when the detection and analysis unit 30 detects the presence of the target particles in the liquid flowing through the detection region 2331, the separation unit 50 is activated and generates a strong magnetic field to perform the enrichment separation of the target particles.
In other embodiments, the separation unit 50 may be built in the microfluidic chip 20, that is, the microfluidic chip 20 has a function of separating target particles.
A method of detecting and separating target particles in a liquid sample, comprising the steps of:
providing a liquid sample and a microfluidic chip, wherein the microfluidic chip is provided with a microfluidic channel,
a filter element and a detection area;
filtering the liquid sample using the filter element to produce a liquid containing target particles
Flows into the microfluidic channel and enters a detection area; and
performing detection analysis on the detection region by using a detection analysis unit to obtain a target micro
Biological information of the particles.
Referring to fig. 3, the specific steps for detecting and separating the target particles in the liquid sample by using the analysis device include the following steps:
s101, a sample injection step, wherein the buffer solution, the liquid sample and the marker flow into and flow out of the microchannel. The buffer solution, the liquid sample and the liquid containing the marker are flowed into the microfluidic chip 20 by using the sample injection unit 10.
S103, a filtering step, namely filtering the liquid sample. The liquid sample is filtered by the filter component 25 of the microfluidic chip 20, the liquid sample contains the target particles after being filtered by the filter component 25, and the liquid containing the target particles flows into the mixed flow channel 233 and enters the detection region 2331.
And S105, a marking step, namely mixing the liquid containing the marker and the liquid sample filtered by the filter element in the second liquid channel and realizing marking. The label may be capable of labeling the target particle. The target particles are labeled by a specific fluorescent reagent, and the specific fluorescent reagent comprises a blue quantum dot modified by a tumor specific antibody of Glypican-1.
S107, a detection step of irradiating and exciting the target particles marked by the fluorescent reagent in the detection area by using a specific light source to emit light, and detecting the target particles. The fluorescent reagent is excited to emit light by a specific light source of the detection and analysis unit 30, and the target particles are detected to generate a detection signal. When the detection and analysis unit 30 finds that the target particles are present in the detection region, the detection and analysis unit 30 performs real-time image capturing and recording on the target particles labeled by the fluorescent reagent in the detection region 2331 of the mixed flow channel 233, generates a detection signal and transmits the detection signal to the control unit 40. When the detection and analysis unit 30 does not find the presence of the target particles in the detection area, no signal is generated to the control unit 40.
And S109, analyzing and calculating. The control unit 40 receives the detection signal transmitted from the detection and analysis unit 30, rapidly calculates and analyzes the fluorescence image of the target particle obtained by the detection and analysis unit 30, generates a control signal, and transmits the control signal to the separation unit 50.
And S111, an enrichment separation step, namely concentrating the target particles with the surfaces connected with the nano magnetic beads at a preset position in the mixed flow channel 233 through a magnetic field, separating the target particles from the liquid sample and realizing the enrichment separation of the target particles. When the separation unit 50 receives the control signal transmitted from the control unit 40, the control signal only targets the cancer cell exosomes labeled with the specific antibody, i.e., the target microparticles, and activates the electromagnetic control system. The target particles connected with the nano magnetic beads are deflected to one side of the mixed flow channel 233 due to the attraction of the magnetic field of the electromagnetic control system, so that the target particles with the nano magnetic beads connected to the surface are concentrated at the preset position in the mixed flow channel 233 and are enriched, thereby realizing the separation and purification of the target particles from the liquid sample, and the separated and purified target particle liquid can flow out from the first sample outlet 28 to obtain exosomes of the cancer cells; and other exosomes of common cells are discharged from the second sample outlet 29 to the other side of the mixed flow channel due to the fact that the exosomes are not attracted by the magnetic field of the electromagnetic control system. In this embodiment, the predetermined position refers to a position of the first sample outlet, and the target particles are separated and purified at an efficiency of 90% or more.
The quantitative analysis of the target microparticles can be performed by counting and calculating the volume of the recovered solution flowing out of the first outlet, the recovered solution being the target microparticles labeled with the labeling substance, and by counting the exosomes of the cancer cells in the flowing-out recovered solution, the specific analysis of the cancer cell exosomes can be performed.
In summary, compared with the prior art, the microfluidic chip of the present invention can be directly used for detecting and separating target particles while performing microfluidic control, the analysis device based on the microfluidic chip of the present invention realizes detection and separation integration, and the separated target particles can be used for subsequent experiments.
In the present invention, the target microparticle is an exosome of a cancer cell, and a specific exosome of a cancer cell can be selected by the analyzer. In addition, the analysis device integrates the microfluidic chip and the detection and analysis unit, so that high integration and automation of the analysis device are realized, and the analysis device is small in size and easy and convenient to operate.
Although the present invention has been described with reference to the above embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention.
Claims (7)
1. A micro-fluidic chip is provided with a micro-fluidic channel and is characterized in that the micro-fluidic chip is also provided with a filter element, a detection area, a first liquid channel and a second liquid channel which are connected with the micro-fluidic channel, the filter element is arranged in the micro-fluidic channel, for filtering the liquid sample to make the liquid containing the target particles flow into the detection area, the first liquid channel is used for introducing a buffer solution to generate sheath flow, the second liquid channel is used for introducing liquid containing the target particles and liquid containing the marker, to label the target particles to cause a liquid containing the labeled target particles to flow into the detection zone, the buffer solution generates a sheath flow to isolate the liquid containing the target particles of the marker in the same plane, separating the target particles in an enrichment manner and allowing the target particles to flow into the detection area in sequence; the microfluidic chip further comprises a separation unit and a mixed flow channel, wherein the separation unit comprises an electromagnetic control system, and when the target particles are detected in the detection area, the electromagnetic control system is started and attracts the target particles to be concentrated at a preset position of the mixed flow channel so as to enrich and separate the target particles.
2. The microfluidic chip of claim 1, wherein: the filter element is a porous membrane, and the pore diameter of the porous membrane is 200-250 nm.
3. The microfluidic chip according to claim 1, wherein the label comprises a fluorescent reagent, the fluorescent reagent is selected from one or more of organic fluorescent molecules, fluorescent liposomes, quantum dots and nanogold, and the label is connected with the target particles to label the target particles.
4. The microfluidic chip according to claim 3, wherein the liquid sample is a body fluid of a cancer patient, the target particles are exosomes of cancer cells, and quantum dots containing tumor-specific antibodies are connected to the surfaces of the target particles.
5. An analysis device, comprising:
the microfluidic chip of any one of claims 1-4; and
and the detection and analysis unit is used for detecting and analyzing whether the target particles exist in the detection area of the microfluidic chip.
6. The device according to claim 5, wherein the detection and analysis unit guides light to the detection region to detect the presence or absence of the target particles, and the detection and analysis unit is further provided with an imaging device for taking a photograph of the detected target particles in real time.
7. A method of analyzing target particles in a liquid sample, comprising the steps of:
providing a liquid sample and a microfluidic chip according to any one of claims 1 to 4; and
and detecting and analyzing whether the target particles exist in the detection area of the microfluidic chip by using a detection and analysis unit.
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| CN109628277B (en) * | 2019-01-23 | 2022-02-01 | 东南大学 | System and method for separating and detecting tumor marker miRNA in exosome |
| CN111518742B (en) * | 2020-05-07 | 2022-02-11 | 西安交通大学 | Nano-scale single exosome separation method |
| CN111774106B (en) * | 2020-06-11 | 2021-12-07 | 复旦大学 | Protein enrichment detection device based on light-operated fluid transportation and magnetic control sample separation |
| CN114214177A (en) * | 2021-12-07 | 2022-03-22 | 广州国家实验室 | Microfluidic chip, detection kit and exosome detection method |
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