CN112023989A - Microfluidic detection integrated chip and sample detection method - Google Patents

Abstract

The present invention provides a microfluidic detection chip and a method for detecting a sample, comprising a substrate, a cover sheet and a detection area on the substrate, wherein the substrate is also provided with a first liquid reservoir, a second liquid reservoir and a waste liquid tank, and first, second, third and fourth flow channels; the cover sheet seals the first liquid reservoir, the second liquid reservoir and the waste liquid tank, and the first, second, third and fourth flow channels; the cover sheet covering the first liquid reservoir, the second liquid reservoir and the waste liquid tank is provided with a first, second and third opening respectively; the first liquid reservoir is in liquid communication with the detection area through the first flow channel and the third flow channel, the second liquid reservoir is in liquid communication with the detection area through the second flow channel and the third flow channel, and the detection area is in liquid communication with the waste liquid tank through the fourth flow channel; by adjusting the surface tension of the first, second, third and fourth flow channels, the liquids in the first liquid reservoir and the second liquid reservoir flow through the detection area and reach the waste liquid tank in sequence and in turn under the action of gravity. It can be realized that the fluid flow is driven without external power.

Description

A microfluidic detection integrated chip and method for detecting samples

technical field

The invention belongs to the technical field of medical diagnostic articles, and relates to a microfluidic detection integrated chip and a method for detecting samples.

Background technique

In the fields of biomedical analysis and disease diagnosis, the emergence of microfluidic technology has promoted the development of the portable rapid diagnosis (point-of-care) industry. Compared with the traditional rapid diagnosis technology in the past, the microfluidic chip has the following advantages. For example, in the previous POCT detection equipment, the calibration liquid, quality control liquid and other liquids are externally placed in the equipment, resulting in the large volume of the detection equipment and the pipelines. Complex, difficult to maintain, easy to contaminate and other problems, and the previous POCT products due to the characteristics of the detection principle, it is difficult to simultaneously detect multiple indicators while performing rapid and accurate quantitative analysis, thereby increasing the consumption of samples to be tested and human errors. On the contrary, the biggest advantage of microfluidic detection technology is that it can perform automatic and rapid detection of multiple indicators at the same time and obtain accurate results under the consumption of microliter-level blood samples. At the same time, the square centimeter-sized microfluidic chip can contain all the functional units of conventional laboratories such as quantitative injection, mixing, reaction, calibration, reagent storage, detection, waste liquid collection, etc., and features such as automatic operation to achieve high integration, A new generation of POCT products with energy saving, convenience and small error.

Fluid control is the core of microfluidic chip design, and all functions of the microfluidic chip depend on the unique design of the microchannel network to achieve. Taking the microfluidic products of several foreign top companies in the industry as an example, from the perspective of fluid power, the power of the microfluidic chip can be air pump (US8986527B2), syringe (US7842234B), external force extrusion (US5821399A), centrifugal force (US20110124128A1).

The chip powered by the air pump has the following characteristics: First, for the control of more than two fluids, the air pump requires a more complex chip microchannel network design, and relatively more valve designs to realize the sequential flow control of the fluid. Such a complex structure leads to the large size of the instrument, and the high cost and high cost of chip processing. Moreover, the air pump as a power will increase the probability of bubbles in the fluid, and the generated bubbles can hinder the normal operation of the sensor.

For a chip powered by a syringe, firstly, the syringe is required to be sealed and docked with the sample injection port of the chip. Such an operation is difficult and prone to human error; secondly, it is easy to cause the risk of sample or instrument contamination due to operation error.

In the fluid push method of external force extrusion, because the force generated by the extrusion deformation itself is relatively small, the chip size is required to be relatively small. Such "miniature" changes will directly lead to the difficulty of chip processing and assembly, resulting in economic losses.

There are relatively few chip products driven by centrifugal force. In principle, centrifugal force-driven chips can achieve high-integration detection to the greatest extent, and achieve the advantages of in-chip sample purification and equal sampling. However, due to its relatively more complex and fine structure, the surface tension of the material can be very high. To a large extent, the flow rate is affected, resulting in relatively high technical barriers and difficulty in industrialization.

With the surge in the market demand for in vitro diagnostics, the advantages of microfluidic technology in in vitro diagnostic applications have gradually emerged, attracting more and more attention from the industry. In the application of microfluidic chips, the sequential flow of various fluids and the preservation and flow control of the built-in liquid in the test piece are common technical difficulties.

SUMMARY OF THE INVENTION

The present invention provides a microfluidic integrated chip capable of controlling fluid flow only by its own gravity. The microfluidic integrated chip can complete the automatic transmission and detection of multiple fluids without external power equipment such as micropumps, syringe pumps, extrusion devices, centrifugal force devices, etc.

Specifically, a microfluidic detection integrated chip provided by the present invention includes a substrate, a cover sheet and a detection area located on the substrate, and the substrate is further provided with a first liquid storage tank, a second liquid storage tank and a waste liquid tank , and the first, second, third and fourth flow channels; the cover sheet seals the first reservoir, the second reservoir and the waste reservoir, and the first, second, third and fourth flow channels; The cover sheets covering the positions of the first liquid storage tank, the second liquid storage tank and the waste liquid tank are respectively provided with first, second and third openings; The detection area is in liquid communication, the second liquid storage tank is in liquid communication with the detection area through the second flow channel and the third flow channel, and the detection area is in liquid communication with the waste liquid tank through the fourth flow channel; by adjusting the first, second, third and fourth The surface tension of the flow channel causes the liquids in the first liquid storage tank and the second liquid storage tank to flow through the detection area and reach the waste liquid tank in sequence and in turn under the action of gravity.

Usually, in the microfluidic detection integrated chip, by adjusting the flow rates of the liquids in the first liquid storage tank and the second liquid storage tank, the two flow to the detection area in turn and finally reach the waste liquid tank. In the present invention, the liquid flow rates of the first liquid storage tank and the second liquid storage tank are controlled by adjusting the surface tension of the flow channel.

In some preferred embodiments, the first flow channel and the third flow channel are treated with hydrophilic treatment; the second flow channel and the fourth flow channel are treated with hydrophobic treatment.

In some preferred embodiments, the width of the first flow channel is greater than the width of the second flow channel; the width of the third flow channel is greater than the width of the fourth flow channel.

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 includes a curved structure.

In some preferred embodiments, the third flow channel is a straight structure, and the fourth flow channel includes a curved structure.

In some preferred embodiments, the first liquid storage tank, the first flow channel, the third flow channel and the detection area are arranged on the front side of the substrate, and the second liquid storage tank, the second flow channel, the fourth flow channel and the waste liquid tank are arranged on the front side of the substrate. Back of the substrate.

In some preferred embodiments, the cover sheet includes an upper cover sheet covering the front side of the substrate and a lower cover sheet covering the reverse side of the substrate; the base plate and the lower cover sheet are made of hydrophobic material, and the upper cover sheet is made of hydrophilic material.

In some preferred embodiments, the substrate is provided with a first through hole connecting the second flow channel and the third flow channel, and a second through hole connecting the detection area and the fourth flow channel.

In some preferred embodiments, the end of the detection zone is bent upwards. .

In some preferred embodiments, the end of the detection area is bent upwards, extends for a certain distance, and then bent downwards to form an inverted "U" structure.

In some preferred embodiments, the detection area is a curved structure or a folded structure.

In some preferred embodiments, the third flow channel includes a curved structure.

In some preferred embodiments, the first, second and third openings are provided with seals.

In some preferred embodiments, the electrode sensors are located in the detection zone. In a more preferred embodiment, the detection part of the electrode sensor is located in the detection area.

In some preferred embodiments, the liquid in the first liquid storage tank is the calibration liquid; the liquid in the second liquid storage tank is the sample.

In some preferred embodiments, the upper ends of the first liquid storage tank and the first flow channel are covered with a hydrophobic membrane layer.

The present invention also provides a method for detecting a sample with a microfluidic detection integrated chip, including a microfluidic detection chip, the microfluidic detection chip includes a hydrophobic substrate, a hydrophilic upper cover sheet covering the front of the substrate, and a microfluidic detection chip covering the substrate. The hydrophobic upper cover sheet on the reverse side, the front side of the substrate is provided with a first liquid storage tank, a first flow channel, a third flow channel and a detection area, and the reverse side of the substrate is provided with a second liquid storage tank, a second flow channel, a fourth flow channel and The waste liquid tank is provided with first, second and third openings on the cover sheets covering the positions of the first liquid storage tank, the second liquid storage tank and the waste liquid tank; the first liquid storage tank passes through the first flow channel and the The third flow channel is in liquid communication with the detection area, the second liquid storage tank is in liquid communication with the detection area through the second flow channel and the third flow channel, and the detection area is in liquid communication with the waste liquid tank through the fourth flow channel; The calibration solution is sealed, and the detection part of the electrode sensor is located in the detection area. The specific operation steps are as follows:

a inject the sample into the second reservoir;

b The microfluidic detection chip is placed vertically or obliquely so that the first liquid storage tank and the second liquid storage tank are higher than the detection area and the waste liquid tank;

c. The first opening, the second opening and the third opening are not closed, and the calibration liquid in the first liquid storage tank flows into the first flow channel, and then flows through the third flow channel and then enters the detection area; at the same time, the second liquid storage tank The sample in the tank also flows slowly into the second flow channel;

d When the electrode sensor disposed in the detection area detects that its surface is completely covered by the calibration solution, the third opening is closed, or the first and second openings, or the first, second and third openings are closed, and the calibration solution and The sample stops flowing, and the electrode sensor detects the calibration solution;

e After the detection is completed, the previously closed opening is opened, the air pressure in the flow channel is restored to balance, the calibration solution and the sample are restored to flow, and the calibration solution flows into the fourth flow channel through the communication detection area and the second perforation, and finally flows into the waste liquid tank; At the same time, the sample flows into the third flow channel through the first perforation connecting 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 its surface is completely covered by the sample, it closes the third opening, or closes the first and second openings, or closes the first, second and third openings, and the sample stops flowing. to detect;

g After the detection is completed, the opening closed in step f is opened, the air pressure in the flow channel is restored to balance, the flow of the sample is restored, and the sample flows into the fourth flow channel through the second perforation, and finally flows into the waste liquid tank.

On the other hand, the present invention also provides another method for detecting a sample with a microfluidic detection chip, wherein in the steps, only step c and step d are different. Specifically, step c is: making the first opening and the The third opening is not closed, and the second opening is closed. The calibration liquid in the first liquid storage tank flows into the first flow channel, and flows through the third flow channel before entering the detection area. At this time, the sample cannot flow from the pressure difference The second liquid storage tank flows into the second flow channel; and step d: when the electrode sensor disposed in the detection area detects that its surface is completely covered by the calibration liquid, close the third opening or the first opening, or close the first opening And the third opening, the calibration solution stops flowing, and the electrode sensor detects the calibration solution.

In some preferred embodiments, in the above two methods for detecting samples with microfluidic detection chips, the microfluidic detection chips are placed vertically or obliquely in the corresponding detection instruments. 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 area is bent upwards, extends for a certain distance, and then bent downwards to form an inverted "U" structure.

In some preferred embodiments, the upper ends of the first liquid storage tank and the first flow channel are covered with a hydrophobic membrane layer.

beneficial effect

(1) The use of the microfluidic detection integrated chip of the present invention can realize the automatic transmission of multiple fluids without external power equipment such as micropumps, syringe pumps, extrusion devices, centrifugal force devices, etc. in terms of fluid drive. The structure of the detection instrument can be simplified and the energy can be saved. Avoid creating air bubbles in the fluid due to the use of an external power source.

(2) The flow rate and diffusion of liquids such as blood in different regions are controlled by the difference in surface tension and hydrophilicity and hydrophobicity in different regions of the chip. For example, when the flow rate of the blood sample flowing out of the second liquid storage tank in the hydrophobic second flow channel is slower than the flow rate of the blood sample in the hydrophilic detection flow channel.

(3) After opening the flow channels on the front and back sides of the substrate, and using the upper and lower cover sheets with different hydrophilicity and hydrophobicity to be water-tightly pasted on the front and back sides of the substrate, the hydrophilicity and hydrophobicity of the substrate flow channel will be due to the hydrophilicity of the cover sheets. The change. In this way, it is easy to manufacture detection chips with different affinities in different regions.

(4) The use area of the substrate can be reduced by distributing the liquid storage tank and the flow channel on both sides of the substrate, thereby contributing to the miniaturization of the detection chip and the detection instrument.

(5) The setting of the "U"-shaped curve in the detection area can further enhance the deceleration effect of the calibration solution and the sample when they flow to the electrode sensor, so that the calibration solution and the sample have enough time to stay in the detection area for detection.

Description of drawings

FIG. 1 is a schematic front view of the substrate of the microfluidic detection integrated chip of the present invention.

FIG. 2 is a schematic view of the reverse side of the substrate of the microfluidic detection integrated chip of the present invention.

3 is a schematic cross-sectional view of the first through hole of the microfluidic detection integrated chip of the present invention.

FIG. 4 is a schematic front view of another substrate of a microfluidic detection integrated chip.

FIG. 5 is a schematic front view of another substrate of a 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 the substrate of another microfluidic detection integrated chip.

FIG. 8 is a schematic front view of another structure of the substrate of another microfluidic detection integrated chip.

Detailed ways

In the following detailed description, the reference text accompanying the figures is a part hereof and is described by way of illustration of specific embodiments in which the invention may be practiced. We do not exclude that the invention can also be implemented in other specific solutions and the structure of the invention can be changed without departing from the scope of use of the invention.

In the present invention, the microfluidic detection chip uses the surface tension of the flow channel and the gravity of the fluid to control the flow of the liquid. In some specific embodiments, the surface tension of the flow channel is changed by changing the hydrophilicity and hydrophobicity of the flow channel. In other embodiments, the surface tension of the flow channel is changed by changing the size of the flow channel. In still other embodiments, the surface tension of the flow channel 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, while changing the hydrophilicity and hydrophobicity of the flow channel, the size or structure of the flow channel is changed.

In some specific embodiments, the microfluidic detection integrated chip includes a substrate, a cover sheet, and a detection area located on the substrate, and the substrate is further provided with a first liquid storage tank, a second liquid storage tank, a waste liquid tank, and a first liquid storage tank. , second, third and fourth flow channels; cover sheets seal the first reservoir, the second reservoir and the waste reservoir. Among them, the first flow channel and the third flow channel are treated with hydrophilic treatment; the second flow channel and the fourth flow channel are treated with hydrophobic treatment. Alternatively, the width of the first flow channel is greater than the width of the second flow channel; the width of the third flow channel is greater than the width of the fourth flow channel. Alternatively, the length of the first flow channel is smaller than the length of the second flow channel. Alternatively, the first flow channel has a straight structure, and the second flow channel includes a curved structure. Alternatively, the third flow channel has a straight structure, and the fourth flow channel includes a curved structure.

In other embodiments, flow channels are distributed on both sides of the substrate 100, the flow channels on one side have a hydrophilic effect, and the other side has a hydrophobic effect. One side of the flow channel is used as the reverse side, and the flow channels on the two sides are connected through perforations. In addition, the substrate 100 is further provided with a first liquid storage tank 11 , a second liquid storage tank 12 , a waste liquid tank 3 and a detection area 2 . The first liquid storage tank 11 and the second liquid storage tank 12 are respectively located on two sides of the substrate 100 . , the liquid in the first liquid storage tank 11 and the second liquid storage tank 12 flows into the detection area 2 through the flow channel successively, and flows into the waste liquid tank 3 through the flow channel after the detection. Specifically, the first liquid storage tank 11 and the detection area 2 are located on the front side of the substrate, covered with a hydrophilic upper cover sheet, the second liquid storage tank 12 and the waste liquid tank 3 are located on the reverse side of the substrate, and the cover is hydrophobic the lower cover. The first liquid storage tank 11 , the second liquid storage tank 12 and the waste liquid tank 3 are in communication with the atmosphere, which is to balance the air pressure in the flow channel of the substrate 100 so that the liquid can flow smoothly in the flow channel. By distributing the liquid storage tanks and flow channels on the front and back sides of the substrate 100, the usable area of the substrate 100 can be reduced, which is helpful for the miniaturization of the detection chip and the detection instrument, and also enables different processing of the surface tension of different regions of the chip. easier to implement.

The hydrophilicity and hydrophobicity of the substrate and the cover sheet can be achieved by selecting a hydrophilic material and a hydrophobic material, or by performing hydrophilic or hydrophobic treatment on the substrate and the cover sheet.

The cover sheet can optionally be a flexible membrane 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 (tens to hundreds of micrometers), so that the flow of the fluid in the flow channel is not only affected by gravity, but also greatly affected by the hydrophilicity and hydrophobicity of the surface of the flow channel. , the size and shape of the structure can be changed for different effect requirements according to the prior art under the guidance of the idea of the present invention except referring to the following embodiments.

Specifically, as shown in FIG. 1 , the substrate 100 of the microfluidic chip is provided with a first liquid storage tank 11 , a second liquid storage tank 12 , a detection area 2 and a waste liquid tank 3 , which are located on the first liquid storage tank 11 The first opening 110 on the second liquid storage tank 12, the third opening 30 on the waste liquid tank 3, the electrode sensor 400 on the detection area 2, and the first flow channel on the substrate 100 41 , the second flow channel 42 , the third flow channel 43 , the fourth flow channel 44 , the first through hole 61 , and the second through hole 62 .

The substrate 100 is made of a hydrophobic material, and a first liquid storage tank 11 , a first flow channel 41 , a third flow channel 43 and a detection area 2 are distributed on the front surface of the substrate 100 , and are covered by a hydrophilic upper cover sheet 200 . A second liquid storage tank 12 , a second flow channel 42 , a fourth flow channel 44 and a waste liquid tank 3 are distributed on the reverse side of the substrate 100 , and are covered by a hydrophobic lower cover sheet 300 .

According to the relative positional relationship from top to bottom in FIG. 1 , the specific distribution relationship on the front surface of the substrate 100 is as follows: the first liquid storage tank 11 is provided with a first opening 110 for communicating with the outside atmosphere, and the lower end of the first liquid storage tank 11 is connected to the The upper end of the first flow channel 41 communicates with each other, the lower end of the first flow channel 41 and the upper end of the third flow channel 43 communicate with each other, and the connection between the lower end of the first flow channel 41 and the upper end of the third flow channel 43 is provided with a first penetrating substrate 100 . The perforation 61, the first perforation 61 is connected to the 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 area 2, the part of the electrode sensor 400 for detecting liquid is set in the detection area 2, the detection area 2 A second through hole 62 extending through the substrate 100 is provided at the lower end of the .

According to the relative positional relationship from top to bottom in FIG. 2 , the specific distribution relationship on the reverse surface of the substrate 100 is as follows: the second liquid storage tank 12 is provided with a second opening 120 that communicates with the outside atmosphere, and the lower end of the second liquid storage tank 12 is connected to the second liquid storage tank 12 . The upper ends of the second flow channels 42 are connected, and the lower end of the second flow channel 42 is provided with a first perforation 61 (for the connection relationship between the first perforation 61, the first flow channel 41, the second flow channel 42 and the third flow channel, please refer to FIG. 3) , the fourth flow channel 44 is located below the second flow channel 42, the fourth flow channel 44 is not directly connected with the second flow channel 42, and the upper end of the fourth flow channel 44 is provided with a second perforation 62, so that the detection area 2 The fourth flow channel 44 is communicated with each other through the second perforation 62, the lower end of the fourth flow channel 44 is communicated with the waste liquid tank 3, and the waste liquid tank 3 is provided with the third perforation 30 communicated with the outside atmosphere. , the liquid in the substrate 100 does not flow out of the waste liquid tank 3 , so that the position of the third through hole 30 is preferably set at the top of the waste liquid tank 3 .

The upper end in the embodiment can be understood as the front end having the same meaning, and the upper end and the front end can be replaced with each other. Likewise, the lower end and the end are interchangeable.

Preferably, the width of the first flow channel 41 , the second flow channel 42 , the third flow channel 43 , the fourth flow channel 44 and the detection area 2 on the substrate is 200-800 micrometers (μm), and the depth is 200-600 μm. Specifically, for example, the thickness of the substrate is 0.4-5 millimeters (mm), the width of the first flow channel 41 , the second flow channel 42 , the third flow channel 43 , the fourth flow channel 44 and the detection area 2 is 400 μm, and the depth is 300 μm. 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 area 2 can be continuously (discontinuously) changed within a limited range, or can be maintained at a certain value. Change.

The initial substrate 100 may be filled with a calibration solution for correcting the electrode sensor 400 in the first liquid storage tank 11, and the first opening 110 is sealed by a sealing member to prevent the calibration liquid from flowing into the flow channel from the liquid storage tank. The seal may be a membrane that can be pierced, or a plug that can be removed, the seal being broken or removed during use. The second opening 120 can be selectively sealed by the sealing member. When the sealing member is a film, when the sealing member is sealed by the film, after the film needs to be pierced, the sample is injected into the second liquid storage tank 12 through a syringe; when the sealing member is a rubber stopper When waiting for the plug body that can be pulled out, after removing the plug body, inject the sample into the second liquid storage tank 12 through a syringe; when the second opening 120 is not sealed by a seal, the second liquid storage tank 12 can be directly passed through the second opening 120. A sample is injected into the tank 12 . The third opening 30 is optionally covered by a seal, which can be removed or destroyed during use. By sealing these openings, firstly, the calibration solution in the substrate 100 can be kept out of communication with the outside atmosphere, thereby increasing its shelf life. Second, it can prevent the calibration liquid from flowing into the flow channel in advance before it is used (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, the air in the space below the liquid will be compressed, This will cause the air pressure to rise and the air pressure in the reservoir to decrease, which will prevent the calibrator from flowing further in the flow path). The third is to prevent tiny pollutants such as dust from entering the substrate 100 .

In addition, the resistance to flow can be enhanced by changing the length, shape or size of the second flow channel 42 , so as to prolong the flow time of the sample in the second flow channel 42 .

When the chip is used for sample detection, the substrate 100 is placed vertically or obliquely in the corresponding detection instrument. At this time, the first liquid storage tank and the second liquid storage tank are higher than the positions of the detection area and the waste liquid tank, and 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 liquid storage tank 12 , and the calibration solution flows into the first flow channel 41 from the first liquid storage tank 12 and continues to flow (flow rate significantly faster than the flow rate of the sample). The calibration solution enters the detection zone 2 after passing through the third flow channel 43 , while the sample does not enter the third flow channel 43 .

When the electrode sensor 400 detects that the surface of its detection part is completely covered by the calibration liquid, 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 detection area 2 The calibrator will continue to flow for a short distance, making the air pressure at the front of the calibrator unbalanced with the air pressure at the back of the calibrator, resulting in a pressure difference and preventing the calibrator from continuing to flow, and the sample also stops flowing due to the pressure difference. . At this time, the electrode sensor 400 has a suitable time to detect the calibrating solution that has stopped flowing.

After the detection is completed, the previously closed opening is opened, the air pressure in the flow channel is restored to equilibrium, and the flow of the calibration solution and the sample is restored. After the calibration liquid passes through the second perforation 62 , it finally flows into the waste liquid tank 30 . After the sample flows into the hydrophilic third flow channel 43 through the first perforation 61, the flow speed starts to increase under the pulling of the hydrophilic tension. When the electrode sensor 400 detects that the surface of its 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 is under the action of the air pressure difference. Stop the flow, at this time, the electrode sensor 400 has a suitable time to detect the stopped flow of the sample. After the detection is completed, the opening to be closed is opened, the liquid in the flow channel begins 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 successively.

In another detection method, the substrate 100 is placed vertically or obliquely on the corresponding detection instrument, so that the first liquid storage tank and the second liquid storage tank are higher than the detection area and the waste liquid tank, and the detection instrument closes the second opening 120, However, the first opening 110 and the third opening 30 are not closed, and the sample cannot flow in the second flow channel 42 under the action of the pressure difference. The calibration liquid flows in the first flow channel 41 and enters the detection area 2 after passing through the third flow channel 43 .

When the electrode sensor 400 detects that the surface of its detection part is completely covered by the calibration solution, the third opening 30 is closed (or the first opening 110 is closed, or all openings are closed), and the calibration solution in the detection area 2 is closed. The flow is stopped 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 that was closed at the beginning) are opened, the air pressure in the flow channel is restored to equilibrium, and the calibration solution and the sample begin to flow. After the calibration liquid passes through the second perforation 62 , it finally flows into the waste liquid tank 30 . The sample flows into the hydrophilic third flow channel 43 through the first perforation 61, and the flow speed begins to increase under the tension. When the electrode sensor 400 in the detection area 2 detects that the surface of its 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), The sample stops flowing under the action of the air pressure difference, and the electrode sensor 400 starts to work at this time. After the detection is completed, the closed opening is opened, the liquid in the flow channel begins 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 successively.

Wherein, the detection area 2 is communicated with the fourth flow channel 44 which is hydrophobic on the back through the second perforation 62, which can greatly reduce the flow rate after the liquid flows to the second perforation 62, so that the detection instrument has enough time to seal the corresponding opening , so that a pressure difference is formed to prevent the liquid in the detection area 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 can also be arranged on the front side of the substrate 100. At this time, there is no second perforation, and the part of the fourth flow channel 44 connected to the detection area 2 needs to be hydrophobicized to slow down the flow into the fourth flow channel. The fluid flow rate of channel 44. Alternatively, if the volumes of the calibration solution and the sample are sufficient, the fourth flow channel 44 and the waste liquid tank 3 are directly arranged on the front surface of the substrate 100 without other processing.

Further, as shown in FIG. 4 , the end of the detection area 2 is bent upwards. More specifically, the end of the detection area 2 shown in FIG. 5 extends upward and then extends downward, so that the tail of the detection area 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 can be reduced, so that the liquid has sufficient time to stay at the electrode sensor 400 in order to close the corresponding opening. When the degree of bulge is relatively large, as shown in FIG. 5 , the calibration liquid cannot flow through the highest part of the bulge and enter the second perforation 62 under the action of hydrophilicity. However, after the sample flows into the third flow channel, it can be squeezed. The air column between the calibration solution and the sample indirectly pushes the calibration solution into the waste liquid tank 3, and the sample will eventually stay 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 so that the liquid stays at the electrode sensor 400 .

In a preferred embodiment, the third flow channel and the detection area can form an arc-shaped bend, as shown in FIG. 4 and FIG. 5 , so as to slow down the flow velocity of the liquid when the third flow channel is a straight structure, so that the liquid can be slowly and efficiently Fully flow through the detection area to ensure the success rate of detection.

Optionally, as shown in FIG. 6 , the detection area 2 has a bend relative to the third flow channel, the end of the detection area 2 extends upward, and the second through hole 62 is located at the end of the extension, so that the flow rate of the liquid is reduced at the extension, Or suspend the flow without subsequent air column extrusion. Since the second perforation 62 is located at the upwardly extending end instead of the downwardly extending end as in FIGS. 4 and 5 , in some cases, after the calibration liquid is pushed into the waste liquid tank 3 , a small amount of calibration will occur. The liquid will flow back to the electrode sensor 400 and affect subsequent detection.

As shown in FIG. 7 , in order to prevent the liquid in the first liquid storage tank 11 from entering the first flow channel 41 too quickly to form bubbles or gas columns, which will affect subsequent detection. A hydrophobic film layer 201 can be covered on the upper ends of the first liquid storage tank 11 and the first flow channel 41, so that the inlets at the upper ends of the first liquid storage tank 11 and the first flow channel 41 are hydrophobic, so that the liquid flows in at a slow speed. The first flow channel 41 avoids the formation of air bubbles or gas columns in the calibration solution.

In other embodiments, only by changing the second perforation 62 (such as the size, roughness, hydrophobicity, etc. of the perforation) and/or the fourth flow channel 44 (such as the thickness, roughness, hydrophobicity, etc. of the flow channel) The residence time of the liquid in the detection zone 2 is extended as much as possible, instead of sealing the opening.

The detection method in the detection area of the present invention may be electrochemical detection, or may be turbidity method, fluorescence method, chemiluminescence method, scattering method, or the like.

In addition to using the electrode sensor 400 for electrochemical detection for quantitative detection, the microfluidic detection chip of the present invention can also perform semi-quantitative or qualitative detection. For example, one or more test strips (which can be blank test strips or test strips pre-added with reagents) are fixed in the detection area 2, and after the calibration solution or sample flows through the detection channel and contacts the test strips, the reagents and samples A color change occurs in the reaction, followed by detection by instrumental or human observation.

In order to cooperate with other detection methods, those skilled in the art need to replace the calibration solution with other detection reagents, such as washing solutions not used for reacting with samples, or enzymes, lysing solutions, antibodies, fluorescent reagents, etc. participating in the reaction. Taking an antibody containing a signal marker (antibody 1) as an example, at this time, the detection area 2 does not have an electrode sensor 400, but an antibody (antibody 2) that can specifically bind to the antigen to be detected in the sample is immobilized. In the second liquid storage tank 12, the sample is added to the first liquid storage tank 11. After the sample flows into the detection area 2, the antigen to be detected in it is captured by the second antibody, and then the first antibody flows into the detection area 2, and the antigen is captured by the second antibody. The captured antigen is specifically bound, and finally, the signal intensity of the antibody-label in the detection zone 2 is measured by the relevant equipment to obtain the final measurement result. Further, as shown in FIG. 8 , a third liquid storage tank 13 for storing rinsing liquid can be added. After the rinsing liquid flows into the detection area 2, the unbound antibody 1 is washed away, thereby avoiding the labeling of the unimmobilized antibody 1. The object interferes with the detection, thereby improving the detection accuracy.

Claims (12)

1. a microfluidic detection integrated chip is characterized in that, comprises substrate, cover sheet and the detection zone positioned on the substrate, the substrate is also provided with the first liquid storage tank, the second liquid storage tank and the waste liquid tank, and 1st, 2nd, 3rd and 4th runners; cover sheet seals the 1st reservoir, 2nd reservoir and waste reservoir, and 1st, 2nd, 3rd and 4th runners; covered in The cover sheets at the positions of the first liquid storage tank, the second liquid storage tank and the waste liquid tank are respectively provided with first, second and third openings; the first liquid storage tank passes through the first flow channel and the third flow channel and the detection area The second liquid storage tank is in liquid communication with the detection area through the second flow channel and the third flow channel, and the detection area is in liquid communication with the waste liquid tank through the fourth flow channel; by adjusting the first, second, third and fourth flow channels The surface tension of the first liquid storage tank and the second liquid storage tank flow through the detection area and reach the waste liquid tank in sequence and in turn under the action of gravity. 2 . The integrated microfluidic detection chip according to claim 1 , wherein the first flow channel and the third flow channel are subjected to hydrophilic treatment; the second flow channel and the fourth flow channel are subjected to hydrophobic treatment. 3 . 3. The integrated microfluidic detection chip according to claim 2, wherein the first liquid storage tank, the first flow channel, the third flow channel and the detection area are arranged on the front of the substrate, the second liquid storage tank, the second liquid storage tank, the second liquid storage tank, the second liquid storage tank, the second liquid storage tank The flow channel, the fourth flow channel and the waste liquid tank are arranged on the reverse side of the substrate. 4 . The integrated microfluidic detection chip according to claim 3 , wherein the cover sheet comprises an upper cover sheet covering the front side of the substrate and a lower cover sheet covering the reverse side of the substrate; the base plate and the lower cover sheet are made of hydrophobic materials, 5 . The cover sheet is made of hydrophilic material. 5 . The integrated microfluidic detection chip according to claim 4 , wherein the substrate is provided with a first through hole connecting the second flow channel and the third flow channel, and a first through hole connecting the detection area and the fourth flow channel. 6 . Two perforations. 6 . The integrated microfluidic detection chip according to claim 5 , wherein the end of the detection area is bent upwards, extends for a certain distance, and then bent downwards to form an inverted "U" structure. 7 . 7 . The integrated microfluidic detection chip according to claim 6 , wherein the detection area is a bending structure or a bending structure. 8 . 8 . The integrated microfluidic detection chip according to claim 5 , wherein the upper ends of the first liquid storage tank and the first flow channel are covered with a hydrophobic membrane layer. 9 . 9. A method for detecting a sample with a microfluidic detection integrated chip, characterized in that it comprises a microfluidic detection chip, and the microfluidic detection chip comprises a hydrophobic substrate, a hydrophilic upper cover sheet covering the front of the substrate, and a covering On the hydrophobic upper cover sheet on the reverse side of the substrate, the front side of the substrate is provided with a first liquid storage tank, a first flow channel, a third flow channel and a detection area, and the reverse side of the substrate is provided with a second liquid storage tank, a second flow channel and a fourth flow channel There are openings on the cover sheets covering the positions of the first liquid storage tank, the second liquid storage tank and the waste liquid tank; the first liquid storage tank passes through the first flow channel and the third flow channel and the detection area The second liquid storage tank is in liquid communication with the detection area through the second flow channel and the third flow channel, and the detection area is in liquid communication with the waste liquid tank through the fourth flow channel; the calibration liquid is sealed in the first liquid storage tank, The specific operation steps are as follows: a inject the sample into the second reservoir; b The chip is placed vertically or inclined so that the first liquid storage tank and the second liquid storage tank are higher than the detection area and the waste liquid tank; c. The first opening, the second opening and the third opening are not closed, and the calibration liquid in the first liquid storage tank flows into the first flow channel, and then flows through the third flow channel and then enters the detection area; at the same time, the second liquid storage tank The sample in the tank also flows slowly to the second flow channel; d When the electrode sensor disposed in the detection area detects that its surface is completely covered by the calibration solution, the third opening is closed, or the first and second openings, or the first, second and third openings are closed, and the calibration solution and The sample stops flowing, and the electrode sensor detects the calibration solution; e After the detection is completed, the previously closed opening is opened, the air pressure in the flow channel is restored to balance, the calibration solution and the sample are restored to flow, and the calibration solution flows into the fourth flow channel through the communication detection area and the second perforation, and finally flows into the waste liquid tank; At the same time, the sample flows into the third flow channel through the first perforation connecting 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 its surface is completely covered by the sample, it closes the third opening, or closes the first and second openings, or closes the first, second and third openings, and the sample stops flowing. to detect; g After the detection is completed, the opening closed in step f is opened, the air pressure in the flow channel is restored to balance, the flow of the sample is restored, and the sample flows into the fourth flow channel through the second perforation, and finally flows into the waste liquid tank. 10. The method for detecting a sample with a microfluidic detection integrated chip according to claim 9, wherein in step c, the first opening and the third opening are not closed, the second opening is closed, and the first liquid storage tank is The calibration liquid flows into the first flow channel, and flows through the third flow channel and then enters the detection area. At this time, the sample cannot flow from the second liquid storage tank to the second flow channel under the action of the pressure difference; and, in step d , when the electrode sensor located in the detection area detects that its surface is completely covered by the calibration solution, it closes the third opening or the first opening, or closes the first and third openings, the calibration solution stops flowing, and the electrode sensor is aligned with the calibration solution. standard solution for detection. 11. The method for detecting a sample with a microfluidic detection integrated chip according to claim 9 or 10, wherein the end of the detection area is bent upwards, extends for a certain distance, and then bends downwards to form an inverted "U" structure. 12 . The method for detecting a sample with a microfluidic detection integrated chip according to claim 9 or 10 , wherein the upper ends of the first liquid storage tank and the first flow channel are covered with a hydrophobic membrane layer. 13 .

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