Biomarker visual quantitative detection device and preparation method thereof

Abstract

The invention provides a biomarker visual quantitative detection device and a preparation method thereof. The device realizes rapid, low-cost and high-sensitivity target molecule concentration detection through innovative design. The device mainly comprises a transparent micro-channel structure, a reaction container, a container cover and scale marks. When the reaction container is placed in the forward direction, air pressure of the reaction container and the micro-channel structure is balanced, after the reaction container is reversed, internal liquid seals the micro-channel due to the gravity effect, air pressure of gas generated by chemical reaction is increased, the liquid is pushed to enter the micro-channel, the length of a liquid column is in direct proportion to target concentration, and naked eye reading is achieved. Compared with the prior art, the invention can perform quantitative detection, has the characteristics of high sensitivity and quick operation, does not need instruments and equipment, is simple to prepare, low in cost and wide in application range, and can detect various biomolecules including nucleic acid and protein. The invention provides a novel efficient detection means for the fields of chemical analysis and biological target detection.

Description

Technical Field

[0001] The present invention relates to the fields of chemical analysis and biomedicine, and in particular to a biomarker visualized quantitative detection device and a preparation method thereof. Background Art

[0002] In biomedical research and clinical diagnosis, rapid and accurate detection of biomarkers is of great significance. Existing detection methods include:

[0003] (1) Catalase detection method

[0004] The concentration / activity detection of catalase is widely used in bacterial identification, cell analysis, etc. At present, the ultraviolet absorption method is usually used to detect catalase: catalase has a characteristic absorption peak at 240nm. Catalase can decompose H2O2, so that the absorbance value at 240nm of the reaction solution decreases with the reaction time. The concentration / activity of catalase can be characterized by the rate of change of absorbance value.

[0005] In bacterial identification, the H2O2 drop observation method can also be used: slowly drop a few drops of 3% H2O2 on the slant, flat bacterial lawn or concentrated bacterial solution, and observe whether bubbles are generated within 5 minutes. If bubbles are generated, it is positive, indicating the presence of catalase.

[0006] (2) Detection methods of biological protein targets

[0007] Existing biological protein target detection methods mainly include enzyme-linked immunosorbent assay, chemiluminescent immunoassay, colloidal gold immunochromatography and other technologies.

[0008] Enzyme-linked immunosorbent assay (ELISA) is a common method for quantitative protein detection. It has a variety of reaction modes: sandwich method, competitive method, indirect method, etc., to suit different types of target molecules. Taking the classic sandwich method as an example, a "solid phase-capture antibody-target protein-enzyme-labeled antibody" sandwich complex is formed with the target protein in the sample, and after washing and removing other unreacted substances, a substrate is added to the system, and the target protein is determined by luminescence or color development. Since ELISA is a solid-phase reaction, its reaction kinetics and detection sensitivity have certain limitations, and the operation is relatively cumbersome and time-consuming (>3h). It has not yet been widely used for exosome protein detection.

[0009] Chemiluminescent immunoassay (CLIA) is the current mainstream high-throughput biological target detection technology. Its basic principle is similar to that of ELISA technology. CLIA technology also includes multiple reaction modes such as sandwich method, competitive method, and indirect method. Taking the classic sandwich method as an example, by forming a "magnetic bead-capture antibody-target protein-enzyme-labeled antibody" sandwich complex with the target protein in the sample, after washing and removing other unreacted substances, an enzymatic luminescent substrate is added to the system, and the target protein can be determined by detecting the intensity of the luminescent signal. CLIA technology has the advantages of high degree of automation and high throughput, but it requires large-scale detection instruments. This method is usually used in large laboratories or large hospital laboratories.

[0010] Colloidal gold immunochromatography technology uses colloidal gold as a tracer marker and nitrocellulose membrane as a carrier, so that the antigen-antibody reaction and washing are carried out on the same permeation membrane. This process uses the capillary action of the microporous membrane to allow the liquid at one end of the membrane to slowly penetrate and move to the other end, thereby driving the movement, binding and washing of the antigen and antibody on the membrane, and finally observing the color change of the detection line to achieve protein target determination. This method is fast and convenient, but because it is a solid-phase reaction, it has low sensitivity and cannot be quantitatively detected.

[0011] The existing related detection technologies mainly have the following disadvantages:

[0012] (1) High detection cost and non-portability: Existing detection technologies for catalase or biological protein targets usually require large instruments such as spectrophotometers, microplate readers, and chemiluminescence analyzers. The detection system is expensive, and large instruments cannot be applied to various instant detection application scenarios, which has great application limitations.

[0013] (2) Complex operation and long detection time: Detection technologies such as ELISA have complex operation procedures and usually take more than 3 hours to detect. Developing a detection device with a short detection cycle and simple operation has important application prospects.

[0014] (3) Low sensitivity and inability to quantify: As an important type of testing application scenario, point-of-care testing usually requires rapidity, sensitivity, and quantification. The existing colloidal gold immunochromatography technology used for point-of-care testing has low sensitivity due to the limitation of solid-phase reaction, and cannot measure certain low-concentration targets in samples. It can only be used for qualitative detection, which further limits the scope of application of this technology.

[0015] In summary, the existing technology has problems such as complex operation, long detection time, low sensitivity, high cost, and high equipment requirements, and cannot meet the needs of use.

[0016] It should be noted that the information disclosed in the above background technology section is only used for understanding the background of the present application, and therefore may include information that does not constitute prior art known to ordinary technicians in the field. Summary of the invention

[0017] The main purpose of the present invention is to overcome the defects existing in the above-mentioned background technology and provide a biomarker visual quantitative detection device and a preparation method thereof.

[0018] To achieve the above object, the present invention adopts the following technical solutions:

[0019] A biomarker visualization and quantitative detection device, comprising:

[0020] Microfluidic structure, made of transparent material, used for measuring liquid flow and liquid column length;

[0021] A reaction container, the upper chamber of which is connected to the upper inlet of the microfluidic structure, and the reaction container is used to load a liquid mixture of a reaction medium and a target to be detected;

[0022] A container cover, which cooperates with the reaction container to form a closed space;

[0023] Scale markings, matched with the microfluidic structure and the reaction container, for naked eye reading;

[0024] When the device is placed forward, the pressure difference between the reaction container and the microfluidic structure is zero, and liquid will not enter the microfluidic structure;

[0025] When the device is placed upside down, the upper chamber of the reaction container is inverted and placed downward, and the liquid in the reaction container is concentrated in the upper chamber of the reaction container under the action of gravity, closing the microfluidic structure, and the gas generated when the reaction medium and the target to be detected undergo a chemical reaction increases the gas pressure in the reaction container, so that the liquid in the container is pushed into the microfluidic structure under the drive of the gas pressure difference;

[0026] The length of the liquid column in the microfluidic channel structure is positively correlated with the concentration of the target to be detected, so that the concentration of the target molecule can be quickly and quantitatively detected by reading the length of the liquid column in the microfluidic channel structure with the naked eye.

[0027] Furthermore, the microfluidic channel structure is made of polydimethylsiloxane (PDMS); preferably, the microfluidic channel structure is designed to be S-shaped.

[0028] Furthermore, it comprises a scale spacer arranged between the microfluidic channel structure and the reaction container, the surface of the scale spacer is provided with scale marks, or the scale marks are directly printed on the side of the reaction container.

[0029] Furthermore, the inlet of the microfluidic channel structure is aligned and connected with the small hole of the reaction container and the small hole of the scale spacer.

[0030] Furthermore, the reaction container is a disposable semi-micro reaction vessel, the container cover is a rubber plug matching the reaction vessel, and the container cover can achieve complete sealing with the reaction vessel.

[0031] Furthermore, the reaction medium is a hydrogen peroxide solution containing a color dye, and the target to be detected is selected from a substance having hydrogen peroxide catalytic activity or a conjugate thereof, wherein the substance having hydrogen peroxide catalytic activity includes catalase (Catalase), platinum nanoparticles (PtNP) and other compounds or materials with similar catalytic activity; the conjugate includes catalase / nanozyme labeled with an antibody or nucleic acid aptamer, which is used to achieve the determination of biological molecules through the specific recognition of the target by the conjugated molecule.

[0032] A method for preparing a biomarker visual quantitative detection device comprises the following steps:

[0033] S1. Prepare a transparent microfluidic structure to form a channel for liquid flow and liquid column length measurement;

[0034] S2, preparing a reaction container, the upper chamber of which is connected to the upper inlet of the microfluidic structure, and the reaction container is used to load a mixture of a reaction medium and a target to be detected;

[0035] S3, configuring a container cover to cooperate with the reaction container to form a closed space;

[0036] S4. Make scale marks that match the microfluidic structure and the reaction container for naked eye reading; wherein, the relationship between the length of the liquid column in the microfluidic structure and the concentration of the target to be measured is calibrated, so that the length of the liquid column in the microfluidic structure can be read by the naked eye to achieve rapid quantitative detection of the concentration of the target molecule.

[0037] Further, step S1 includes:

[0038] S1.1, coating photoresist on the silicon wafer and performing exposure and development to form a flow channel template;

[0039] S1.2, using PDMS material to replicate the flow channel template to form a PDMS flow channel;

[0040] S1.3, post-processing the PDMS channel, including inlet drilling and plasma treatment, and then bonding it with the PDMS film to obtain a complete microfluidic channel structure.

[0041] Furthermore, in step S2, the reaction container is a disposable semi-micro reaction vessel, the container cover is a rubber plug matching the reaction vessel, and a small hole is punched at a corresponding position between the reaction container and the entrance of the microfluidic structure to complete the preparation of the reaction container.

[0042] The flow channel template, in step S4, is used to establish a fitting relationship between the liquid column length and the target concentration, and to produce a scale mark corresponding to the concentration reading.

[0043] The present invention has the following beneficial effects:

[0044] The present invention provides a biomarker visualization quantitative detection device, which can realize rapid, highly sensitive, low-cost and easy-to-operate biomarker quantitative detection without relying on large instruments. The device uses a sophisticated structural design to quantitatively detect the concentration of target molecules by using the change in the length of the liquid column in the microfluidic structure. There is a positive correlation between the length of the liquid column and the concentration of the target to be measured, so that the detection result can be directly read by the naked eye, greatly simplifying the operation process. In addition, the device can respond quickly. After calibrating the gas pressure, only a simple inversion operation is required to trigger the detection reaction, so that the detection process is completed within a few minutes. Due to the use of a microfluidic structure, the device is sensitive to trace gases and can detect low-concentration biomarkers. In addition, the device is not only suitable for direct detection of substances with hydrogen peroxide catalytic activity such as catalase and platinum nanoparticles, but also can realize specific recognition and determination of biological molecules such as nucleic acids and proteins through conjugates, showing extremely strong application expansibility. In short, the detection device of the present invention has a wide range of application prospects in the fields of chemical analysis and biological target detection, can meet the needs of instant detection, and at the same time reduce the detection cost and operation difficulty.

[0045] Compared with the traditional detection device, the main advantages of the present invention are:

[0046] (1) Rapid calibration of the internal and external air pressure of the device: The length of the liquid column pushed out is directly related to the internal and external air pressure of the device. When the gas generation reaction begins, the internal and external air pressure difference of the device is zero (i.e., air pressure calibration is achieved). When the device is in use, the reaction container contains a certain volume of air, and this space is connected to the external atmosphere through the microchannel structure, so the internal and external air pressure difference is zero, ensuring the non-sensitive rapid calibration of the internal and external air pressure difference of the container.

[0047] (2) Extremely simplified operation steps: The designed device structure uses the gravity of the liquid in the container through a simple "inversion" operation to achieve automatic closure of the liquid conduit, thereby forming a closed space in the reaction container, ensuring that the generated gas can effectively increase the internal air pressure of the reaction container to achieve the purpose of detection without the need for additional equipment and operations.

[0048] (3) High-sensitivity detection: With the microfluidic structure, even if only a small volume of gas is generated in the reaction vessel, a long liquid column can be formed in the flow channel due to the small cross-sectional area of ​​the flow channel, thereby achieving high-sensitivity detection of the target.

[0049] (4) Naked-eye quantitative detection: The scale is printed directly on the device, and the quantitative results can be quickly read by naked-eye by observing the scale corresponding to the position of the liquid column in the flow channel.

[0050] This device can be used not only for the direct detection of substances with hydrogen peroxide catalytic activity (such as catalase, nanozyme, etc.), but also for the determination of the conjugates of the above substances (such as catalase / nanozyme labeled with antibody / nucleic acid aptamer), and the determination of biological molecules such as nucleic acids and proteins can be achieved through the specific recognition of the target by the coupled molecules. This device is not only suitable for reaction systems that catalyze the decomposition of hydrogen peroxide to produce oxygen, but also for any reaction system that uses liquid reactions to produce gas to cause changes in the pressure inside and outside the container. This method has many advantages such as no need for instruments, naked eye reading, high sensitivity, quantitative determination, rapid detection, simple operation, low cost, etc., and has strong scalability. It is expected to be used in many fields such as chemical analysis and biological target detection, and has a wide range of application value.

[0051] This device can be directly used for the determination of substances with hydrogen peroxide catalytic activity such as catalase and platinum nanoparticles, or for the specific recognition of other target molecules through the conjugates of the above substances, so as to achieve the determination of more types of targets (such as nucleic acids and proteins). Among them, the determination of catalase and biological protein targets has wide application value in the biomedical field.

[0052] Other beneficial effects of the embodiments of the present invention will be further described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Schematic diagram of the structure of a visual quantitative detection device for biomarkers according to an embodiment of the present invention.

[0054] Figure 2 Schematic diagram of the operating method of the detection device according to an embodiment of the present invention.

[0055] Figure 3 Schematic diagram of the preparation process of the PDMS microfluidic structure according to an embodiment of the present invention.

[0056] Figure 4 Schematic diagram of the PDMS microfluidic channel structure of an embodiment of the present invention.

[0057] Figure 5 4 is a calibration curve of the catalase concentration and the liquid column movement distance of the embodiment of the present invention.

[0058] Figure 6 This is a real picture of the detection device according to an embodiment of the present invention used for the determination of catalase. DETAILED DESCRIPTION

[0059] The following is a detailed description of the embodiments of the present invention. It should be emphasized that the following description is only exemplary and is not intended to limit the scope and application of the present invention.

[0060] It should be noted that when an element is referred to as being "fixed to" or "disposed on" another element, it can be directly on the other element or indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element. In addition, connection can be used for fixing as well as for coupling or communication.

[0061] It should be understood that the orientation or position relationship indicated by terms such as "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside" and "outside" are based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0062] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the embodiments of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0063] See also Figures 1 to 6 , an embodiment of the present invention provides a biomarker visualization quantitative detection device, which can be used for detecting active substances that catalyze gas generation and their labeled biological targets.

[0064] See also Figure 1 , Figure 2 and Figure 4 The detection device comprises: a microfluidic structure 1, which is made of a transparent material and is used for measuring liquid flow and liquid column length; a reaction container 3, whose upper chamber is connected to the upper inlet 5 of the microfluidic structure 1, and the reaction container 3 is used to load a liquid mixture of a reaction medium and a target to be detected; a container cover 4, which cooperates with the reaction container 3 to form a closed space; a scale mark, which cooperates with the microfluidic structure 1 and the reaction container 3, and is used for naked eye reading; Figure 2As shown on the left side, when the device is placed forward, the pressure difference between the reaction container 3 and the microfluidic structure 1 is zero, and the liquid 6 will not enter the microfluidic structure 1; Figure 2 As shown on the right side, when the device is placed upside down, the upper chamber of the reaction container 3 is inverted downward, and the liquid 6 in the reaction container 3 is concentrated in the upper chamber of the reaction container 3 under the action of gravity, closing the microfluidic structure 1. The gas generated when the reaction medium chemically reacts with the target to be measured increases the air pressure of the air 7 in the reaction container 3, so that the liquid in the container 3 is pushed into the microfluidic structure 1 under the drive of the air pressure difference; wherein, the length of the liquid column in the microfluidic structure 1 is positively correlated with the concentration of the target to be measured, so that the concentration of the target molecule can be quickly and quantitatively detected by reading the length of the liquid column in the microfluidic structure 1 with the naked eye.

[0065] In a preferred embodiment, the microfluidic channel structure 1 is made of polydimethylsiloxane (PDMS), but the present invention does not limit the specific material of the channel structure. Preferably, the microfluidic channel structure 1 is designed to be S-shaped.

[0066] like Figure 1 As shown, in some embodiments, the detection device includes a scale partition 2 disposed between the microfluidic structure 1 and the reaction container 3, and a scale mark is disposed on the surface of the scale partition 2. The inlet 5 of the microfluidic structure 1 is aligned and connected with the small hole of the reaction container 3 and the small hole of the scale partition 2.

[0067] In other embodiments, the scale markings may be directly printed on the side of the reaction container 3 .

[0068] like Figure 1 As shown, in some embodiments, the reaction container 3 is a disposable semi-micro reaction vessel, the container cover 4 is a rubber plug matching the reaction vessel, and the container cover 4 can achieve complete sealing with the reaction container 3.

[0069] In some embodiments, the reaction medium can be a hydrogen peroxide solution containing a color dye, and the target to be detected can be selected from a substance having hydrogen peroxide catalytic activity or a conjugate thereof, wherein the substance having hydrogen peroxide catalytic activity includes but is not limited to catalase (Catalase), platinum nanoparticles (PtNP) and other compounds or materials with similar catalytic activity; the conjugate includes catalase / nanozyme labeled with an antibody or nucleic acid aptamer, which is used to achieve the determination of biological molecules such as nucleic acids and proteins through specific recognition of the target by the conjugated molecule, wherein the conjugate can achieve high specificity detection of specific biological molecules by forming a "magnetic bead-capture antibody-target protein-catalase labeled antibody" sandwich complex similar to the CLIA method.

[0070] The biomarker visualization quantitative detection device provided by the present invention connects the microfluidic structure with the upper part of a reaction container, and uses the length of the liquid column in the flow channel as the reading index, so as to cleverly form a detection device for naked eye reading. Among them, there is a certain volume of air in the upper part of the container, and the lower part of the container is filled with a mixture of hydrogen peroxide (which may contain a color dye) and a target to be detected. When the above-mentioned device is placed forward, since the inside and outside of the container are connected through the microfluidic structure, the internal and external air pressure difference is zero; when the above-mentioned device is placed upside down, the internal liquid will be concentrated under the container (the air is concentrated above) due to gravity, and the flow channel will be closed. Since there is no air pressure difference between the inside and outside of the reaction container, the liquid will not enter the microfluidic structure at this time. As the reaction proceeds, the substance to be tested with hydrogen peroxide catalytic activity (such as catalase (Catalase), platinum nanoparticles (PtNP), or its conjugate) will catalyze the decomposition of hydrogen peroxide in the container to produce water and oxygen. The continuously generated oxygen gathers in the container, causing the air pressure in the container to increase, and finally the liquid in the container is pushed into the microfluidic structure driven by the air pressure difference. In the same time, the length of the liquid column in the flow channel is positively correlated with the concentration of the target to be detected. By reading the length of the liquid column in the microchannel structure with the naked eye, and then according to the corresponding fitting relationship between the length of the liquid column and the target concentration, the concentration of the target molecule can be quickly and quantitatively detected. The present invention can be used for the detection of active substances that catalyze gas generation.

[0071] This detection device can be used not only for the direct detection of substances with hydrogen peroxide catalytic activity (such as catalase, nanozyme, etc.), but also for the determination of the above-mentioned substance conjugates (such as catalase / nanozyme labeled with antibody / nucleic acid aptamer), through the specific recognition of the target by the coupled molecule, so as to achieve the determination of biological molecules such as nucleic acids and proteins. This device is not only suitable for reaction systems that catalyze the decomposition of hydrogen peroxide to produce oxygen, but also for any reaction system that uses liquid reaction to produce gas to cause pressure changes inside and outside the container. The present invention has many advantages such as no need for instruments, naked eye reading, high sensitivity, quantitative determination, rapid detection, simple operation, low cost, etc., and has extremely strong expansibility, and is expected to be used in many fields such as chemical analysis and biological target detection, and has a wide range of application value.

[0072] The present invention also provides a method for preparing a biomarker visualization quantitative detection device, comprising the following steps:

[0073] S1. Prepare a transparent microfluidic structure to form a channel for liquid flow and liquid column length measurement;

[0074] S2, preparing a reaction container, the upper chamber of which is connected to the upper inlet of the microfluidic structure, and the reaction container is used to load a mixture of a reaction medium and a target to be detected;

[0075] S3, configuring a container cover to cooperate with the reaction container to form a closed space;

[0076] S4. Make scale marks that match the microfluidic structure and the reaction container for naked eye reading; wherein, the relationship between the length of the liquid column in the microfluidic structure and the concentration of the target to be measured is calibrated, so that the length of the liquid column in the microfluidic structure can be read by the naked eye to achieve rapid quantitative detection of the concentration of the target molecule.

[0077] The specific embodiments of the present invention are further described below.

[0078] The structural design of the detection device

[0079] The detection device mainly includes four parts: microfluidic structure 1, scale partition 2, reaction container 3 and container cover. Figure 1 As shown. The detection device is based on a reaction container (including a container cover), and there is a small hole on the upper side of the reaction container. The above-mentioned side of the reaction container is pasted together with an opaque partition layer with a small hole and a scale at the corresponding position, and the scale partition layer (with a distance or concentration scale printed on the surface) is then pasted together with a polydimethylsiloxane (PDMS) microfluidic structure. The small hole of the reaction container, the small hole of the scale partition layer and the inlet 5 of the PDMS microfluidic structure are aligned, so that the interior of the assembled reaction container is connected to the external atmosphere through the small hole of the scale partition layer and the microfluidic structure.

[0080] The above structures can be optimized as follows:

[0081] ① The above-mentioned microfluidic channel structure is designed to be S-shaped to maximize the channel length, and can also be designed to other shapes; the channel can adopt different widths / heights (or a combination of different widths / heights) to meet the requirements of different sensitivities and detection ranges: reducing the channel width / height (i.e., cross-sectional area) can improve the detection sensitivity, and increasing the channel width / height can increase the detection range.

[0082] ②The above-mentioned microfluidic structure is made of PDMS material, which can also be replaced by other materials.

[0083] ③ The above reaction container is a disposable semi-micro reaction dish, and the container cover is a rubber plug matching the reaction dish, and the container cover can achieve complete sealing with the reaction container. The above reaction container and container cover can also be replaced with other functional devices of other materials, sizes or shapes.

[0084] ④ The scale spacer pattern can also be directly printed on the side of the reaction container, thereby omitting the scale spacer.

[0085] How to operate the detection device

[0086] An appropriate volume of hydrogen peroxide solution containing a color dye and a certain volume of a test solution of catalase with an unknown concentration are mixed and added to the reaction container. The height of the liquid in the reaction container must not exceed the height of the small hole, and the container lid is closed. Since the air inside and outside the reaction container is connected, the pressure difference between the inside and outside of the container is zero. At this time, the detection device is inverted, and the liquid in the reaction container will be concentrated at the bottom of the container due to gravity, and the flow channel will be closed. The catalase in the solution will catalyze the decomposition of H2O2 in the reaction container to produce water and oxygen. The generated oxygen gathers in the closed container, causing the air pressure in the container to increase. Finally, the liquid in the reaction container is pushed into the microfluidic structure from the flow channel entrance driven by the air pressure difference. Since the pushed liquid contains a color dye, the length of the liquid column in the flow channel can be directly observed by the naked eye. In the same period of time, the length of the liquid column in the flow channel is positively correlated with the concentration of catalase. By establishing a fitting relationship between the length of the liquid column and the concentration of catalase, rapid quantitative detection of target molecules can be achieved. The operation method is as follows. Figure 2 shown. Figure 2 The device is shown in perspective without the graduated compartment.

[0087] The above components can be optimized as follows:

[0088] ① The analyte (catalase) can be replaced with other substances with catalase-catalyzed decomposition activity (such as platinum nanoparticles), thereby achieving the concentration determination of the substance.

[0089] ② The above-mentioned test substances can be replaced with corresponding conjugates (such as molecules labeled with antibody / nucleic acid aptamers), and the specific recognition of the target by the antibody / nucleic acid on the conjugate in the pre-reaction (such as forming a "magnetic bead-capture antibody-target protein-catalase labeled antibody" sandwich complex similar to the CLIA method) can be achieved to achieve the determination of biological molecules such as proteins and nucleic acids.

[0090] ③ The above-mentioned reaction system for catalyzing the decomposition of hydrogen peroxide to produce oxygen can be replaced by any other reaction system that utilizes liquid reaction to produce gas and causes changes in the pressure inside and outside the container.

[0091] Examples

[0092] The detection device of the embodiment of the present invention is manufactured, and the detection device is used to measure the unknown concentration of catalase solution. The specific implementation is as follows:

[0093] ① Preparation of the detection device:

[0094] 1. Preparation of microfluidic structure: Add 2mL SU-8 photoresist on a 3-inch silicon wafer, and use a photoresist spreader to evenly spread the photoresist on the surface of the silicon wafer. Place the silicon wafer after the photoresist is spread on a dryer and dry for 7 minutes (65°C) and 30 minutes (95°C), respectively. The expected photolithography height is 170μm. Turn on the photolithography machine, place a photomask on the silicon wafer (the width of the flow channel in the front half is designed to be 0.3mm, and the width of the flow channel in the back half is designed to be 0.6mm), and expose for 35 seconds under 2.14V parameters. Then place the silicon wafer on a dryer and dry for 5 minutes (65°C) and 12 minutes (95°C), respectively. Soak the dried silicon wafer in the developer for 12 minutes, and then rinse the silicon wafer with ethanol and developer alternately for 3 rounds. Place the cleaned silicon wafer on a dryer and dry for 10 minutes (150°C) to complete the preparation of the flow channel template. Place the flow channel template in a round culture dish, weigh 15g of PDMS component 1 and 1.5g of component 2 (cross-linker component), stir evenly with a glass rod, and pour onto the silicon wafer template. After the bubbles evaporate, place the above materials on a 65°C hot plate and heat for 3 hours. After the PDMS is cured, peel it off from the silicon wafer template (re-mold), and then cut it into a 40mm×12mm PDMS flow channel template. Make a small hole at the PDMS flow channel inlet corresponding to the PDMS film of the same size, and place it in a plasma machine for 5 minutes together with the above PDMS flow channel. After the above PDMS flow channel is bonded to the PDMS film, heat it at 65°C for 2 hours to obtain a complete microfluidic structure. The overall preparation process of the PDMS flow channel is as follows: Figure 3 As shown in Figure 2, the microchannel structure is as follows: Figure 4 shown.

[0095] Figure 3 The preparation process of PDMS microfluidic structure is shown, including operations such as photolithography, molding and plasma bonding. Figure 4 The PDMS microfluidic channel structure is shown, including a microfluidic channel 1 , a microfluidic channel structure inlet 5 and a microfluidic channel structure outlet 8 .

[0096] 2. Preparation of reaction container: Use a disposable semi-micro reaction vessel as the reaction vessel, and use the rubber plug provided with the reaction vessel as the container cover. Make a small hole at the corresponding position between the reaction vessel and the entrance of the microfluidic structure, and the preparation of the reaction vessel is completed.

[0097] 3. Overall assembly of the detection device: Use waterproof paper to print out the scale mark: For easy reading, divide the length of each row of flow channels into 5 parts and use black dots as scale marks. Each length is defined as 1 relative length unit (au), and a small hole is punched at the corresponding position of the scale partition and the entrance of the microfluidic structure. Use double-sided tape to stick the microfluidic structure, scale partition and reaction vessel together in sequence: align the small hole of the reaction vessel, the small hole of the scale partition and the entrance of the PDMS microfluidic structure, so that the interior of the assembled reaction vessel can be connected to the outside through the small hole of the scale partition and the microfluidic structure. The overall assembly of this detection device is completed.

[0098] ②Use this detection device to measure the concentration of catalase:

[0099] 1. Drawing of calibration curve: First, use phosphate buffered saline (PBS) as a buffer to prepare a series of catalase solutions with known concentrations (0, 2, 4, 6, 8, 10 μg / mL, respectively). Take 1 mL of each of the above catalase solutions of different concentrations, mix with 1 mL of H2O2 solution (20 mM) containing dark ink, and add to the assembled reaction container, cover the lid to seal the reaction container. Then quickly turn the reaction device upside down, react at 25°C for 5 minutes, and read the length of the liquid pushed out of the flow channel. Each sample was tested three times and the average was taken. The "concentration-length" fitting curve was established based on the relationship between the concentration of the catalase solution with a known concentration gradient and the length reading. The positive correlation satisfied the 4-parameter fitting equation: y = A2 + (A1-A2) / (1 + (x / x0)^p), where y is the distance the liquid column moves, x is the catalase concentration, and the other fitting parameters are: A1 = 3.73, A2 = 237836.28, x0 = 101.32, p = 3.26, and the correlation coefficient R 2 =0.9938. The calibration curve after fitting is as follows Figure 5 Finally, according to the fitting curve, the length reading is converted into the concentration of the catalase solution, and a scale partition directly marking the corresponding concentration reading is made, and the detection device is assembled to detect the sample to be tested.

[0100] 2. Determination of catalase samples of unknown concentration: Use PBS as a buffer, take 1mL of the unknown concentration of the sample to be tested and mix it with 1mL of H2O2 solution (20mM) containing dark ink, add it to the assembled reaction device with concentration scale, cover the reaction container with a lid to seal it. Then quickly turn the reaction device upside down, react at 25℃ for 5 minutes, and read the concentration reading corresponding to the position of the liquid pushed out in the flow channel directly with the naked eye (such as the actual object Figure 6 ), which is the concentration of catalase in the sample to be tested. Figure 6A physical diagram of a detection device for catalase determination is shown, with scale length markings 9 and scale number markings 10 .

[0101] Advantages of this detection device:

[0102] This detection device can achieve rapid determination of catalase concentration within 5 minutes. It realizes simple operation, no instrumentation, high sensitivity, naked eye quantitative, and rapid determination of catalase concentration at low cost. In addition, this method has extremely high scalability. By replacing catalase with a conjugate with a substance having catalase activity and a substance having a target recognition function (such as catalase / platinum nanoparticles labeled with antibodies / nucleic acid aptamers), the specific recognition of the target molecule by the conjugate can achieve the determination of biological molecules such as proteins and nucleic acids. This device is not only suitable for reaction systems that catalyze the decomposition of hydrogen peroxide to produce oxygen, but also for any reaction system that uses liquid reactions to produce gas to cause changes in the pressure inside and outside the container. It is expected to be used in many fields such as chemical analysis and biological target detection, and has a wide range of application value.

[0103] Compared with the prior art, the present invention has the following technical advantages:

[0104] (1) Quantitative detection: Traditional colloidal gold immunochromatography can only detect target molecules qualitatively. The present invention uses the corresponding relationship between the length of the liquid column pushed out and the concentration of the analyte to achieve quantitative detection of target molecules. The quantitative results are of great practical significance for the determination of many targets, so quantitative detection has a wider application prospect.

[0105] (2) Highly sensitive detection: The reaction process of the present invention is carried out in a solution, which has better reaction kinetics than immunochromatography, making the reaction process faster and more thorough. On the other hand, since the cross-sectional area of ​​the microfluidic structure is very small, the trace amount of gas generated in the reaction container can also push out a longer liquid column, which is conducive to the high-sensitivity determination of low-concentration samples.

[0106] (3) Rapid detection and simple operation: The present invention can achieve rapid detection of the target within a few minutes, and the operation is very simple. After adding the sample, the device only needs to be "inverted", which greatly improves the ease of use and practicality of the device.

[0107] (4) No instrument required, low cost: This method does not require any instrument or equipment, involves a small number of raw materials with low prices (such as disposable reaction vessels, hydrogen peroxide solution), and a simple preparation process. The overall detection cost is significantly lower than that of traditional detection technology.

[0108] (5) Strong application expandability: By replacing catalase with a conjugate of a substance with catalase activity and a substance with target recognition function (such as catalase / platinum nanoparticles labeled with antibodies / nucleic acid aptamers), the conjugated molecule can achieve specific recognition of the target, thereby realizing the determination of biological molecules such as nucleic acids and proteins. This device is not only suitable for reaction systems that catalyze the decomposition of hydrogen peroxide to produce oxygen, but also for any reaction system that uses liquid reactions to produce gas to cause changes in the pressure inside and outside the container, and has extremely strong application expandability.

[0109] The above content is a further detailed description of the present invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, without departing from the concept of the present invention, it can also make several substitutions or modifications to these described embodiments, and these substitutions or modifications should be regarded as belonging to the protection scope of the present invention. In the description of this specification, the description of the reference terms "an embodiment", "some embodiments", "preferred embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily target the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In the absence of mutual contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions and modifications can be made herein without departing from the scope of protection of the patent application.

Claims

1. A biomarker visualization quantitative detection device, characterized in that: include: Microfluidic structure, made of transparent material, used for measuring liquid flow and liquid column length; A reaction container, the upper chamber of which is connected to the upper inlet of the microfluidic structure, and the reaction container is used to load a liquid mixture of a reaction medium and a target to be detected; A container cover, which cooperates with the reaction container to form a closed space; Scale markings, matched with the microfluidic structure and the reaction container, for naked eye reading; When the device is placed forward, the pressure difference between the reaction container and the microfluidic structure is zero, and liquid will not enter the microfluidic structure; When the device is placed upside down, the upper chamber of the reaction container is inverted and placed downward, and the liquid in the reaction container is concentrated in the upper chamber of the reaction container under the action of gravity, closing the microfluidic structure, and the gas generated when the reaction medium and the target to be detected undergo a chemical reaction increases the gas pressure in the reaction container, so that the liquid in the container is pushed into the microfluidic structure under the drive of the gas pressure difference; The length of the liquid column in the microfluidic channel structure is positively correlated with the concentration of the target to be detected, so that the concentration of the target molecule can be quickly and quantitatively detected by reading the length of the liquid column in the microfluidic channel structure with the naked eye.

2. The biomarker visualization and quantitative detection device according to claim 1, characterized in that: The microfluidic channel structure is made of polydimethylsiloxane (PDMS); preferably, the microfluidic channel structure is designed to be S-shaped.

3. The biomarker visualization and quantitative detection device according to claim 1 or 2, characterized in that: It comprises a scale spacer arranged between the microchannel structure and the reaction container, the surface of the scale spacer is provided with scale marks, or the scale marks are directly printed on the side of the reaction container.

4. The biomarker visualization and quantitative detection device according to any one of claims 1 to 3, characterized in that: The inlet of the microfluidic channel structure is aligned and communicated with the small hole of the reaction container and the small hole of the scale partition.

5. The biomarker visualization and quantitative detection device according to any one of claims 1 to 4, characterized in that: The reaction container is a disposable semi-micro reaction vessel, the container cover is a rubber plug matched with the reaction vessel, and the container cover can achieve complete sealing with the reaction vessel.

6. The biomarker visualization and quantitative detection device according to any one of claims 1 to 5, characterized in that: The reaction medium is a hydrogen peroxide solution containing a color dye, and the target to be detected is selected from a substance with hydrogen peroxide catalytic activity or a conjugate thereof, wherein the substance with hydrogen peroxide catalytic activity includes catalase (Catalase), platinum nanoparticles (PtNP) and other compounds or materials with similar catalytic activity; the conjugate includes catalase / nanozyme labeled with an antibody or a nucleic acid aptamer, which is used to realize the determination of biological molecules through the specific recognition of the target by the conjugated molecule.

7. A method for preparing a biomarker visualization quantitative detection device, characterized in that: The following steps are involved: S1. Prepare a transparent microfluidic structure to form a channel for liquid flow and liquid column length measurement; S2, preparing a reaction container, the upper chamber of which is connected to the upper inlet of the microfluidic structure, and the reaction container is used to load a mixture of a reaction medium and a target to be detected; S3, configuring a container cover to cooperate with the reaction container to form a closed space; S4. Make scale marks that match the microfluidic structure and the reaction container for naked eye reading; wherein, the relationship between the length of the liquid column in the microfluidic structure and the concentration of the target to be measured is calibrated, so that the length of the liquid column in the microfluidic structure can be read by the naked eye to achieve rapid quantitative detection of the concentration of the target molecule.

8. The method for preparing the biomarker visualization and quantitative detection device according to claim 7, characterized in that: Step S1 includes: S1.1, coating photoresist on the silicon wafer and performing exposure and development to form a flow channel template; S1.2, using PDMS material to replicate the flow channel template to form a PDMS flow channel; S1.3, post-processing the PDMS channel, including inlet drilling and plasma treatment, and then bonding it with the PDMS film to obtain a complete microfluidic channel structure.

9. The method for preparing the biomarker visualization and quantitative detection device according to claim 7 or 8, characterized in that: In step S2, the reaction container is a disposable semi-micro reaction vessel, the container cover is a rubber plug matching the reaction vessel, and a small hole is punched at a position corresponding to the entrance of the reaction container and the microfluidic structure to complete the preparation of the reaction container.

10. The method for preparing the biomarker visualization and quantitative detection device according to any one of claims 7 to 9, characterized in that: In step S4, a fitting relationship between the liquid column length and the target concentration is established to produce a scale mark corresponding to the concentration reading.

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