Composite micro-fluidic chip for inflammation multi-factor diagnosis

Abstract

The invention discloses a composite micro-fluidic chip for inflammation multi-factor diagnosis, and relates to the technical field of human body disease detection. The composite micro-fluidic chip comprises an upper cover plate, a runner interlayer, a paper chip layer and a bottom layer, the flow channel interlayer comprises an interlayer body, and a sample adding area, a primary reaction area, a mixing area, a paper chip area and a reaction hole which are etched on the interlayer body and are communicated in sequence; the runner interlayer is arranged between the upper cover plate and the bottom layer; the paper chip layer is arranged in the paper chip area and the reaction hole; the upper cover plate comprises a sample adding hole, the sample adding hole and the sample adding area are correspondingly arranged, a sample is dropwise added into the sample adding area through the sample adding hole, and the sample sequentially passes through the sample adding area, the primary reaction area, the mixing area, a paper chip layer in the paper chip area and a paper chip layer in the reaction hole to complete inflammation multi-factor diagnosis. The kit can detect various inflammatory factors at the same time, improves the detection speed, and has sensitivity and specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of human disease detection, and in particular to a composite microfluidic chip for multi-factor diagnosis of inflammation. Background Art

[0002] Infectious diseases are common and frequently occurring diseases in clinic, threatening human health. In particular, systemic bacterial infectious diseases account for a considerable proportion of patients in intensive care units, with atypical clinical symptoms, rapid progression, and serious harm, so early diagnosis is particularly important. Therefore, finding laboratory test indicators that can diagnose early, monitor efficacy, and have high specificity is of great significance in helping clinicians to make timely and accurate early diagnosis, early effective treatment, reduce mortality, and avoid the abuse of antibiotics and reduce the development of bacterial resistance.

[0003] However, existing inflammation detection methods are limited by current methodology. Whether it is instant testing or luminescence assembly line, they can only detect inflammatory factors one by one, and the detection speed is slow. Summary of the invention

[0004] The purpose of the present invention is to provide a composite microfluidic chip for multi-factor diagnosis of inflammation, which can detect multiple inflammatory factors at the same time, improve the detection speed, and have sensitivity and specificity.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] A composite microfluidic chip for multi-factor diagnosis of inflammation, comprising:

[0007] An upper cover plate, a flow channel sandwich, a paper chip layer and a bottom layer; the flow channel sandwich includes a sandwich body and a sample addition area, a primary reaction area, a mixing area, a paper chip area and a reaction hole etched on the sandwich body and connected in sequence;

[0008] The flow channel interlayer is arranged between the upper cover plate and the bottom layer; the paper chip layer is arranged in the paper chip area and the reaction hole; the upper cover plate includes a sample addition hole, and the sample addition hole is arranged corresponding to the sample addition area. The sample is dripped into the sample addition area through the sample addition hole, and the sample passes through the sample addition area, the primary reaction area, the mixing area, the paper chip layer in the paper chip area and the paper chip layer in the reaction hole in sequence to complete the multi-factor diagnosis of inflammation.

[0009] Optionally, the bottom layer includes: a lower cover plate and a waste liquid recovery device; a penetration hole is arranged on the lower cover plate; the position of the penetration hole corresponds to the position of the reaction hole; the waste liquid recovery device is arranged on the lower bottom surface of the lower cover plate; the sample after completing the multi-factor diagnosis of inflammation penetrates into the waste liquid recovery device through the penetration hole.

[0010] Optionally, the upper cover plate is colorless and transparent.

[0011] Optionally, the upper cover plate is made of PMMA material or COC material.

[0012] Optionally, the paper core layer is prepared from polyacrylonitrile nanofiber membrane or nitrocellulose membrane using electrospinning technology.

[0013] Optionally, the mixing zone is a serpentine channel.

[0014] Optionally, the thickness of the upper cover plate is 20mm-30mm.

[0015] Optionally, the thickness of the interlayer body is 0.05mm-0.1mm.

[0016] Optionally, the thickness of the lower cover plate is 30mm-40mm.

[0017] According to the specific embodiments provided by the present invention, the present invention discloses the following technical effects:

[0018] The present invention sets up a composite microfluidic chip for multi-factor diagnosis of inflammation through a plastic-paper composite microfluidic method, which can specifically detect multiple inflammatory factors at the same time, realizes the simultaneous detection of multiple test objects, and has extremely high sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0020] Figure 1 A schematic diagram of the results of a composite microfluidic chip for multi-factor diagnosis of inflammation provided by an embodiment of the present invention;

[0021] Figure 2 A schematic diagram of the structure of an upper cover plate provided in an embodiment of the present invention;

[0022] Figure 3 A schematic diagram of the structure of a flow channel interlayer provided in an embodiment of the present invention;

[0023] Figure 4 A schematic diagram of the structure of a paper core layer provided in an embodiment of the present invention;

[0024] Figure 5 A schematic structural diagram of a base plate provided in an embodiment of the present invention.

[0025] Explanation of symbols:

[0026] Sample loading hole-1, primary reaction area-2, mixing area-3, paper chip area-4, reaction hole-5, sample loading area-6, permeation hole-7, waste liquid recovery device-8. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] Microfluidics is a technology that precisely controls microscale fluids at the submicron scale, integrates basic operating units such as sample preparation, reaction, separation, and detection into a micron-scale chip to automatically complete the entire analysis process. It is referred to as a chip laboratory. This is a new research field that integrates life sciences, chemical analysis, fluid mechanics, optics, polymer materials, mold design, and mechanical manufacturing. Due to the micron-scale structure of the channels required for microfluidics, the biggest feature is the small volume of test samples, low reagent consumption and energy consumption, fast biochemical reaction speed, easy industrial expansion of production, and good batch stability. Paper-based microfluidic chips are the concept of "microfluidic paper-based analytical devices" (μPADs). Through various micro-machining technologies, fluid channel networks and related analytical devices are constructed on filter paper to establish a "paper micro-laboratory" (lab-on-paper). μPADs combines the advantages of filter paper, such as cheap and easy availability and good biocompatibility, with the traditional advantages of microfluidic chips, solving the problems of batch preparation of traditional microfluidic chips. Based on this, the embodiment of the present invention provides a composite microfluidic chip for multi-factor diagnosis of inflammation, such as Figures 1 to 4 As shown, the composite microfluidic chip for multi-factor diagnosis of inflammation provided by the embodiment of the present invention includes:

[0030] An upper cover plate, a flow channel interlayer, a paper chip layer and a bottom layer; the flow channel interlayer includes an interlayer body and a sample addition area 6, a primary reaction area 2, a mixing area 3, a paper chip area 4 and a reaction hole 5 etched on the interlayer body and connected in sequence; the flow channel interlayer is arranged between the upper cover plate and the bottom layer; the paper chip layer is arranged in the paper chip area 4 and the reaction hole 5; the upper cover plate includes a sample addition hole 1, and the sample addition hole 1 is arranged corresponding to the sample addition area 6. The sample is dripped into the sample addition area 6 through the sample addition hole 1, and the sample passes through the sample addition area 6, the primary reaction area 2, the mixing area 3, the paper chip layer in the paper chip area 4 and the paper chip layer in the reaction hole 5 in sequence to complete the multi-factor diagnosis of inflammation.

[0031] As an optional implementation, Figure 5 As shown, the bottom layer includes: a lower cover plate and a waste liquid recovery device 8; a penetration hole 7 is arranged on the lower cover plate; the position of the penetration hole 7 corresponds to the position of the reaction hole 5; the waste liquid recovery device 8 is arranged on the lower bottom surface of the lower cover plate; the sample after completing the multi-factor diagnosis of inflammation penetrates into the waste liquid recovery device 8 through the penetration hole 7, and the interior of the waste liquid recovery device 8 is a 10mm hollow structure, which can store the recovered waste liquid.

[0032] As an optional implementation, the upper cover is colorless and transparent, has good light transmittance, and has a circular or semicircular sample addition hole 1 .

[0033] As an optional implementation, the upper cover plate is made of PMMA material or COC material.

[0034] As an optional embodiment, the paper core layer is made of a highly absorbent material such as polyacrylonitrile nanofiber membrane or nitrocellulose membrane using electrostatic spinning technology; after spinning, it is directly sprayed into a membrane material with the same thickness as the flow channel interlayer, and then rolled and dried to form. Further, the dried and formed paper material is cut.

[0035] As an optional implementation, the mixing zone 3 is a serpentine channel.

[0036] As an optional implementation, the thickness of the upper cover plate is 20 mm-30 mm.

[0037] As an optional implementation, the interlayer body is made of colorless PS material with a thickness of 0.05mm-0.1mm, and is coated with uniform pressure-sensitive glue on both sides for later pasting and fixing of the upper cover and the lower cover.

[0038] As an optional implementation, the lower cover plate is made of ABS or other materials and has a thickness of 30 mm-40 mm.

[0039] The present invention aims at the simultaneous detection of multiple inflammatory factors in the human body. Through a plastic-paper composite microfluidic method, multiple inflammatory factors can be specifically detected simultaneously, thereby achieving simultaneous detection of multiple analytes with extremely high sensitivity and specificity.

[0040] The steps for using the composite microfluidic chip for multi-factor diagnosis of inflammation provided by the embodiment of the present invention are as follows:

[0041] 1. After adding the sample (test specimen) into the sample adding hole 1, the sample enters the primary reaction zone 2 along the pipeline under the action of capillary action.

[0042] 2. In the primary reaction area 2, there is a pre-freeze-dried and stored reagent powder; the sample first reacts with the reagent powder.

[0043] 3. After the reaction is completed, the reacted sample solution continues to be mixed and fully reacted along the serpentine channel (mixing area 3) under the capillary action until it reaches the paper chip area 4.

[0044] 4. In the paper chip area 4, it further penetrates into the reaction hole 5 along the etched flow path, undergoes a secondary reaction with the paper chip layer coated in the reaction hole 5 and develops color. The reagent on the paper in the paper chip area 4 is different from the reagent in the previous primary reaction area 2.

[0045] 5. Excess liquid percolates to the bottom of the reaction well 5 and enters the waste liquid recovery device 8 at the bottom of the chip through the permeation hole 7.

[0046] 6. Insert the chip into the matching reader to read the concentration value.

[0047] The present invention provides a more specific embodiment to introduce the above-mentioned composite microfluidic chip for multi-factor diagnosis of inflammation in detail:

[0048] 1. Antibody labeling: 10mL (content 0.1%) of fluorescent microspheres (20nm) were first ultrasonically dispersed for 5min using an ultrasonic disperser, then centrifuged at 15000rpm for 30 minutes, and the clean supernatant was aspirated; then 10mL of 50mg / ml EDC and NHS mixed solution (prepared with 0.1M MES buffer (PH6.0)) was added, ultrasonically dispersed, resuspended and reacted for 10min; then centrifuged at 15000rpm for 30 minutes again, the supernatant was extracted, and 10mL of 0.01M PBS was added to resuspend. Then 100uL of antibody (CRP / PCT / SAA / IL-6) was slowly added dropwise, and the antibody dosage was 0.10mg; stirred for 1h~2h at 2℃-8℃, and 5M glycine was added for blocking for 0.5h; centrifuged at 15000rpm for 30 minutes again, the supernatant was extracted, and then the preservation solution was added and sealed in a dark place at 4℃ for future use.

[0049] 2. Antibody packaging and freeze drying: dilute the labeled antibody to the working concentration, use a spotter to inject 10uL of liquid along the injection hole of the primary reaction area 2 of the chip, and then seal the injection hole with tape. Then put the chip into a freeze dryer to prepare freeze-dried powder, seal it and store it at 4°C for later use.

[0050] 3. Preparation of standard curve: Use negative serum to prepare standard samples (CRP / PCT / SAA / IL-6) of different working concentrations, then take 100uL of the prepared solution and add it to the chip for reaction, read the fluorescence value of the reaction, and establish a standard curve.

[0051] Compared with traditional microfluidic chips, this detection chip has the following advantages:

[0052] a) Paper is abundant and can be mass-produced at low cost.

[0053] b) The detection background is low, which is more conducive to photometric detection.

[0054] c) Good biocompatibility, no need to change the properties of paper and plastic chips through chemical modification.

[0055] d) Disposable portable analysis, easy to operate, even without the need for professional operators.

[0056] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the system disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0057] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A composite microfluidic chip for multi-factor diagnosis of inflammation, characterized in that: include: An upper cover plate, a flow channel sandwich, a paper chip layer and a bottom layer; the flow channel sandwich includes a sandwich body and a sample addition area, a primary reaction area, a mixing area, a paper chip area and a reaction hole etched on the sandwich body and connected in sequence; The flow channel interlayer is arranged between the upper cover plate and the bottom layer; the paper chip layer is arranged in the paper chip area and the reaction hole; the upper cover plate includes a sample addition hole, and the sample addition hole is arranged corresponding to the sample addition area. The sample is dripped into the sample addition area through the sample addition hole, and the sample passes through the sample addition area, the primary reaction area, the mixing area, the paper chip layer in the paper chip area and the paper chip layer in the reaction hole in sequence to complete the multi-factor diagnosis of inflammation.

2. The composite microfluidic chip for multi-factor diagnosis of inflammation according to claim 1, characterized in that: The bottom layer includes: a lower cover plate and a waste liquid recovery device; a penetration hole is arranged on the lower cover plate; the position of the penetration hole corresponds to the position of the reaction hole; the waste liquid recovery device is arranged on the lower bottom surface of the lower cover plate; the sample after completing the multi-factor diagnosis of inflammation penetrates into the waste liquid recovery device through the penetration hole.

3. The composite microfluidic chip for multi-factor diagnosis of inflammation according to claim 1, characterized in that: The upper cover plate is colorless and transparent.

4. The composite microfluidic chip for multi-factor diagnosis of inflammation according to claim 1, characterized in that: The upper cover plate is made of PMMA or COC.

5. The composite microfluidic chip for multi-factor diagnosis of inflammation according to claim 1, characterized in that: The paper core layer is prepared from polyacrylonitrile nanofiber membrane or nitrocellulose membrane by using electrostatic spinning technology.

6. The composite microfluidic chip for multi-factor diagnosis of inflammation according to claim 1, characterized in that: The mixing zone is a serpentine channel.

7. The composite microfluidic chip for multi-factor diagnosis of inflammation according to claim 1, characterized in that: The thickness of the upper cover plate is 20mm-30mm.

8. The composite microfluidic chip for multi-factor diagnosis of inflammation according to claim 1, characterized in that: The thickness of the sandwich body is 0.05mm-0.1mm.

9. The composite microfluidic chip for multi-factor diagnosis of inflammation according to claim 2, characterized in that: The thickness of the lower cover plate is 30mm-40mm.

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