Micro-fluidic chip with step-by-step control and fluorescence enhancement functions

Abstract

The utility model discloses a micro-fluidic chip with step-by-step control and fluorescence enhancement functions. The micro-fluidic chip comprises a shell and a micro-channel system in the shell, the shell is enclosed by a bottom plate and a cover plate, and the cover plate comprises a sample adding hole and an observation hole; the micro-channel system comprises a sample adding area, a sample separating area, a dissolving area, a time control area, a mixing channel area and a detection area which are sequentially connected in the flowing direction of a sample solution; the detection area comprises a sample detection area and a quality control detection area which are connected in parallel; and a fluorescent marker is coated in the dissolving area. The kit has the advantages that 1) the sensitivity and the accuracy of disease detection can be obviously improved; (2) a reverse cleaning system is adopted, and blow-drying is introduced, so that better conditions can be provided for fluorescent signal reading.

Description

TECHNICAL FIELD

[0001] The utility model relates to a kind of in vitro immune detection technology, especially a kind of immune detection microfluidic chip. BACKGROUND

[0002] Microfluidic technology is a kind of technology system based on micron scale fluid control, by integrating biological / chemical experimental function module in square centimeter chip, with the help of active or passive fluid driving mechanism, the flow rate, flow direction and filling volume of microliter / nanoliter level liquid can be realized Precise control.Compared with traditional immunochromatography test paper detection card, this technology has the advantages of less sample consumption (usually 1 / 10-1 / 100 of traditional method), high detection sensitivity (up to fg / mL level) and operation automation, and has become the core technology path in the field of point-of-care testing (POCT).

[0003] Through the research on the existing immune fluorescence detection microfluidic chip, the following key defects need to be solved:

[0004] 1, signal sensitivity is limited: the existing scheme is mostly dependent on the biological reagent coating of preset function area, and the detection is realized by directly collecting fluorescent dye emission signal. Due to the low intensity of fluorescent signal and the interference of background, the lower limit of detection is high (usually >1ng / mL), which is difficult to meet the detection demand of trace biomarker;

[0005] 2, reaction mode defect: the second antibody immune reaction depends on the dynamic flow of fluid to transport the first antibody reaction combination to the T line and C line marking area arranged in series for capture and fixation. This mode is affected by the coupling of fluid stability, material surface characteristics and reagent efficiency, which easily leads to the fluctuation of target capture rate, and further causes false positive / false negative problem;

[0006] 3, lack of function module: the existing chip is mostly integrated with sample adding, mixing, reaction and waste liquid collecting module, lacks key cleaning function unit, which leads to non-specific binding residue and reagent background signal interference (signal-to-noise ratio decreases by 30%-50%), and seriously reduces the detection precision. UTILITY MODEL CONTENT

[0007] In order to simplify the chip structure, and improve the detection ability of weak fluorescent signal and the precision of quantitative analysis, the utility model provides a kind of microfluidic chip with step-by-step control and fluorescence enhancement function.

[0008] The utility model discloses a technical scheme that solves its technical problems: microfluidic chip with step-by-step control and fluorescence enhancement function, including casing and microfluid channel system in casing, the casing is enclosed by bottom plate and cover, the cover includes sample adding hole and observation hole, the microfluid channel system includes sample adding area, sample separation area, dissolving area, time control area, mixing channel area and detection area that are sequentially connected by microfluid channel along sample solution flow direction, the detection area includes sample detection area and quality control detection area that are parallel to each other, and the fluorescence marker is coated in the dissolving area.

[0009] It is easy to understand that the function of time control area is to prolong the residence time of sample solution in microchannel system, especially in dissolving area, so that the elongated microfluid channel can be usually used, for example, the flow channel width of time control area can be less than mixing area, and can be designed as 'S' shape or other similar forms, as long as the above-mentioned purposes can be achieved.

[0010] As a further improvement of the utility model, the microfluid channel system further includes a cleaning and drying access end, the sample detection area and the quality control detection area are connected with the cleaning and drying access end through the microfluid channel, and a cleaning and drying access opening corresponding to the cleaning and drying access end is formed in the cover. The embedded cleaning unit can reduce background interference and improve signal-to-noise ratio, and the detailed principle and operation method can be referred to the specific embodiment part.

[0011] As a further improvement of the utility model, the microfluid channel system further includes a waste liquid area for collecting waste liquid generated by cleaning, and the sample adding area is communicated with the waste liquid area through the microfluid channel. As shown in the specific embodiment, the waste liquid area can be distributed along the periphery of the bottom plate, and a material with strong water absorption performance, such as sponge or water absorption paper, is preferably selected, so that the capillary force of the waste liquid area is greater than that of other microfluid channel parts, so as to avoid backflow of waste liquid.

[0012] As a further improvement of the utility model, the cover is provided with an exhaust hole communicated with the microfluid channel system. During cleaning, the exhaust hole communicates the microfluid channel with the atmosphere, so that the cleaning liquid pumped through the cleaning and drying opening can smoothly enter the waste liquid area.

[0013] In order to prevent the sample solution from flowing to the waste liquid area, a first step stop valve for cutting off the sample solution into the waste liquid area is arranged on the microfluid channel between the waste liquid area and the sample adding area. Referring to Figure 8 , the step stop valve is realized by vertical expansion in the depth direction of the microchannel. When the liquid reaches the step stop valve, the capillary contact angle changes, so as to cut off the liquid.

[0014] As a further improvement of the utility model, the mixed flow channel area is sequentially first mixed flow channel area, second mixed flow channel area and third mixed flow channel area along the sample solution flow direction, and the first mixed flow channel micro flow channel depth < second mixed flow channel micro flow channel depth = third mixed flow channel micro flow channel depth < detection area micro flow channel depth; this flow channel design scheme can make the sample solution slowly enter the detection area to prevent the generation of bubbles; easily understood, the width of the third mixed flow channel can also be designed to be less than the width of the first and second mixed flow channels; or the third mixed flow channel is designed to gradually reduce the flow area along the sample solution flow direction, so that the detection area can be further ensured to slowly enter the liquid without bubble residue.

[0015] The first mixed flow channel and the second mixed flow channel, and the third mixed flow channel and the detection area are provided with flow valves for breaking the capillary cutoff effect. It is easily understood that the flow valve and the step cutoff valve have opposite effects, and the purpose is to make the liquid continue to flow at the step (for example Figure 9 The U-shaped flow valve can break the potential energy between liquid molecules, so that the liquid can continue to fall to the bottom of the step at the step). The person skilled in the art can select the flow valve according to the needs, and as a preferred scheme, the U-shaped flow valve shown in Figure 9 is adopted between the first mixed flow channel and the second mixed flow channel, and the stepped micro-step flow valve shown in Figure 10 is adopted between the third mixed flow channel and the detection area.

[0016] As a further improvement of the utility model, the second step cutoff valve for cutting off the continuous flow of the sample solution is arranged on the micro flow channel at the end of the sample detection area and the quality control detection area. The function of the second step cutoff valve is to prevent the sample solution from continuing to flow through the detection area, so that the sample is static in the detection area. In this scheme, the static and parallel micro-reaction chamber design is used to replace the dynamic series flow reaction mode in the prior art, which enhances the controllability, independence and stability of the reaction, and eliminates the dynamic flow interference of the traditional immunochromatography or microfluidic scheme.

[0017] As a further improvement of the utility model, the sample splitting area is composed of at least three parallel microchannels connecting the sample adding area and the dissolution area, and the connecting end of the sample adding area is arranged in an arc shape.

[0018] As a further improvement of the utility model, the dissolving area is provided with a cross microarray structure for driving the sample solution to flow in the microchannel. The cross microarray structure can not only enhance the self-driven crawling ability of the sample solution in the microchannel system, but also enhance the surface area of the fluorescent marker coating area, so that the sample solution and the fluorescent marker can be fully contacted. It is easy to understand that the cross microarray or other forms of flow structure such as a wave shape can also be arranged in the first mixing area, so that the sample solution and the fluorescent marker can be better mixed.

[0019] As a further improvement of the utility model, the detection area is provided with a fluorescent enhancement reaction unit. The specific fluorescent enhancement structure is preset in the detection area, and the narrowband filter system of the instrument is matched, so that the problem of insufficient detection ability of the existing fluorescent detection scheme for weak fluorescent signals is solved. The fluorescent reflection unit can adopt a dot matrix structure, for example, a 3*3 or 5*5 photonic crystal, to reflect specific wavelength fluorescent light, and the fluorescent reflection unit is also marked with a capture antigen or an antibody.

[0020] The cover plate of the utility model can also cover a label, and the label is provided with a scanning window, wherein the part corresponding to the reaction detection area is transparent, and the remaining area is non-transparent, and the best structure of the non-transparent area is black frosted, so as to reduce the background signal noise.

[0021] The utility model discloses a microfluidic immunochromatography reagent card, adopts passive drive scheme, can accurately control the stop and flow of fluid in each functional area through the function valve arranged at different positions, guarantees full reaction, and can greatly enhance the immunofluorescence signal intensity and improve the sensitivity and accuracy of disease detection in combination with the fluorescent reflection material or structure arranged in the area. 2) The utility model adopts reverse cleaning operation, and the cleaning liquid is washed back from the detection area to the sample adding area, can guarantee that the substances participating in the reaction are removed in time, avoids background signal interference, and through the introduction of blow drying, the cleaning liquid in the detection area after cleaning can be discharged, and better conditions are provided for the reading of the fluorescent signal. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a microfluidic chip surface structure diagram of the specific embodiment.

[0023] Figure 2 It is a structure explosion diagram of the microfluidic chip of the specific embodiment.

[0024] Figure 3 It is a microfluidic channel system structure schematic diagram of the microfluidic chip of the specific embodiment.

[0025] Figure 4 It is a mixing flow channel area structure schematic diagram of the microfluidic chip of the specific embodiment.

[0026] Figure 5 is a schematic diagram of a mixed flow channel area of the microfluidic chip of the specific embodiment.

[0027] Figure 6 is a schematic diagram of a cover plate structure of the microfluidic chip of the specific embodiment.

[0028] Figure 7 is a sectional view of a waste liquid area of the microfluidic chip of the specific embodiment.

[0029] Figure 8 is a schematic diagram of a step cut-off valve structure of the microfluidic chip of the specific embodiment.

[0030] Figure 9 is a schematic diagram of a U-shaped flow-through valve structure of the microfluidic chip of the specific embodiment.

[0031] Figure 10 is a schematic diagram of a stepped micro-step flow-through valve structure of the microfluidic chip of the specific embodiment.

[0032] Figure 11 is a schematic diagram of a cross microarray structure of the microfluidic chip of the specific embodiment.

[0033] In the figure, 1 is a bottom plate, 2 is a cover plate, 201 is a sample adding hole, 202 is an observation hole, 203 is a cleaning and drying access port, 204 is an exhaust hole, 205 is a gas permeation valve, 3 is a sample adding area, 4 is a sample dividing area, 5 is a dissolving area, 6 is a timing area, 7 is a mixed flow channel area, 701 is a first mixed flow channel, 702 is a second mixed flow channel, 703 is a third mixed flow channel, 8 is a sample detection area, 9 is a quality control detection area, 10 is a cleaning and drying access end, 11 is a waste liquid area, 12 is a first step cut-off valve, 13 is a U-shaped flow-through valve, 14 is a stepped micro-step flow-through valve, 15 is a second step cut-off valve, 16 is a fluorescence enhancement reaction unit, 17 is a cleaning liquid buffer area, 18 is a label, 1801 is a scanning window. Specific Embodiment

[0034] The utility model is further described below in combination with the drawings and examples.

[0035] As Figures 1-11As shown, the microfluidic chip with step-by-step control and fluorescence enhancement function of the utility model, utilize the capillary tension of sample solution in microchannel to flow, including the casing and the micro flow passage system inside the casing;The casing is enclosed by the way of bonding by bottom plate 1 and cover plate 2, and cover plate 2 covers label 18. Cover plate 2 includes sample adding hole 201 and observation hole 202;The micro flow passage system includes sample adding area 3, sample separation area 4, dissolution area 5, time control area 6, mixing flow passage area 7 and detection area sequentially connected along the flow direction of sample solution by micro flow passage;The detection area includes sample detection area 8 and quality control detection area 9 which are parallel to each other;Fluorescent marker is coated in the dissolution area 5;Dissolution area 5 is further provided with cross microarray for driving sample solution to flow in micro flow passage;The detection area is provided with fluorescence enhancement reaction unit 16.

[0036] The micro flow passage system further includes cleaning and drying access end 10, and the detection area is connected with the cleaning and drying access end 10 through micro flow passage;Cover plate 2 is provided with cleaning and drying access hole 203 corresponding to the cleaning and drying access end 10. The micro flow passage system further includes waste liquid area 11 for collecting waste liquid generated by cleaning;The sample adding area 3 is communicated with the waste liquid area 11 through micro flow passage. Cover plate 2 is provided with exhaust hole 204 communicated with the micro flow passage system and air permeable valve 205 for preventing waste liquid from overflowing reagent card. First step stop valve 12 is arranged on the micro flow passage between the waste liquid area 11 and the sample adding area 3 for stopping sample solution from entering the waste liquid area 11.

[0037] The mixing flow passage area 7 sequentially includes first mixing flow passage 701 (with micro column array eddy structure), second mixing flow passage 702 and third mixing flow passage 703 along the flow direction of sample solution. Since the detection area needs to ensure sufficient reaction system (the reaction system of the present scheme is 20 μl), considering that sample solution has sufficient capillary driving capacity before entering the detection area, the flow passage depth from the mixing area to the detection area has a gradient relationship: the micro flow passage depth of the first mixing flow passage 701 < the micro flow passage depth of the second mixing flow passage 702 = the micro flow passage depth of the third mixing flow passage 703 < the micro flow passage depth of the detection area, and the width of the third mixing flow passage is smaller than the width of the first and second mixing flow passages so as to ensure that the fluid slowly enters the reaction detection area and avoid generating air bubbles. U-shaped flow valve 13 and stepped micro-step flow valve 14 are arranged between the first mixing flow passage 701 and the second mixing flow passage 702 and between the third mixing flow passage 702 and the detection area respectively for breaking the capillary stop effect, so as to realize step-by-step breaking of the capillary stop effect and ensure that sample solution smoothly enters the detection area. Second step stop valve 15 is arranged on the micro flow passage at the end of the sample detection area 8 and the quality control detection area 9 for stopping sample solution from continuously flowing. The sample separation area 4 is composed of multiple micro channels parallel to each other and connecting the sample adding area 3 and the dissolution area 5, and the connection end of the sample adding area 3 is arranged in arc shape.

[0038] The detection process is as follows:

[0039] When the user performs the detection, first, the blood sample is pre-filtered and diluted, and a certain amount of sample solution is added from the sample adding hole 201 to the sample adding area 3 by means of a sample adding tool. Under the capillary force, the sample starts to flow. Since the first step stop valve 12 exists, the blood sample flows to the valve and is automatically stopped, thereby ensuring that the sample can flow to the sample dividing area 4. After the action of the sample dividing area 4, the sample solution is evenly spread along the width direction of the flow channel, and then enters the dissolving area 5. The area is pre-embedded with a fluorescently labeled substance coated with a primary antibody. By means of the staggered distribution of the micro-column array features, the reconstitution and mixing of the fluorescent standard substance are realized. Then, the mixed solution flows through the S-shaped time control area 6 flow channel (width of 400 um, height of 100 um). The flow slows down, further ensuring that the sample solution has sufficient time to stay in the dissolving area 5 and mix with the fluorescently labeled substance arranged in the dissolving area 5. In order to improve the mixing of the sample and the fluorescently labeled substance, a mixing flow channel area 7 is further arranged behind the time control area 6 flow channel. After dynamic mixing, the sample mixed solution flows into the reaction detection area 8 and the quality control detection area 9, and is stopped at the second step stop valve 15, realizing static reaction. Finally, the microfluidic chip is inserted into the instrument, and the instrument automatically seals the sample adding hole 201. At the same time, the interface on the instrument is connected with the cleaning and drying interface 203. After the mixed sample is fully reacted in the reaction detection area 8 and the quality control detection area 9, the user or the instrument automatically adds cleaning solution to the cleaning solution buffer area 17, and then adds positive pressure to the cleaning solution buffer area 17. The cleaning solution enters the reaction detection area 8 and the quality control detection area 9 under the action of pressure, and pushes the reagent in the cavity which does not participate in the immune reaction to flow in the opposite direction of the sample adding operation, and finally breaks through the first step stop valve 12 and flows into the waste liquid area 11. In order to ensure that the cleaning solution can smoothly enter the waste liquid area 11, the exhaust hole 204 and the air permeable valve 205 are specially set. The air permeable valve 205 can prevent the waste liquid in the waste liquid area 11 from overflowing the reagent card and causing pollution. Finally, when the cleaning solution is completely discharged, the cleaning and drying interface 203 is continuously filled with gas, and the gas blows the cleaning liquid in the cavities and micro-flow channels into the waste liquid area 11, thereby ensuring that there is no liquid residue in the reaction detection area 8 and the quality control detection area 9, and improving the sensitivity of the fluorescence signal detection.

Claims

1. A microfluidic chip with step-by-step control and fluorescence enhancement function, comprising a shell and a microfluidic channel system inside the shell; the shell is enclosed by a bottom plate (1) and a cover plate (2), the cover plate (2) comprises a sample hole (201) and an observation hole (202); characterized in that: The micro-channel system comprises, in sequence along the sample solution flow direction, a sample adding area (3), a sample dividing area (4), a dissolving area (5), a time control area (6), a mixing channel area (7) and a detection area; the detection area comprises a sample detection area (8) and a quality control detection area (9) in parallel with each other; a fluorescent marker is coated on the dissolving area (5).

2. The microfluidic chip with step-by-step control and fluorescence enhancement function according to claim 1, characterized in that: The micro-channel system further comprises a cleaning and drying access end (10), and the detection area is connected with the cleaning and drying access end (10) through a micro-channel; the cover plate (2) is provided with a cleaning and drying access hole (203) corresponding to the cleaning and drying access end (10). 3.The microfluidic chip with step-by-step control and fluorescence enhancement function according to claim 2, characterized in that: The micro-channel system further comprises a waste liquid area (11) for collecting waste liquid generated by cleaning; the sample adding area (3) is communicated with the waste liquid area (11) through a micro-channel.

4. The microfluidic chip with step-by-step control and fluorescence enhancement function according to claim 3, characterized in that: The cover plate (2) is provided with an exhaust hole (204) communicated with the micro-channel system.

5. The microfluidic chip with step-by-step control and fluorescence enhancement function according to claim 3, characterized in that: A first step-off valve (12) for cutting off the sample solution from entering the waste liquid area (11) is arranged on the micro-channel between the waste liquid area (11) and the sample adding area (3).

6. The microfluidic chip with step-by-step control and fluorescence enhancement function according to any one of claims 1-5, characterized in that: The mixing channel area (7) comprises, in sequence along the sample solution flow direction, a first mixing channel (701), a second mixing channel (702) and a third mixing channel (703), and the micro-channel depth of the first mixing channel (701) < the micro-channel depth of the second mixing channel (702) = the micro-channel depth of the third mixing channel (703) < the micro-channel depth of the detection area; a flow-through valve for breaking the capillary cutoff effect is arranged between the first mixing channel (701) and the second mixing channel (702), and between the third mixing channel (703) and the detection area.

7. The microfluidic chip with step-by-step control and fluorescence enhancement function according to any one of claims 2-5, characterized in that: A second step-off valve (15) for cutting off the sample solution from continuing to flow is arranged on the micro-channel at the end of the sample detection area (8) and the quality control detection area (9).

8. The microfluidic chip with step-by-step control and fluorescence enhancement function according to any one of claims 1-5, characterized in that: The sample dividing area (4) is composed of at least three parallel micro-channels connecting the sample adding area (3) and the dissolving area (5), and the connecting end of the sample adding area (3) is arranged in an arc shape.

9. The microfluidic chip with step-by-step control and fluorescence enhancement function according to any one of claims 1-5, characterized in that: The dissolving area (5) is provided with a cross-microarray structure for driving the sample solution to flow in the micro-channel.

10. The microfluidic chip with step-by-step control and fluorescence enhancement function according to any one of claims 1-5, characterized in that: The detection area is provided with a fluorescent enhancement reaction unit (16).

Images