A microfluidic chip and its preparation method and application

Abstract

The present invention discloses a microfluidic chip, its preparation method, and application. The microfluidic chip includes a base layer; a detection layer disposed on the surface of the base layer and having a microfluidic channel disposed therein; and a sample feed strip disposed between the base layer and the detection layer, intersecting the microfluidic channel. The number of sample feed strips is at least one. The microfluidic chip of the present invention utilizes only one liquid drive control to perform biochemical testing on at least one sample, simplifying the microfluidic chip and reducing its liquid drive control requirements.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis, and in particular to a microfluidic chip and a preparation method and application thereof. Background Art

[0002] Biological / chemical testing (abbreviated as biochemical testing) is mostly based on the biochemical reaction between liquid or gaseous samples and sensitive substances in the detector, resulting in a signal output related to the sample concentration. Due to the need to test a variety of different samples or conduct multiple parallel tests on the same sample, biochemical testing generally requires the ability to simultaneously test multiple samples. With the increasing demand for on-site, point-of-care testing in outdoor and home settings, biochemical testing currently typically uses miniaturized, portable solutions based on microfluidic chips. In related technologies, multi-sample testing often requires multiple microchannels and multiple independent liquid drives (such as pressure rods and micropumps).

[0003] Therefore, it is necessary to develop a microfluidic chip with a simple structure and low requirements for liquid drive control. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a microfluidic chip with a simple structure and low requirements for liquid drive control.

[0005] The present invention also provides a method for preparing the microfluidic chip.

[0006] The present invention also provides applications of the microfluidic chip.

[0007] The first aspect of the present invention provides a microfluidic chip, the microfluidic chip comprising

[0008] basal layer;

[0009] A detection layer, the detection layer is disposed on the surface of the base layer, and a microfluidic channel is provided in the detection layer;

[0010] The sample introduction strip is provided between the base layer and the detection layer, the sample introduction strip intersects with the microfluidic channel, and the number of the sample introduction strip is at least one.

[0011] The microfluidic chip of the present invention has a single flow channel and does not require a liquid storage and reaction chamber structure, and has a simple structure.

[0012] According to some embodiments of the present invention, the constituent materials of the base layer and the detection layer are independently selected from the following materials: one of glass, quartz, polycarbonate, cycloolefin copolymer, cycloolefin polymer, polyester, PMMA (polymethyl methacrylate), PS (polystyrene material), PEEK (polyetheretherketone) and PDMS (polydimethylsiloxane); the constituent materials of the base layer and the detection layer are the same or different.

[0013] According to some embodiments of the present invention, the base layer and the detection layer are combined in a manner including bonding, adhesion and mechanical bonding.

[0014] According to some embodiments of the present invention, the bonding is thermocompression bonding.

[0015] According to some embodiments of the present invention, the bonding adhesive is epoxy resin, UV glue and hot melt glue.

[0016] According to some embodiments of the present invention, matching screw holes are provided on the base layer and the detection layer, and the base layer and the detection layer are mechanically combined by bolts.

[0017] According to some embodiments of the present invention, the base layer and the detection layer are connected via a clamp to achieve the mechanical bonding.

[0018] According to some embodiments of the present invention, the microfluidic channel is a spiral microfluidic channel or a radial microfluidic channel.

[0019] According to some embodiments of the present invention, the detection layer is provided with a liquid outlet and a liquid inlet.

[0020] According to some embodiments of the present invention, when the microfluidic channel is a spiral microfluidic channel, one end of the spiral microfluidic channel is a liquid inlet, and the other end is a liquid outlet.

[0021] According to some embodiments of the present invention, the spiral microfluidic channel has at least 2 spiral turns.

[0022] The present invention realizes parallel testing based on the intersection of microfluidic channels and sample feeding strips, thus avoiding the randomness of single tests. One spiral ring and the sample feeding strip are crossed once for one test, and multiple parallel tests can be realized only when two or more spiral rings are crossed with the sample feeding strips.

[0023] According to some embodiments of the present invention, the radial microfluidic channel is focused on the liquid inlet and radiates outward to form branches; the number of the branches is at least 2.

[0024] According to some embodiments of the present invention, when the microfluidic channel is a radial microfluidic channel, the liquid inlet is connected to the focal end of the radial microfluidic channel, and the liquid outlet is connected to the non-focal end of the radial microfluidic channel.

[0025] One branch crosses the sample feed strip once for one test, and more than two branches cross the sample feed strip to achieve multiple parallel tests.

[0026] According to some embodiments of the present invention, the injection strip is made of a hydrophilic material.

[0027] According to some embodiments of the present invention, the water contact angle of the hydrophilic material is less than 90°.

[0028] According to some embodiments of the present invention, the water contact angle of the hydrophilic material is 60° to 90°.

[0029] According to some embodiments of the present invention, the composition material of the sample feeding strip is one of nitrocellulose and polyester.

[0030] The sample injection strip has requirements on the wettability of the material and is easy to absorb and store the solution.

[0031] According to some embodiments of the present invention, the number of the liquid outlet is at least one.

[0032] According to some embodiments of the present invention, the thickness of the sample feeding strip is less than 50 μm; preferably, the thickness of the sample feeding strip is 10 μm to 30 μm.

[0033] If the thickness of the sample strip is too high, it will affect the bonding effect and cause the solution to leak from the chip; if the thickness is too thin, the amount of sample liquid absorbed will be small, resulting in a weak signal that is difficult to detect.

[0034] According to some embodiments of the present invention, the sample injection strip and the microfluidic channel have at least two intersection points.

[0035] According to some embodiments of the present invention, the intersection angle between the injection strip and the microfluidic channel is vertical or non-vertical.

[0036] A second aspect of the present invention provides a method for preparing a microfluidic chip, comprising the following steps: combining the base layer, the sample injection strip layer and the detection layer to obtain the microfluidic chip.

[0037] A third aspect of the present invention provides an application of a microfluidic chip in multi-sample detection.

[0038] A fourth aspect of the present invention provides a method for multi-sample detection, comprising the following steps:

[0039] S1, contacting the sample with the exposed end of the sample introduction strip;

[0040] S2. Add a detection reagent from the liquid inlet, and the detection reagent reacts with the sample; after the reaction, perform detection.

[0041] According to some embodiments of the present invention, the reaction in step S2 includes a color development reaction or a luminescence reaction.

[0042] The present invention has at least the following beneficial effects: The microfluidic chip of the present invention comprises a sandwich structure composed of a base layer, a sample feed strip, and a detection layer. Samples are injected through the exposed end of the sample feed strip, eliminating the need for additional structures such as a reagent storage chamber. Furthermore, the microchannels, by forming a cross-matrix structure with the sample feed strip, enable the chip to achieve biochemical detection of at least one sample using a single liquid drive, simplifying the microfluidic chip and reducing its liquid drive control requirements. In this application, the reagent reaction and signal detection processes are continuous, making the detection process more flexible and providing more complete information. Compared to related technologies (such as centrifugally driven chips), where each step must be performed stepwise, the detection signal obtained is the terminal signal after a certain reaction period. For transient colorimetric or luminescent reactions, a signal is generated immediately upon contact between the sample and the reaction solution. Waiting until the chip has completely stopped rotating before performing detection can easily miss the detection window or lose information on key nodes such as reaction initiation, acceleration, saturation, and decay. For each sample, this application provides parallel experiments formed at multiple intersections between the sample feed strip and the microfluidic channel, avoiding the randomness associated with single experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a multi-sample detection process in an embodiment of the present invention;

[0044] Figure 2 Schematic diagram of the microfluidic chip structure in Example 1 of the present invention;

[0045] Figure 3 Schematic diagram of the microfluidic chip structure in Example 2 of the present invention;

[0046] Figure 4 Schematic diagram of the microfluidic chip structure in Example 3 of the present invention;

[0047] Figure 5 The results of multiple parallel tests of multiple samples in Example 1 of the present invention are as follows;

[0048] Figure 6 The results of multiple parallel tests of multiple samples in Example 2 of the present invention are as follows;

[0049] Figure 7 This is the result of multiple parallel tests on a single sample in Example 3 of the present invention.

[0050] Reference numerals:

[0051] 1. Basal layer; 2. Annular sample strip; 21. Sample strip I; 22. Sample strip II; 23. Sample strip III; 3. Detection layer; 31. Liquid inlet; 32. Microfluidic channel; 33. Liquid outlet; 34. Exposure hole. DETAILED DESCRIPTION

[0052] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0053] The microfluidic chip in the embodiment of the present invention includes:

[0054] basal layer;

[0055] A detection layer, the detection layer being disposed on the surface of the base layer, wherein a microfluidic channel is disposed in the detection layer, one end of the microfluidic channel being connected to the liquid inlet, and the other end being connected to the liquid outlet;

[0056] A sample introduction strip, the sample introduction strip is provided between the base layer and the detection layer, the sample introduction strip intersects with the microfluidic channel, and the number of the sample introduction strip is at least one;

[0057] The base layer, the sample injection strip and the detection layer are stacked in sequence to form a sealed microfluidic chip with a sandwich structure.

[0058] The substrate layer and the detection layer are made of one of the following materials: glass, quartz, polycarbonate (PC), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polyester (PET), polymethyl methacrylate (PMMA), polystyrene (PS), polyetheretherketone (PEEK) and polydimethylsiloxane (PDMS).

[0059] The materials selected for the base layer and the detection layer are the same or different.

[0060] The base layer and the detection layer are tightly combined together, and the self-priming sample strip is sandwiched in the middle; the bonding method is one of bonding (thermocompression bonding), adhesion (adhesives such as epoxy resin, UV glue and EVA hot melt adhesive) or mechanical locking (screws or clamps).

[0061] The detection layer includes a liquid inlet, at least one liquid outlet, and a microfluidic channel connected to the liquid inlet and the liquid outlet. The microfluidic channel includes a spiral microfluidic channel or a radial microfluidic channel; the above-mentioned spiral microfluidic channel includes at least 2 complete spiral rings, and the above-mentioned radial microfluidic channel includes at least 2 branches radiating outward from the center.

[0062] The present invention realizes parallel testing through the intersection of microfluidic channels and sample feeding strips, thus avoiding the randomness of single tests. One spiral ring and the sample feeding strip are crossed once for one test, and more than two spiral rings and the sample feeding strip are crossed to realize multiple parallel tests.

[0063] Each branch crosses the sample strip once for a single test. Multiple parallel tests can only be performed when two or more branches cross the sample strip. The self-priming sample strip is in the form of a thin, thin sheet or ring, and is made of nitrocellulose membrane (NC membrane) or polyester membrane.

[0064] The self-priming sample strip has hydrophilic properties (static water contact angle is less than 90°) and is easy to absorb aqueous solutions. Otherwise, the sample liquid will find it difficult to self-prime and reach the detection area.

[0065] When the self-priming sample introduction strip is in the shape of a thin and slender sheet, one end of the self-priming sample introduction strip is exposed to the air for receiving the sample solution to be tested, and the other end is clamped between the base and the upper cover, intersecting with the microfluidic channel, for guiding the sample to be tested to react with the reagent in the microfluidic channel.

[0066] When the self-priming sample introduction strip is ring-shaped, an exposure hole is also provided on the above-mentioned detection layer. The exposure hole allows one end of the self-priming sample introduction strip to be exposed to the air for receiving the sample solution to be tested, and the other end is clamped between the base and the upper cover, intersecting with the microfluidic channel, for guiding the sample to be tested to react with the reagent in the microfluidic channel.

[0067] The number of the sample injection strips is at least one, and each self-priming sample injection strip intersects with the microfluidic channel at least at two points, and the intersection angle is vertical or non-vertical.

[0068] The thickness of the self-priming sample strip is 10μm~30μm. If the thickness is too high, it will affect the bonding effect and cause the solution to leak from the chip. If the thickness is too thin, the amount of sample liquid absorbed will be small, resulting in a weak signal that is difficult to detect.

[0069] The microfluidic chip in the embodiment of the present invention is used to perform multi-sample detection. The detection process is as follows: Figure 1 As shown, the following steps are included:

[0070] S1. Prepare the sample solution to be tested;

[0071] S2. Add the sample solution to be tested dropwise to each aspiration sample strip;

[0072] S3, after each sample is saturated with the test solution, the reaction solution is introduced into the microfluidic channel from the liquid inlet;

[0073] S4, the reaction solution and the sample to be tested undergo a color or light reaction at the intersection matrix;

[0074] S5. After the reaction is completed, signal acquisition and processing are performed to complete the multi-sample detection.

[0075] Example 1

[0076] This embodiment is a microfluidic chip, the structure of which is as follows Figure 2 Shown: Includes:

[0077] basal layer 1;

[0078] The detection layer 3 is provided on the surface of the base layer 1. A microfluidic channel 32 is provided in the detection layer 3. The microfluidic channel 32 is a spiral microfluidic channel. One end of the spiral microfluidic channel is connected to the liquid inlet 31, and the other end is connected to the liquid outlet 33.

[0079] The microfluidic channel 32 contains four spiral rings.

[0080] Inlet strips I 21, II 22, and III 23 are positioned between the base layer 1 and the detection layer 3, intersecting the microfluidic channel 32. Base layer 1 is a glass layer; detection layer 3 is a PDMS layer; and inlet strips I 21, II 22, and III 23 are all nitrocellulose membranes (NC membranes) with a thickness of 30 μm and a static water contact angle of 70°.

[0081] The bonding method of the base layer 1 and the detection layer 3 is hot pressing bonding after plasma surface treatment: first, the surfaces of the base layer 1 and the detection layer 3 to be bonded are placed in a plasma cleaning apparatus (Harrick Plasma, USA, model PDC-002) and evacuated for 5 minutes, then the plasma is turned on for surface treatment for 2 minutes. After taking them out, the sample bars I 21, II 22 and III 23 are placed between the two bonding surfaces of the base layer 1 and the detection layer 3, aligned and clamped, and placed under pressure annealing at 90 degrees Celsius for 30 minutes to complete the bonding.

[0082] A multi-sample multiple parallel detection method comprises the following steps:

[0083] S1. Dissolve horseradish peroxidase lyophilized powder (Shanghai Aladdin Biochemical Technology Co., Ltd., model P105528) in phosphate buffer (1x PBS, pH=7.4) to prepare three horseradish peroxidase solutions with a concentration of 10 μg / mL. Use a pipette to drop 10 μL of horseradish peroxidase solution onto the exposed ends of self-priming sample strips I21, II22, and III23 in sequence; allow the self-priming sample strips to fully absorb the horseradish peroxidase solution until saturated after 10 minutes.

[0084] S2. The undiluted BeyoECL MoonA and BeyoECL MoonB stock solutions of the chemiluminescent detection reagent (Beyotime Biotechnology Co., Ltd., Shanghai) with luminol as the substrate were mixed in a volume ratio of 1:1 and stirred evenly. The mixture was then pumped into the spiral flow channel of the microfluidic chip from the liquid inlet 31 using a microinjection pump. The mixture then flowed through the intersection nodes with the self-priming sample strips I 21, II 22, and III 23 in sequence to react.

[0085] S3. Under light-proof conditions, the luminescence signal is collected through a CCD detector (charge-coupled device detector) to complete the detection.

[0086] Example 2

[0087] This embodiment is a microfluidic chip, the structure of which is as follows Figure 3 Shown: Includes:

[0088] basal layer 1;

[0089] The detection layer 3 is provided on the surface of the base layer 1. A microfluidic channel 32 is provided in the detection layer 3. The microfluidic channel 32 is a spiral microfluidic channel. One end of the spiral microfluidic channel is connected to the liquid inlet 31, and the other end is connected to the liquid outlet 33.

[0090] The spiral microfluidic channel contains four spiral rings.

[0091] The sample introduction strip I 21 and the sample introduction strip II 22 are arranged between the base layer 1 and the detection layer 3 , and the sample introduction strip I 21 and the sample introduction strip II 22 intersect with the spiral microfluidic channel.

[0092] The base layer 1 is a quartz glass layer; the detection layer 3 is a PDMS layer; the injection strip I 21 and the injection strip II 22 are both nitrocellulose membranes (NC membranes) with a thickness of 20 μm and a static water contact angle of 70°.

[0093] The bonding method of the base layer 1 and the detection layer 3 is hot pressing bonding after plasma surface treatment: first, the surfaces of the base layer 1 and the detection layer 3 to be bonded are placed in a plasma cleaning chamber (Harrick Plasma, USA, model PDC-002) and evacuated for 5 minutes, then the plasma is turned on for surface treatment for 2 minutes. After taking them out, the sample strips I 21 and II 22 are placed between the two bonding surfaces of the base layer 1 and the detection layer 3, aligned and clamped, and placed at 90 degrees Celsius for pressure annealing for 30 minutes to complete the bonding.

[0094] A multi-sample multiple parallel detection method comprises the following steps:

[0095] S1. Dissolve horseradish peroxidase lyophilized powder (Shanghai Aladdin Biochemical Technology Co., Ltd., model number P105528) in common phosphate buffer (1x PBS, pH=7.4) to prepare two horseradish peroxidase solutions with a concentration of 50 μg / mL. Use a pipette to drop 10 μL of the horseradish peroxidase solution onto the exposed ends of the self-priming sample strips I21 and II22. Allow the self-priming sample strips to fully absorb the horseradish peroxidase solution until saturated after 10 minutes.

[0096] S2. The undiluted BeyoECL MoonA and BeyoECL MoonB stock solutions of the chemiluminescent detection reagent with luminol as the substrate (provided by Shanghai Beyotime Biotechnology Co., Ltd., model number BeyoECL Moon) were mixed in a volume ratio of 1:1 and stirred evenly. The mixture was then pumped from the liquid inlet 31 into the spiral flow channel of the microfluidic chip using a microinjection pump. The mixture then flowed through the intersection nodes with the self-priming sample strip I 21 and the self-priming sample strip II 22 in sequence to react.

[0097] S3. Under light-proof conditions, the luminescence signal is collected through a CCD detector (charge-coupled device detector) to complete the detection.

[0098] Example 3

[0099] This embodiment is a microfluidic chip, the structure of which is as follows Figure 4 As shown:

[0100] include:

[0101] basal layer 1;

[0102] The detection layer 3 is provided on the surface of the base layer 1. A microfluidic channel 32 is provided in the detection layer 3. The microfluidic channel 32 is a radial microfluidic channel. One end of the radial microfluidic channel is connected to the liquid inlet 31, and the other end is connected to the liquid outlet 33.

[0103] The radial microfluidic channel consists of three branches.

[0104] The annular sample injection strip 2 is provided between the base layer 1 and the detection layer 3 , and the annular sample injection strip 2 intersects with the microfluidic channel 32 .

[0105] The detection layer 3 is further provided with an exposure hole 34 .

[0106] The exposed hole 34 provides an exposed end for the annular sample strip 2. The base layer 1 is a glass layer of polymethyl methacrylate (PMMA); the detection layer 3 is a PDMS layer; and the self-priming sample strip is a polyester film with a thickness of 25 μm and a static water contact angle of 75°.

[0107] The base layer 1 and the detection layer 3 are bonded by epoxy resin.

[0108] A single-sample multiple parallel detection method comprises the following steps:

[0109] S1. Dissolve horseradish peroxidase lyophilized powder (provided by Shanghai Aladdin Biochemical Technology Co., Ltd., model number P105528) in common phosphate buffer (1x PBS, pH=7.4) to prepare a horseradish peroxidase solution with a concentration of 100 μg / mL. Use a pipette to add 10 μL of horseradish peroxidase solution dropwise to the exposed hole of the detection layer and contact the circular self-priming sample strip; after 10 minutes, the self-priming sample strip fully absorbs the horseradish peroxidase solution to saturation.

[0110] S2. Mix the undiluted BeyoECL MoonA and BeyoECL MoonB stock solutions of the luminol-based chemiluminescent detection reagent (provided by Shanghai Beyotime Biotechnology Co., Ltd., model number BeyoECL Moon) in a 1:1 volume ratio and stir evenly. Pump the solution through the inlet into the radial flow channel of the microfluidic chip using a microinjection pump, and allow the solution to flow through the intersection nodes with the annular self-priming inlet strips to react.

[0111] S3. Under light-proof conditions, the luminescence signal is collected through a CCD detector (charge-coupled device detector) to complete the detection.

[0112] The test results in Examples 1 to 3 of the present invention are shown in Figures 5-7 ,from Figures 5 and 6 It is known that: Example 1 and Example 2 of the present invention only use one liquid drive control to achieve multiple parallel biochemical tests of multiple samples; Figure 7 It is known that Example 3 of the present invention realizes multiple parallel biochemical detections of a single sample with only one liquid drive control. From the test results of Examples 1 to 3, it is known that the present invention realizes multiple parallel biochemical detections of at least one sample with only one liquid drive control, which simplifies the microfluidic chip and reduces its liquid drive control requirements.

[0113] In summary, the microfluidic chip of the present invention forms a sandwich structure with a base layer, a sample feed strip, and a detection layer. Samples are injected through the exposed end of the sample feed strip, eliminating the need for additional structures such as a reagent storage chamber. Furthermore, the microchannels form a cross-matrix structure with the sample feed strip, enabling the chip to achieve multi-sample biochemical testing with a single liquid drive, simplifying the microfluidic chip and reducing its liquid drive control requirements. In the present invention, the reagent reaction and signal detection processes are continuous, making the detection process more flexible and the information reflected more complete. Compared to related art methods where each step must be performed step by step (such as centrifugally driven chips), the detection signal obtained is the terminal signal after a period of reaction. For transient color or luminescence reactions, the signal is generated immediately upon contact between the sample and the reaction solution. Waiting until the chip completely stops rotating before performing detection can easily miss the detection window or lose information on key nodes such as reaction initiation, acceleration, saturation, and decay. For each sample, the present invention provides parallel experiments formed by multiple intersections of the strips and the flow channel, avoiding the randomness of single experiments.

[0114] While the embodiments of the present invention have been described in detail above in conjunction with the specification and accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A microfluidic chip, characterized in that: include basal layer; A detection layer, the detection layer is provided on the surface of the base layer, and a microfluidic channel is provided in the detection layer, and the microfluidic channel is a spiral microfluidic channel or a radial microfluidic channel; A sample feeding strip, the sample feeding strip being provided between the base layer and the detection layer, the sample feeding strip intersecting with the microfluidic channel, and the number of the sample feeding strip being at least one; The detection layer is provided with a liquid outlet and a liquid inlet; when the microfluidic channel is a radial microfluidic channel, the liquid inlet is used as a focus and branches are formed radiating outward; the number of the branches is at least 2; When the microfluidic channel is a spiral microfluidic channel, one end of the spiral microfluidic channel is a liquid inlet, and the other end is a liquid outlet; the number of spiral turns of the spiral microfluidic channel is at least 2; The sample feeding strip is a self-priming sample feeding strip, the constituent material of the sample feeding strip is one of nitrocellulose and polyester, and the thickness of the sample feeding strip is 10 μm to 30 μm.

2. A microfluidic chip according to claim 1, characterized in that: The bonding methods of the base layer and the detection layer include bonding, adhesion and mechanical bonding.

3. The microfluidic chip according to claim 2, wherein: The bonding is thermal compression bonding.

4. The microfluidic chip according to claim 2, wherein: The adhesives used for the bonding are epoxy resin, UV glue and hot melt glue.

5. The microfluidic chip according to claim 2, characterized in that: The base layer and the detection layer are provided with matching screw holes, and the base layer and the detection layer are mechanically combined by bolts.

6. The microfluidic chip according to claim 2, wherein: The base layer and the detection layer are connected via a clamp to achieve the mechanical combination.

7. A method for preparing the microfluidic chip according to any one of claims 1 to 6, characterized in that: The following steps are involved: The microfluidic chip is obtained by combining the base layer, the sample injection strip and the detection layer.

8. Use of the microfluidic chip according to any one of claims 1 to 6 in multi-sample detection.

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