3D paper-based microfluidic osteoprotegerin detection device, preparation method and application

Abstract

The application discloses a 3D paper-based microfluidic osteoprotegerin detection device, a preparation method and application, and relates to the paper-based microfluidic field.The device comprises a paper-based flow guide layer, a paper-based detection layer and an absorption layer;the paper-based flow guide layer is provided with a hydrophilic pattern area with a periphery hydrophobic based on a wax printing heating method;the hydrophilic pattern area is used for receiving a mixed fluid;the mixed fluid comprises a sample to be detected and an antibody-color developing solution;the paper-based detection layer is provided with a standard indication area based on a wax printing heating method, and a detection area corresponding to the hydrophilic pattern area and used for receiving the mixed fluid permeated through the hydrophilic pattern area;the detection area is fixed with a human osteoprotegerin antibody, and the standard indication area is dyed with a standard concentration detection color development used for indicating a human osteoprotegerin concentration gradient;the absorption layer is used for collecting the waste liquid permeated through the paper-based detection layer after detection.Compared with the prior art, the application can realize instant detection of a human osteoprotegerin sample, has low production cost, high portability, no dependence on large instruments, and wide application scenarios.

Description

Technical Field

[0001] The present invention relates to the field of paper-based microfluidic technology, and more specifically, to a 3D paper-based microfluidic osteoprotegerin detection device, a preparation method, and applications. Background Art

[0002] Osteoprotegerin, a tumor necrosis factor (TNF) receptor, promotes cell survival by inhibiting TNF-induced apoptosis. Its mechanism of action is direct or indirect binding of osteoprotegerin to its ligands. Osteoprotegerin binds to NF-κB receptor activator protein ligands and tumor necrosis factor-related apoptosis-inducing ligands, activating osteoblast expression and promoting the formation of mature osteoclasts. This suggests that osteoprotegerin has a significant impact on bone regeneration, and that osteoprotegerin levels in the human body can reflect and regulate the apoptosis-regeneration state of bone tissue. Furthermore, studies have shown that osteoprotegerin is associated with a variety of diseases, including coronary artery calcification, cerebral ischemia, multiple sclerosis, brain tumors, autoimmune diseases, renal failure, and malignant tumors. Therefore, osteoprotegerin has great potential as a biomarker and therapeutic target, and has long been the focus of many medical institutions.

[0003] At present, mainstream osteoprotegerin testing needs to be conducted in professional testing medical institutions. This type of testing limits the testing scenarios, and centralized clinical testing will bring additional burdens to high-speed operating testing medical institutions, resulting in high testing costs. Summary of the Invention

[0004] In order to overcome the defect of limited osteoprotegerin detection scenarios described in the above-mentioned prior art, the present invention provides a 3D paper-based microfluidic osteoprotegerin detection device, preparation method and application.

[0005] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0006] In a first aspect, a 3D paper-based microfluidic osteoprotegerin detection device comprises, in sequence, a paper-based flow guide layer, a paper-based detection layer, and an absorption layer;

[0007] The paper-based flow-guiding layer is provided with a hydrophilic pattern area with a hydrophobic periphery based on a wax printing heating method; wherein the hydrophilic pattern area is used to receive a mixed fluid; the mixed fluid includes a sample to be detected and an antibody-color developing solution;

[0008] The paper-based detection layer is provided with a standard indicator area based on a wax printing heating method, and a detection area corresponding to the hydrophilic pattern area for receiving the mixed fluid that permeates through the hydrophilic pattern area; wherein the detection area is immobilized with human osteoprotegerin antibodies, and the standard indicator area is stained with a standard concentration detection color for indicating the concentration gradient of human osteoprotegerin;

[0009] The absorption layer is used to collect the waste liquid remaining after detection and permeated through the paper-based detection layer.

[0010] In a second aspect, a method for preparing a 3D paper-based microfluidic osteoprotegerin detection device comprises:

[0011] Providing a first paper-based substrate as a guide layer, and printing a first preset pattern on the first paper-based substrate using a wax printing heating method to form a hydrophilic pattern area with a hydrophobic periphery;

[0012] Providing a second paper-based substrate as a paper-based detection layer, and printing a second preset pattern on the second paper-based substrate using a wax printing heating method to form an initial standard indicator area and an initial detection area, dripping and incubating human osteoprotegerin antibodies in the detection area to functionalize the detection area, and staining the standard indicator area to indicate a human osteoprotegerin concentration gradient;

[0013] An absorption layer is provided, and is sequentially combined with the paper-based detection layer and the paper-based guide layer to form the 3D paper-based microfluidic osteoprotegerin detection device.

[0014] In a third aspect, a method for detecting human osteoprotegerin, using the device described in the first aspect, comprises:

[0015] Adding the sample to be tested and the antibody-color development solution to the hydrophilic pattern area on the paper-based flow-guiding layer of the device;

[0016] The mixed fluid is transported by capillary action of the hydrophilic patterned area of ​​the paper-based guide layer, and the mixed fluid is infiltrated through the paper-based guide layer through the microchannel to the detection area on the paper-based detection layer of the device, so that the sample to be detected is combined with the human osteoprotegerin antibody fixed in the detection area, and color is developed based on the antibody-color development solution;

[0017] Determine the concentration of human osteoprotegerin in the sample to be tested based on the color development result and the standard concentration of the standard indicator area on the paper-based detection layer;

[0018] The waste liquid formed after the detection that permeates the paper-based detection layer is absorbed by the absorption layer of the device.

[0019] Compared with the prior art, the beneficial effects of the technical solution of the present invention are:

[0020] The present invention provides a 3D paper-based microfluidic osteoprotegerin detection device, preparation method, and application. The device is based on wax printing technology, forming a hydrophilic pattern on the paper-based guide layer and a detection area on the paper-based detection layer. Due to the hydrophobicity of the wax, the fluid will be restricted to flow in the area not printed by the wax, forming a micro-sized fluid channel that can guide the fluid (including the sample to be tested) to flow to the detection area, thereby achieving real-time detection of human osteoprotegerin samples and effectively reducing the amount of reagents required for real-time detection. Compared with the existing technology, the present invention has low production cost, strong portability, no dependence on large-scale instruments, a wide range of application scenarios, and is suitable for industrial promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is an exploded view of the 3D paper-based microfluidic osteoprotegerin detection device in Example 1 of the present application;

[0022] Figure 2 This is a schematic diagram of a physical sample of the 3D paper-based microfluidic osteoprotegerin detection device in Example 1 of the present application;

[0023] Figure 3 Schematic diagram of the structure of the paper-based detection layer in Example 1 of the present application;

[0024] Figure 4 This is a schematic structural diagram of the paper-based guide layer in Example 1 of the present application;

[0025] Figure 5 This is a schematic structural diagram of the upper portion of the protective housing in Example 1 of the present application;

[0026] Figure 6 This is a schematic structural diagram of the lower portion of the protective housing in Example 1 of the present application;

[0027] Figure 7 Schematic diagram of the process for preparing the 3D paper-based microfluidic osteoprotegerin detection device in Example 2 of the present application;

[0028] Figure 8 This is a schematic flow chart of the method for detecting human osteoprotegerin in Example 3 of the present application;

[0029] The accompanying drawings are numerals as follows:

[0030] 1- protective shell upper part; 2- paper-based flow guide layer; 3- paper-based detection layer; 4- absorption layer; 5- protective shell lower part;

[0031] 101-sample entry window; 102-observation window; 201-hydrophilic pattern area; 202-limiting hole;

[0032] 2011- diversion channel; 2012- diversion port; 301- detection area; 302- standard indication area;

[0033] 501-limiting column; 502-operating window. DETAILED DESCRIPTION

[0034] The terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequential order. It should be understood that the terms used in this way can be interchangeable under appropriate circumstances, and this is merely a way of distinguishing the objects of the same attributes when describing them in the embodiments of the present application. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions, so that the process, method, system, product or equipment comprising a series of units need not be limited to those units, but may include other units that are not clearly listed or inherent to these processes, methods, products or equipment.

[0035] The accompanying drawings are for illustrative purposes only and are not to be construed as limiting this patent;

[0036] In order to better illustrate this embodiment, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product size;

[0037] It is understandable to those skilled in the art that some well-known structures and descriptions thereof may be omitted in the drawings.

[0038] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0039] Example 1

[0040] This embodiment provides a 3D paper-based microfluidic osteoprotegerin detection device, see Figure 1-Figure 3 , sequentially comprising: a paper-based flow guide layer, a paper-based detection layer and an absorption layer; wherein,

[0041] The paper-based flow-guiding layer is provided with a hydrophilic pattern area with a hydrophobic periphery based on a wax printing heating method; wherein the hydrophilic pattern area is used to receive a mixed fluid; the mixed fluid includes a sample to be detected and an antibody-color developing solution;

[0042] The paper-based detection layer is provided with a standard indicator area based on a wax printing heating method, and a detection area corresponding to the hydrophilic pattern area for receiving the mixed fluid that permeates through the hydrophilic pattern area; wherein the detection area is immobilized with human osteoprotegerin antibodies, and the standard indicator area is stained with a standard concentration detection color for indicating the concentration gradient of human osteoprotegerin;

[0043] The absorption layer is used to collect the waste liquid remaining after detection and permeated through the paper-based detection layer.

[0044] In this embodiment, based on wax printing technology, a hydrophilic pattern is formed on the paper-based guide layer and a detection area is formed on the paper-based detection layer. Due to the hydrophobicity of the wax, the fluid will be restricted to flow in the area not printed by the wax, forming a micro-sized fluid channel that can guide the fluid (including the sample to be tested) to flow to the detection area, thereby realizing the instant detection of human osteoprotegerin samples and effectively reducing the amount of reagents required for instant detection.

[0045] It should be noted that by controlling the degree of overlap between the detection area and the hydrophilic pattern area, the number of detection areas working simultaneously can be controlled. Based on this, the device also has the characteristics of high repeatability and low detection cost.

[0046] Furthermore, the paper-based guide layer, the paper-based detection layer and the absorption layer are tightly combined.

[0047] In some examples, a plurality of standard indicator areas are formed on the paper-based detection layer to be stained with standard concentrations corresponding to different concentration gradients of human osteoprotegerin for color development.

[0048] In other examples, a standard indicator area is formed on the paper-based detection layer, and the standard concentration detection colors corresponding to different human osteoprotegerin concentration gradients are all arranged in the same standard indicator area.

[0049] In some preferred embodiments, see Figure 4 The hydrophilic pattern area includes at least two diversion channels and a diversion port; wherein the diversion port is used to receive the mixed fluid, the diversion channels are connected through the diversion port, and the position and number of the diversion channels correspond to the detection area.

[0050] It should be noted that the diversion port can simultaneously guide the mixed fluid to flow rapidly in several diversion channels, and the amount of liquid lost is very small, which significantly reduces the cost of detection reagents.

[0051] In some examples, the flow guiding channel is in the shape of an elongated strip, and the mixed fluid can evenly penetrate through the microchannel to reach the detection area.

[0052] In some examples, the detection area is in a capsule shape, and the standard indicator area is in a circular shape.

[0053] In some examples, the number of the guiding channels is the same as the detection area, and the orthographic projection of the guiding channels on the paper-based detection layer may coincide with the detection area.

[0054] In a specific implementation process, the paper-based guide layer, paper-based detection layer and absorption layer are all circular. For each semicircular area of ​​the paper-based guide layer, multiple guide channels are distributed in a fan shape around the center of the circle, with a width of 2 mm, and the angle between adjacent guide channels is 16°.

[0055] In some preferred embodiments, the standard indicator area is not located on an extension line of a connecting line between the two farthest ends of the detection area.

[0056] In some examples, the orthographic projection of the diversion channel on the paper-based detection layer may coincide with the standard indication area or the detection area, respectively.

[0057] In a specific implementation process, the angle between the standard indicator area and the detection area is 8°, and there is no overlap.

[0058] In some preferred embodiments, the paper-based guide layer and the paper-based detection layer use chromatography paper as a substrate.

[0059] In some optional embodiments, the chromatography paper is a nitrocellulose membrane with a thickness of 105 μm-140 μm and a pore size of 0.45 μm.

[0060] In some preferred embodiments, the device can detect human osteoprotegerin in the range of 2 pg / mL to 1 mg / mL.

[0061] In some preferred embodiments, see Figure 5 、 Figure 6 The device further comprises an upper protective shell portion and a lower protective shell portion that are rotatably matched to accommodate the paper-based guide layer, the paper-based detection layer and the absorption layer; wherein,

[0062] The upper portion of the protective housing is provided with a sample entry window and an observation window which are rotated and adjusted to correspond to the hydrophilic pattern area;

[0063] A limiting column is provided at the center of the inner surface of the upper part of the protective shell or the lower part of the protective shell, and a limiting hole adapted to the limiting column is opened at the center of the paper-based guide layer, the paper-based detection layer and the absorption layer.

[0064] In some optional embodiments, an operation window is provided on the side wall of the lower portion of the protective shell for operating the paper-based guide layer, the paper-based detection layer and / or the absorption layer.

[0065] In some examples, a 180° operating window is formed on the outer side wall of the protective housing along its circumference.

[0066] Furthermore, a sliding groove is provided on the inner side of the side wall of the lower part of the protective shell, and the upper part of the protective shell is slidably connected with the lower part of the protective shell through the sliding groove to achieve rotational cooperation, and the rotation range is limited by the cooperation between the limit piece extending out of the operating window and the operating window.

[0067] In some examples, the protective shell upper portion and the protective shell lower portion are made by 3D printing.

[0068] Example 2

[0069] This embodiment provides a method for preparing a 3D paper-based microfluidic osteoprotegerin detection device. Figure 7 ,include:

[0070] Providing a first paper-based substrate as a paper-based guide layer, and printing a first preset pattern on the first paper-based substrate using a wax printing heating method to form a hydrophilic pattern area with a hydrophobic periphery;

[0071] Providing a second paper-based substrate as a paper-based detection layer, and printing a second preset pattern on the second paper-based substrate using a wax printing heating method to form an initial standard indicator area and an initial detection area, dripping and incubating human osteoprotegerin antibodies in the detection area to functionalize the detection area, and staining the standard indicator area to indicate a human osteoprotegerin concentration gradient;

[0072] An absorption layer is provided, and is sequentially combined with the paper-based detection layer and the paper-based guide layer to form the 3D paper-based microfluidic osteoprotegerin detection device.

[0073] In some examples, a medical cotton absorbent sheet is used as the absorbent layer.

[0074] It should be noted that the method described in this embodiment is used to prepare a 3D paper-based microfluidic osteoprotegerin detection device, which has a simple and reliable process, low production cost, and is suitable for industrial promotion.

[0075] In some preferred embodiments, the wax printing heating method includes printing wax patterns on the first paper-based substrate or the second paper-based substrate using screen printing or wax jet printing, and heating to 80° C. to form a hydrophobic surface.

[0076] In a specific implementation process, a screen printing method is used to transfer a pattern (i.e., the first preset pattern or the second preset pattern) to a screen printing plate, and then the printing plate is placed on a nitrocellulose membrane. The wax is heated to a liquid state, and the liquid wax is printed on the nitrocellulose membrane to obtain a hydrophobic pattern. The obtained pattern is then cut and heated so that the wax completely seals the entire cross-section covered by the pattern, thereby obtaining the paper-based guide layer or the paper-based detection layer.

[0077] In some preferred embodiments, the functionalization process of the detection zone includes:

[0078] A quantitative, equal-concentration human osteoprotegerin antibody solution is added dropwise to the detection area, incubated at room temperature to human body temperature (36°C-38°C), rinsed with Tween 20 solution and deionized water, and finally dried at room temperature to human body temperature.

[0079] It should be noted that the double washing process ensures that no antibodies bound to the paper substrate are washed away.

[0080] In a specific implementation process, 4 μL of human osteoprotegerin antibody with a concentration of 40 μg / mL is added to the unfunctionalized detection area, and after incubation at 37°C for one hour, it is washed with 0.05% Tween 20 Tris-HCl (50 mM, pH 7.4) solution for 10 seconds, and then washed with deionized water for 10 seconds. After drying at 37°C, a highly sensitive human osteoprotegerin detection area can be obtained.

[0081] Example 3

[0082] This embodiment provides a method for detecting osteoprotegerin, using the device described in Example 1, see Figure 8 ,include:

[0083] Adding the sample to be tested and the antibody-color development solution to the hydrophilic pattern area on the paper-based flow-guiding layer of the device;

[0084] The mixed fluid is transported by capillary action of the hydrophilic patterned area of ​​the paper-based guide layer, and the mixed fluid is infiltrated through the paper-based guide layer through the microchannel to the detection area on the paper-based detection layer of the device, so that the sample to be detected is combined with the human osteoprotegerin antibody fixed in the detection area, and color is developed based on the antibody-color development solution;

[0085] Determine the concentration of human osteoprotegerin in the sample to be tested based on the color development result and the standard concentration of the standard indicator area on the paper-based detection layer;

[0086] The waste liquid formed after the detection that permeates the paper-based detection layer is absorbed by the absorption layer of the device.

[0087] It can be understood that the options in the above embodiment 1 are also applicable to this embodiment, so they will not be described again here.

[0088] The same or similar reference numerals correspond to the same or similar components;

[0089] The terms used in the drawings to describe positional relationships are for illustrative purposes only and are not to be construed as limiting the present application.

[0090] It should be noted that, unless there is any conflict, the embodiments and features in the embodiments of this application can be combined with each other.

[0091] In different specific implementations, the method or system described in this application can be implemented in software, hardware or a combination thereof. In addition, the order of the steps of the method can be changed, and various elements can be added, reordered, combined, omitted, modified, etc.

[0092] Obviously, the above embodiments of the present application are merely examples for clearly illustrating the present application, and are not intended to limit the implementation methods of the present application, and are not intended to limit the present application. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. Each discrete structural / functional module or unit can be integrated together to form an independent part, or each module can exist alone, or two or more modules can be integrated to form an independent part, and the structure and function of the discrete components can be implemented as a combined structure or component. It is not necessary and impossible to enumerate all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the scope of protection of the claims of the present application.

Claims

1. A 3D paper-based microfluidic osteoprotegerin detection device, characterized in that: It includes a paper-based guide layer, a paper-based detection layer and an absorption layer in sequence, and also includes a protective shell upper part and a protective shell lower part that are rotatably matched to accommodate the paper-based guide layer, the paper-based detection layer and the absorption layer; wherein, The paper-based flow-guiding layer is provided with a hydrophilic pattern area with a hydrophobic periphery based on a wax printing heating method; wherein the hydrophilic pattern area is used to receive a mixed fluid; the mixed fluid includes a sample to be detected and an antibody-color developing solution; The paper-based detection layer is formed with a standard indicator area based on a wax printing heating method, and a detection area corresponding to the hydrophilic pattern area for receiving the mixed fluid that permeates through the hydrophilic pattern area; wherein the detection area is immobilized with human osteoprotegerin antibodies, and the standard indicator area is dyed with a standard concentration detection color for indicating the concentration gradient of human osteoprotegerin; by controlling the degree of overlap between the detection area and the hydrophilic pattern area, the number of detection areas working simultaneously is controlled; The absorption layer is used to collect the waste liquid remaining after the test that has penetrated through the paper-based detection layer; wherein the hydrophilic pattern area comprises at least two diversion channels and a diversion port; wherein the diversion port is used to receive the mixed fluid, the diversion channels are connected via the diversion port, and the positions and numbers of the diversion channels correspond to the detection area; and, The upper portion of the protective housing is provided with a sample entry window and an observation window which are rotated and adjusted to correspond to the hydrophilic pattern area; A limiting post is provided at the center of the inner surface of the upper part of the protective shell or the lower part of the protective shell, and a limiting hole adapted to the limiting post is provided at the center of each of the paper-based guide layer, the paper-based detection layer and the absorption layer; An operation window is provided on the side wall of the lower portion of the protective shell for operating the paper-based guide layer, the paper-based detection layer and / or the absorption layer.

2. A 3D paper-based microfluidic osteoprotegerin detection device according to claim 1, characterized in that: The paper-based flow-guiding layer and the paper-based detection layer use nitrocellulose membrane filter paper as a substrate.

3. The 3D paper-based microfluidic osteoprotegerin detection device according to claim 2, characterized in that: The nitrocellulose membrane filter paper is a nitrocellulose membrane with a thickness of 105 μm-140 μm and a pore size of 0.45 μm.

4. A method for preparing a 3D paper-based microfluidic osteoprotegerin detection device, used to prepare the device according to any one of claims 1 to 3, characterized in that: include: Providing a first paper-based substrate as a paper-based guide layer, and printing a first preset pattern on the first paper-based substrate using a wax printing heating method to form a hydrophilic pattern area with a hydrophobic periphery; Providing a second paper-based substrate as a paper-based detection layer, and printing a second preset pattern on the second paper-based substrate using a wax printing heating method to form an initial standard indicator area and an initial detection area, dripping and incubating human osteoprotegerin antibodies in the detection area to functionalize the detection area, and staining the standard indicator area to indicate a human osteoprotegerin concentration gradient; An absorption layer is provided, and is sequentially combined with the paper-based detection layer and the paper-based guide layer to form the 3D paper-based microfluidic osteoprotegerin detection device.

5. The method for preparing a 3D paper-based microfluidic osteoprotegerin detection device according to claim 4, characterized in that: The wax printing heating method includes printing wax patterns on the first paper-based substrate or the second paper-based substrate by screen printing or wax jet printing, and heating to 80° C. to form a hydrophobic surface.

6. The method for preparing a 3D paper-based microfluidic osteoprotegerin detection device according to any one of claims 4-5, characterized in that: The functionalization process of the detection zone includes: A quantitative and equal-concentration human osteoprotegerin antibody solution is added dropwise to the detection area, incubated at room temperature to human body-like temperature, rinsed with Tween 20 solution and deionized water, and finally dried at room temperature to human body-like temperature.

7. A method for detecting human osteoprotegerin, using the device according to any one of claims 1 to 3, characterized in that: include: Adding the sample to be tested and the antibody-color development solution to the hydrophilic pattern area on the paper-based flow-guiding layer of the device; The mixed fluid is transported by capillary action of the hydrophilic patterned area of ​​the paper-based guide layer, and the mixed fluid is infiltrated through the paper-based guide layer through the microchannel to the detection area on the paper-based detection layer of the device, so that the sample to be detected is combined with the human osteoprotegerin antibody fixed in the detection area, and color is developed based on the antibody-color development solution; Determine the concentration of human osteoprotegerin in the sample to be tested based on the color development result and the standard concentration of the standard indicator area on the paper-based detection layer; The waste liquid formed after the detection that permeates the paper-based detection layer is absorbed by the absorption layer of the device.

Images