CN111289762A - Micro-fluidic chip sample adding device and testing method - Google Patents
Micro-fluidic chip sample adding device and testing method Download PDFInfo
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- CN111289762A CN111289762A CN202010255113.1A CN202010255113A CN111289762A CN 111289762 A CN111289762 A CN 111289762A CN 202010255113 A CN202010255113 A CN 202010255113A CN 111289762 A CN111289762 A CN 111289762A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/00584—Control arrangements for automatic analysers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
- G01N35/1004—Cleaning sample transfer devices
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
- G01N35/1009—Characterised by arrangements for controlling the aspiration or dispense of liquids
- G01N35/1016—Control of the volume dispensed or introduced
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N2035/00178—Special arrangements of analysers
- G01N2035/00237—Handling microquantities of analyte, e.g. microvalves, capillary networks
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N2035/00178—Special arrangements of analysers
- G01N2035/00277—Special precautions to avoid contamination (e.g. enclosures, glove- boxes, sealed sample carriers, disposal of contaminated material)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
- G01N2035/1027—General features of the devices
- G01N2035/1034—Transferring microquantities of liquid
Abstract
The invention discloses a microfluidic chip sample adding device, which comprises a conical sample adding pool, wherein the bottom of the sample adding pool is tightly contacted with a chip inlet; the top of the sample adding pool is provided with a pressing piece, the bottom of the sample adding pool is provided with a small hole, and the side wall of the sample adding pool is provided with a liquid discharge pipe; and the pressing piece is provided with a pressure discharge pipe and a sample adding header pipe, and the sample adding header pipe is respectively connected with the antibody collecting pipe, the buffer solution collecting pipe and the air pump. Its advantage lies in, can compromise the application of sample requirement of the blood sample of large capacity and micro-antibody reagent, and the bubble in the automatic discharge pipeline before the chip entry improves experiment precision and degree of automation.
Description
Technical Field
The invention belongs to the field of microfluidic chips, and particularly relates to a sample adding device which is connected with a chip inlet and can automatically complete gas-liquid conversion and reagent replacement.
Background
Due to the advantages of high efficiency, portability and integration, the microfluidic technology has huge application prospects in aspects of mass spectrometry, antigen detection, cell culture, cell sorting, drug analysis and the like, and is expected to change detection and analysis items which are originally high in price, large in size and high in environmental requirements to portability, low in cost and bedside. However, one test item often requires sequential addition of multiple chemical reagents for incubation and reaction, which involves removing residual reagents from the inlet line and washing or replacing with new reagents after a reaction is completed. Looking up patents and literature, it can be known that there are many ways for reagent change, and the time and the volume of application of sample can be finely tuned according to specific conditions in a flexible way in manual application of sample, guarantees the seamless connection of reagent conversion, but has high requirements for the professional level of operating personnel, can not realize automation, and is multipurpose for the laboratory scene. The other solution is computer-based automatic sample adding by a machine, and the consumption of the microfluidic liquid is in micro-liter or nano-scale, so that high-precision equipment such as induction identification and motion control and complex computer operation are required, the equipment cost and environmental requirements are greatly improved, the advantages of low cost and low reagent loss of the microfluidic chip are weakened, and the bedside application is difficult to realize. The on-chip control is mainly realized by the valves on the multilayer structure, the manufacturing process of the on-chip valve structure is complex, the yield is low, the pressure resistance, the blocking property and the stability are poor, the standardized and large-scale production cannot be realized, and the popularization is not facilitated. On the other hand, when the liquid descends along the side wall of the container, the liquid drops due to the friction and surface tension, and when the diameter of the liquid drops is larger than the inner diameter of the container, the liquid seal effect is generated on the gas at the lower section. This often leads to the introduction of air bubbles during the loading process of the microfluidic chip, which is still a significant reason for the limitation of the automation. Therefore, how to conveniently realize the conversion of multiple reagents becomes a technical bottleneck for converting the microfluidic chip into automatic and standardized equipment.
Disclosure of Invention
In order to overcome the problems, the invention provides a device and a method which can solve the problem of gas-liquid conversion in a sample adding pool, ensure less residual quantity, effectively prevent air bubbles from being introduced and realize automatic multi-reagent sequential sample adding. The technical proposal is that the method comprises the following steps,
a microfluidic chip sample adding device comprises a conical sample adding pool, wherein the bottom of the sample adding pool is tightly contacted with a chip inlet; the top of the sample adding pool is provided with a pressing piece, the bottom of the sample adding pool is provided with a small hole, and the side wall of the sample adding pool is provided with a liquid discharge pipe; and the pressing piece is provided with a pressure discharge pipe and a sample adding header pipe, and the sample adding header pipe is respectively connected with the antibody collecting pipe, the buffer solution collecting pipe and the air pump.
Furthermore, the bottom of the sample adding pool is provided with a connecting pipe, and the connecting pipe is in close contact with the chip inlet.
Furthermore, electromagnetic valves are arranged at the outlet of the liquid discharge pipe, the pressure discharge pipe, the outlet of the air pump, the antibody collecting pipe, the buffer liquid collecting pipe and the chip; at least one antibody collection tube.
Furthermore, a first electromagnetic valve and a second electromagnetic valve are arranged on the liquid discharge pipe and the pressure discharge pipe; a third electromagnetic valve is arranged at the outlet of the air pump; when the antibody collecting tube is one, a fourth electromagnetic valve is arranged on the antibody collecting tube; the buffer solution collecting pipe is provided with a fifth electromagnetic valve; and a sixth electromagnetic valve is arranged at the outlet of the chip.
Furthermore, the vertical distance from the bottom of the liquid discharge pipe to the bottom of the sample adding pool is 1-3 mm.
Further, the vertical distance between the bottom of the pressure discharge pipe and the bottom of the pressing piece is not larger than the vertical distance between the bottom of the sample loading manifold and the bottom of the pressing piece.
A microfluidic chip sample application testing method is characterized by comprising the following steps:
s1, exhausting before an experiment begins; firstly, a first electromagnetic valve, a second electromagnetic valve, a third electromagnetic valve, a fourth electromagnetic valve and a fifth electromagnetic valve are in a closed state, and a sixth electromagnetic valve is in an open state; opening a fifth electromagnetic valve during air exhaust, and exhausting the chip after the buffer solution enters the inlet of the chip through the sample adding pool;
s2, discharging liquid from the sample adding pool; closing the fifth electromagnetic valve and the sixth electromagnetic valve; opening the first electromagnetic valve and the third battery valve, when the liquid level of the buffer solution in the sample adding pool is lower than the bottom of the liquid discharge pipe, allowing gas to enter the liquid discharge pipe, stopping the liquid level of the buffer solution from descending, finishing liquid discharge, and closing the third electromagnetic valve and the first electromagnetic valve;
s3, adding a blood sample; opening a second battery valve, opening a pressing piece, directly adding the blood sample into the sample adding pool, and closing the pressing piece; closing the second battery valve, opening the third battery valve, driving the blood sample to enter the chip inlet at constant pressure, and then entering the chip detection area;
s4, washing the sample adding pool; closing the third cell valve, opening the fifth cell valve, squeezing the blood sample to the chip detection area by the buffer solution, and repeating S2;
s5, adding an antibody; opening a second electromagnetic valve and a fourth electromagnetic valve, and injecting the antibody into the sample adding pool; opening a third battery valve and a sixth electromagnetic valve, driving the antibody to a chip detection area, closing all the electromagnetic valves, and incubating in a dark place; repeating the step S2 after the incubation is finished, and draining liquid;
s6, washing the chip; and opening the second electromagnetic valve and the fifth electromagnetic valve, enabling the buffer solution to enter the sample adding pool, opening the third electromagnetic valve and the sixth electromagnetic valve, and washing the unbound antibody in the chip.
Further, in step S3, the blood sample can be continuously added at a preset speed by using a syringe pump.
Further, in step S4, the antibody incubation staining is performed by any one of flowing, static or shaking.
Advantageous effects
The invention provides a novel sample adding device with an external inlet, which can discharge liquid in a sample adding pool through a bypass in an air pressure driving mode, then add a new reagent to finish liquid replacement, and the amount of the residual liquid is flexible and controllable. Therefore, the designed sample adding device does not depend on a sensor and motion control to realize accurate control of the liquid level in the sample adding pool, solves the problem of gas-liquid conversion in the sample adding pool by utilizing the funnel principle, and can effectively prevent air bubbles from being introduced while ensuring less residual quantity, thereby realizing automatic multi-reagent sequential sample adding. And the device is connected and air pressure drive based on the mounting completely, low cost, easily operation, easily implement more and promote.
Drawings
FIG. 1 is a schematic structural diagram of the present application;
wherein: 1-sample adding pool; 2-pressing piece; 3-a liquid discharge pipe; 4-pressure discharge pipe; 5-loading manifold; 6-chip detection area; 7-an air pump; 8-buffer solution; 9-a syringe pump; 10-connecting pipe; 11-chip inlet; 12-chip outlet; 13-sixth battery valve; 14-a second battery valve; 15-a first solenoid valve; 16-a fourth solenoid valve; 17-a fifth solenoid valve; 18-a third solenoid valve; d 1-vertical distance from the bottom of the drain to the bottom of the sample well; d 2-vertical distance between the bottom of the pressure bar and the bottom of the compression element; d 3-vertical distance between the bottom of the loading manifold to the bottom of the compression member.
Detailed Description
The following further description of the technology will be provided in conjunction with fig. 1 and the specific embodiments to aid in understanding the present invention.
A microfluidic chip sample adding device comprises a conical sample adding pool 1, wherein the bottom of the sample adding pool 1 is tightly contacted with a chip inlet 11; the top of the sample adding pool 1 is provided with a pressing piece 2, the bottom is provided with a small hole, and the side wall of the sample adding pool is provided with a liquid discharge pipe 3; the pressing piece 2 is provided with a pressure discharge pipe 4 and a sample adding header pipe 5, and the sample adding header pipe 5 is respectively connected with an antibody collecting pipe, a buffer solution collecting pipe and an air pump 7; electromagnetic valves are arranged at the positions of the liquid discharge pipe 3, the pressure discharge pipe 4, the outlet of the air pump 7, the antibody collecting pipe, the buffer liquid collecting pipe and the chip outlet 11; at least one antibody collection tube is provided, a plurality of antibody collection tubes are provided in the embodiment, and the antibody collection tubes are provided with battery valves, so that various samples can be added into the sample adding pool 1 according to experiment requirements, and a specific embodiment takes one antibody collection tube as an example. The drain pipe 3 and the drain pipe 4 are provided with a first electromagnetic valve 15 and a second electromagnetic valve 14; the outlet of the air pump 7 is provided with a third electromagnetic valve 18; the antibody collecting tube is provided with a fourth electromagnetic valve 16 which is connected with the injection pump 9; the buffer solution collecting tube is connected with the buffer solution reagent bottle through a fifth electromagnetic valve 17; a sixth electromagnetic valve 17 is arranged at the chip outlet 11. The buffer solution reagent bottle is positive pressure, the fifth electromagnetic valve 17 is opened and then drives the buffer solution to enter the sample adding pool 1 by means of positive pressure, and the buffer solution adopts phosphate buffer salt solution.
One end of the liquid discharge pipe 3 extending into the sample adding pool 1 is a stainless steel thin pipe with the inner diameter of 0.2mm, and the other end of the liquid discharge pipe is connected with a waste liquid pool through a polytetrafluoroethylene thin pipe and is controlled by a first electromagnetic valve 15. The vertical distance d1 between the bottom of the drain 4 and the bottom of the sample addition well 1 is 1-3mm, preferably 2 mm.
One end of the pressure discharge pipe 4 adopts a stainless steel pipe with the inner diameter of 0.75mm, penetrates through the pressing piece 2 from the top to enter the sample adding pool 1, and is fixed on the pressing piece 2 by means of tight combination with the sealing unit, and the other end of the pressure discharge pipe 4 is communicated with the external atmospheric environment through a polytetrafluoroethylene tubule and is controlled by the second electromagnetic valve 14, so that the pressure discharge pipe is opened when liquid is replaced, and the balance of the internal pressure and the external pressure of the sample adding pool 1 is maintained. The vertical distance d2 between the bottom of the pressure discharge tube and the bottom of the pressure element is not more than the vertical distance d3 between the bottom of the loading manifold and the bottom of the pressure element, wherein d2 is 2 mm; d3 is 5mm, which is beneficial to reducing the pollution probability caused by contacting with reagent during exhausting.
The small hole at the bottom of the sample adding pool 5 is connected with a connecting pipe 10, and the connecting pipe 10 is tightly contacted with the chip inlet 11; the sample adding pool 5 and the connecting pipe 10 can be of an integral structure, both adopt hydrophobic materials, preferably polytetrafluoroethylene tubules, and the diameter of the connecting pipe 10 is 0.5-1mm, preferably 0.7 mm.
A microfluidic chip sample adding test method comprises the following specific steps:
s1, exhausting before an experiment begins; firstly, the first electromagnetic valve 15, the second electromagnetic valve 14, the third electromagnetic valve 18, the fourth electromagnetic valve 16 and the fifth electromagnetic valve 17 are in a closed state, and the sixth electromagnetic valve 13 is in an open state; when exhausting, the fifth electromagnetic valve 17 is opened, and after the buffer solution enters the chip inlet 11 through the sample adding pool 1, the exhausting time is 5 minutes;
s2, discharging liquid from the sample adding pool; closing the fifth electromagnetic valve 17 and the sixth electromagnetic valve 13; opening the first electromagnetic valve 15 and the third battery valve 18, adjusting the pressure of the air pump to 10Kpa, when the liquid level of the buffer solution in the sample adding pool 1 is lower than the bottom of the liquid discharge pipe 3, allowing the air to enter the liquid discharge pipe 3, stopping the liquid level of the buffer solution from descending, finishing liquid discharge, and closing the third electromagnetic valve 18 and the first electromagnetic valve 15;
s3, adding a blood sample; opening the second battery valve 14, opening the pressing piece 2, directly adding the blood sample into the sample adding pool 1, and closing the pressing piece 2; closing the second battery valve 14 and opening the third battery valve 18 to drive the blood sample into the chip inlet 11 at a constant pressure set at 20Kpa for 25 minutes; or continuously adding the blood sample at a preset speed by adopting a syringe pump;
s4, washing the sample adding pool; closing the third cell valve 18, opening the fifth cell valve 17, washing for 5 minutes, squeezing the blood sample into the chip detection zone 6 with the buffer solution, and then repeating S2;
s5, adding an antibody; opening the second electromagnetic valve 14 and the fourth electromagnetic valve 16, and injecting the antibody into the sample adding pool 1, wherein the buffer solution is replaced by the antibody; opening a third battery valve 18 and a sixth electromagnetic valve 13, pushing the antibody into the chip detection area 6, closing all the electromagnetic valves, incubating for 1 hour in a dark place, and performing antibody incubation dyeing by adopting one of flowing, static and oscillating modes, wherein static dyeing is selected in the example; repeating the step S2 after the incubation is finished;
s5, washing the chip; opening the second electromagnetic valve 14 and the fifth electromagnetic valve 17, allowing the buffer solution to enter the sample adding cell 1, opening the third electromagnetic valve 18 and the sixth battery valve 13, washing the unbound antibodies in the chip, setting the pressure of the air pump 7 at 20Kpa for 20 minutes, and repeating the step S2.
The liquid sample of the invention is composed of human or animal blood samples, nematode samples, cell suspension, various chemical reagents and the like, under the condition that the detection item is the capture and identification of circulating tumor cells, the sample to be detected is whole blood, and the chemical reagents comprise buffer solution and antibodies. In the present example, the liquid sample is a blood sample of a colon cancer patient, which is collected and stored in an EDTA anticoagulant blood collection tube, the buffer solution is Phosphate Buffered Saline (PBS), and the antibody is a specific fluorescent antibody mouse anti-human EpCAM-FITC, mouse anti-human CD45-PE/CY5, DAPI.
Of course, the above description is not intended to limit the present technology, and the present technology is not limited to the above examples, and those skilled in the art may make variations, modifications, additions or substitutions within the spirit and scope of the present invention. The terms front, rear, left, and right are used for clarity and should not be construed as limiting the technology of the present application.
Claims (9)
1. The microfluidic chip sample adding device is characterized by comprising a conical sample adding pool, wherein the bottom of the sample adding pool is tightly contacted with a chip inlet; the top of the sample adding pool is provided with a pressing piece, the bottom of the sample adding pool is provided with a small hole, and the side wall of the sample adding pool is provided with a liquid discharge pipe; and the pressing piece is provided with a pressure discharge pipe and a sample adding header pipe, and the sample adding header pipe is respectively connected with the antibody collecting pipe, the buffer solution collecting pipe and the air pump.
2. The microfluidic chip sample adding device according to claim 1, wherein a connecting tube is disposed at the bottom of the sample adding cell, and the connecting tube is in close contact with the chip inlet.
3. The microfluidic chip sample adding device according to claim 1, wherein the liquid discharge pipe, the pressure discharge pipe, the air pump outlet, the antibody collection pipe, the buffer solution collection pipe and the chip outlet are respectively provided with an electromagnetic valve; at least one antibody collection tube.
4. The microfluidic chip sample adding device according to claim 1, wherein the drain pipe and the pressure discharge pipe are provided with a first solenoid valve and a second solenoid valve; a third electromagnetic valve is arranged at the outlet of the air pump; when the antibody collecting tube is one, a fourth electromagnetic valve is arranged on the antibody collecting tube; the buffer solution collecting pipe is provided with a fifth electromagnetic valve; and a sixth electromagnetic valve is arranged at the outlet of the chip.
5. The microfluidic chip sample application device according to claim 1, wherein the vertical distance from the bottom of the drain to the bottom of the sample application well is 1-3 mm.
6. The microfluidic chip loading device according to claim 1, wherein the vertical distance from the bottom of the pressure discharge tube to the bottom of the pressing member is not greater than the vertical distance from the bottom of the loading manifold to the bottom of the pressing member.
7. A microfluidic chip sample application testing method is characterized by comprising the following steps:
s1, exhausting before an experiment begins; firstly, a first electromagnetic valve, a second electromagnetic valve, a third electromagnetic valve, a fourth electromagnetic valve and a fifth electromagnetic valve are in a closed state, and a sixth electromagnetic valve is in an open state; opening a fifth electromagnetic valve during air exhaust, and exhausting the chip after the buffer solution enters the inlet of the chip through the sample adding pool;
s2, discharging liquid from the sample adding pool; closing the fifth electromagnetic valve and the sixth electromagnetic valve; opening the first electromagnetic valve and the third electromagnetic valve, when the liquid level of the buffer solution in the sample adding pool is lower than the bottom of the liquid discharge pipe, allowing gas to enter the liquid discharge pipe, stopping the liquid level of the buffer solution from descending, finishing liquid discharge, and closing the third electromagnetic valve and the first electromagnetic valve;
s3, adding a blood sample; opening a second battery valve, opening a pressing piece, directly adding the blood sample into the sample adding pool, and closing the pressing piece; closing the second battery valve, opening the third battery valve, driving the blood sample to enter the chip inlet at constant pressure, and then entering the chip detection area;
s4, washing the sample adding pool; closing the third cell valve, opening the fifth cell valve, squeezing the blood sample to the chip detection area by the buffer solution, and repeating S2;
s5, adding an antibody; opening a second electromagnetic valve and a fourth electromagnetic valve, and injecting the antibody into the sample adding pool; opening a third battery valve and a sixth electromagnetic valve, driving the antibody to a chip detection area, closing all the electromagnetic valves, and incubating in a dark place; repeating the step S2 after the incubation is finished, and draining liquid;
s6, washing the chip; and opening the second electromagnetic valve and the fifth electromagnetic valve, enabling the buffer solution to enter the sample adding pool, opening the third electromagnetic valve and the sixth electromagnetic valve, and washing the unbound antibody in the chip.
8. The method for testing sample application of a microfluidic chip as claimed in claim 7, wherein in step S3, the blood sample can be continuously applied at a predetermined speed by using a syringe pump.
9. The method for testing sample application on a microfluidic chip according to claim 7, wherein in step S4, the antibody incubation staining is performed in any one of a flowing, static or oscillating manner.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010255113.1A CN111289762A (en) | 2020-04-02 | 2020-04-02 | Micro-fluidic chip sample adding device and testing method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111929460A (en) * | 2020-08-19 | 2020-11-13 | 河南科技大学 | Sampling, chip and liquid inlet control device suitable for microfluidic automatic detection |
| WO2022062995A1 (en) * | 2020-09-27 | 2022-03-31 | 青岛海尔电冰箱有限公司 | Microfluidic testing system and control method therefor, and refrigerator |
-
2020
- 2020-04-02 CN CN202010255113.1A patent/CN111289762A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111929460A (en) * | 2020-08-19 | 2020-11-13 | 河南科技大学 | Sampling, chip and liquid inlet control device suitable for microfluidic automatic detection |
| CN111929460B (en) * | 2020-08-19 | 2024-03-01 | 河南科技大学 | Sampling, chip and liquid inlet control device suitable for microfluidic automatic detection |
| WO2022062995A1 (en) * | 2020-09-27 | 2022-03-31 | 青岛海尔电冰箱有限公司 | Microfluidic testing system and control method therefor, and refrigerator |
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