Micro-fluidic chip and testing method thereof

Abstract

The present disclosure relates to a micro-fluidic chip, comprising a substrate; a cover piece, the cover piece comprising a groove extending in the length direction; and a micro-channel defined by the substrate and the groove, wherein the groove of the cover piece comprises a channel area, the channel area is sequentially provided with a sample adding area, a reaction area and a control area in the length direction, and the sample adding area, the reaction area and the control area are in fluid communication with each other, wherein a control valve is arranged in the control area, the control valve is configured to control the flow of liquid in the reaction area to the control area, the height of the reaction area is lower than that of the control area, and the width of the reaction area is smaller than the width of the control area. The control valve can enable the liquid in the reaction area to flow towards the control area at the required time, so that the time in the sample reaction area is controlled, and the liquid flows into the control area after full reaction and is not retained in a channel of the reaction area.

Description

A microfluidic chip and detection method thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese invention patent application with application number 202311230967.4 and invention name “A microfluidic chip” submitted to the China Patent Office on September 22, 2023, and the Chinese invention patent application with application number 202311230921.2 and invention name “A microfluidic chip and its detection method”, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present invention relates to the technical field of in vitro diagnosis, and in particular to a microfluidic chip and a detection method thereof. Background Art

[0004] Microfluidic chips are the primary platform for implementing microfluidic technology. They can integrate essential operational units for biological, chemical, and medical analysis, including sample preparation, reaction, separation, and detection, onto a single, compact chip. Microchannels automate the entire analytical process, enabling the various functions of conventional chemical or biological laboratories. Microfluidic chips offer advantages such as compact size, minimal sample and reagent requirements, rapid reaction speed, massive parallel processing capabilities, and disposable design. They hold enormous potential in fields such as biology, chemistry, and medicine. In recent years, they have developed into a new research field at the intersection of biology, chemistry, medicine, fluidics, electronics, materials, and mechanics.

[0005] Microfluidic chips generally add a fixed volume of reaction liquid. Using its special structure, the sample flows through the microchannel through capillary action after addition and finally reaches the waste liquid area, thus completing the automatic reaction process. It is then tested with an optical analyzer to analyze the test results.

[0006] In known microfluidic chips, the sample loading hole and the buffer hole are usually arranged upstream of the labeling area, and the labeling area is arranged upstream of the detection area. Therefore, after the sample passes through the sample loading hole, it will first react in the labeling area before entering the detection area. For samples with high viscosity, they cannot push the signal molecules in the labeling area to the detection area by themselves, and subsequent buffer is required to push them. However, this method will dilute the sample, resulting in inaccurate test results. At the same time, other substances contained in the sample may also interfere with the signal molecules in the labeling area.

[0007] The flow and control of samples within microfluidic chips is a key concern. One known microfluidic chip with controllable reaction time embeds a water-absorbing material at the sample outlet. This material contacts the microchannel and absorbs the sample trapped within the channel. However, this method cannot control the timing of sample adsorption within the channel, which can easily lead to incomplete reactions within the channel.

[0008] Another known microfluidic chip includes a microchannel of defined height and width formed by a substrate and a cover sheet. The sample fluid can flow toward the outlet of the microchannel under the capillary force of the microchannel. The height of the top wall of the microchannel is lower than the height of the contact surface between the substrate and the cover sheet. However, this method requires ensuring the injection volume of the sample liquid, and leakage may still occur during operation, resulting in inaccurate test results.

[0009] Summary of the Invention

[0010] The purpose of the present invention is to provide a microfluidic chip and a detection method thereof to address the deficiencies in the prior art, so as to solve the technical problems raised in the above-mentioned background technology.

[0011] On the one hand, to achieve the above-mentioned purpose, the present invention proposes a microfluidic chip, comprising a substrate, a cover sheet, and a microchannel formed by the substrate and the cover sheet, wherein the microchannel comprises a buffer loading area, a reaction area, and a control area, wherein the buffer loading area, the reaction area, and the control area are connected in sequence along the length direction, and a movable water-absorbing material is provided in the control area, and the water-absorbing material can contact or move away from the reaction area, and wherein the reaction area comprises a labeling area, a detection area, and a sample loading area, and the detection area and the sample loading area are both arranged on the same side of the labeling area.

[0012] In some examples, the cover sheet is provided with a groove along its length, and the groove and the substrate together form the microchannel.

[0013] In some examples, the detection area is sequentially provided with detection sites and quality control sites along the length direction, and the number of the detection sites and quality control sites is one or more.

[0014] In some examples, the labeling area includes antibodies\antigens labeled with signal molecules, the detection sites in the detection area are coated with antibodies\antigens that can bind to the target analyte, the target analyte can simultaneously bind to the antibodies\antigens in the labeling area and the detection site to form a double antigen\antibody sandwich complex, and the quality control sites in the detection area are coated with antibodies\antigens that can bind to the antibodies\antigens labeled with the signal molecules in the labeling area.

[0015] In some examples, a braking hole is provided on a side of the control zone opposite to the reaction zone, and the braking hole is a rectangular through hole that passes through the substrate and the cover plate, and at least partially extends to the water-absorbing material in the control zone, wherein a through hole is provided at one end of the water-absorbing material away from the reaction zone, and the through hole is within the braking hole.

[0016] In some examples, the buffer loading area is provided with a buffer hole connecting the upper and lower surfaces of the cover sheet, and the sample loading area is provided with a sample loading hole connecting the upper and lower surfaces of the cover sheet.

[0017] In some examples, the sample addition area and the labeling area are respectively disposed on both sides of the detection area.

[0018] In some examples, the sample addition area is disposed between the labeling area and the detection area.

[0019] In some examples, the signal molecule-labeled antigen\antibody is a signal molecule-labeled antigen\antibody molecule.

[0020] On the other hand, the present invention also provides a microfluidic chip detection method, which uses the microfluidic chip as described above for detection, and the method includes at least the following steps:

[0021] Adding the sample into the microchannel through the sample adding area and keeping the water-absorbing material away from the reaction area;

[0022] After the sample flows through the detection zone in the reverse direction due to capillary action and meets the first judgment condition, the water-absorbing material is moved to contact the reaction zone to absorb excess sample until the flow is cut off, and the sample retained in the microchannel reacts with the detection zone; then, the water-absorbing material is separated from or continues to contact the reaction zone;

[0023] After the second reaction period, the buffer solution is added to the microchannel through the buffer solution addition area. The buffer solution passes through the labeling area under capillary action, washes the antigen / antibody coated with the signal molecule in the labeling area, and then enters the detection area. When the signal molecules in the reaction area channel merge with the sample, the water-absorbing material in the control area is moved to contact the reaction area, and the excess sample is adsorbed to the control area through the water-absorbing material.

[0024] After the excess sample in the channel is absorbed by the water-absorbing material and the signal molecules in the labeling area are dissolved and spread throughout the reaction area channel, the water-absorbing material is moved away from the reaction area to break contact with the reaction area channel;

[0025] After the third reaction period, the water-absorbing material in the control area is moved to contact the detection area, and the excess liquid including the signal molecules is absorbed into the control area through the water-absorbing material;

[0026] The signal value of the detection zone is measured.

[0027] In some examples, the first determination condition includes: a first time period has passed, the amount of the sample liquid is greater than or equal to a predetermined amount, or the signal intensity of the signal molecule in the detection zone is greater than or equal to a threshold.

[0028] In some examples, the first time period is in the range of 0 to 60 seconds, the predetermined volume is in the range of 15 μL to 50 μL, and the threshold is above a reasonable background range of the chip. The signal intensity range varies for different signal molecules and corresponding detection devices. The reasonable background range referred to herein refers to an intensity greater than the background signal, i.e., an intensity that clearly indicates the presence of the corresponding signal molecule.

[0029] In some examples, the second time period is in a range of 10 seconds to 300 seconds, and the third time period is in a range of 30 seconds to 5 minutes.

[0030] The microfluidic chip and detection method obtained by the above technical solution have the following beneficial effects:

[0031] 1. Since the sample addition area is closer to the detection area, the flow distance of the sample in the microchannel is reduced, which reduces the amount of sample used. In addition, the sample can be directly added to react with the detection area. After the sample reacts in the detection area, the buffer is used to push the signal molecule, avoiding the dilution of the sample by directly pushing the sample with the buffer. The sensitivity is high and the detection results are more accurate.

[0032] 2. It can directly test whole blood or samples with high viscosity, and has a wide range of application. The signal molecules in the labeled area are pushed into the reaction area by the buffer, rather than directly by the sample, thus avoiding contamination of the channel by whole blood or samples with high viscosity adhering to the channel surface, which may affect the accuracy of subsequent detection.

[0033] 3. The excess sample is absorbed by the water-absorbing material after reacting in the reaction area, avoiding the influence of other substances in the sample on the signal molecules.

[0034] 4. It can overcome the hook effect of immune response and avoid the situation where the target analyte content in some samples is too high (far higher than the number of antigens / antibodies labeled with signal molecules), resulting in the target analyte occupying the reaction sites in the entire reaction area, while the immune complexes formed by the target analyte and the signal molecule cannot or rarely react with the antigens / antibodies in the detection area, making it impossible for the detection area to accurately detect the signal value, affecting the accuracy of the test results.

[0035] On the other hand, the present invention provides a microfluidic chip, comprising a substrate; a cover plate, the cover plate comprising a groove extending along the length direction; and a microchannel formed by the substrate and the groove, wherein the groove of the cover plate comprises a channel area, the channel area is sequentially provided with a sample addition area, a reaction area and a control area along the length direction, the sample addition area, the reaction area and the control area are fluidically connected to each other, wherein a control valve is provided in the control area, the control valve is configured to control the flow of liquid from the reaction area to the control area, and wherein the height of the reaction area is lower than the height of the control area, and the width of the reaction area is smaller than the width of the control area.

[0036] In some examples, the control zone gradually narrows from the middle to the reaction zone.

[0037] In some examples, the control valve includes a movable fluid suction member and a brake hole, wherein the fluid suction member can contact or move away from the reaction zone, and the brake hole is arranged on the side away from the reaction zone, wherein the brake hole is a rectangular through hole passing through the substrate and the cover plate, and a through hole is arranged at one end of the fluid suction member away from the reaction zone, and the through hole is within the brake hole.

[0038] In some examples, the control zone is provided with a stopper configured to hold the fluid suction member away from the reaction zone.

[0039] In some examples, the locking portion includes a convex portion arranged along the width direction, and the fluid suction member is correspondingly provided with a concave portion that cooperates with the convex portion.

[0040] In some examples, the cover sheet is provided with a retention portion at the connection between the control zone and the reaction zone, and the retention portion at least partially extends into the reaction zone, so that when the fluid suction member contacts the channel of the reaction zone, at least a portion of the fluid suction member is inserted into the retention portion.

[0041] In some examples, the retention portion is a tooth portion, which is constructed as a concave arc extending into the reaction zone, and the lower end of the arc is provided with a sawtooth or wave structure, wherein the fluid suction member gradually narrows on the side close to the reaction zone, and is provided with a notch in the middle of the side close to the reaction zone, so that when the fluid suction member contacts the channel of the reaction zone, the notch is inserted into the tooth portion.

[0042] In some examples, the retentate portion is an inclined trough inclined toward the reaction zone.

[0043] In some examples, the fluid absorbent comprises a water-absorbing material, wherein the water-absorbing material comprises at least one of a desiccant, an antioxidant, and a moisture absorption indicator.

[0044] In some examples, the sample loading area is provided with a sample loading hole connecting the upper and lower surfaces of the cover plate, and the height of the sample loading hole at the lower surface of the cover plate is on the same level as the height of the reaction area.

[0045] In some examples, the groove is rectangular, the shape of the substrate matches the shape of the groove, and the substrate is placed in the groove and is on the same horizontal plane as the surface of the cover sheet.

[0046] In some examples, a channel area, a barrier area, and a pressing area are respectively provided in the groove of the cover sheet from the middle to the outside along the width direction, and the heights of the channel area and the barrier area are higher than the height of the pressing area, so that the substrate is placed in the groove on the lower surface of the cover sheet and contacts the pressing area, and a gap is formed between the channel area and the barrier area.

[0047] In some examples, the barrier region is disposed at the periphery of the sample loading region and / or the reaction region of the channel region, and in the length direction, the barrier region covers at least a portion of the reaction region, or covers the sample loading region and / or the reaction region.

[0048] In some examples, the base sheet is coated with a hydrophobic material at locations corresponding to the barrier regions within the recesses of the cover sheet.

[0049] In some examples, the reaction zone and the barrier zone are on the same level, and the barrier zone is also coated with a hydrophobic material.

[0050] In some examples, the substrate is laser-processed at locations corresponding to the barrier regions within the grooves of the cover sheet to form a hydrophobic surface.

[0051] In some examples, the reaction area and the barrier area are on the same horizontal plane, and the barrier area is laser-treated to form a hydrophobic surface.

[0052] In some examples, the height of the reaction zone is lower than that of the barrier zone, so that the sample does not enter the barrier zone due to gravity when flowing along the reaction zone.

[0053] The microfluidic chip obtained by the above technical solution has the following beneficial effects:

[0054] 1. A control valve is provided in the control area to control the flow of liquid from the reaction area to the control area, so that the liquid in the reaction area flows to the control area when needed, thereby controlling the time the sample spends in the reaction area so that it flows to the control area after sufficient reaction and does not remain in the reaction area channel.

[0055] 2. The teeth of the control valve are used so that when the fluid aspirator is inserted into the control and contacts the reaction zone, the top portion is inserted into the arc structure of the reaction zone, and the bottom portion contacts the serrated or wavy structure at the bottom of the arc structure. The fibers inside the fluid aspirator are connected with the serrated or wavy structure after contact, so that the bottom portion of the fluid aspirator is squeezed and thickened, and better contacts the channel of the reaction zone, thereby achieving the purpose of adsorbing the sample retained in the channel.

[0056] 3. The control valve can be used multiple times in one experiment to open and close the channel between the reaction zone and the control zone as needed, and the liquid in the channel can be adsorbed multiple times at appropriate times to increase the accuracy of the detection.

[0057] 4. The substrate is placed in the corresponding barrier area in the groove of the cover sheet and coated with a hydrophobic material or laser treated so that during the sample addition process, the bottom of the sample is restricted to flow in the corresponding reaction area and will not flow into the corresponding barrier area.

[0058] 5. The barrier area on the cover sheet is higher than the reaction area, so that the sample will not enter the barrier area due to gravity when flowing along the reaction area, thereby achieving the purpose of sample flowing in the reaction area.

[0059] 6. When the barrier area and the reaction area are at the same height, the barrier area on the cover sheet is coated with a hydrophobic material or laser treated to similarly restrict the sample flow to the reaction area. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present disclosure, rather than limiting the present disclosure.

[0061] FIG1 is a schematic top view of a microfluidic chip according to at least one embodiment of the present invention;

[0062] FIG2 is a schematic structural diagram of a cover sheet of a microfluidic chip according to at least one embodiment of the present invention;

[0063] FIG3 is a schematic cross-sectional view of the structure taken along line AA in FIG2 ;

[0064] FIG4 is an enlarged view of point B in FIG2 , showing the retention portion of the tooth portion;

[0065] FIG5 is an enlarged view of point C in FIG3 , showing a cross-sectional structure of the retention portion;

[0066] FIG6 is a schematic structural diagram of a cover sheet of a microfluidic chip according to another embodiment of the present invention;

[0067] FIG7 is a schematic cross-sectional view of the structure taken along line DD in FIG6 ;

[0068] FIG8 is an enlarged view of point E in FIG6 ;

[0069] FIG9 is an enlarged view of point F in FIG7 ;

[0070] FIG10 is a schematic structural diagram of a substrate according to at least one embodiment of the present invention;

[0071] FIG11 is a schematic side cross-sectional view of the microfluidic chip according to at least one embodiment of the present invention, showing a groove of a cover sheet;

[0072] FIG12 is a schematic side cross-sectional view of a microfluidic chip according to another embodiment of the present invention; FIG12 is a schematic side cross-sectional view of a microfluidic chip according to another embodiment of the present invention;

[0073] 13 is a schematic structural diagram of a cover sheet at a groove position according to another embodiment of the present invention;

[0074] FIG14 is a schematic side cross-sectional view of a microfluidic chip according to another embodiment of the present invention; it shows the groove of the cover sheet;

[0075] FIG15 is a schematic structural diagram of a microfluidic chip according to at least one embodiment of the present invention;

[0076] FIG16 is a schematic cross-sectional view of a microfluidic chip according to at least one embodiment of the present invention;

[0077] FIG17 is a schematic structural diagram of a microfluidic chip according to another embodiment of the present invention.

[0078] In Figures 1 to 14, 1 is a substrate; 2 is a cover plate; 21 is a groove; 211 is a channel area; 2111 is a sample adding area; 2112 is a reaction area; 2113 is a control area; 2112a is a control valve; 2113a1 is a fluid suction part; 2113a2 is a tooth part; 2113a3 is a brake hole; 212 is a barrier area; 213 is a pressing area; 3 is a microchannel; 4 is a through hole; 5 is a stop part.

[0079] In Figures 15 to 17, 1-substrate; 2-cover sheet; 3-microchannel; 4-water-absorbing material; 31-buffer loading area; 32-reaction area; 33-control area; 321-labeling area; 322-detection area; 323-sample loading area. DETAILED DESCRIPTION

[0080] To address the shortcomings of the prior art, the present invention proposes a novel microfluidic chip and detection method thereof. The present invention is described in detail below with reference to the accompanying drawings. It should be noted that in the accompanying drawings, components having substantially the same or similar structures and functions are assigned the same reference numerals, and their repeated descriptions are omitted.

[0081] Compared to the embodiments shown in the drawings, feasible embodiments within the scope of protection of the present invention may have fewer components, other components not shown in the drawings, different components, differently arranged components, or differently connected components, etc. In addition, without departing from the concept of the present invention, two or more components in the drawings may be implemented in a single component, or a single component shown in the drawings may be implemented as multiple separate components.

[0082] As shown in Figures 1 to 5, a microfluidic chip according to at least one embodiment of the present invention includes a substrate 1, a cover plate 2, and a microchannel formed by the substrate 1 and the cover plate 2. A groove 21 is provided in the middle of the lower surface of the cover plate 2, and the groove 21 extends along the length direction L.

[0083] It should be noted that the length direction L in the accompanying drawings refers to the direction along the length of the microfluidic chip, which is exemplarily the horizontal rightward direction in the drawings. The width direction W in the accompanying drawings refers to the direction along the width of the microfluidic chip, which is exemplarily the vertical downward direction in the drawings.

[0084] As shown in Figure 1, the groove 21 of the cover plate 2 is provided with a channel area 211, a barrier area 212 and a pressing area 213 from the middle to the outside along the width direction W. The heights of the channel area 211 and the barrier area 212 are higher than the height of the pressing area 213, so that the substrate 1 is placed in the groove 21 on the lower surface of the cover plate 2 and contacts the pressing area 213, and a gap is formed between the channel area 211 and the barrier area 212.

[0085] As shown in FIG2 , the channel region 211 is sequentially provided with a sample addition region 2111, a reaction region 2112, and a control region 2113 along the liquid flow direction (lengthwise L). The sample addition region 2111, the reaction region 2112, and the control region 2113 are interconnected. The height of the reaction region 2112 is lower than that of the control region 2113, and the width of the control region 2113 is greater than that of the reaction region 2112. Combined with the features of the control valve 2113a and the retention portion 2113a2 described below, the height difference between the reaction region 2112 and the control region 2113 helps maintain the fluid aspirating member 2113a1 in a stable contact position with the reaction region 2112. Furthermore, the smaller width of the reaction region 2112 than that of the control region 2113 helps excess liquid be absorbed into and contained within the control region 2113.

[0086] Illustratively, the control zone 2113 may gradually narrow from the middle to the reaction zone 2112 .

[0087] In this embodiment, a control valve 2113a is provided in the control area 2113, and the control valve 2113a is configured to control the flow of liquid in the reaction area 2112 to the control area 2113, so that the liquid in the reaction area 2112 flows to the control area when needed, thereby controlling the time in the sample reaction area 2112 so that it flows to the control area 2113 after sufficient reaction and does not remain in the channel of the reaction area 2112.

[0088] Specifically, the control valve 2113a can be implemented in a variety of ways. For example, in one embodiment shown in Figures 2 to 5, the control valve 2113a can include a stopper hole 2113a3 and a movable fluid suction member 2113a1, which can be moved into or out of contact with the reaction zone 2112. When the fluid suction member 2113a1 is in contact with the reaction zone 2112, the fluid suction member 2113a1 can draw liquid from the reaction zone 2112 into the control zone 2113. Conversely, when the fluid suction member 2113a1 is away from the reaction zone 2112 or not in contact with the reaction zone 2112, the liquid remains in the reaction zone 2112 and does not flow into the control zone 2113.

[0089] Referring again to FIG. 2 , to prevent the fluid-absorbing member 2113a1 from accidentally contacting the reaction zone 2112, the control zone 2113 is provided with a retaining portion 5. The retaining portion 5 is configured to retain the fluid-absorbing member 2113a1 away from the reaction zone 2112. In this embodiment, the retaining portion 5 includes a protrusion arranged along the width direction W, and the fluid-absorbing member 2113a1 is correspondingly provided with a concave portion that cooperates with the protrusion. The protrusions may be arcuate or rectangular and may be aligned or staggered along the length.

[0090] Alternatively, the locking portion 5 may also be other possible stopping structures, such as a snap structure or an elastic reset member that biases the fluid suction member 2113a1 toward the reaction area 2112.

[0091] The cover sheet 2 is provided with a retention portion 2113a2 at the connection between the control area 2113 and the reaction area 2112. The retention portion 2113a2 at least partially extends into the reaction area 2112, so that when the fluid suction piece 2113a1 contacts the channel of the reaction area 2112, at least a portion of the fluid suction piece 2113a1 is inserted into the retention portion 2113a2.

[0092] As shown in Figures 4 and 5 , the retention portion 2113a2 may be a toothed portion, formed as a concave arc extending into the reaction zone 2112. The lower end of the arc may be provided with serrations. The fluid suction member 2113a1 gradually narrows on the side closest to the reaction zone 2112 and has a notch in the middle of the side closest to the reaction zone 2112. When the fluid suction member 2113a1 contacts the channel of the reaction zone 2112, the notch is inserted into the toothed portion.

[0093] Alternatively, in an embodiment not shown, the lower end of the arc can also be configured as a wave structure. Different from a sawtooth structure, the wave structure can be an undulating curve.

[0094] Therefore, when the fluid suction piece 2113a1 is inserted into the control area 2113 and contacts the reaction area 2112, the top is inserted into the arc structure of the reaction area 2112, and the bottom contacts the serrations at the bottom of the arc structure. The fibers inside the fluid suction piece 2113a1 come into contact with the serrations and then interconnect, causing the bottom of the fluid suction piece 2113a1 to be squeezed and thickened, thereby better contacting the channel of the reaction area 2112, thereby achieving the purpose of adsorbing the sample retained in the channel.

[0095] Movement of the fluid suction member 2113a1 is achieved through the structure of the braking hole 2113a3 and the through hole 4. As shown in Figure 2, the braking hole 2113a3 is a rectangular through-hole extending through the substrate cover. The end of the fluid suction member 2113a1, away from the reaction zone, may be provided with a through hole 4, positioned within the range of the braking hole 2113a3. For example, a braking rod can be inserted into the braking hole 2113a3 and passed through the through hole 4 of the fluid suction member 2113a1 to drive the fluid suction member 2113a1, causing the other end of the fluid suction member 2113a1 to contact or remove from the reaction zone 2112. This allows for controlled adsorption of liquid within the reaction zone 2112 onto the fluid suction member 2113a1 at the appropriate time.

[0096] Illustratively, Figures 6 to 9 also illustrate a cover structure of a microfluidic chip according to another embodiment of the present invention. It should be noted that the following description of the embodiment is primarily directed to the differences between it and the aforementioned embodiment, and the description of identical or similar features will be omitted. Unlike the aforementioned embodiment, this embodiment employs a retention portion with a simpler structure and a retaining portion with another structure. As shown in Figures 6 to 9, the retention portion 2113a2 is an inclined groove inclined toward the reaction zone 2112. The inclined groove can also accommodate and cross-link the fiber structure of the fluid suction member 2113a1. Even though its effect may not be as good as the tooth portion, its processing is simpler and the cost is lower.

[0097] Furthermore, in this embodiment, the convex portion of the retaining portion 5 is rectangular, and the concave portion of the fluid absorbing member 2113a1 is a concave portion that mates with the rectangular convex portion. Because the rectangular width of the concave portion is relatively small, it is depicted as a straight line in FIG6 . This is because it is recommended that the width of the rectangular concave portion not be too large, otherwise it would affect the movement of the fluid absorbing member 2113a1.

[0098] Alternatively, the control valve 2113a may also use a pneumatic valve or plunger pump or other structures known in the art to control the flow of the liquid, which will not be described in detail here.

[0099] Exemplarily, the fluid absorbing member 2113a1 is a water-absorbing material, and the water-absorbing material may include or be added with one or more of a desiccant, an antioxidant, and a moisture absorption indicator.

[0100] For another example, the water-absorbing material can be polyester fiber, water-absorbing resin, water-absorbing gelatin, papermaking pulp or other materials with water-absorbing characteristics.

[0101] Illustratively, the reaction area 2112 is sequentially provided with a labeling area, a detection area, and a quality control area along the length direction L, which may also be referred to as the sample flow direction.

[0102] The groove 21 in the middle of the cover plate 2 is rectangular, and the shape of the substrate 1 matches the groove 21 . The substrate 1 is placed in the groove 21 and is on the same horizontal plane as the surface of the cover plate 2 .

[0103] The sample adding area 2111 is provided with a sample adding hole connecting the upper and lower surfaces of the cover sheet 2. The height of the sample adding hole on the lower surface of the cover sheet 2 is on the same level as the height of the reaction area 2112, so that the sample flows through the sample adding hole to the reaction area 2112 on the right.

[0104] The barrier region 212 is disposed outside the reaction region 2112 in the channel region 211 and covers the entire reaction region 2112 in the length direction L. As shown in FIG10 , the substrate 1 can be coated with a hydrophobic material at the location corresponding to the barrier region 212 within the groove 21 of the cover plate 2. This restricts the flow of the sample from the bottom portion of the substrate 1 to the reaction region 2112 during sample loading and prevents the sample from flowing into the barrier region 212.

[0105] Alternatively, in an embodiment not shown, the barrier area 212 may only cover part of the reaction area 2112, or be only arranged in the sample adding area 2111 and cover the sample adding area 2111, or be arranged around the periphery of the sample adding area 2111 and the reaction area 2112 and cover the sample adding area 2111 and the reaction area 2112.

[0106] As shown in Figures 13 and 14 , the reaction zone 2112 and the barrier zone 212 are on the same horizontal plane. The barrier zone 212 is also coated with a hydrophobic material, which restricts the sample flow to the reaction zone 2112. By coating the barrier zone 212 with a hydrophobic material, during the reaction, the sample liquid is confined to the channel of the reaction zone 2112 and does not leak or flow into non-reaction areas, such as the barrier zone 212, thereby improving reaction sensitivity.

[0107] For example, the hydrophobic material may be polytetrafluoroethylene ink, silane coupling agent ink, fluoride polymer ink or other materials with hydrophobic characteristics.

[0108] As shown in FIG11 , the height of the reaction zone 2112 is lower than that of the barrier zone 212 , so that when the sample flows along the reaction zone 2112 , it will not enter the barrier zone 212 due to gravity. Therefore, there is no need to add a hydrophobic material to the barrier zone 212 , and the flow of the sample in the reaction zone 2112 can also be restricted.

[0109] It should be noted that the solid color filled portions in Figures 11 and 13 show the coated hydrophobic material, which is shown as thicker for ease of description. In fact, the coated hydrophobic material can be a very thin layer of coating.

[0110] Alternatively, instead of applying the hydrophobic material at the locations shown in Figures 10 and 11 , laser treatment can be used to remove the existing hydrophilic layer to form a hydrophobic surface, as shown in Figure 12 . Laser treatment can include, for example, laser sintering or laser marking. The locations and dimensions of the laser treatment can remain consistent with those of the locations shown in the previous embodiments where the hydrophobic material was applied.

[0111] After laser treatment, the original hydrophilic layer is removed, and a concave structure is formed, which is inherently hydrophobic. In addition, due to the high temperature of the laser, the concave structure is dissolved on the side closest to the channel, forming a protrusion, further forming a physical barrier that prevents liquid from flowing into the concave structure.

[0112] Alternatively, in embodiments where the reaction region 2112 and the barrier region 212 are on the same horizontal plane, the barrier region 212 can be directly laser treated to form a hydrophobic surface. In this case, the substrate 1 can be coated with a hydrophobic material at the location corresponding to the barrier region 212, or laser treated as shown in FIG12 .

[0113] The following shows several examples of experiments using the microfluidic chip of the present invention to verify and illustrate the technical effects claimed in the present invention.

[0114] Example 1

[0115] Experimental purpose: To compare the effect of adding a control valve in the waste liquid area (control area) on the interpretation results.

[0116] 1. Material preparation

[0117] Microfluidic chip No. 1 before improvement and microfluidic chip No. 2 after improvement with a control valve. Compared with chip No. 1 before improvement, the improved chip No. 2 has an additional control valve in the waste liquid zone (control zone) at the end of the reaction zone. All of the above are produced by Shandong Maiwei Microbiology Technology Co., Ltd.

[0118] RSV clinical samples were obtained from relevant hospitals;

[0119] Shandong Maiwei Microbiology Technology Co., Ltd. produces fluorescent immunoassay analyzers, timers (such as stopwatches) and pipettes.

[0120] 2. Coating site

[0121] Coated spot 1 is located in the detection area and is coated with RSV antibodies;

[0122] The second coating point is located in the quality control area and is coated with secondary antibody.

[0123] The fluorescent labeling area is immobilized with dried fluorescently labeled paired antibodies.

[0124] 3. Detection method

[0125] 3.1 Improved microfluidic chip with control valve (chip 2)

[0126] The improved microfluidic chip with a control valve (chip No. 2) was placed flat on the laboratory bench, and RSV standard solution samples with concentrations of 100 ng / ml, 5 ng / ml, and 1 ng / ml were added to the sample wells of the microfluidic chip. After timing for 90 seconds, the control valve was used to allow the water-absorbing material to contact the reaction area, allowing the liquid in the reaction area to flow to the control area. The chip was interpreted using a fluorescent immunoassay analyzer, and the test results of the IgE samples were recorded. Each sample was tested twice, and the test signal value of each sample, the signal value of the quality control area (C line), and the signal value of the test area (T line) were recorded.

[0127] 3.2 Microfluidic chip before improvement (chip No. 1)

[0128] The microfluidic chip before improvement (chip No. 1) was placed flat on the laboratory bench. RSV standard solution samples with concentrations of 100 ng / ml, 5 ng / ml, and 1 ng / ml were added to the sample wells of the microfluidic chip. After the reaction, the samples naturally flowed to the waste liquid area at the end of the reaction area and were adsorbed by the absorbent material. The chip was interpreted by a fluorescent immunoassay analyzer, and the test results of the IgE samples were recorded. Each sample was tested twice, and the test signal value of each sample, the signal value of the quality control area (C line) and the test area (T line) were recorded.

[0129] 4. Results

[0130] As shown in Table 1, the improved microfluidic chip No. 2 with a control valve uses the control valve to control the reaction and then connects the reaction area and the control area 90 seconds after the reaction, so that the sample can fully react in the reaction area. The detected T-line and C-line signal values ​​are higher than those of the microfluidic chip No. 1 before the improvement.

[0131] Table 1 Sample test results before and after microfluidic chip improvement

[0132] Experimental conclusion: A control valve is added to the waste liquid area (control area) at the end of the reaction area to control the flow of liquid from the reaction area to the control area, thereby controlling the time in the sample reaction area so that the liquid in the reaction area flows to the control area after sufficient reaction, resulting in higher detected signal values ​​and higher accuracy.

[0133] Example 2

[0134] Experimental purpose: To compare the water absorption rate after adding a retention portion at the end of the reaction zone and its effect on the interpretation results.

[0135] 1. Material preparation

[0136] The improved IgE No. 3 microfluidic chip before improvement and the improved tooth-shaped IgE No. 4 microfluidic chip after improvement. Compared with the improved No. 3 chip before improvement, the improved No. 4 chip has a retention portion (a tooth portion in the embodiment) added to the upper cover of the channel at the end of the reaction zone (near the water-absorbing material end). All of the above are produced by Shandong Maiwei Microbiology Technology Co., Ltd.

[0137] IgE clinical serum sample S1 was obtained from the relevant hospital;

[0138] Shandong Maiwei Microbiology Technology Co., Ltd. produces fluorescent immunoassay analyzers, timers (such as stopwatches) and pipettes.

[0139] 2. Coating site

[0140] Coating point 1 is located in the detection area and is coated with IgE antibodies;

[0141] The second coating point is located in the quality control area and is coated with secondary antibody.

[0142] The fluorescent labeling area is fixed with dried IgE fluorescent labeling paired antibody.

[0143] 3. Detection method

[0144] 3.1 Improved tooth IgE microfluidic chip

[0145] The improved tooth-shaped microfluidic chip (chip #4) was placed flat on the laboratory bench. A 35 μL sample was added to the sample well of the microfluidic chip. After 90 seconds, the absorbent material was allowed to contact the channel and absorb water. The time it took for the absorbent material to absorb the liquid until the flow stopped was recorded. The chip was then analyzed using a fluorescent immunoassay analyzer and the IgE sample test results were recorded. Each sample was tested three times, and the signal value, peak value of the control zone (line C), and peak value of the test zone (line T) were recorded for each sample.

[0146] 3.2 IgE microfluidic chip before improvement

[0147] Before improvement, the IgE microfluidic chip (chip #3) was placed flat on the laboratory bench. A 35 μL sample was added to the sample well of the microfluidic chip. After 90 seconds, the absorbent material was allowed to contact the channel to absorb water. The time it took for the absorbent material to absorb the liquid until the flow stopped was recorded. The chip was then read using a fluorescent immunoassay analyzer, and the IgE sample test results were recorded. Each sample was tested three times, and the test signal value, peak value of the control zone (line C), and peak value of the test zone (line T) were recorded for each sample.

[0148] 4. Results

[0149] As shown in Table 2, the water absorption time for chip #4 was within 2 minutes and 30 seconds, while that for chip #3 was approximately 3 minutes. There was no significant difference in signal values. The improved chip reduced water absorption time by 30 seconds compared to the original chip.

[0150] Table 2 Sample test results before and after microfluidic chip improvement

[0151] Experimental Conclusion: Adding teeth to the end of the reaction zone diverts the liquid, increasing the contact surface between the liquid and the absorbent material. This increases the water absorption rate compared to the original structure and has little effect on the reading results. This can optimize the reading speed of the instrument.

[0152] Example 3

[0153] Experimental purpose: To compare and verify the effect of coating hydrophobic material on both sides of the substrate corresponding to the upper cover channel position on the interpretation results.

[0154] 1. Material preparation

[0155] Before the improvement, the No. 5 microfluidic chip for the quadruple test (RSV / FluA / FluB / COVID-19) and the No. 6 microfluidic chip for the quadruple test (same test items as above) coated with hydrophobic material were compared. After the No. 5 chip was assembled with the cover, the substrate of the No. 6 chip was sprayed with a hydrophobic coating at the corresponding channel position to prevent the sample from leaking from the channel or the sides of the reaction zone when the sample is overloaded or the concentration of microspheres is too high, which would affect the test results. All of the above are produced by Shandong Maiwei Microbiology Technology Co., Ltd.

[0156] RSV clinical sample S2 was obtained from a relevant hospital;

[0157] Shandong Maiwei Microbiology Technology Co., Ltd. produces fluorescent immunoassay analyzers, timers (such as stopwatches) and pipettes.

[0158] 2. Coating site

[0159] Coated spot 1 is located in the detection area and is coated with RSV antibodies;

[0160] Coating point 2 is located in the detection area and is coated with FluA antibody;

[0161] Coating point three is located in the detection area and is coated with FluB antibody;

[0162] Coating point four is located in the testing area and is coated with COVID-19 antibodies;

[0163] Coating point five is located in the quality control area and is coated with secondary antibody.

[0164] The fluorescent labeling area is immobilized with dried fluorescently labeled paired antibodies.

[0165] 3. Detection method

[0166] 3.1 Quadruple microfluidic chip coated with hydrophobic material

[0167] A quadruple microfluidic chip (chip #6) coated with hydrophobic material was placed flat on the laboratory bench. 35 μL of RSV sample was added to the sample well. After a 90-second count, a control valve was used to allow the absorbent material to absorb water for 3 minutes. The chip was then analyzed using a fluorescence immunoassay analyzer and the RSV test results were recorded. Each sample was tested three times, and any leakage was observed after sample addition. The signal value for each sample, including the peak value in the control zone (line C) and the peak value in the test zone (line T), was recorded.

[0168] 3.2 Improvement of the front quadruple test microfluidic chip

[0169] Before the improvement, the quadruple microfluidic chip (chip #5) was placed flat on the laboratory bench. 35 μL of RSV sample was added to the sample well of the microfluidic chip. After a 90-second count, a control valve was used to allow the absorbent material to absorb water for 3 minutes. The chip was then analyzed using a fluorescence immunoassay analyzer and the RSV sample test results were recorded. Each sample was tested three times, and any leakage was observed after sample addition. The test signal value for each sample, as well as the peak value of the control zone (line C) and the peak value of the test zone (line T) were recorded.

[0170] 4. Results

[0171] As shown in Table 3, even if liquid leaks occur in the quadruple test chip No. 6 coated with a hydrophobic material, the hydrophobic material intercepts the leaks, preventing the microspheres from scattering around the channel and affecting the detection background, nor does it leak into the reaction zone, affecting the flow of microspheres into the channel. The absorbent material absorbs the intercepted liquid and does not affect the test results. Before the improvement, the quadruple test chip No. 5 also leaked liquid, with microspheres leaking into the reaction zone, invalidating the results. Although the absorbent material can absorb the leaked liquid, nonspecific adsorption of microspheres in incorrect locations can still affect the interpretation.

[0172] Table 3 Sample test results before and after microfluidic chip improvement

[0173] Experimental conclusion: There is a certain probability of leakage. Coating with hydrophobic material can effectively intercept the leaked liquid and ensure the interpretation effect.

[0174] Example 4

[0175] Experimental purpose: To verify the advantages of multiple (more than two times) contact / detachment of the control area of ​​the dual-drive microfluidic chip for detecting IgE in plasma samples.

[0176] 1. Material preparation

[0177] Improved IgE microfluidic chips No. 7 and No. 8 were tested using a one-step method (chip No. 7): the control area contacted the channel once, and a two-step method (chip No. 8): the control area contacted the channel twice. Both were manufactured by Shandong Maiwei Biotechnology Co., Ltd.

[0178] IgE clinical plasma sample S3 was obtained from the relevant hospital;

[0179] Shandong Maiwei Microbiology Technology Co., Ltd. produces fluorescent immunoassay analyzers, timers (such as stopwatches) and pipettes.

[0180] 2. Coating site

[0181] Coating point 1 is located in the detection area and is coated with anti-human IgE antibodies;

[0182] The second coating point is located in the quality control area and is coated with biotinylated IgE antigen.

[0183] The labeling area is fixed with dried IgE fluorescently labeled paired antibodies.

[0184] 3. Detection method

[0185] 3.1 Dual-drive microfluidic chip control area filter twice separated from / contacted the channel to control the reaction process

[0186] An IgE microfluidic chip (chip #8) was placed flat on the laboratory bench. A 20 μL IgE plasma sample was added to the sample well. After 10 seconds, the plasma flowed through the detection and quality control zones and reached the top of the channel. A control valve was then activated to allow the absorbent material to contact the channel, absorbing excess plasma until the flow stopped (first contact). The plasma sample remaining in the channel reacted with the antibodies in the detection zone for 90 seconds. 35 μL of sheep serum was then added to the sample well as a buffer. After the buffer and the plasma in the channel merged, the sample continued to flow toward the control zone for approximately 10 seconds. Once the plasma in the channel had been absorbed and the fluorescent microspheres had dissolved and filled the channel, the control valve was activated to disconnect the absorbent material from the channel (absorbent material detachment). Two minutes later, the absorbent material was allowed to absorb water again for another 2 minutes (second contact). The chip was then read using a fluorescent immunoassay, and the IgE sample test results were recorded. Each sample was tested three times, and the instrument readings for each sample were recorded for the peak values ​​in the quality control zone (line C) and the detection zone (line T).

[0187] 3.2 Dual-drive microfluidic chip control area filter one-time separation / contact channel to control the reaction process

[0188] The IgE microfluidic chip (chip #7) was placed flat on the laboratory bench. A 20 μL plasma sample was added to the micro-wells. After 10 seconds, it flowed through the test and quality control zones. A control valve then allowed the absorbent material to contact the channel, absorbing excess plasma until the flow stopped (first contact). The plasma sample remaining in the channel reacted with the antibody for 90 seconds. 35 μL of sheep serum was added to the wells as a buffer. The fluorescent microspheres dissolved, flowed through the channel, and then continued to enter the absorbent material in the control zone. After absorbing water for 2 minutes, the chip was read using a fluorescent immunoassay analyzer, and the IgE sample test results were recorded. Each sample was tested three times, and the instrument-interpreted peak values ​​in the quality control zone (line C) and the test zone (line T) were recorded for each sample.

[0189] 4. Results

[0190] As shown in Table 4, the C-line peak and T-line peak of IgE at 6 min for the two-step control microfluidic chip (chip No. 8) were relatively consistent. The C-line peak of IgE detected by the one-step control microfluidic chip (chip No. 7) was lower (probably the result of the remaining IgE competing with the IgE antigen in the C region for binding to the antibody on the fluorescent microspheres), and the T-line value of the two-step method was higher than that of the one-step method.

[0191] Table 4 Sample test results before and after microfluidic chip improvement

[0192] Experimental conclusion: The detection results using two-step contact / disengagement control reaction are significantly better than those using one-step control.

[0193] Example 5

[0194] Experimental purpose: To compare and verify the effects of coating the substrate with hydrophobic material on both sides of the upper cover channel and laser treatment on the interpretation results.

[0195] 1. Material preparation

[0196] A quadruple test (same test items as above) of microfluidic chip No. 9 coated with hydrophobic material and microfluidic chip No. 10 after laser treatment. The difference between chip No. 10 and chip No. 9 is whether chip No. 10 is coated with hydrophobic material or laser sintered. All of the above are produced by Shandong Maiwei Microbiology Technology Co., Ltd.

[0197] RSV clinical sample S2 was obtained from a relevant hospital.

[0198] 2. Detection method

[0199] 2.1 Quadruple microfluidic chip coated with hydrophobic material

[0200] A quadruple microfluidic chip (chip #9) coated with hydrophobic material was placed flat on the laboratory bench. A 35μL RSV sample was added to the sample wells. The flow time of the sample in the microchannel was recorded. The time it took for the control valve to allow the water-absorbing material to absorb water was then recorded. The sample was then allowed to sit for 90 minutes. Each sample was tested three times, and any leakage was observed after addition.

[0201] 3.2 Laser-processed quadruple microfluidic chip

[0202] A laser-treated quadruple microfluidic chip (chip #10) was placed flat on the lab bench. A 35μL RSV sample was added to the sample well. The flow time of the sample in the microchannel was recorded. The time it took for the control valve to allow the water-absorbing material to absorb water was then recorded. The sample was then allowed to sit for 90 minutes. Each sample was tested three times, and any leakage was observed after addition.

[0203] 4. Results

[0204] As shown in Table 5, the hydrophobic-coated quadruple microfluidic chip No. 9 and the laser-treated quadruple microfluidic chip No. 10 both exhibited consistent liquid storage capacity, with no leakage. Laser-treated chip No. 10 did not affect liquid flow within the channels and required significantly shorter water absorption times.

[0205] Table 5 Sample test results before and after microfluidic chip improvement

[0206] Experimental conclusion: The laser-treated hydrophobic surface's liquid retention capacity matches that of a chip coated with hydrophobic material. After 90 minutes of standing, no leakage occurred, and the flow of liquid through the channel was unaffected. Both processes were completed in approximately 15 seconds. Furthermore, the laser-engraved hydrophobic surface significantly shortened subsequent water absorption time.

[0207] In known microfluidic chips, since the sample loading hole is set upstream of the labeling area, the sample will first react in the labeling area after passing through the loading hole, and then enter the detection area. For samples with high viscosity, they cannot push the signal molecules in the labeling area to the detection area by themselves, and subsequent buffer solution is required to push them. However, this method will dilute the sample, resulting in inaccurate test results. At the same time, other substances contained in the sample may also interfere with the signal molecules in the labeling area.

[0208] Furthermore, the "hook effect" of immune responses is another pressing issue that needs to be addressed. This phenomenon, also known as the "hook effect," refers to a false-negative phenomenon caused by an inappropriate antigen-antibody ratio. The detection signal intensity decreases with increasing antigen concentration. This is because the target analyte occupies the entire reaction zone, while the immune complex formed by the target analyte and the signal molecule is unable to react, or reacts only minimally, with the antigen / antibody in the detection zone, making it impossible to accurately detect the signal value.

[0209] In another aspect of the present invention, in response to the shortcomings of the prior art, the present invention provides a novel microfluidic chip and a detection method thereof. The present invention is described in detail below with reference to the accompanying drawings. It should be noted that in the accompanying drawings, components having substantially the same or similar structures and functions are assigned the same reference numerals, and their repeated descriptions are omitted.

[0210] As shown in Figures 15 and 16, a microfluidic chip according to at least one embodiment of the present invention includes a substrate 1, a cover sheet 2, and a microchannel 3 enclosed by the substrate 1 and the cover sheet 2. The grooves provided on the cover sheet 2 along its length direction L enclose the substrate 1 to form a microchannel. The microchannel 3 includes a buffer loading area 31, a reaction area 32, and a control area 33. The buffer loading area 31, the reaction area 32, and the control area 33 are sequentially connected from left to right along the length direction L. A movable water-absorbing material 4 is provided in the control area 33. The water-absorbing material 4 can be moved to contact or away from the reaction zone 32, and can be moved to contact the reaction zone 32 within a suitable time as needed to absorb excess sample in the microchannel 3. The reaction zone 32 includes a labeling zone 321, a detection zone 322 and a sample loading zone 323. The detection zone 322 and the sample loading zone 323 are both arranged on the same side of the labeling zone 321 (as shown in Figure 15, on the right side of the labeling zone 321), so that after the sample enters the microchannel through the sample loading zone 33, it first flows into the detection zone 32.

[0211] It should be noted that the length direction L in the drawings refers to the direction along the length of the microfluidic chip, which is exemplarily the horizontal rightward direction in the drawings.

[0212] Specifically, the detection area 322 is provided with detection sites and quality control sites in sequence from left to right along the length direction L. The number of the detection sites and quality control sites is one or more. The detection sites are used to detect the presence or concentration of the target analyte, and the quality control sites are used to verify the effectiveness of the reaction.

[0213] Specifically, the labeling area 321 includes antibodies\antigens labeled with signal molecules, the detection sites of the detection area 322 are coated with antibodies\antigens that can bind to the target analyte, the target analyte can simultaneously bind to the antibodies\antigens in the labeling area 321 and the detection sites to form a double antigen\antibody sandwich complex, and the quality control sites of the detection area 322 are coated with antibodies\antigens that can bind to the antibodies\antigens labeled with the signal molecules in the labeling area.

[0214] A braking hole 331 is provided on the right side of the control zone 33, extending at least partially to where the control zone 33 contacts the water-absorbing material 4. This braking hole 331 is a rectangular through-hole extending through the substrate 1 and the cover plate 2. A through-hole on the end of the water-absorbing material 4 facing away from the reaction zone 32 is positioned within this braking hole 331 to control the movement of the water-absorbing material 4. For example, a braking mechanism can be inserted into the braking hole 331 and passed through the through-hole in the water-absorbing material 4, thereby controlling the movement of the braking mechanism or the movement of the microfluidic chip, thereby controlling the movement of the water-absorbing material 4.

[0215] The water-absorbing material 4 can be made of polyester fiber, water-absorbing resin, water-absorbing gelatin, papermaking pulp or other materials with water-absorbing characteristics.

[0216] The buffer loading area 31 is provided with a buffer hole connecting the upper and lower surfaces of the cover sheet 2, and the sample loading area 323 is provided with a loading hole connecting the upper and lower surfaces of the cover sheet 2. Buffer and sample can be added to the microchannel 3 through the buffer hole and loading hole respectively.

[0217] The sample loading area 323 and the labeling area 321 are respectively arranged on both sides of the detection area 322. Therefore, after the sample enters the microchannel 3 through the sample loading area 323 or the loading hole, as shown in Figure 15, it flows to the left to the detection area 322 due to capillary action and combines with the detection area 322. The excess sample can be adsorbed by the water-absorbing material 4.

[0218] Alternatively, as shown in FIG17 , the sample loading area 323 may also be provided between the labeling area and the detection area 322. After the sample enters the microchannel 3 through the sample loading area 323 or the loading hole, it moves toward the left and right ends of the microchannel 3 due to capillary action. When flowing to the right, it flows to the detection area 322. After the reaction is completed, the excess sample is adsorbed by the water-absorbing material 4. However, this structure requires controlling the amount of sample added so that the sample is just combined with the detection area 322 and not with the labeling area 321.

[0219] For example, the antigen / antibody labeled with a signal molecule can be an antigen / antibody labeled with a signal molecule. The signal molecule can be, for example, a fluorescent protein, a fluorescent dye, a fluorescent microsphere, or a nanoparticle, such as a quantum dot.

[0220] On the other hand, the present invention also provides a microfluidic chip detection method, which uses the microfluidic chip as described above for detection, and the method includes at least the following steps:

[0221] 1) Add the sample into the microchannel 3 through the sample adding area 323 and keep the water-absorbing material 4 away from the reaction area 32;

[0222] 2) Waiting for a first period of time, after the sample has reversely flowed through the detection zone 322 due to capillary action, the water-absorbing material 4 is moved to contact the reaction zone 32 to absorb excess sample until the flow is cut off, and the sample retained in the microchannel 3 reacts with the detection zone 322;

[0223] 3) After the second reaction period, a buffer solution is added to the microchannel 3 through the buffer solution addition area 31. The buffer solution passes through the labeling area 321 under capillary action, flushing the antigen / antibody coated with the signal molecule in the labeling area 321 and then entering the detection area 322. When the signal molecules in the channel of the reaction area 32 merge with the sample, the water-absorbing material 4 in the control area 33 is moved to contact the reaction area 32, and the excess sample is adsorbed by the water-absorbing material 4 to the control area 33.

[0224] 4) After the excess sample in the channel is absorbed by the water-absorbing material 4 and the signal molecules in the labeling area are dissolved and spread throughout the channel of the reaction zone 32, the water-absorbing material 4 is moved away from the reaction zone 32 to break contact with the channel of the reaction zone 32;

[0225] 5) After the third reaction period, the water-absorbing material 4 in the control area 33 is moved to contact the detection area 322, and the excess liquid including the signal molecules is adsorbed into the control area 33 by the water-absorbing material 4;

[0226] 6) Finally, the signal value of the detection zone 322 is measured. For example, a detector can be used to measure the signal value of the detection zone for subsequent analysis (the detector is a conventional technology, such as a fluorescence detector, which is consistent with the signal carried by the antigen / antibody coated with the signal molecule).

[0227] For example, the first time period, the second time period, and the third time period can be adjusted according to needs and sample conditions. For example, the first time period can be in the range of 0 to 60 seconds, the second time period can be in the range of 10 to 300 seconds, and the third time period can be in the range of 30 seconds to 5 minutes.

[0228] The detection method can also use a first judgment condition to determine when to move the water-absorbing material 4 into contact with the reaction zone 32 to absorb excess sample until the flow is cut off. The above example illustrates the first time period. Alternatively, the first judgment condition can include the sample liquid volume being greater than or equal to a predetermined amount, where the predetermined amount is in the range of 15 μL to 50 μL; or the signal intensity of the signal molecule in the detection zone 322 being greater than or equal to a threshold value above the chip's reasonable background range.

[0229] Several examples of experiments using the microfluidic chip of the present invention are shown below to verify and illustrate the technical effects claimed in the present invention.

[0230] It should be noted that the detection method proposed in the present invention differs from the conventional microfluidic chip loading method (i.e., the sample and buffer are added simultaneously, also known as the "one-step method") because the detection area and sample loading area of ​​the chip used are both located on the same side of the labeling area. The detection method proposed in the present invention requires the separate addition of sample and buffer, and after the sample is added, the sample must flow into the detection area, hence the term "two-step method." In the embodiments described below, the term "one-step method" refers to the conventional microfluidic chip (also known as the pre-improvement chip), and the term "two-step method" refers to the microfluidic chip proposed in the present invention (also known as the improved chip).

[0231] Example 6

[0232] Experimental purpose: To verify whether the one-step and two-step microfluidic chips can use whole blood as the test sample.

[0233] 1. Material preparation

[0234] The improved IgE microfluidic chip 1 and the improved IgE two-step microfluidic chip 2. Compared with the improved one-step microfluidic chip, the improved two-step microfluidic chip has an additional micro-sample injection hole on the cover of the reaction zone channel 0.5 cm to the left of the control zone. Both are produced by Shandong Maiwei Microbiology Technology Co., Ltd.

[0235] IgE clinical whole blood sample S2 was obtained from the relevant hospital;

[0236] Shandong Maiwei Microbiology Technology Co., Ltd. produces fluorescent immunoassay analyzers, timers (such as stopwatches) and pipettes.

[0237] 2. Coating site

[0238] The coated spot 1 is located at the detection site 322 of the detection area and is coated with IgE antibodies;

[0239] The second coating point is located at the quality control site 322 in the detection area and is coated with a secondary antibody.

[0240] The labeling area 321 is immobilized with dried IgE fluorescently labeled paired antibodies.

[0241] 3. Detection method

[0242] 3.1 Improved IgE two-step microfluidic chip

[0243] The improved two-step IgE microfluidic chip is placed flat on the laboratory bench. A 10μL whole blood sample is added to the micro-sample well. After waiting 10 seconds for the whole blood sample to flow back through the quality control and detection sites, the absorbent material (4) is brought into contact with the channel to absorb excess blood until the flow is cut off. The remaining whole blood sample reacts with the antibody for 90 seconds. 35μL of sheep serum is added to the buffer well as a buffer. After the buffer and the whole blood in the channel are combined, the absorbent material (4) is allowed to absorb water for 10 seconds. Once the blood in the channel is absorbed and the signal molecules have dissolved and filled the channel, the absorbent material (4) is disconnected from the channel. After a two-minute reaction, the absorbent material (4) is again used to absorb water for another two minutes. The chip is then analyzed using a fluorescent immunoassay analyzer, and the IgE sample test results are recorded. Each sample is tested three times, and the signal values ​​at the quality control and detection sites are recorded for each sample.

[0244] 3.2 IgE microfluidic chip before improvement

[0245] Before the improvement, the IgE microfluidic chip was placed flat on the laboratory bench. A 35μL whole blood sample was added to the microfluidic chip sample wells and allowed to react for 2 minutes. Water was then absorbed using absorbent material 4 for 3 minutes. The chip was then analyzed using a fluorescent immunoassay analyzer, and the IgE sample test results were recorded. Each sample was tested three times, and the signal values ​​at the quality control and test sites were recorded for each sample.

[0246] 4. Results

[0247] As shown in Table 6, the IgE detection signal value, quality control site peak value, and detection site peak value for the two-step microfluidic chip 2 at 6 minutes were relatively consistent. However, the IgE detection results for the one-step microfluidic chip 1 were invalid within 5 minutes due to significant background interference from whole blood, and no quality control site peak value could be detected. Furthermore, the two-step method achieved higher detection site values ​​than the one-step method.

[0248] Table 6 Sample test results before and after microfluidic chip improvement

[0249] Experimental Conclusion: The two-step method significantly outperforms the one-step method. The addition of whole blood to the one-step method can lead to hemolysis, resulting in red background contamination and loss of detection of quality control sites, leading to invalid test results. The two-step method involves the sample first reacting with the antibody and then with the signal molecule dissolved in the buffer, flushing the whole blood sample from the channel and eliminating background contamination. The two-step method is feasible for whole blood samples, while the one-step method is not.

[0250] Example 7

[0251] Experimental purpose: To compare and verify whether the one-step method and the two-step method have the barb effect in detecting IgE serum samples.

[0252] 1. Material preparation

[0253] The improved IgE microfluidic chip 3 and the improved IgE two-step microfluidic chip 4. Compared with the improved one-step microfluidic chip, the improved two-step microfluidic chip has an additional micro-sample injection hole on the cover of the reaction zone channel 0.5 cm to the left of the control zone. All of the above are produced by Shandong Maiwei Microbiology Technology Co., Ltd.

[0254] IgE clinical serum sample S3 (concentration > 5000 ng / ml) was obtained from the relevant hospital;

[0255] Shandong Maiwei Microbiology Technology Co., Ltd. produces fluorescent immunoassay analyzers, timers (such as stopwatches) and pipettes.

[0256] 2. Coating site

[0257] Coated point 1 is located at the detection site of the detection area and is coated with IgE antibodies;

[0258] The second coating point is located at the quality control site of the detection area and is coated with a secondary antibody.

[0259] The labeling area is fixed with dried IgE fluorescently labeled paired antibodies.

[0260] 3. Detection method

[0261] 3.1 Improved IgE two-step microfluidic chip

[0262] The improved two-step IgE microfluidic chip is placed flat on the laboratory bench. 10 μL of sample is added to the micro-sample well. After waiting for 5 seconds for the sample to flow back through the quality control and detection sites, the absorbent material (4) is brought into contact with the channel to absorb excess sample until the flow is cut off. The sample remaining in the channel reacts with the antibody for 90 seconds. 35 μL of sheep serum is added to the buffer well as a buffer. After the buffer and the whole blood in the channel are combined, the absorbent material (4) is allowed to absorb water for 10 seconds. After the residual sample in the channel is absorbed and the signal molecules have dissolved and filled the channel, the absorbent material (4) is disconnected from the channel. After a 2-minute reaction, the absorbent material (4) is again used to absorb water for another 2 minutes. The chip is then read using a fluorescent immunoassay analyzer, and the IgE sample test results are recorded. Each sample is tested three times, and the signal values ​​at the quality control and detection sites are recorded for each sample.

[0263] 3.2 IgE microfluidic chip before improvement

[0264] Before the improvement, the IgE microfluidic chip was placed flat on the laboratory bench. A 35μL sample was placed into the microfluidic chip sample well for 2 minutes. Water was then absorbed using absorbent material 4 for 3 minutes. The chip was then analyzed using a fluorescent immunoassay analyzer, and the IgE sample test results were recorded. Each sample was tested three times, and the signal values ​​at the quality control and test sites were recorded for each sample.

[0265] 3.3 Dilute the S3 sample 10-fold with sheep serum and repeat the test process of 3.1 (chip 6) and 3.2 (chip 5), and record the relevant data.

[0266] 4. Results

[0267] As shown in Table 7, comparing the detection results of Chip 5 and Chip 3, when the one-step detection method was used, the peak value of the detection site decreased after the sample concentration increased 10-fold, and a hook appeared; comparing the detection results of Chip 6 and Chip 4, when the two-step detection method was used, the peak value of the detection site increased 10-fold after the sample concentration increased, and no hook appeared.

[0268] Table 7 Sample test results before and after microfluidic chip improvement

[0269] Experimental conclusion: The two-step method does not produce barbs when testing high-concentration samples, while the one-step method does. The two-step method is more accurate than the one-step method.

[0270] Example 8

[0271] 1. Material preparation

[0272] Pre-improved IgE microfluidic chip 7 and improved IgE two-step microfluidic chip 8. Compared to the pre-improved one-step microfluidic chip, the improved two-step microfluidic chip has an additional micro-sample well on the cover slip of the reaction zone channel, 0.5 cm to the left of the control zone. Both are manufactured by Shandong Maiwei Biotechnology Co., Ltd.

[0273] IgE clinical whole blood sample S1 was obtained from the relevant hospital;

[0274] Shandong Maiwei Microbiology Technology Co., Ltd. produces fluorescent immunoassay analyzers, timers (such as stopwatches) and pipettes.

[0275] 2. Coating site

[0276] The coated spot 1 is located at the detection site 322 of the detection area and is coated with IgE antibodies;

[0277] The second coating point is located at the quality control site 322 in the detection area and is coated with a secondary antibody.

[0278] The labeling area 321 is immobilized with dried IgE fluorescently labeled paired antibodies.

[0279] 3. Detection method

[0280] 3.1 Improved IgE two-step microfluidic chip

[0281] The improved two-step IgE microfluidic chip was placed flat on the laboratory bench. A 6μL sample was added to the micro-sample well. After a 30-second count, 35μL of sheep serum was added to the buffer well as a buffer. After the buffer and the whole blood in the channel were combined, the channel was then absorbed with absorbent material 4 for 10 seconds. Once the blood in the channel was absorbed and the signal molecules dissolved and filled the channel, the absorbent material 4 was disconnected from the channel. After a 2-minute reaction, the channel was again absorbed with absorbent material 4 for 2.5 minutes. The chip was then analyzed using a fluorescent immunoassay analyzer, and the IgE sample test results were recorded. Each sample was tested three times, and the signal value at each detection site was recorded, and the mean and deviation values ​​were calculated.

[0282] 3.2 IgE microfluidic chip before improvement

[0283] Before the improvement, the IgE microfluidic chip was placed flat on the laboratory bench. A 10μL sample, diluted approximately 10-fold with diluent (containing goat serum), was added to the microfluidic chip sample well for 2 minutes. Water was then absorbed using absorbent material 4 for 3 minutes. The chip was then analyzed using a fluorescent immunoassay analyzer, and the IgE sample test results were recorded. Each sample was tested three times, and the signal value at each sample detection site was recorded, with the mean and deviation calculated.

[0284] 4. Results

[0285] As shown in Table 8, the deviation of the IgE detection results of the improved IgE two-step microfluidic chip 8 at 6 minutes was within 3, and the deviation of the IgE detection results of the IgE microfluidic chip 7 before the improvement at 5 minutes was within 7. In terms of signal value, the two-step method is higher than the one-step method. The deviation of the improved detection results is significantly smaller than that before the improvement. The reaction order between the sample, the antibody and the signal molecule leads to different signal values, indicating that the improved chip detection results are more accurate and the signal is stronger.

[0286] Table 8 Sample test results before and after microfluidic chip improvement

[0287] Example 9

[0288] 1. Material preparation

[0289] The improved quadruple test (RSV / FluA / FluB / COVID-19) microfluidic chip 9 and the improved quadruple test (same test items as above) two-step microfluidic chip 10. Compared with the improved one-step microfluidic chip, the improved two-step microfluidic chip has an additional micro-sample injection hole on the cover of the reaction zone channel 0.5 cm to the left of the control zone. All of the above are produced by Shandong Maiwei Microbiology Technology Co., Ltd.

[0290] RSV clinical sample S1 was obtained from a relevant hospital;

[0291] Shandong Maiwei Microbiology Technology Co., Ltd. produces fluorescent immunoassay analyzers, timers (such as stopwatches) and pipettes.

[0292] 2. Coating site

[0293] Coating point one is located at detection site 1 in detection area 322 and is coated with RSV antibody; coating point two is located at detection site 2 in detection area 322 and is coated with FluA antibody; coating point three is located at detection site 3 in detection area 322 and is coated with FluB antibody; coating point four is located at detection site 4 in detection area 322 and is coated with COVID-19 antibody; coating point five is located at quality control site 322 in detection area and is coated with secondary antibody.

[0294] The fluorescent labeling region 321 is immobilized with dried fluorescent labeling paired antibodies.

[0295] 3. Detection method

[0296] 3.1 Improved quadruple two-step microfluidic chip

[0297] The improved quadruple two-step microfluidic chip was placed flat on the laboratory bench. 35 μL of RSV sample was added to the micro-wells. After waiting for 0 seconds, the RSV sample flowed back through the quality control site and the detection site. Then, the absorbent material 4 was brought into contact with the channel to absorb excess sample until the flow stopped. The RSV sample remaining in the channel reacted with the antibody for 90 seconds. Once the sample was absorbed and the signal molecules dissolved and filled the channel, the absorbent material 4 was disconnected from the channel. After a 2-minute reaction, the absorbent material 4 was again used to absorb water for 2.5 minutes. The chip was then analyzed using a fluorescent immunoassay analyzer and the RSV sample test results were recorded. Each sample was tested three times. The signal value at detection site 1 for each sample was recorded, and the mean and deviation values ​​were calculated.

[0298] 3.2 Improvement of the front quadruple test microfluidic chip

[0299] Before the improvement, the quadruple microfluidic chip was placed flat on the laboratory bench. 35 μL of RSV sample was added to the microfluidic chip sample wells and allowed to react with the antibodies for 2 minutes. Water was then absorbed using absorbent material 4 for 3 minutes. The chip was then analyzed using a fluorescent immunoassay analyzer and the RSV test results recorded. Each sample was tested three times, and the signal value at detection site 1 for each sample was recorded, with the mean and deviation calculated.

[0300] 4. Results

[0301] As shown in Table 9, the RSV test results of the improved quadruple two-step microfluidic chip 10 at 6 minutes had a deviation of less than 3, while the RSV test results of the quadruple microfluidic chip 9 before the improvement had a deviation of less than 4 at 5 minutes. Furthermore, the signal value of the two-step method was higher than that of the one-step method. The deviation of the improved test results was smaller than that before the improvement. The reaction order between the sample, the antibody, and the signal molecule resulted in different signal values, indicating that the improved chip test results are more accurate and the signal is stronger.

[0302] Table 9 Sample test results before and after microfluidic chip improvement

[0303] The above technical solutions only represent preferred technical solutions of the present invention. However, those skilled in the art will appreciate that various modifications and variations may be made to the above specific embodiments without departing from the principles of the present invention. Furthermore, various combinations of the various technical features and structures proposed in various aspects of the present invention may be made without departing from the scope of protection of the present invention, which is determined by the appended claims.

[0304] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0305] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, features defined as "first", "second", etc. may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0306] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "installed," "connected," "connected," and "disposed" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

Claims

1. A microfluidic chip, comprising: Substrate (1); A cover plate (2) comprising a groove (21) extending along a length direction (L); as well as a microchannel (3) formed by enclosing the substrate (1) and the groove (21), The invention is characterized in that the groove (21) of the cover sheet (2) comprises a channel area (211), and the channel area (211) is provided with a sample adding area (2111), a reaction area (2112) and a control area (2113) in sequence along the length direction (L), and the sample adding area (2111), the reaction area (2112) and the control area (2113) are fluidically connected to each other. The control zone (2113) is provided with a control valve (2113a), and the control valve (2113a) is configured to control the flow of the liquid in the reaction zone (2112) to the control zone (2113), and Wherein, the height of the reaction zone (2112) is lower than the height of the control zone (2113), and the width of the reaction zone (2112) is smaller than the width of the control zone (2113).

2. The microfluidic chip according to claim 1, characterized in that: The control zone (2113) gradually narrows from the middle to the reaction zone (2112) to make it easier to contact the reaction zone liquid and maintain a detached state.

3. The microfluidic chip according to claim 1, characterized in that: The control valve (2113a) comprises: A movable fluid suction member (2113a1), the fluid suction member (2113a1) being able to contact or move away from the reaction zone (2112); and A braking hole (2113a3), wherein the braking hole (2113a3) is arranged on a side away from the reaction zone (2112), wherein the braking hole (2113a3) is a rectangular through hole that passes through the substrate (1) and the cover plate (2), and a through hole (4) is arranged on one end of the fluid suction member (2113a1) away from the reaction zone, and the through hole (4) is inside the braking hole (2113a3).

4. The microfluidic chip according to claim 3, characterized in that: The control area (2113) is provided with a stopper (5), and the stopper (5) is configured to keep the fluid suction member (2113a1) at a position away from the reaction area (2112).

5. The microfluidic chip according to claim 4, characterized in that: The locking portion (5) includes a convex portion arranged along the width direction (W), and the fluid suction member (2113a1) is correspondingly provided with a concave portion that cooperates with the convex portion.

6. The microfluidic chip according to claim 3, characterized in that: The cover sheet (2) is provided with a retention portion (2113a2) at the connection between the control zone (2113) and the reaction zone (2112), and the retention portion (2113a2) at least partially extends into the reaction zone (2112), so that when the fluid absorption member (2113a1) contacts the channel of the reaction zone (2112), at least a portion of the fluid absorption member (2113a1) is inserted into the retention portion (2113a2).

7. The microfluidic chip according to claim 6, characterized in that: The retention portion (2113a2) is a tooth portion, which is constructed as a concave arc extending into the reaction zone (2112), and the lower end of the arc is provided with a sawtooth or wave structure, wherein the fluid suction piece (2113a1) gradually narrows on the side close to the reaction zone (2112), and a notch is provided in the middle of the side close to the reaction zone (2112), so that when the fluid suction piece (2113a1) contacts the channel of the reaction zone (2112), the notch is inserted into the tooth portion (2113a2).

8. The microfluidic chip according to claim 6, characterized in that: The retention portion (2113a2) is an inclined trough inclined toward the reaction zone (2112).

9. The microfluidic chip according to any one of claims 3 to 8, characterized in that: The fluid absorbent (2113a1) comprises a water-absorbent material, wherein the water-absorbent material comprises at least one of a desiccant, an antioxidant, and a moisture absorption indicator.

10. The microfluidic chip according to claim 1, characterized in that: The sample addition area (2111) is provided with a sample addition hole connecting the upper and lower surfaces of the cover sheet (2); the height of the sample addition hole at the lower surface of the cover sheet (2) is on the same horizontal plane as the height of the reaction area (2112).

11. The microfluidic chip according to claim 1, characterized in that: The groove (21) is rectangular, the shape of the substrate (1) matches the groove (21), and the substrate (1) is placed in the groove (21) and is on the same horizontal plane as the surface of the cover plate (2).

12. The microfluidic chip according to claim 1, characterized in that: A channel area (211), a barrier area (212) and a pressing area (213) are respectively arranged in the groove (21) of the cover sheet (2) from the middle to the outside along the width direction (W); the heights of the channel area (211) and the barrier area (212) are higher than the height of the pressing area (213), so that the substrate (1) is placed in the groove (21) on the lower surface of the cover sheet (2) and contacts the pressing area (213), and a gap is formed between the channel area (211) and the barrier area (212).

13. The microfluidic chip according to claim 12, characterized in that: The barrier zone (212) is arranged at the periphery of the sample adding zone (2111) and / or the reaction zone (2112) of the channel zone (211), and in the length direction (L), the barrier zone (212) covers at least a portion of the reaction zone (2112), or covers the sample adding zone (2111) and / or the reaction zone (2112).

14. The microfluidic chip according to claim 12, characterized in that: in, The base sheet (1) is coated with a hydrophobic material at the position of the corresponding barrier area (212) in the groove (21) of the cover sheet (2).

15. The microfluidic chip according to claim 14, characterized in that: The reaction zone (2112) and the barrier zone (212) are on the same horizontal plane, and the barrier zone (212) is also coated with a hydrophobic material.

16. The microfluidic chip according to claim 12, characterized in that: in, The substrate (1) is laser-processed at the position of the corresponding barrier area (212) in the groove (21) of the cover sheet (2) to form a hydrophobic surface.

17. The microfluidic chip according to claim 14 or 16, characterized in that: The reaction area (2112) and the barrier area (212) are on the same horizontal plane, and the barrier area (212) is laser-treated to form a hydrophobic surface.

18. The microfluidic chip according to claim 14, characterized in that: The height of the reaction zone (2112) is lower than the height of the barrier zone (212), so that the sample will not enter the barrier zone (212) due to gravity when flowing along the reaction zone (2112).

19. A microfluidic chip, comprising: Substrate (1); Cover sheet (2); as well as A microchannel (3) formed by enclosing a substrate (1) and a cover sheet (2), The microchannel (3) comprises a buffer solution adding area (31), a reaction area (32) and a control area (33), wherein the buffer solution adding area (31), the reaction area (32) and the control area (33) are sequentially connected along the length direction (L), a movable water-absorbing material (4) is arranged in the control area (33), and the water-absorbing material (4) can contact or move away from the reaction area (32), and The reaction area (32) includes a labeling area (321), a detection area (322) and a sample adding area (323), and the detection area (322) and the sample adding area (323) are both arranged on the same side of the labeling area (321).

20. The microfluidic chip according to claim 19, characterized in that: The cover sheet (2) is provided with a groove along the length direction (L), and the groove and the base sheet (1) are combined to form the microchannel (3).

21. The microfluidic chip according to claim 19, characterized in that: The detection area (322) is provided with detection sites and quality control sites in sequence along the length direction (L), and the number of the detection sites and quality control sites is one or more.

22. The microfluidic chip according to claim 19, characterized in that: The labeling area (321) includes antibodies\antigens labeled with signal molecules, and the detection sites of the detection area (322) are coated with antibodies\antigens that can bind to the target analyte. The target analyte can simultaneously bind to the antibodies\antigens in the labeling area (321) and the detection sites to form a double antigen\antibody sandwich complex, and the quality control sites of the detection area (322) are coated with antibodies\antigens that can bind to the antibodies\antigens labeled with the signal molecules in the labeling area (321).

23. The microfluidic chip according to claim 19, characterized in that: A braking hole (331) is provided on the side of the control zone (33) opposite to the reaction zone (32), and the braking hole (331) is a rectangular through hole that penetrates the substrate (1) and the cover plate (2), and at least partially extends to the water-absorbing material (4) in the control zone (33), wherein a through hole is provided at one end of the water-absorbing material (4) away from the reaction zone, and the through hole is within the braking hole (331).

24. The microfluidic chip according to claim 19, characterized in that: The buffer solution loading area (31) is provided with a buffer solution hole connecting the upper and lower surfaces of the cover sheet (2), and the sample loading area (323) is provided with a loading hole connecting the upper and lower surfaces of the cover sheet (2).

25. The microfluidic chip according to claim 19, characterized in that: The sample addition area (323) and the labeling area (321) are respectively arranged on two sides of the detection area (322).

26. The microfluidic chip according to claim 19, characterized in that: The sample addition area (323) is arranged between the labeling area (321) and the detection area (322).

27. The microfluidic chip according to claim 22, characterized in that: The antigen\antibody labeled with the signal molecule is an antigen\antibody labeled with a signal molecule.

28. A detection method for a microfluidic chip, characterized in that: The microfluidic chip according to any one of claims 1 to 9 is used for detection, and the method comprises at least the following steps: Adding a sample into the microchannel (3) through the sample adding area (323), and keeping the water-absorbing material (4) away from the reaction area (32); After the sample flows through the detection zone (322) in the reverse direction under the capillary action and satisfies the first judgment condition, the water-absorbing material (4) is moved to contact the reaction zone (32) to absorb the excess sample until the flow is cut off, and the sample retained in the microchannel (3) reacts with the detection zone (322); then, the water-absorbing material (4) is separated from or continues to contact the reaction zone (32); After the second reaction period, a buffer solution is added into the microchannel (3) through the buffer solution adding area (31). The buffer solution passes through the labeling area (321) under the capillary action, washes the antigen\antibody coated with the signal molecule in the labeling area (321) and enters the detection area (322). When the signal molecule in the channel of the reaction area (32) merges with the sample, the water-absorbing material (4) in the control area (33) is moved to contact the reaction area (32), and the excess sample is adsorbed to the control area (33) through the water-absorbing material (4). After the excess sample in the channel is absorbed by the water-absorbing material (4) and the signal molecules in the labeling area (321) are dissolved and spread over the channel of the reaction area (32), the water-absorbing material (4) is moved away from the reaction area (32) so that the water-absorbing material (4) is disconnected from the channel of the reaction area (32); After the third reaction period, the water-absorbing material (4) in the control area (33) is moved to contact the detection area (322), and the excess liquid including the signal molecule is adsorbed to the control area (33) through the water-absorbing material (4); The signal value of the detection zone (322) is measured.

29. The detection method according to claim 28, characterized in that: The first judgment condition includes: a first time period has passed or the amount of sample liquid is greater than or equal to a predetermined amount or the signal intensity of the signal molecule in the detection area (322) is greater than or equal to a threshold.

30. The detection method according to claim 29, characterized in that: The first time period is in the range of 0 to 60 seconds, and the predetermined amount is in the range of 15 μL to 50 μL.

31. The detection method according to claim 28 or 29, characterized in that: The second time period is in the range of 10 seconds to 300 seconds, and the third time period is in the range of 30 seconds to 5 minutes.

Images