CN112023989A - Microfluidic detection integrated chip and sample detection method - Google Patents
Microfluidic detection integrated chip and sample detection method Download PDFInfo
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- CN112023989A CN112023989A CN201910478285.2A CN201910478285A CN112023989A CN 112023989 A CN112023989 A CN 112023989A CN 201910478285 A CN201910478285 A CN 201910478285A CN 112023989 A CN112023989 A CN 112023989A
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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
- 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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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
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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
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0887—Laminated structure
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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
Abstract
The invention provides a microfluidic detection chip and a method for detecting a sample, which comprise a substrate, a cover plate and a detection area positioned on the substrate, wherein the substrate is also provided with a first liquid storage tank, a second liquid storage tank, a waste liquid tank, a first flow channel, a second flow channel, a third flow channel and a fourth flow channel; the cover plate seals the first liquid storage tank, the second liquid storage tank, the waste liquid tank, the first flow channel, the second flow channel, the third flow channel and the fourth flow channel; the cover plate covering the first liquid storage tank, the second liquid storage tank and the waste liquid tank is respectively provided with a first opening, a second opening and a third opening; the first liquid storage tank is in liquid communication with the detection area through a first flow channel and a third flow channel, the second liquid storage tank is in liquid communication with the detection area through a second flow channel and a third flow channel, and the detection area is in liquid communication with the waste liquid tank through a fourth flow channel; the liquid in the first liquid storage tank and the liquid in the second liquid storage tank sequentially flow through the detection area under the action of gravity and reach the waste liquid tank by adjusting the surface tension of the first flow channel, the second flow channel, the third flow channel and the fourth flow channel. The fluid flow can be driven without external power.
Description
Technical Field
The invention belongs to the technical field of medical diagnosis articles, and relates to a microfluidic detection integrated chip and a method for detecting a sample.
Background
The development of the portable-of-care (point-of-care) industry has been driven by the advent of microfluidic technology in the fields of biomedical analysis, disease diagnosis. Compared with the traditional quick diagnosis technology, the microfluidic chip has the following advantages that for example, in the traditional POCT detection equipment, liquid such as calibration liquid, quality control liquid and the like are externally arranged in the equipment, so that the detection equipment has the problems of large volume, complex pipelines, difficult maintenance, easy pollution and the like, and in addition, due to the detection principle characteristics of the traditional POCT product, the simultaneous detection of a plurality of indexes is difficult to realize while the rapid and accurate quantitative analysis is carried out, and further, the consumption and the human error of a sample to be detected are increased. On the contrary, the microfluidic detection technology has the greatest advantage that the full-automatic rapid detection of multiple indexes can be simultaneously carried out under the condition of microliter-level blood sample consumption, and accurate results can be obtained. Meanwhile, the microfluidic chip with the size of square centimeter can contain all functional units of conventional laboratories such as quantitative sample introduction, mixing, reaction, calibration, reagent storage, detection, waste liquid collection and the like, and the characteristics of full-automatic operation and the like realize the new generation of POCT products with high integration, energy conservation, convenience and small error.
Fluid control is the core of microfluidic chip design, and all functions of the microfluidic chip are realized by means of the unique design of the microchannel network. Taking the microfluidic products of several foreign trade-leading enterprises as examples, the microfluidic chip power is divided from fluid power, and can be air pump (US8986527B2), injector (US7842234B), external force extrusion (US5821399A) and centrifugal force (US20110124128A 1).
The chip using the air pump as power has the following characteristics: firstly, for more than two fluid control, the air pump requires a more complex chip micro-channel network design, and realizes sequential flow control of the fluid by relatively more valve designs. The complicated structure leads to the characteristics that the volume of the instrument is often larger, and the chip processing requirement is high in cost. Moreover, the air pump as power can increase the probability of generating air bubbles in the fluid, and the generated air bubbles can prevent the sensor from working normally.
The chip taking the injector as power firstly requires the injector to be in sealed butt joint with the sample adding port of the chip in operation, so that the operation is difficult and human errors are easily introduced; and secondly, the risk of contamination of the sample or instrument due to operational errors.
In the fluid pushing mode of external force extrusion, the force generated by extrusion deformation is small, so that the size of a chip is required to be small, and the difficulty in processing and assembling the chip can be directly caused by the change of micro-size, so that economic loss is caused.
The chip products driven by the centrifugal force are fewer, the centrifugal force driving chip can realize high-integration detection to the greatest extent in principle, the purification of samples in the chip is realized, and the advantages of equal sample division are realized.
With the rapid market demand for in vitro diagnosis, the advantages of microfluidic technology in vitro diagnostic applications are gradually highlighted, and the microfluidic technology is receiving more and more attention from the industry. In the application of the microfluidic chip, the sequential flow of various fluids and the preservation and flow control of the liquid in the test strip are the common technical difficulties at present.
Disclosure of Invention
The invention provides a microfluidic integrated chip for controlling fluid flow only by self gravity. The microfluidic integrated chip can finish automatic transmission and detection of a plurality of fluids without additional power equipment such as a micro pump, an injection pump, an extrusion device, a centrifugal force device and the like.
Specifically, the microfluidic detection integrated chip provided by the invention comprises a substrate, a cover plate and a detection area positioned on the substrate, wherein the substrate is also provided with a first liquid storage tank, a second liquid storage tank, a waste liquid tank, a first flow channel, a second flow channel, a third flow channel and a fourth flow channel; the cover plate seals the first liquid storage tank, the second liquid storage tank, the waste liquid tank, the first flow channel, the second flow channel, the third flow channel and the fourth flow channel; the cover plate covering the first liquid storage tank, the second liquid storage tank and the waste liquid tank is respectively provided with a first opening, a second opening and a third opening; the first liquid storage tank is in liquid communication with the detection area through a first flow channel and a third flow channel, the second liquid storage tank is in liquid communication with the detection area through a second flow channel and a third flow channel, and the detection area is in liquid communication with the waste liquid tank through a fourth flow channel; the liquid in the first liquid storage tank and the liquid in the second liquid storage tank sequentially flow through the detection area under the action of gravity and reach the waste liquid tank by adjusting the surface tension of the first flow channel, the second flow channel, the third flow channel and the fourth flow channel.
Generally, in the microfluidic detection integrated chip, the first liquid storage tank and the second liquid storage tank sequentially flow to the detection area and finally reach the waste liquid tank by adjusting the flow rate of the liquids in the two liquid storage tanks. In the invention, the flow rate of the liquid in the first liquid storage tank and the second liquid storage tank is controlled by adjusting the surface tension of the flow channel.
In some preferred embodiments, the first flow channel and the third flow channel are subjected to hydrophilic treatment; and performing hydrophobic treatment on the second flow channel and the fourth flow channel.
In some preferred embodiments, the first channel width is greater than the second channel width; the third flow channel width is greater than the fourth flow channel width.
In some preferred embodiments, the length of the first flow channel is less than the length of the second flow channel.
In some preferred embodiments, the first flow channel is a straight structure and the second flow channel comprises a curved structure.
In some preferred embodiments, the third flow channel is a straight structure and the fourth flow channel comprises a curved structure.
In some preferred embodiments, the first reservoir, the first channel, the third channel and the detection region are disposed on the front surface of the substrate, and the second reservoir, the second channel, the fourth channel and the waste liquid channel are disposed on the back surface of the substrate.
In some preferred embodiments, the cover sheet comprises an upper cover sheet covering the obverse side of the base sheet and a lower cover sheet covering the reverse side of the base sheet; the base plate and the lower cover plate are made of hydrophobic materials, and the upper cover plate is made of hydrophilic materials.
In some preferred embodiments, the substrate is provided with a first through hole for communicating the second flow channel with the third flow channel, and a second through hole for communicating the detection region with the fourth flow channel.
In some preferred embodiments, the end of the detection zone is bent upward. .
In some preferred embodiments, the end of the detection region is bent upward and then extended for a distance and then bent downward to form an inverted "U" structure.
In some preferred embodiments, the detection zone is a curved or bent structure.
In some preferred embodiments, the third flow channel comprises a curved structure.
In some preferred embodiments, the first, second and third openings are provided with seals.
In some preferred embodiments, the electrode sensor is located in the detection zone. In a more preferred embodiment, the detection portion of the electrode sensor is located in the detection zone.
In some preferred embodiments, the liquid in the first reservoir is a calibration liquid; the liquid in the second reservoir is the sample.
In some preferred embodiments, the upper ends of the first reservoir and the first channel are covered with a hydrophobic membrane layer.
The invention also provides a method for detecting a sample by the micro-fluidic detection integrated chip, which comprises a micro-fluidic detection chip, wherein the micro-fluidic detection chip comprises a hydrophobic substrate, a hydrophilic upper cover plate covering the front surface of the substrate and a hydrophobic upper cover plate covering the back surface of the substrate, the front surface of the substrate is provided with a first liquid storage tank, a first flow channel, a third flow channel and a detection area, the back surface of the substrate is provided with a second liquid storage tank, a second flow channel, a fourth flow channel and a waste liquid tank, and the cover plates covering the positions of the first liquid storage tank, the second liquid storage tank and the waste liquid tank are respectively provided with a first opening, a second opening and a; the first liquid storage tank is in liquid communication with the detection area through a first flow channel and a third flow channel, the second liquid storage tank is in liquid communication with the detection area through a second flow channel and a third flow channel, and the detection area is in liquid communication with the waste liquid tank through a fourth flow channel; the first liquid storage tank is sealed with calibration liquid, the detection part of the electrode sensor is positioned in the detection area, and the specific operation steps are as follows:
a, injecting a sample into a second liquid storage tank;
b, vertically or obliquely placing the microfluidic detection chip to enable the first liquid storage tank and the second liquid storage tank to be higher than the detection area and the waste liquid tank;
c, enabling the first opening, the second opening and the third opening to be not closed, enabling the calibration solution in the first liquid storage tank to flow into the first flow channel and enter the detection area after flowing through the third flow channel; meanwhile, the sample in the second liquid storage tank slowly flows into the second flow channel;
d, when the electrode sensor arranged in the detection area detects that the surface of the electrode sensor is completely covered by the calibration solution, closing the third opening, or closing the first opening and the second opening, or closing the first opening, the second opening and the third opening, stopping the flow of the calibration solution and the sample, and detecting the calibration solution by the electrode sensor;
e, after the detection is finished, opening the opening which is closed before, restoring the balance of the air pressure in the flow channel, restoring the flow of the calibration liquid and the sample, and finally enabling the calibration liquid to flow into a waste liquid tank after the calibration liquid flows into a fourth flow channel through a communication detection zone and the second through hole; meanwhile, the sample flows into the third flow channel through the first perforation which is communicated with the second flow channel and the third flow channel and then flows into the detection area;
f, when the electrode sensor in the detection area detects that the surface of the electrode sensor is completely covered by the sample, the third opening is closed, or the first opening and the second opening are closed, or the first opening, the second opening and the third opening are closed, the sample stops flowing, and the electrode sensor detects the sample;
g, after the detection is finished, opening the opening closed in the step f, restoring the balance of air pressure in the flow channel, restoring the flow of the sample, and enabling the sample to flow into the fourth flow channel through the second through hole and finally flow into the waste liquid tank.
On the other hand, the invention also provides another method for detecting a sample by using the microfluidic detection chip, wherein in the steps, only the step c is different from the step d, and specifically, the step c is as follows: the first opening and the third opening are not closed, the second opening is closed, the calibration solution in the first liquid storage tank flows into the first flow channel and flows into the detection area after flowing through the third flow channel, and at the moment, the sample cannot flow into the second flow channel from the second liquid storage tank under the action of pressure difference; and step d: when the electrode sensor arranged in the detection area detects that the surface of the electrode sensor is completely covered by the calibration solution, the third opening or the first opening and the third opening are closed, the calibration solution stops flowing, and the electrode sensor detects the calibration solution.
In some preferred embodiments, in the two methods for detecting a sample by using a microfluidic detection chip, the microfluidic detection chip is vertically or obliquely arranged in the corresponding detection instrument. The detection result of the electrode sensor of the microfluidic detection chip is read and analyzed by the corresponding detection instrument.
In some preferred embodiments, the end of the detection region is bent upward and then extended for a distance and then bent downward to form an inverted "U" structure.
In some preferred embodiments, the upper ends of the first reservoir and the first channel are covered with a hydrophobic membrane layer.
Advantageous effects
(1) The microfluidic detection integrated chip can realize the automatic transmission of a plurality of fluids without external power equipment such as a micropump, an injection pump, an extrusion device, a centrifugal force device and the like in the aspect of fluid driving. The structure of the detection instrument can be simplified, and energy is saved. The generation of air bubbles in the fluid due to the use of an external power source is avoided.
(2) The flow rate and diffusion condition of liquid such as blood in different areas can be controlled by the difference of surface tension and hydrophilicity and hydrophobicity of different areas of the chip. For example, when the flow rate of the blood sample from the second reservoir in the hydrophobic second flow channel is slower than the flow rate of the blood sample in the hydrophilic detection flow channel.
(3) The flow channels are respectively arranged on the front and back surfaces of the base plate, and after the upper and lower cover plates with different hydrophilicity and hydrophobicity are pasted on the front and back surfaces of the base plate in a watertight manner, the hydrophilicity and hydrophobicity of the flow channels of the base plate can be changed correspondingly due to the hydrophilicity and hydrophobicity of the cover plates. In this way, it is easy to manufacture a detection chip having different affinities and affinities in different regions.
(4) The liquid storage tanks and the flow channels are distributed on the two square surfaces of the substrate, so that the use area of the substrate can be reduced, and the miniaturization of the detection chip and the detection instrument is facilitated.
(5) The setting of detection zone "U" type bend can further strengthen the speed reduction effect when calibration solution and sample flow to electrode sensor, makes calibration solution and sample have sufficient time to stay in the detection zone and detects.
Drawings
Fig. 1 is a schematic front view of a substrate of a microfluidic detection integrated chip according to the present invention.
Fig. 2 is a schematic reverse side view of a substrate of the microfluidic detection integrated chip of the present invention.
Fig. 3 is a schematic cross-sectional view of a first through-hole of a microfluidic detection integrated chip according to the present invention.
Fig. 4 is a schematic front view of a substrate of another microfluidic detection integrated chip.
Fig. 5 is a schematic front view of a substrate of another microfluidic detection integrated chip.
Fig. 6 is a schematic front view of a substrate of another microfluidic detection integrated chip.
Fig. 7 is a schematic front view of another structure of a substrate of another microfluidic detection integrated chip.
Fig. 8 is a schematic front view of another structure of a substrate of another microfluidic detection integrated chip.
Detailed Description
In the following detailed description, reference is made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration specific embodiments in which the invention may be practiced. It is not excluded that the invention can also be implemented in other embodiments and that the structure of the invention can be varied without departing from the scope of use of the invention.
In the invention, the micro-fluidic detection chip controls the flow of the liquid by using the surface tension of the flow channel and the self gravity of the fluid. In some embodiments, the surface tension of the flow channels is altered by altering the hydrophilicity and hydrophobicity of the flow channels. In other embodiments, the flow channel surface tension is changed by changing the size of the flow channel. In still other embodiments, the flow channel surface tension is changed by changing the structure of the flow channel. Or the above embodiments are superimposed to change the surface tension of the flow channel, for example, the size or structure of the flow channel is changed while the hydrophilicity and hydrophobicity of the flow channel are changed.
In some specific embodiments, the microfluidic detection integrated chip comprises a substrate, a cover plate and a detection area located on the substrate, wherein the substrate is further provided with a first liquid storage tank, a second liquid storage tank, a waste liquid tank, a first flow channel, a second flow channel, a third flow channel and a fourth flow channel; the cover plate seals the first liquid storage tank, the second liquid storage tank and the waste liquid tank. Wherein, the first flow channel and the third flow channel are subjected to hydrophilic treatment; and performing hydrophobic treatment on the second flow channel and the fourth flow channel. Or the width of the first flow channel is larger than that of the second flow channel; the third flow channel width is greater than the fourth flow channel width. Alternatively, the length of the first flow passage is less than the length of the second flow passage. Alternatively, the first flow channel is a straight structure and the second flow channel comprises a curved structure. Or the third flow channel is a straight line structure, and the fourth flow channel comprises a bent structure.
In other embodiments, the substrate 100 has channels on both sides, the channels on one side have a hydrophilic effect, and the channels on the other side have a hydrophobic effect, wherein the hydrophilic channels are disposed on the side as the front side, the hydrophobic channels are disposed on the side as the back side, and the channels on the two sides are connected through the through holes. In addition, still be equipped with first reservoir 11, second reservoir 12, waste liquid groove 3 and detection zone 2 on the base plate 100, first reservoir 11 and second reservoir 12 are located the both sides of base plate 100 respectively, and the liquid in first reservoir 11 and the second reservoir 12 flows into detection zone 2 through the runner successively, flows into waste liquid groove 3 through the runner after the detection. Specifically, the first reservoir 11 and the detection region 2 are located on the front side of the substrate, and are covered with a hydrophilic upper cover sheet, the second reservoir 12 and the waste liquid tank 3 are located on the back side of the substrate, and are covered with a hydrophobic lower cover sheet. The first reservoir 11, the second reservoir 12, and the waste liquid tank 3 are connected to the atmosphere in order to balance the air pressure in the flow channel of the substrate 100 and to allow the liquid to smoothly flow through the flow channel. The liquid storage tanks and the flow channels are distributed on the front surface and the back surface of the substrate 100, so that the use area of the substrate 100 can be reduced, the miniaturization of a detection chip and a detection instrument is facilitated, and meanwhile, different processing of surface tension of different areas of the chip is easier to realize.
The hydrophilicity and the hydrophobicity of the base plate and the cover plate can be realized by selecting hydrophilic materials and hydrophobic materials, and can also be realized by performing hydrophilic or hydrophobic treatment on the base plate and the cover plate.
The cover sheet may alternatively be a flexible film material or a rigid sheet material.
On the substrate 100, at least one dimension of the cross section of the flow channel is in the micrometer scale (several tens to several hundreds of micrometers), so that the flow of the fluid in the flow channel is influenced by gravity, and is greatly influenced by the hydrophilicity and hydrophobicity of the surface of the flow channel, and the size and shape structure of the flow channel can be changed by those skilled in the art according to the present technology and with the guidance of the present invention.
Specifically, as shown in fig. 1, a substrate 100 of the microfluidic chip is provided with a first reservoir 11, a second reservoir 12, a detection area 2, a waste liquid tank 3, a first opening 110 located on the first reservoir 11, a second opening 120 located on the second reservoir 12, a third opening 30 located on the waste liquid tank 3, an electrode sensor 400 located on the detection area 2, and a first flow channel 41, a second flow channel 42, a third flow channel 43, a fourth flow channel 44, a first perforation 61, and a second perforation 62 located on the substrate 100.
The substrate 100 is made of a hydrophobic material, and the first reservoir 11, the first channel 41, the third channel 43 and the detection region 2 are distributed on the front surface of the substrate 100 and covered by the hydrophilic upper cover 200. The second reservoir 12, the second channel 42, the fourth channel 44 and the waste liquid tank 3 are distributed on the reverse side of the substrate 100, and are covered by the hydrophobic lower cover plate 300.
According to the relative position relationship from top to bottom in fig. 1, the specific distribution relationship on the front surface of the substrate 100 is: the first reservoir 11 is provided with a first opening 110 for communicating with the outside atmosphere, the lower end of the first reservoir 11 is communicated with the upper end of the first flow channel 41, the lower end of the first flow channel 41 is communicated with the upper end of the third flow channel 43, a first perforation 61 penetrating through the substrate 100 is arranged at the joint of the lower end of the first flow channel 41 and the upper end of the third flow channel 43, the first perforation 61 is connected with the tail end of the second flow channel 42, the lower end of the third flow channel 43 is communicated with the upper end of the detection zone 2, the part of the electrode sensor 400 for detecting liquid is arranged in the detection zone 2, and the lower end of the detection zone 2 is provided with a second perforation 62 penetrating through the substrate 100.
According to the relative position relationship from top to bottom in fig. 2, the specific distribution relationship on the reverse side of the substrate 100 is: the second reservoir 12 is provided with a second opening 120 communicating with the outside atmosphere, the lower end of the second reservoir 12 communicates with the upper end of the second flow path 42, the lower end of the second flow path 42 is provided with a first through hole 61 (the first through hole 61, the first flow path 41, the connection relationship between the second flow path 42 and the third flow path can refer to fig. 3), the lower end of the second flow path 42 is provided with a fourth flow path 44, the fourth flow path 44 does not directly communicate with the second flow path 42, the upper end of the fourth flow path 44 is provided with a second through hole 62, so that the detection region 2 and the fourth flow path 44 communicate with each other through the second through hole 62, the lower end of the fourth flow path 44 communicates with the waste liquid tank 3, the waste liquid tank 3 is provided with a third through hole 30 communicating with the outside atmosphere, and the position of the third through hole 30 is preferably set at the uppermost position of the waste liquid tank 3 so that the liquid in the substrate 100 does not flow out of the.
The upper end in the embodiment may be understood as a front end having the same meaning, and the upper end and the front end may be replaced with each other. Likewise, the lower end and the distal end may be replaced with each other.
Preferably, the first flow channel 41, the second flow channel 42, the third flow channel 43, the fourth flow channel 44 and the detection region 2 have a width of 200-. Specifically, for example, the thickness of the substrate is 0.4 to 5 millimeters (mm), and the width and depth of the first flow channel 41, the second flow channel 42, the third flow channel 43, the fourth flow channel 44, and the detection section 2 are 400 μm and 300 μm, respectively. The widths and depths of the first flow channel 41, the second flow channel 42, the third flow channel 43, the fourth flow channel 44 and the detection region 2 may be continuously (discontinuously) varied within a limited range, or may be maintained at a certain value.
The initial substrate 100 may be filled with the calibration solution for calibrating the electrode sensor 400 in the first reservoir 11, and the first opening 110 is sealed by a sealing member for preventing the calibration solution from flowing from the reservoir into the flow path. The seal may be a pierceable membrane or may be a removable plug, which is broken or removed in use. The second opening 120 can be selectively sealed by a sealing member, and when the sealing member is a thin film, the sample can be injected into the second reservoir 12 through a syringe after the thin film needs to be punctured when the sealing member is sealed by the thin film; when the sealing element is a plug body such as a rubber plug which can be pulled out, after the plug body is removed, a sample is injected into the second liquid storage tank 12 through a syringe; when the second opening 120 is not sealed by a seal, the sample can be directly injected into the second reservoir 12 through the second opening 120. The third opening 30 may optionally be covered by a seal, which may be removed or broken in use. By sealing these openings, the calibration liquid in the substrate 100 is not communicated with the outside atmosphere, and the shelf life thereof is increased. Secondly, the calibration liquid can be prevented from flowing into the flow channel in advance before use (due to the airtight space in the substrate 100, when a small part of the calibration liquid flows into the flow channel from the liquid storage tank, air in the space below the liquid is compressed, so that air pressure rises, air pressure in the liquid storage tank is reduced, and the air pressure difference can prevent the calibration liquid from flowing further in the flow channel). Thirdly, minute contaminants such as dust can be prevented from entering the substrate 100.
In addition, the resistance to flow can be enhanced by changing the length, shape or hydrophilicity/hydrophobicity of the second flow channel 42, and the flow time of the sample in the second flow channel 42 can be prolonged.
When the chip is used for detecting a sample, the substrate 100 is vertically or obliquely placed in a corresponding detection instrument, at this time, the first reservoir and the second reservoir are higher than the detection area and the waste liquid tank, the first opening 110, the second opening 120 and the third opening 30 are not closed, the sample slowly flows into the second flow channel 42 from the second reservoir 12, and the calibration solution flows into the first flow channel 41 from the first reservoir 12 and continues to flow (the flow rate is significantly faster than the flow rate of the sample). The calibration solution passes through the third flow channel 43 and then enters the detection region 2, while the sample does not enter the third flow channel 43.
When the electrode sensor 400 detects that the surface of the detection portion is completely covered by the calibration solution, the third opening 30 is closed (or the first opening 110 and the second opening 120 are closed, or all the openings are closed), and the calibration solution in the detection area 2 continues to flow for a short distance, so that the air pressure at the front end of the calibration solution is unbalanced with the air pressure at the rear end of the calibration solution, thereby causing a pressure difference and preventing the calibration solution from continuing to flow, and the sample stops flowing under the action of the pressure difference. At this time, the electrode sensor 400 has a proper time to detect the stopped flow of the calibration solution.
After the detection is finished, the opening which is closed before is opened, the air pressure in the flow channel is restored to balance, and the calibration liquid and the sample are restored to flow. The calibration solution finally flows into the waste liquid tank 30 after passing through the second through hole 62. After the sample flows into the hydrophilic third flow channel 43 through the first through hole 61, the flow rate starts to increase under the pulling of the hydrophilic tension. When the electrode sensor 400 detects that the surface of the detection part is completely covered by the sample, the third opening 30 is closed, or the first opening 110 and the second opening 120 are closed, or all the openings are closed, and the sample stops flowing under the action of the air pressure difference, at this time, the electrode sensor 400 has a proper time to detect the sample which stops flowing. After the detection is completed, the opening to be closed is opened, the liquid in the flow channel starts to flow again, and finally all the liquid flows to the waste liquid tank 3 after passing through the second perforation 62 and the fourth flow channel 44 in sequence.
In another detection method, after the substrate 100 is vertically or obliquely placed in the corresponding detection instrument, the first reservoir and the second reservoir are higher than the detection area and the waste liquid groove, the detection instrument closes the second opening 120, but the first opening 110 and the third opening 30 are not closed, and the sample cannot flow in the second channel 42 under the action of pressure difference. The calibration solution flows through the first flow channel 41, passes through the third flow channel 43, and enters the detection region 2.
When the electrode sensor 400 detects that the surface of the detection portion thereof is completely covered with the calibration solution, the third opening 30 is closed (or the first opening 110 is closed, or all the openings are closed), and the calibration solution in the detection area 2 stops flowing due to the pressure difference. At this time, the electrode sensor 400 starts to detect the calibration solution.
After the detection is completed, all the closed openings (including the second opening 120 which is closed at the beginning) are opened, the air pressure in the flow channel is balanced, and the flow of the calibration solution and the sample is started. The calibration solution finally flows into the waste liquid tank 30 after passing through the second through hole 62. The sample flows into the hydrophilic third flow channel 43 through the first perforations 61 and the flow rate starts to increase under tensile pull. When the electrode sensor 400 in the detection area 2 detects that the surface of the detection portion thereof is completely covered with the sample, the third opening 30 is closed (or the first opening 110 and the second opening 120 are closed, or all the openings are closed), the sample stops flowing under the action of the air pressure difference, and the electrode sensor 400 starts to operate. After the detection is finished, the closed opening is opened, the liquid in the flow channel starts to flow again, and finally all the liquid flows to the waste liquid tank 3 after passing through the second perforation 62 and the fourth flow channel 44 in sequence.
Wherein the detection region 2 is communicated with the fourth flow channel 44 with the hydrophobic back surface through the second perforation 62, which can greatly reduce the flow rate of the liquid flowing to the second perforation 62, so that the detection instrument has enough time to seal the corresponding opening, thereby forming a pressure difference to prevent the liquid in the detection region 2 from flowing further, so that the electrode sensor 400 has enough time to complete the detection. Of course, the fourth flow channel 44 and the waste liquid tank 3 may be disposed on the front surface of the substrate 100, and the portion of the fourth flow channel 44 communicating with the detection region 2 needs to be subjected to hydrophobic treatment to slow down the flow rate of the fluid flowing into the fourth flow channel 44. Alternatively, in the case where the volumes of the calibration liquid and the sample are sufficient, the fourth flow path 44 and the waste liquid tank 3 are provided directly on the front surface of the substrate 100 without any other processing.
Further, as shown in FIG. 4, the end of the detection zone 2 is bent upward. More specifically, the end of the detection region 2 shown in fig. 5 extends upward and then extends downward, so that the tail of the detection region 2 protrudes upward to form a shape similar to an inverted "U". When the degree of protrusion is low, as shown in fig. 4, the speed of the liquid flowing to the upward portion may be reduced, thereby allowing the liquid sufficient time to stay at the electrode sensor 400 for closing the corresponding opening. When the degree of the projection is large, as shown in fig. 5, the calibration solution cannot flow through the highest point of the projection into the second through hole 62 under the hydrophilic action, however, after the sample flows into the third flow channel, the gas column between the calibration solution and the sample can be pressed, the calibration solution is indirectly pushed into the waste liquid tank 3, and the sample finally stays at the electrode sensor 400 to complete the detection. Therefore, as long as the time when the sample enters the third flow channel is controlled, it is not necessary to close the corresponding opening to allow the liquid to stay at the electrode sensor 400.
In a preferred embodiment, the third flow channel and the detection area may form an arc-shaped curve, as shown in fig. 4 and 5, to slow down the flow rate of the liquid when the third flow channel is in a straight structure, so that the liquid slowly and sufficiently flows through the detection area, thereby ensuring the detection success rate.
Alternatively, as shown in FIG. 6, the detection zone 2 may have a bend relative to the third flow channel, with the end of the detection zone 2 extending upward and the second perforation 62 located at the end of the extension, thereby allowing the liquid to reduce its flow rate at the extension or to halt its flow without subsequent compression by the gas column. Since the second through hole 62 is located at the end extending upward, not at the end extending downward as shown in fig. 4 and 5, a small amount of the calibration solution may flow back to the electrode sensor 400 after the calibration solution is pushed into the waste liquid tank 3 in some cases, and the subsequent detection may be affected.
As shown in fig. 7, in order to prevent the liquid in the first reservoir tank 11 from entering the first flow channel 41 too fast, a bubble or a gas column is formed, which affects the subsequent detection. A hydrophobic film 201 may be coated on the upper ends of the first reservoir 11 and the first flow channel 41, so that the inlets on the upper ends of the first reservoir 11 and the first flow channel 41 are hydrophobic, thereby allowing the liquid to flow into the first flow channel 41 at a slow speed and avoiding the formation of bubbles or air columns in the calibration solution.
In other embodiments, the residence time of the liquid in the detection zone 2 is increased as much as possible by merely changing the second perforations 62 (e.g., the size, roughness, hydrophobicity, etc. of the perforations) and/or the fourth flow channel 44 (e.g., the thickness, roughness, hydrophobicity, etc. of the flow channel) without using a closed-open approach.
The detection method in the detection zone of the present invention may be electrochemical detection, or may be a turbidity method, a fluorescence method, a chemiluminescence method, a scattering method, or the like.
The microfluidic detection chip of the invention can perform semi-quantitative or qualitative detection besides quantitative detection by using the electrode sensor 400 for electrochemical detection. For example, one or more test strips (either blank test strips or test strips with pre-added reagents) are fixed on the detection area 2, and after a calibration solution or a sample flows through the detection flow channel and contacts with the test strips, the reagent reacts with the sample to generate a color change, and then the detection result is obtained through instrument or artificial observation.
In order to cooperate with other detection methods, the skilled person needs to replace the calibration solution with other detection reagents, such as washing solutions not used for reaction with the sample, or enzymes, lysis solutions, antibodies, fluorescent reagents, etc. that take part in the reaction. Taking an antibody (antibody one) containing a signal marker as an example, when the detection area 2 does not have the electrode sensor 400, but an antibody (antibody two) capable of specifically binding with an antigen to be detected in the sample is fixed, the antibody one is added into the second reservoir 12, the sample is added into the first reservoir 11, after the sample flows into the detection area 2, the antigen to be detected in the sample is captured by the antibody two, then the antibody one flows into the detection area 2 and specifically binds with the antigen captured by the antibody two, and finally, the signal intensity of the antibody one marker in the detection area 2 is measured by a relevant device, so as to obtain a final measurement result. Further, as shown in fig. 8, a third reservoir 13 for storing a washing liquid may be added, and after the washing liquid flows into the detection region 2, the unbound first antibody is washed away, so as to prevent the label of the unbound first antibody from interfering with the detection, thereby improving the accuracy of the detection.
Claims (12)
1. A micro-fluidic detection integrated chip is characterized by comprising a substrate, a cover plate and a detection area positioned on the substrate, wherein the substrate is also provided with a first liquid storage tank, a second liquid storage tank, a waste liquid tank, a first flow channel, a second flow channel, a third flow channel and a fourth flow channel; the cover plate seals the first liquid storage tank, the second liquid storage tank, the waste liquid tank, the first flow channel, the second flow channel, the third flow channel and the fourth flow channel; the cover plate covering the first liquid storage tank, the second liquid storage tank and the waste liquid tank is respectively provided with a first opening, a second opening and a third opening; the first liquid storage tank is in liquid communication with the detection area through a first flow channel and a third flow channel, the second liquid storage tank is in liquid communication with the detection area through a second flow channel and a third flow channel, and the detection area is in liquid communication with the waste liquid tank through a fourth flow channel; the liquid in the first liquid storage tank and the liquid in the second liquid storage tank sequentially flow through the detection area under the action of gravity and reach the waste liquid tank by adjusting the surface tension of the first flow channel, the second flow channel, the third flow channel and the fourth flow channel.
2. The microfluidic detection integrated chip of claim 1, wherein the first flow channel and the third flow channel are subjected to hydrophilic treatment; and performing hydrophobic treatment on the second flow channel and the fourth flow channel.
3. The microfluidic detection integrated chip of claim 2, wherein the first reservoir, the first channel, the third channel and the detection region are disposed on the front surface of the substrate, and the second reservoir, the second channel, the fourth channel and the waste liquid channel are disposed on the back surface of the substrate.
4. The microfluidic detection integrated chip of claim 3, wherein the cover plate comprises an upper cover plate covering the front surface of the substrate and a lower cover plate covering the back surface of the substrate; the base plate and the lower cover plate are made of hydrophobic materials, and the upper cover plate is made of hydrophilic materials.
5. The microfluidic detection integrated chip of claim 4, wherein the substrate has a first through hole for communicating the second flow channel with the third flow channel, and a second through hole for communicating the detection region with the fourth flow channel.
6. The microfluidic detection integrated chip of claim 5, wherein the end of the detection region is bent upward, extended for a distance, and bent downward to form an inverted "U" structure.
7. The microfluidic detection integrated chip of claim 6, wherein the detection region has a curved structure or a bent structure.
8. The microfluidic detection integrated chip of claim 5, wherein the upper ends of the first reservoir and the first flow channel are covered with a hydrophobic membrane layer.
9. A method for detecting a sample by a microfluidic detection integrated chip is characterized by comprising a microfluidic detection chip, wherein the microfluidic detection chip comprises a hydrophobic substrate, a hydrophilic upper cover plate covering the front surface of the substrate and a hydrophobic upper cover plate covering the back surface of the substrate, the front surface of the substrate is provided with a first liquid storage tank, a first flow channel, a third flow channel and a detection area, the back surface of the substrate is provided with a second liquid storage tank, a second flow channel, a fourth flow channel and a waste liquid tank, and the cover plates covering the positions of the first liquid storage tank, the second liquid storage tank and the waste liquid tank are all provided with openings; the first liquid storage tank is in liquid communication with the detection area through a first flow channel and a third flow channel, the second liquid storage tank is in liquid communication with the detection area through a second flow channel and a third flow channel, and the detection area is in liquid communication with the waste liquid tank through a fourth flow channel; the first liquid storage tank is sealed with calibration liquid, and the specific operation steps are as follows:
a, injecting a sample into a second liquid storage tank;
b, vertically or obliquely placing the chip to enable the first liquid storage tank and the second liquid storage tank to be higher than the detection area and the waste liquid tank;
c, enabling the first opening, the second opening and the third opening to be not closed, enabling the calibration solution in the first liquid storage tank to flow into the first flow channel and enter the detection area after flowing through the third flow channel; meanwhile, the sample in the second liquid storage tank slowly flows to the second flow channel;
d, when the electrode sensor arranged in the detection area detects that the surface of the electrode sensor is completely covered by the calibration solution, closing the third opening, or closing the first opening and the second opening, or closing the first opening, the second opening and the third opening, stopping the flow of the calibration solution and the sample, and detecting the calibration solution by the electrode sensor;
e, after the detection is finished, opening the opening which is closed before, restoring the balance of the air pressure in the flow channel, restoring the flow of the calibration liquid and the sample, and finally enabling the calibration liquid to flow into a waste liquid tank after the calibration liquid flows into a fourth flow channel through a communication detection zone and the second through hole; meanwhile, the sample flows into the third flow channel through the first perforation which is communicated with the second flow channel and the third flow channel and then flows into the detection area;
f, when the electrode sensor in the detection area detects that the surface of the electrode sensor is completely covered by the sample, the third opening is closed, or the first opening and the second opening are closed, or the first opening, the second opening and the third opening are closed, the sample stops flowing, and the electrode sensor detects the sample;
g, after the detection is finished, opening the opening closed in the step f, restoring the balance of air pressure in the flow channel, restoring the flow of the sample, and enabling the sample to flow into the fourth flow channel through the second through hole and finally flow into the waste liquid tank.
10. The method for detecting the sample by the microfluidic detection integrated chip according to claim 9, wherein in the step c, the first opening and the third opening are not closed, and the second opening is closed, and the calibration solution in the first reservoir flows into the first channel and flows through the third channel to enter the detection area, at this time, the sample cannot flow from the second reservoir to the second channel under the action of the pressure difference; and in the step d, after the electrode sensor arranged in the detection area detects that the surface of the electrode sensor is completely covered by the calibration solution, the third opening or the first and third openings are closed, the calibration solution stops flowing, and the electrode sensor detects the calibration solution.
11. The method for detecting the sample by the microfluidic detection integrated chip according to claim 9 or 10, wherein the tail end of the detection area is bent upwards, then extends for a certain distance and then is bent downwards to form an inverted U-shaped structure.
12. The method for detecting the sample by the microfluidic detection integrated chip according to claim 9 or 10, wherein the upper ends of the first reservoir and the first flow channel are covered with a hydrophobic membrane layer.
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