CN113275047A - Microfluidic chip and application thereof - Google Patents
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Abstract
The invention discloses a micro-fluidic chip and application thereof, comprising a reaction chip and a filter sheet which is positioned above the reaction chip and is used for filtering and collecting objects to be detected, wherein the reaction chip is provided with a sample outlet, at least one sample inlet and a plurality of functional cavities which are communicated through flow channels, each sample inlet is respectively and correspondingly communicated with a liquid inlet, and the liquid inlet leads corresponding liquid into the functional cavities or the flow channels. Based on the micro-fluidic chip technology, the invention combines the affinity enrichment method and the physical characteristic method, integrates the two technical means on the micro-fluidic chip, separates, captures and enriches the rare cells in the blood sample to obtain enriched cell sample liquid with higher purity, and accurately counts the rare cells in the blood sample by the immunofluorescence staining method; meanwhile, the immunofluorescence staining detection method is integrated on the microfluidic chip, so that the interference of human factors is avoided, the manual error is reduced, the detection accuracy is improved, the detection speed is increased, the reagent cost and the equipment cost are reduced, and the detection engineering is favorably realized.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis, in particular to a micro-fluidic chip and application thereof, and especially application and a method of the micro-fluidic chip in detecting rare cells.
Background
Rare Cells (rare Cells) refer to Cells less than 1000 per ml of sample, and mainly include Circulating Tumor Cells (CTCs), Circulating Endothelial Cells (CECs), Circulating Endothelial progenitor Cells (cEPCs), stem Cells and fetal Cells in maternal blood.
The number of rare cells in the circulatory system of different individuals is different, and the human body has difference in the number of rare cells in the body under the healthy state, different disease states and different stages of the same disease, and the rare cells are closely related to the disease, for example, CTCs and Circulating Tumor Microemboli (CTM) are considered as important reasons and markers of tumor metastasis and recurrence, the existence of the CTCs and the circulating tumor microemboli not only represents the invasive capability of primary tumor, but also predicts the possibility of forming a metastasis at a far end, has important clinical significance, provides important basis for the accurate detection of the number of CTCs and CTM in blood in unit volume, namely, provides important basis for early screening of cancer, and also can provide dynamic monitoring and prognosis basis for the disease development of tumor patients; meanwhile, accurate screening, enrichment and detection of CTCs and CTMs can provide a research window for tumor cell pathogenic mechanism and drug resistance mechanism.
Therefore, the separation, purification, enrichment and detection analysis of the rare cells have important significance for clinical medicine, and compared with other normal blood cells existing in peripheral blood, the concentration of the rare cells is extremely low, and the detection difficulty is high.
The accurate screening, enrichment and release of target cells are the precondition for realizing reliable detection, and the current detection method aiming at rare cells mainly comprises a flow cytometry detection method, a fluorescence in situ hybridization method, a real-time quantitative PCR method and an immunofluorescence staining method.
The flow cytometry detection method has high detection speed, can simultaneously carry out multi-channel detection, but has low detection sensitivity, higher requirement on the number of cells and can not observe the cell morphology; the fluorescence in situ hybridization method can realize the detection of molecular level, the clinical application is mature, however, the phenomenon of low hybridization efficiency is easy to cause when the selected probe is short, and the accuracy is low; the real-time quantitative PCR can detect RNA in a target cell, has high detection sensitivity, is limited by the characteristics of easy degradation and easy pollution of the RNA, can cause higher probability of false positive, and limits the clinical application and popularization of the RNA.
The immunofluorescence staining method can observe clear cell morphology, has high detection sensitivity, is the most common detection method at present, but has various observed cell morphologies, and has lower accuracy of results due to the fact that the statistical counting accuracy is greatly influenced by the purity of a sample and the volume of liquid of the sample.
Therefore, the existing detection methods have the problem of low accuracy in detecting rare cells.
Disclosure of Invention
The invention aims to provide a micro-fluidic chip and application thereof, which can separate, capture and enrich rare cells in a blood sample, obtain enriched cell sample liquid with higher purity, accelerate the detection speed and reduce the reagent cost and the equipment cost.
In order to achieve the above objects, according to one aspect of the present invention, there is provided a microfluidic chip including a reaction chip and a filter sheet disposed above the reaction chip, the filter sheet being used for filtering and collecting an object to be measured; the reaction chip is provided with a sample outlet, at least one sample inlet and a plurality of functional cavities, the functional cavities are connected through a flow channel, each sample inlet corresponds to a liquid inlet in communication respectively, and the liquid inlets introduce corresponding liquid into the functional cavities or the flow channels.
According to the invention, the functional cavity is one or more of a sample adding functional cavity, a reaction functional cavity, a filtering functional cavity, a detection functional cavity and a mixing functional cavity; preferably, the functional cavity comprises an enrichment cavity, a specificity reaction cavity and an elution cavity, a first flow channel is arranged between the enrichment cavity and the specificity reaction cavity, and a second flow channel is arranged between the specificity reaction cavity and the elution cavity; preferably, the first flow channel and the second flow channel are micro flow channels or capillary flow channels. Preferably, the first and second flow passages comprise one or more flow passages. Preferably, the first flow channel and the second flow channel are formed by arranging a plurality of flow channels in parallel; preferably, the width and depth of the first flow channel and the second flow channel are the same or different. Preferably, the widths of the flow channels at different positions on the first flow channel and the second flow channel are the same or different. Preferably, the first flow channel and the second flow channel are in a straight line structure, a broken line structure or a curve structure, and more preferably in a spiral structure or a serpentine structure.
According to the invention, the regions where the enrichment chamber and the specificity reaction chamber are located are both provided with magnetic fields. Preferably, a magnetic field is arranged below the enrichment cavity, and a magnetic field is arranged above and below the specificity reaction cavity. Preferably, the upper and lower magnetic fields are generated by standard Ru FeB permanent magnets, the magnets have the same maximum energy product of 20-60 MGeo, the upper and lower magnets are parallel, the long side is perpendicular to the specific reaction chamber 3, and the distances between the upper and lower magnets and the central plane of the channel of the specific reaction chamber are equal.
According to the invention, the number of the sample inlets is 1 or more, preferably 2, 3, 4 or more than or equal to 5, and the positions of the sample inlets and the sample outlets are arranged on the flow channel or connected to the reaction cavity; preferably, the reaction chip is provided with a treatment solution inlet, a specific solution inlet and a waste liquid outlet; the elution cavity is connected with a treatment liquid inlet, the enrichment cavity is connected with a waste liquid outlet, and the second flow channel is connected with a specific solution inlet; preferably, the specific solution inlet is arranged close to the specific reaction cavity; preferably, the treatment solution inlet and the specific solution inlet both comprise a sample adding hole, and the sample adding hole is communicated with the elution cavity and the enrichment cavity through corresponding flow channels.
According to the invention, the filter membrane is arranged on the filter disc, and the part of the filter disc corresponding to the filter membrane is of a cavity structure; when the top surface of the filter membrane faces the reaction chip, at least one part of the cavity structure is communicated with the enrichment cavity.
According to the invention, an encapsulation sheet is arranged between the reaction chip and the filter sheet, an opening is arranged at the position of the encapsulation sheet corresponding to the filter membrane, the opening is sealed by an encapsulation membrane, and reversible encapsulation is carried out between the encapsulation membrane and the encapsulation sheet. Preferably, the top of the filter disc is provided with a sample adding disc, the sample adding disc is provided with a sample adding hole, and the sample adding hole is positioned on the upper side of the filter disc and corresponds to the filter membrane area on the filter disc.
Preferably, the packaging film is a film material adhered to the opening or is obtained by coating an elastic layer on a packaging sheet, the thickness of the elastic layer on the packaging sheet is 0.05-0.5 mm, and preferably, the thickness of the elastic layer is larger than the thickness of the packaging sheet; illustratively, the elastic layer has a thickness of 0.06mm, 0.08mm, 0.1mm, 0.12mm, 0.14mm, 0.16mm, 0.18mm, 0.20 mm. Preferably, the top and the bottom of the filter sheet are both provided with elastic layers with the thickness of less than 1 mm; preferably, the thickness of the elastic layer on the filter sheet is less than 0.9mm, preferably less than 0.8mm, preferably less than 0.6mm, by way of example 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 0.95 mm.
According to another aspect of the present invention, there is also provided the use of any one of the above microfluidic chips in antigen, antibody, nucleic acid, protein, and cell detection.
According to another aspect of the invention, the application of any one of the microfluidic chips in cell separation, enrichment, purification, enrichment, screening and detection is also provided; preferably in rare cell detection.
According to still another aspect of the present invention, there is provided a method for detecting rare cells by using any one of the above microfluidic chips, comprising the steps of: filtering a sample to be detected on a filter membrane, and intercepting an object to be detected; adding specific magnetic particles into the specific reaction cavity, wherein the specific magnetic particles can perform specific reaction with the intercepted substance to be detected; and enriching the products after reaction, and counting the enriched cells.
According to the invention, after staining the specific expression protein and the cell nucleus, the enriched cells are counted by using a fluorescence microscope or a flow cytometer. Preferably, after the enriching and before the dyeing, the method further comprises the following steps: performing membrane rupture and sealing on the cells; preferably, after the enrichment and before the rupture and the sealing of the cell, the method further comprises the following steps: fixing and washing the cells; preferably, the magnetic particles are immunomagnetic beads and aptamer magnetic beads, and the immunomagnetic beads comprise one or more of folic acid immunomagnetic beads, epithelial cell adhesion factor immunomagnetic beads and epidermal growth factor immunomagnetic beads.
According to the present invention, the method for detecting rare cells using a microfluidic chip further comprises the steps of: s1, adding the sample to be tested on a filter membrane of a filter plate positioned above the reaction chip, filtering and intercepting the object to be tested; s2, introducing magnetic particles capable of being specifically combined with the sample to be detected into the specific reaction cavity; s3, eluting the trapped substance to be detected and introducing the eluted substance to a specific reaction cavity for reaction; s4, transferring the reacted substances to an enrichment cavity for enrichment; s5, staining and counting the cells;
preferably, step S5 includes: cell staining and counting are realized in the microfluidic chip, and the enriched liquid is led out to the outside for cell staining and counting; preferably, the implementation of cell staining and counting in the microfluidic chip comprises the following steps: s501, fixing and cleaning the reacted cells; s502, cell rupture and cell sealing are carried out; s503, dyeing the cell specific expression protein and the cell nucleus in sequence; and S504, collecting the solution in the enrichment cavity and counting cells.
Preferably, the step of guiding the enriched liquid to the outside for cell staining and counting specifically comprises the following steps: s601, leading out the enriched liquid, filtering, and removing enriched magnetic beads; s602, fixing and cleaning cells; s603, cell rupture and cell sealing are carried out; s604, staining cell specific expression protein and cell nucleus in sequence; and S605, counting the cells.
Preferably, the specific steps of step S1 are: will add the application of sample piece and filter plate laminating and centre gripping fixed, the sample that awaits measuring filters through the filter membrane through the application of sample hole on the application of sample piece, holds back the great target cell of size on the filter membrane during filtration, and the waste liquid after the filtration can be retrieved and is used for other detections.
Preferably, the specific steps of step S2 are: keeping the treatment liquid inlet in a closed state, and the packaging sheet in a packaging state, introducing a magnetic particle solution from the specific solution inlet, wherein the treatment liquid inlet is in the closed state, the flow resistance of a flow channel between the treatment liquid inlet and the specific solution inlet is large, the solution flows from the specific solution inlet to the specific reaction cavity, the magnetic particles are fixed in the current region by a magnetic field existing in the region of the specific reaction cavity, and the residual solution passes through the elution cavity and reaches the waste liquid outlet; the solution flow rate of the treatment solution inlet is 50-500 mu L/min; preferably, the solution flow rate is 100-450 muL/min; preferably, the solution flow-through rate is 150 to 400. mu.L/min, more preferably 200 to 350. mu.L/min, and most preferably 250 to 300. mu.L/min.
Preferably, the reaction chip and the filter plate are made of polydimethylsiloxane, polymethyl methacrylate, glass, silicon, polycarbonate, polypropylene, polystyrene, cyclic olefin copolymer or cyclic olefin polymer, and preferably made of PMMA, PC and PP.
Preferably, the filter membrane is made of parylene (parylene), Polycarbonate (PC), polyethylene terephthalate (TETP), Polyethersulfone (PES) or Polyetherimide (PEI), preferably, xylene, polycarbonate or polyethylene terephthalate.
The invention has the beneficial effects that:
based on the microfluidic chip technology, the method integrates two technical means on the microfluidic chip by combining an affinity enrichment method and a physical characteristic method, separates, captures and enriches rare cells in a blood sample to obtain enriched cell sample liquid with higher purity, and accurately counts the rare cells in the blood sample by an immunofluorescence staining method; meanwhile, the immunofluorescence staining detection method is integrated on the microfluidic chip, so that the interference of human factors is avoided, the manual error is reduced, the detection accuracy is improved, the detection speed is increased, the reagent cost and the equipment cost are reduced, and the detection engineering is favorably realized.
Drawings
FIG. 1 is a schematic structural diagram of a microfluidic chip according to an embodiment of the present invention;
FIG. 2 is a sectional view of a reaction chip in an embodiment of the present invention;
FIG. 3 is a cross-sectional view of a filter plate in an embodiment of the invention;
FIG. 4 is a cross-sectional view of a sample addition piece in an embodiment of the present invention;
FIG. 5 is a flowchart of a method for detecting rare cells in the present invention.
In the figure: 1-reaction chip, 2-enrichment cavity, 3-specificity reaction cavity, 4-elution cavity, 5-first flow channel, 6-second flow channel, 7-treatment liquid inlet, 8-waste liquid outlet, 9-specificity solution inlet, 10-filter plate, 11-filter membrane, 12-packaging plate, 13-opening, 14-packaging membrane, 15-sample adding plate and 16-sample adding hole.
Detailed Description
The microfluidic chip and its application will be described in further detail with reference to the accompanying drawings and specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Referring to fig. 1 to 4, the present invention provides a microfluidic chip including a reaction chip 1 and a filter sheet 10 disposed thereon for filtering and collecting an analyte. The reaction chip 1 is provided with a sample outlet, at least one sample inlet and a plurality of functional cavities, the functional cavities are connected through a flow channel, each sample inlet is respectively corresponding to one liquid inlet in communication, and the liquid inlets lead corresponding liquid into the functional cavities or the flow channels.
The liquid is driven to sample through a power mechanism, the power mechanism is arranged between the sample inlet and the power source, and the power mechanism can be a constant pressure pump, an air compressor, a piezoelectric driver or an ultrasonic driver.
According to the invention, the number and size of the functional cavities are set according to actual needs, and can be one or more of functional cavities for sample adding, reaction, filtration, detection, mixing and the like; the functional cavity and the flow channel can be prepared by photoetching, numerical control, hot pressing, injection molding and other methods on a chip material.
As shown in FIG. 2, the functional cavity comprises an enrichment cavity 2, a specificity reaction cavity 3 and an elution cavity 4, a first flow channel 5 is arranged between the enrichment cavity 2 and the specificity reaction cavity 3, and a second flow channel 6 is arranged between the specificity reaction cavity 3 and the elution cavity 4.
According to the present invention, the first flow channel 5 and the second flow channel 6 are micro flow channels or capillary flow channels, wherein the first flow channel 5 and the second flow channel 6 include but are not limited to one flow channel. Preferably, the first flow channel 5 and the second flow channel 6 are formed by arranging a plurality of flow channels in parallel, the widths and depths of the first flow channel 5 and the second flow channel 6 may be the same or different, when the first flow channel 5 and the second flow channel 6 are formed by arranging a plurality of flow channels in parallel, the widths of different flow channels may be the same or different, and the widths of different positions of the same flow channel may be the same or different.
Preferably, the first flow channel 5 and the second flow channel 6 may have a straight line structure, a broken line structure or a curved line structure, and more preferably, a spiral structure or a serpentine structure.
Wherein, the areas of the enrichment cavity 2 and the specificity reaction cavity 3 are both provided with magnetic fields. For example, a magnetic field may be provided below the enrichment chamber 2 and a magnetic field may be provided above and below the specific reaction chamber 3. The upper and lower magnetic fields are generated by standard Ru iron boron permanent magnets, the magnets have the same maximum energy product which is 20-60 MGeo, the upper and lower magnets are parallel, the long edges are vertical to the specific reaction cavity 3, and the distances between the upper and lower magnets and the central plane of the channel of the specific reaction cavity 3 are equal.
The number of the sample inlets is set according to actual needs, and can be 1 or more, preferably 2, 3, 4 or more than or equal to 5, and the positions of the sample inlets and the sample outlets are set according to needs and can be arranged on the flow channel or connected on the reaction cavity.
Preferably, two sample inlets, a treatment solution inlet 7 and a specificity solution inlet 9, and one sample outlet waste outlet 8 are included. The functional cavity, the flow channel, the sample inlet and the sample outlet are specifically arranged as follows: the elution cavity 4 is connected with a treatment liquid inlet 7, the enrichment cavity 3 is connected with a waste liquid outlet 8, and the second flow channel 7 is connected with a specific solution inlet 9. Preferably, the specific solution inlet 9 is disposed near the specific reaction chamber 3.
Preferably, the treatment solution inlet 7 and the specific solution inlet 9 both comprise a sample adding hole, the sample adding hole is communicated with the elution cavity 4 and the enrichment cavity 2 through corresponding flow channels, and when the solution is dripped into the sample adding hole, the solution can be added manually through a pipette and other devices, and can also be accurately extracted and added through a pump body.
Preferably, the reaction chip 1 and the filter plate 10 are made of Polydimethylsiloxane (PDMS), Polymethylmethacrylate (PMMA), glass, silicon, Polycarbonate (PC), polypropylene (PP), Polystyrene (PS), Cyclic Olefin Copolymer (COC) or Cyclic Olefin Polymer (COP), and because they need to have good biocompatibility and weak adsorption capacity to biological materials when in use, they have low cost and are compatible with mass production methods of processing, bonding and integration, and preferably made of PMMA, PC and PP.
The filter sheet 10 is provided with a filter membrane 11, and the part of the filter sheet 10 corresponding to the filter membrane 11 is a cavity structure, when the top surface of the filter membrane 11 faces the reaction chip 1, at least one part of the cavity structure is communicated with the enrichment cavity 3, and the liquid in the enrichment cavity 3 can enter the cavity structure to wash the filter membrane 11. Preferably, the filter membrane 11 is made of parylene (parylene), Polycarbonate (PC), polyethylene terephthalate (TETP), Polyethersulfone (PES) or Polyetherimide (PEI), preferably, xylene, polycarbonate or polyethylene terephthalate.
Wherein, an encapsulation sheet 12 is arranged between the reaction chip 1 and the filter sheet 10, an opening 13 is arranged at the position where the encapsulation sheet 12 corresponds to the filter membrane 11, the opening 13 is sealed by an encapsulation membrane 14, and reversible encapsulation is carried out between the encapsulation membrane 14 and the encapsulation sheet 12. The top surface of the filter chip faces the reaction chip, and the other side of the filter chip is packaged by the attached packaging sheet. The sealing film 14 may be a film material adhered to the opening 13, or may be obtained by coating a film material.
For simplicity of operation, the encapsulating sheet 12 may be coated with an elastic layer having a thickness of 0.05 to 0.5mm, preferably 0.1 to 0.4mm, more preferably 0.2 to 0.3mm, most preferably 0.15mm, and illustratively 0.06mm, 0.08mm, 0.1mm, 0.12mm, 0.14mm, 0.16mm, 0.18mm, 0.20 mm.
In order to ensure the sealing performance of the micro flow channel, the packaging sheet 12 and the reaction chip 1 are packaged irreversibly, for example, by thermocompression bonding or laser bonding. The filter sheet 10 and the reaction chip 1 are packaged irreversibly, for example, the filter sheet 10 and the reaction chip 1 can be packaged and fixed integrally by using a clamping device to improve the sealing property.
According to the invention, an elastic layer with a thickness of less than 1mm is provided on both the top and the bottom of the filter sheet 10. Preferably, the elastic layer is obtained by coating or directly adhering an elastic material, for example by coating a solvent-based acrylic material. Preferably, the thickness of the elastic layer on the filter sheet 10 is less than 0.9mm, preferably the thickness of the elastic layer is less than 0.8mm, and more preferably the thickness of the elastic layer is less than 0.6 mm. By way of example, the thickness of the elastic layer may be 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 0.95 mm.
In this embodiment, the top of the filter sheet 10 is provided with a sample adding sheet 15, the sample adding sheet 15 is provided with a sample adding hole 16, and the sample adding 16 is located on the upper side of the filter sheet 10 and corresponds to the region of the filter membrane 11 on the filter sheet 10.
In order to reduce the phenomenon of cell adhesion, the surfaces of the first flow channel 5 and the second flow channel 6 and the inner surface of the opening 13 of the encapsulating sheet 12 can be subjected to hydrophobic treatment or hydrophobic materials are preferably selected to prepare the reaction chip 1 and the encapsulating sheet 12, and when the hydrophobic treatment is carried out, the hydrophobic materials can be attached to the surfaces in a coating, spraying or self-assembly mode, so that the hydrophobic effect is achieved, and the cell adhesion is reduced.
According to another aspect of the present invention, there is also provided a method for detecting rare cells using the above microfluidic chip, comprising the steps of: filtering a sample to be detected on a filter membrane 11, intercepting an object to be detected, adding specific magnetic particles into a specific reaction cavity 3, wherein the specific magnetic particles can perform specific reaction with the object to be detected, enriching the intercepted object to be detected after the reaction with the specific magnetic particles, and counting the enriched cells.
Wherein, the counting can be carried out by staining the specific expression protein and cell nucleus, and then counting the cells by using a fluorescence microscope or a flow cytometer. After enrichment and before staining, the method also comprises the steps of membrane rupture and blocking of the cells. Preferably, after the enrichment and before the rupture and sealing of the cell, the steps of fixing and washing the cell are also included.
Specifically, referring to fig. 5, the method for detecting rare cells on a microfluidic chip comprises the following steps:
s1, adding the sample to be tested to the filter membrane 11 for filtration and trapping the substance to be tested.
S2, introducing magnetic particles capable of being specifically combined with the sample to be detected into the specific reaction cavity 3.
S3, eluting the trapped analyte and introducing the eluted analyte into the specific reaction cavity 3 for reaction.
And S4, transferring the reacted substances to the enrichment cavity 2 for enrichment.
S5, staining and counting the cells.
Step S5 includes the following two implementation manners: cell staining and counting are realized in the microfluidic chip, and the enriched liquid is led out to the outside for cell staining and counting.
The method for realizing cell staining and counting in the microfluidic chip comprises the following steps:
s501, fixing and washing the reacted cells.
And S502, cell rupture and cell sealing are carried out.
S503, staining the cell specificity expression protein and the cell nucleus in sequence.
S504, collecting the solution in the enrichment cavity 3 and counting cells.
The method for guiding the enriched liquid out to perform cell staining and counting specifically comprises the following steps:
s601, leading out the enriched liquid, filtering, and removing the enriched magnetic beads.
S602, fixing and washing the cells.
And S603, cell rupture and cell sealing are carried out.
And S604, sequentially staining cell specific expression proteins and cell nuclei.
And S605, counting the cells.
The specific steps of step S1 are: the sample adding sheet is attached to the filter sheet 10 and clamped and fixed, a sample to be detected is filtered through the filter membrane 11 through the sample adding hole 16 on the sample adding sheet 15, target cells with larger sizes are intercepted on the filter membrane 11 during filtering, and waste liquid after filtering can be recycled for other detection.
In this embodiment, to ensure complete filtration, a buffer solution (such as phosphate PBS buffer solution) with the same amount as the sample is introduced into the opening 13 for 1-5 times to wash the filter membrane 11.
The specific steps of step S2 are: : keeping the treatment liquid inlet 7 in a closed state, the packaging sheet 12 in a packaging state, and introducing the magnetic particle solution from the specific solution inlet 9, wherein the treatment liquid inlet 7 is in the closed state, the flow resistance of a flow channel between the treatment liquid inlet 7 and the specific solution inlet 9 is large, the solution flows from the specific solution inlet 9 to the specific reaction cavity 3, the magnetic particles are fixed in the current region by a magnetic field existing in the region of the specific reaction cavity 3, and the residual solution reaches the waste liquid outlet 8 through the enrichment cavity 2.
In this embodiment, the solution flowing rate at the treatment solution inlet 7 is 50 μ L/mL to 500 μ L/min, and this rate range can ensure that the magnetic particles are distributed more stably while maintaining the process efficiency. Preferably, the solution flow rate is 100 to 450. mu.L/min, more preferably 150 to 400. mu.L/min, even more preferably 200 to 350. mu.L/min, and most preferably 250 to 300. mu.L/min.
The magnetic particles of the present invention include, but are not limited to, immunomagnetic beads, aptamer magnetic beads, etc., and the immunomagnetic beads include, but are not limited to, folic acid immunomagnetic beads, epithelial cell adhesion factor (EpCAM) immunomagnetic beads, epidermal growth factor (EGFR1) immunomagnetic beads, etc., as examples of CTCs.
The specific steps of step S1 are: firstly, the packaging film 14 on the packaging sheet 12 is removed, the filter sheet 10 is turned over to the upper surface to be attached to the reaction chip 1, and the reaction chip is clamped and fixed, and at the moment, the surface of the filter film 11 with the target cells faces the elution cavity 4.
And then closing the waste liquid outlet 8 and the specific solution inlet 9, introducing flushing liquid and gas with the same volume from the treatment liquid inlet 7, filling the liquid into the elution cavity 4 under the combined action of flow resistance and gravity, then enabling the liquid to flow through the filter membrane 11, drawing the flushing liquid back after the flushing liquid flows through the filter membrane 11, repeating the process for 2-10 times, and transferring the target cells from the filter membrane 11 to the flushing liquid at the elution cavity 4 to form the eluent.
Opening the waste liquid outlet 8, closing the lower side of the filter sheet 10 to enable the corresponding elution cavity to be in a closed state, removing the magnetic field above the specific reaction cavity 3, introducing the flushing liquid at the treatment liquid inlet 7 until the front end of the eluent reaches the specific reaction cavity 3, controlling the flow at 100-.
Step S3 specifically includes the following steps: keeping the treatment fluid inlet 7 open, the specific solution inlet 9 closed, the waste fluid outlet 8 open, resetting the magnet above the anisotropic reaction chamber 3, maintaining the enrichment chamber 2 in a strong magnetic field, introducing flushing fluid from the treatment fluid inlet 7 until the rear end of the eluent leaves the specific reaction chamber 3, adopting low-speed feeding type, stepping type, intermittent type or extraction type sample introduction to ensure that the cells are fully contacted with the specific magnetic particle area, controlling the flux at 25-200 muL/min, and then, discharging waste fluid from the waste fluid outlet 8.
Step S4 specifically includes the following steps:
when the capturing process is finished, namely the rear end of the eluent leaves the enrichment cavity 3, the magnetic field at the specific reaction cavity 3 is removed, the flushing liquid is introduced from the treatment liquid inlet 7, the process is stopped after the magnetic particles in the specific reaction cavity 3 are transferred to the enrichment cavity 2, and the enrichment cavity 2 is under the strong magnetic field, so that the cells, the magnetic particles and the magnetic particles can be retained in the enrichment cavity 3.
Step S501 includes the steps of:
keeping the treatment fluid inlet 7 open, closing the specific solution inlet 9, opening the waste liquid outlet 8, introducing gas from the treatment fluid inlet 7 to discharge all waste liquid, introducing fixing fluid (4% paraformaldehyde) from the treatment fluid inlet 7, stopping liquid inlet after the enrichment cavity 2 is filled with the fixing fluid, sealing the treatment fluid inlet 7 and the waste liquid outlet 8, and standing at room temperature for 10 min.
Keeping the treatment fluid inlet 7 open, the specific solution inlet 9 closed and the waste fluid outlet 8 open, introducing gas from the treatment fluid inlet 7 to discharge all waste fluid, introducing cleaning fluid (phosphate buffered saline (PBS) can be selected) from the treatment fluid inlet 7, stopping liquid inlet after the enrichment cavity 2 is filled with the cleaning fluid, sealing the treatment fluid inlet 7 and the waste fluid outlet 8, standing for 5min at room temperature, repeating for 3 times, and finishing cleaning.
Step S502 specifically includes the following steps:
cell membrane rupture: keeping the treatment fluid inlet 7 open, the specific solution inlet 9 closed and the waste liquid outlet 8 open, introducing gas from the treatment fluid inlet 7 to discharge all waste liquid, introducing 0.1% Triton-100X from the treatment fluid inlet 7, stopping liquid inlet after the enrichment cavity 2 is filled with the solution, sealing the treatment fluid inlet 7 and the waste liquid outlet 8, and standing at room temperature for 10 min.
Cell sealing: and (3) repeating the steps S1-S5, keeping the treatment fluid inlet 7 open, closing the specific solution inlet 9, opening the waste fluid outlet 8, introducing gas from the treatment fluid inlet 7 to discharge all waste fluid, introducing 1% bovine serum albumin solution (1% BSA solution) from the treatment fluid inlet 7, stopping liquid inlet after the enrichment cavity 2 is filled with the solution, sealing the treatment fluid inlet 7 and the waste fluid outlet 8, and standing at room temperature for 1 h.
Step S503 specifically includes the following steps:
keeping a treatment fluid inlet 7 open, a specific solution inlet 9 closed, a waste liquid outlet 8 open, introducing gas from the treatment fluid inlet 7 to discharge all waste liquid, introducing a direct standard antibody solution (the concentration can be prepared according to the instructions of reagents) of target cell specific expression protein and impurity cell specific expression protein with a certain concentration from the treatment fluid inlet 7, stopping liquid inlet after the enrichment cavity 2 is filled with the solution, sealing the treatment fluid inlet 7 and the waste liquid outlet 8, standing for 2-5 h at room temperature, or taking out the chip from a clamping device carefully, keeping the treatment fluid inlet 7, the specific solution inlet 9 and the waste liquid outlet 8 in a sealed state, and keeping the treatment fluid inlet 7, the specific solution inlet 9 and the waste liquid outlet 8 in a dark state at the temperature of 4 ℃ for 12-20 h.
Packaging the chip, repeating the steps S1-S5, keeping the treatment liquid inlet 7 open, closing the specific solution inlet 9, opening the waste liquid outlet 8, introducing gas from the treatment liquid inlet 7 to discharge all waste liquid, introducing a cell nucleus staining solution with a certain concentration (the concentration can be prepared according to the instructions of the reagent) from the treatment liquid inlet 7, stopping liquid inlet after the enrichment cavity 3 is filled with the solution, closing the treatment liquid inlet 7 and the waste liquid outlet 8, standing for 10min to realize cell nucleus staining, wherein the waste liquid is collected at the waste liquid outlet 8 in the step.
Step S504 specifically includes the following steps: and counting the number of target cells and impurity cells in the enrichment solution through a fluorescence inverted microscope or a flow cytometer.
Step S601 specifically includes the following steps: and (4) leading out the enriched liquid, and filtering the enriched liquid through a microporous filter membrane to remove the enriched magnetic beads.
Step S602 specifically includes the following steps: placing the microporous filter membrane for bearing the cells in a culture dish containing a proper amount of 4% paraformaldehyde, standing at room temperature for 10min, and then sucking out the fixing solution.
Step S603 specifically includes the following steps: adding a proper amount of 0.1% Triton-100X into the fixed cells in the S602, standing for 10min at room temperature, and sucking off the 0.1% Triton-100X to complete cell membrane rupture; an appropriate amount of 1% BSA solution was added to the fixed cells in S602, and the mixture was allowed to stand at room temperature for 1 hour, and the 1% BSA solution was aspirated to complete cell blocking.
Step S604 specifically includes the following steps: introducing a proper amount of direct-labeling antibody solution of target cell specific expression protein and impurity cell specific expression protein with certain concentration until the solution submerges the microporous filter membrane after cell sealing, standing for 2-5 h at room temperature or standing for 12-20 h at 4 ℃, absorbing the direct-labeling antibody solution, and finishing specific protein dyeing; adding a proper amount of DAPI staining solution or Hoechst staining solution (the volume ratio of the staining solution to the PBS buffer solution is 1: 500-1: 1000) into the fixed cells, standing for 10min at room temperature, and sucking off the staining solution to complete cell nucleus staining.
Step S605 specifically includes the following steps: spreading and flatly placing the microporous filter membrane with the stained cells on a glass slide, dripping a proper amount of anti-quenching agent, covering a cover glass, sealing and storing by using a sealing tablet, and observing and counting the cells carried on the microporous filter membrane by an inverted fluorescence microscope.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The microfluidic chip is characterized by comprising a reaction chip (1) and a filter (10) positioned above the reaction chip, wherein the filter (10) is used for filtering and collecting an object to be detected; the reaction chip (1) is provided with a sample outlet, at least one sample inlet and a plurality of functional cavities, the functional cavities are connected through a flow channel, each sample inlet corresponds to a liquid inlet of the communication, and the liquid inlets lead corresponding liquid into the functional cavities or the flow channels.
2. The microfluidic chip according to claim 1, wherein the functional cavity is one or more of a sample adding, a reaction, a filtration, a detection and a mixing functional cavity;
preferably, the functional cavity comprises an enrichment cavity (2), a specific reaction cavity (3) and an elution cavity (4), a first flow channel (5) is arranged between the enrichment cavity (2) and the specific reaction cavity (3), and a second flow channel (6) is arranged between the specific reaction cavity (3) and the elution cavity (4);
preferably, the first flow channel (5) and the second flow channel (6) are micro flow channels or capillary flow channels.
Preferably, the first flow channel (5) and the second flow channel (6) comprise one or more flow channels.
Preferably, the first flow channel (5) and the second flow channel (6) are formed by arranging a plurality of flow channels in parallel;
preferably, the widths and the depths of the first flow channel (5) and the second flow channel (6) are the same or different.
Preferably, the widths of the flow channels at different positions on the first flow channel (5) and the second flow channel (6) are the same or different.
Preferably, the first flow channel (5) and the second flow channel (6) are in a straight line structure, a broken line structure or a curve structure, and more preferably in a spiral structure or a serpentine structure.
3. The microfluidic chip according to claim 2, wherein the enrichment chamber (2) and the specific reaction chamber (3) are provided with magnetic fields in their areas.
Preferably, a magnetic field is arranged below the enrichment cavity (2), and magnetic fields are arranged above and below the specificity reaction cavity (3);
preferably, the upper and lower magnetic fields are generated by standard Ru FeB permanent magnets, the magnets have the same maximum energy product of 20-60 MGeo, the upper and lower magnets are parallel, the long sides are vertical to the specific reaction cavity 3, and the distances between the upper and lower magnets and the central plane of the channel of the specific reaction cavity (3) are equal.
4. The microfluidic chip according to claim 2, wherein the number of the sample inlets is 1 or more, preferably 2, 3, 4 or more than or equal to 5, and the positions of the sample inlets and the sample outlets are arranged on the flow channel or connected to the reaction chamber;
preferably, the reaction chip (1) is provided with a treatment liquid inlet (7), a specific solution inlet (9) and a waste liquid outlet (8);
the elution cavity (4) is connected with a treatment liquid inlet (7), the enrichment cavity (2) is connected with a waste liquid outlet (8), and the second flow channel (7) is connected with a specific solution inlet (9);
preferably, the specificity solution inlet (9) is arranged close to the specificity reaction chamber (3);
preferably, the treatment liquid inlet (7) and the specificity solution inlet (9) both comprise sample adding holes, and the sample adding holes are communicated with the elution cavity (4) and the enrichment cavity (2) through corresponding flow channels.
5. The microfluidic chip according to claim 2, wherein the filter (10) is provided with a filter membrane (11), and the filter (10) has a cavity structure corresponding to the filter membrane (11); when the top surface of the filter membrane (11) faces the reaction chip (1), at least a part of the cavity structure is communicated with the enrichment cavity (2).
6. The microfluidic chip according to claim 2, wherein an encapsulation sheet (12) is disposed between the reaction chip (1) and the filter sheet (10), an opening (13) is disposed at a position where the encapsulation sheet (12) corresponds to the filter membrane (11), the opening (13) is sealed by an encapsulation membrane (14), and reversible encapsulation is performed between the encapsulation membrane (14) and the encapsulation sheet (12).
Preferably, the top of the filter sheet (10) is provided with a sample adding sheet (15), the sample adding sheet (15) is provided with sample adding holes (16), and the sample adding holes (16) are positioned on the upper side of the filter sheet (10) and correspond to the area of the upper filter membrane (11) of the filter sheet (10).
Preferably, the packaging film (14) is a film material adhered on the opening (13) or is obtained by coating an elastic layer on the packaging sheet (12), and the thickness of the elastic layer on the packaging sheet (12) is 0.05-0.5 mm, preferably the thickness of the elastic layer;
illustratively, the elastic layer has a thickness of 0.06mm, 0.08mm, 0.1mm, 0.12mm, 0.14mm, 0.16mm, 0.18mm, 0.20 mm.
Preferably, the top and the bottom of the filter sheet (10) are both provided with an elastic layer with the thickness of less than 1 mm;
preferably, the thickness of the elastic layer on the filter sheet (10) is less than 0.9mm, preferably less than 0.8mm, preferably less than 0.6mm, as an example 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 0.95 mm.
7. Use of the microfluidic chip of any one of claims 1 to 6 for antigen, antibody, nucleic acid, protein, and cell detection; or, the use of the microfluidic chip of any one of claims 1 to 6 for cell separation, enrichment, purification, screening and detection; preferably, the microfluidic chip of any one of claims 1 to 6 is used for rare cell detection.
8. A method for detecting rare cells by using the microfluidic chip according to any one of claims 1 to 6, comprising the steps of:
filtering a sample to be detected on a filter membrane (11) and intercepting an object to be detected;
specific magnetic particles are added into the specific reaction cavity (3), and the specific magnetic particles can perform specific reaction with the intercepted substance to be detected;
and enriching the products after reaction, and counting the enriched cells.
9. The method of claim 8, wherein the enriched cells are counted by staining for specific expressed proteins, staining for nuclei, and then using a fluorescence microscope or flow cytometer.
Preferably, after the enriching and before the dyeing, the method further comprises the following steps: performing membrane rupture and sealing on the cells;
preferably, after the enrichment and before the rupture and the sealing of the cell, the method further comprises the following steps: fixing and washing the cells;
preferably, the magnetic particles are immunomagnetic beads and aptamer magnetic beads, and the immunomagnetic beads comprise one or more of folic acid immunomagnetic beads, epithelial cell adhesion factor immunomagnetic beads and epidermal growth factor immunomagnetic beads.
10. The method of claim 8, comprising the steps of:
s1, adding a sample to be detected on a filter membrane (11) of a filter sheet positioned above the reaction chip (1) for filtering and intercepting an object to be detected;
s2, introducing magnetic particles capable of being specifically combined with a sample to be detected into the specific reaction cavity (3);
s3, eluting the trapped substance to be detected and introducing the eluted substance to the specific reaction cavity (3) for reaction;
s4, transferring the reacted substances to an enrichment cavity (2) for enrichment;
s5, staining and counting the cells;
preferably, step S5 includes: cell staining and counting are realized in the microfluidic chip, and the enriched liquid is led out to the outside for cell staining and counting;
preferably, the implementation of cell staining and counting in the microfluidic chip comprises the following steps:
s501, fixing and cleaning the reacted cells;
s502, cell rupture and cell sealing are carried out;
s503, dyeing the cell specific expression protein and the cell nucleus in sequence;
s504, collecting the solution in the enrichment cavity (2) and counting cells;
preferably, the step of guiding the enriched liquid to the outside for cell staining and counting specifically comprises the following steps:
s601, leading out the enriched liquid, filtering, and removing enriched magnetic beads;
s602, fixing and cleaning cells;
s603, cell rupture and cell sealing are carried out;
s604, staining cell specific expression protein and cell nucleus in sequence;
s605, counting cells;
preferably, the specific steps of step S1 are: attaching the sample adding sheet and the filter sheet (10) and clamping and fixing the sample adding sheet and the filter sheet, filtering a sample to be detected through a filter membrane (11) through a sample adding hole (16) on the sample adding sheet (15), intercepting target cells with larger sizes on the filter membrane (11) during filtering, and recycling filtered waste liquid for other detection;
the specific steps of step S2 are: keeping the treatment liquid inlet (7) in a closed state, the packaging sheet (12) in a packaging state, introducing a magnetic particle solution from the specific solution inlet (9), wherein the treatment liquid inlet (7) is in the closed state, the flow resistance of a flow channel between the treatment liquid inlet (7) and the specific solution inlet (9) is large, the solution flows from the specific solution inlet (9) to the specific reaction cavity (3), the magnetic particles are fixed in the current region by a magnetic field existing in the region of the specific reaction cavity (3), and the residual solution reaches the waste liquid outlet (8) through the elution cavity (4);
the solution flow rate of the treatment solution inlet (7) is 50-500 mu L/min;
preferably, the solution flow rate is 100-450 muL/min;
preferably, the solution flow-through rate is 150 to 400. mu.L/min, more preferably 200 to 350. mu.L/min, and most preferably 250 to 300. mu.L/min.
Preferably, the reaction chip (1) and the filter (10) are made of polydimethylsiloxane, polymethyl methacrylate, glass, silicon, polycarbonate, polypropylene, polystyrene, cyclic olefin copolymer or cyclic olefin polymer, preferably PMMA, PC, PP.
Preferably, the filter membrane (11) is made of parylene (parylene), Polycarbonate (PC), polyethylene terephthalate (TETP), Polyethersulfone (PES) or Polyetherimide (PEI), preferably, xylene, polycarbonate or polyethylene terephthalate.
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