A Visual Quantitative Detection Device for Biomarkers and Its Preparation Method

Abstract

This invention provides a visual quantitative detection device for biomarkers and its preparation method. This device, through innovative design, achieves rapid, low-cost, and highly sensitive detection of target molecule concentrations. The device mainly includes a transparent microfluidic structure, a reaction container, a container lid, and graduated markings. When placed upright, the gas pressure of the reaction container and the microfluidic structure is balanced. When inverted, the internal liquid seals the microfluidic channel due to gravity, and the gas generated by the chemical reaction increases the gas pressure, pushing the liquid into the microfluidic channel. The length of the liquid column is proportional to the target concentration, enabling naked-eye readings. Compared to existing technologies, this invention enables quantitative detection, features high sensitivity and rapid operation, requires no instruments, is simple to prepare, low in cost, and has a wide range of applications, capable of detecting various biomolecules, including nucleic acids and proteins. This invention provides a new and efficient detection method for the fields of chemical analysis and biological target detection.

Description

Technical Field

[0001] This invention relates to the fields of chemical analysis and biomedicine, and in particular to a device for visual quantitative detection of biomarkers and its preparation method. Background Technology

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

[0003] (1) Detection method of catalase

[0004] The concentration / activity detection of catalase is widely used in bacterial identification and cell analysis. Currently, ultraviolet absorption is commonly used to detect catalase: catalase has a characteristic absorption peak at 240 nm. Catalase can decompose H2O2, causing the absorbance of the reaction solution at 240 nm to decrease with reaction time. The concentration / activity of catalase can be characterized by the rate of change of absorbance.

[0005] In bacterial identification, the H2O2 addition observation method can also be used: slowly add a few drops of 3% H2O2 to the slant, plate bacterial growth, or concentrated bacterial solution, and observe whether bubbles are produced within 5 minutes. The presence of bubbles indicates a positive result, signifying the presence of catalase.

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

[0007] Existing methods for detecting biological protein targets mainly include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, and colloidal gold immunochromatography.

[0008] Enzyme-linked immunosorbent assay (ELISA) is a commonly used method for protein quantification. It employs various reaction modes, including sandwich, competitive, and indirect methods, to suit different types of target molecules. Taking the classic sandwich method as an example, a "solid-capture antibody-target protein-enzyme-labeled antibody" sandwich complex is formed between the antibody and the target protein in the sample. After washing away unreacted substances, a substrate is added to the system, and the target protein is measured through luminescence or color development. Because ELISA is a solid-phase reaction, its reaction kinetics and detection sensitivity are limited, and the operation is relatively cumbersome and time-consuming (>3 hours), thus it is not yet widely used for exosome protein detection.

[0009] Chemiluminescent immunoassay (CLIA) is currently the mainstream high-throughput biomarker detection technology. Its basic principle is similar to ELISA, and CLIA also includes various reaction modes such as sandwich, competitive, and indirect methods. Taking the classic sandwich method as an example, a "magnetic bead-capture antibody-target protein-enzyme-labeled antibody" sandwich complex is formed between the bead and the target protein in the sample. After washing away unreacted substances, an enzyme-catalyzed 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 advantages such as high automation and high throughput, but it requires large-scale detection instruments and is typically used in large laboratories or hospital clinical laboratories.

[0010] Colloidal gold immunochromatography uses colloidal gold as a tracer and nitrocellulose membrane as a carrier, allowing antigen-antibody reaction and washing to occur on the same permeate membrane. This process utilizes the capillary action of the microporous membrane, allowing liquid to slowly permeate from one end to the other, thereby facilitating the movement, binding, and washing of antigens and antibodies on the membrane. The final result is the observation of color changes in the detection line, enabling protein target assay. This method is rapid and convenient, but because it is a solid-phase reaction, its sensitivity is relatively low and it cannot provide quantitative detection.

[0011] Existing detection technologies have the following main drawbacks:

[0012] (1) High detection cost and lack of portability: Existing detection technologies for catalase or biological protein targets usually require large instruments such as spectrophotometers, enzyme-linked immunosorbent assay (ELISA) readers, and chemiluminescence immunoassay analyzers. The detection system is expensive, and the large instruments are not suitable for various point-of-care testing applications, resulting in significant limitations in application.

[0013] (2) Complex operation and long detection time: Detection technologies such as ELISA have complex operation procedures and the detection time usually takes more than 3 hours. 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 testing application scenario, point-of-care testing usually requires speed, sensitivity, and quantification. Existing colloidal gold immunochromatography technology for point-of-care testing has low sensitivity due to the limitations of solid-phase reaction, making it unable to measure certain low-concentration targets in samples, and it can only be used for qualitative detection, further limiting the application scope of this technology.

[0015] In summary, existing technologies suffer from problems such as complex operation, long detection time, low sensitivity, high cost, and high equipment requirements, and cannot adequately meet the needs of users.

[0016] It should be noted that the information disclosed in the background section above is only for understanding the background of this application, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0017] The main objective of this invention is to overcome the deficiencies in the aforementioned background technology and provide a device for visual quantitative detection of biomarkers and its preparation method.

[0018] To achieve the above objectives, the present invention adopts the following technical solution:

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

[0020] Microchannel structures, made of transparent materials, are used for measuring liquid flow and liquid column length;

[0021] A reaction vessel, the upper chamber of which is connected to the upper inlet of the microfluidic structure, and the reaction vessel is used to hold a liquid mixture of reaction medium and target to be tested.

[0022] The container lid fits into the reaction vessel to form a sealed space;

[0023] The scale markings, in conjunction with the microfluidic structure and reaction vessel, are for naked-eye reading.

[0024] When the device is placed upright, the pressure difference between the reaction vessel and the microchannel structure is zero, and at this time, liquid will not enter the microchannel structure.

[0025] When the device is inverted, the upper chamber of the reaction vessel is upside down. The liquid in the reaction vessel is concentrated in the upper chamber of the reaction vessel under the action of gravity, which seals the microchannel structure. When the reaction medium reacts with the target, the gas generated increases the gas pressure in the reaction vessel, which pushes the liquid in the vessel into the microchannel structure under the pressure difference.

[0026] The length of the liquid column in the microfluidic structure is positively correlated with the concentration of the target, thus enabling rapid quantitative detection of the target molecule concentration by visually measuring the length of the liquid column in the microfluidic structure.

[0027] Furthermore, the microchannel structure is made of polydimethylsiloxane (PDMS); preferably, the microchannel structure is designed in an S-shape.

[0028] Furthermore, it includes a graduated partition disposed between the microfluidic structure and the reaction vessel, wherein the surface of the graduated partition is provided with graduated markings, or the graduated markings are directly printed on the side of the reaction vessel.

[0029] Furthermore, the inlet of the microchannel structure is aligned and connected with the orifice of the reaction vessel and the orifice of the graduated partition.

[0030] Furthermore, the reaction vessel is a disposable semi-micro reaction dish, and the container lid is a rubber stopper that matches the reaction dish, and the container lid can achieve a complete seal with the reaction vessel.

[0031] Furthermore, the reaction medium is a hydrogen peroxide solution containing a colored dye, and the target to be tested is selected from substances with hydrogen peroxide catalytic activity or their conjugates. The substances with hydrogen peroxide catalytic activity include catalase, platinum nanoparticles (PtNP), and other compounds or materials with similar catalytic activity. The conjugates include antibody- or nucleic acid aptamer-labeled catalase / nanozymes, which are used to achieve the determination of biomolecules through the specific recognition of the target by the conjugate molecules.

[0032] A method for preparing a biomarker visualization and quantitative detection device includes the following steps:

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

[0034] S2. Prepare 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 reaction medium and target to be tested.

[0035] S3. Configure a container lid to fit the reaction vessel and form a sealed space;

[0036] S4. Create scale markings that fit the microfluidic structure and reaction vessel for naked-eye reading; wherein, the relationship between the liquid column length in the microfluidic structure and the concentration of the target to be measured is calibrated, so that the target molecule concentration can be rapidly quantitatively detected by reading the liquid column length in the microfluidic structure with the naked eye.

[0037] Further, step S1 includes:

[0038] S1.1. Coat a silicon wafer with photoresist and expose and develop it to form a flow channel template;

[0039] S1.2. Use PDMS material to replicate the flow channel template to form PDMS flow channels;

[0040] S1.3. Post-processing of the PDMS channel, including inlet perforation and plasma treatment, is performed, and then it is bonded to the PDMS film to obtain a complete microchannel structure.

[0041] Further, in step S2, the reaction vessel is a disposable semi-micro reaction dish, the container lid is a rubber stopper that matches the reaction dish, and a small hole is drilled at the corresponding position of the inlet of the reaction vessel and the microchannel structure to complete the preparation of the reaction vessel.

[0042] In step S4, the flow channel template is used to create a scale marking for the corresponding concentration reading by establishing a fitting relationship between the liquid column length and the target concentration.

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

[0044] This invention provides a visual quantitative detection device for biomarkers, enabling rapid, highly sensitive, low-cost, and easy-to-operate quantitative detection of biomarkers without relying on large instruments. Through its ingenious structural design, the device utilizes changes in the length of the liquid column within a microfluidic structure to quantitatively detect the concentration of target molecules. A positive correlation exists between the liquid column length and the concentration of the target molecule, allowing the detection results to be read directly with the naked eye, greatly simplifying the operational process. Furthermore, the device offers rapid response; after calibrating the gas pressure, a simple inversion triggers the detection reaction, completing the detection process within minutes. Due to the microfluidic structure, the device is highly sensitive to trace amounts of gas, enabling the detection of low concentrations of biomarkers. Moreover, this device is not only suitable for the direct detection of substances with hydrogen peroxide catalytic activity, such as catalase and platinum nanoparticles, but can also achieve specific recognition and measurement of biomolecules such as nucleic acids and proteins through conjugates, demonstrating strong application scalability. In summary, the detection device of this invention has broad application prospects in the fields of chemical analysis and biological target detection, meeting the needs of point-of-care testing while reducing detection costs and operational complexity.

[0045] Compared with traditional detection devices, the main advantages of this invention are:

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

[0047] (2) Extremely simplified operation steps: The designed device structure uses a simple "inverted" operation to achieve automatic sealing of the liquid flow channel by the gravity of the liquid inside the container, thereby forming a sealed space inside the reaction container. This ensures that the generated gas can effectively increase the internal gas pressure of the reaction container and achieve the detection purpose without the need for additional devices and operations.

[0048] (3) High sensitivity detection: The microchannel structure is adopted. Even if only a small volume of gas is generated in the reaction vessel, a long liquid column can be formed in the channel due to the small cross-sectional area of ​​the 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 naked-eye quantitative results can be quickly read 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 and nanozymes), but also for the determination of conjugates of these substances (such as antibody / nucleic acid aptamer-labeled catalase / nanozymes). Through the specific recognition of the target by the conjugate molecules, the determination of biomolecules such as nucleic acids and proteins can be achieved. This device is suitable not only for reaction systems that catalyze the decomposition of hydrogen peroxide to produce oxygen, but also for any reaction system that utilizes a liquid reaction to generate gas, resulting in a change in pressure inside and outside the container. This method has many advantages, including no instrument required, naked-eye reading, high sensitivity, quantitative determination, rapid detection, simple operation, and low cost. It also has strong scalability and is expected to be used in many fields such as chemical analysis and biological target detection, demonstrating broad application value.

[0051] This device can be directly used to determine substances with hydrogen peroxide catalytic activity, such as catalase and platinum nanoparticles, or to determine other target molecules through conjugates of these substances, enabling the determination of a wider range of targets (such as nucleic acids and proteins). The determination of catalase and biological protein targets has broad application value in the biomedical field.

[0052] Other beneficial effects of the embodiments of the present invention will be further described below. Attached Figure Description

[0053] Figure 1 This is a schematic diagram of the structure of a biomarker visualization and quantitative detection device according to an embodiment of the present invention.

[0054] Figure 2 This is a schematic diagram of the operation method of the detection device according to an embodiment of the present invention.

[0055] Figure 3 This is a schematic diagram of the PDMS microfluidic structure fabrication process according to an embodiment of the present invention.

[0056] Figure 4 This is a schematic diagram of the PDMS microchannel structure according to an embodiment of the present invention.

[0057] Figure 5 This is a calibration curve showing the relationship between catalase concentration and liquid column movement distance in an embodiment of the present invention.

[0058] Figure 6 This is a physical image of the detection device used for catalase determination in an embodiment of the present invention. Detailed Implementation

[0059] The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary and is not intended to limit the scope and application of the present invention.

[0060] It should be noted that when a component is referred to as "fixed to" or "set on" another component, it can be directly on or indirectly on that other component. When a component is referred to as "connected to" another component, it can be directly connected to or indirectly connected to that other component. Furthermore, a connection can be used for fixing, coupling, or communication.

[0061] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They 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 device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention.

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

[0063] See Figures 1 to 6 This invention provides a biomarker visualization and quantitative detection device, which can be used for the detection of active substances with catalytic gas generation and their labeled biological targets.

[0064] See Figure 1 , Figure 2 and Figure 4 This detection device includes: a microfluidic structure 1, made of transparent material, used for measuring liquid flow and liquid column length; a reaction container 3, the upper chamber of which is connected to the upper inlet 5 of the microfluidic structure 1, the reaction container 3 being used to hold a liquid mixture of reaction medium and the target to be tested; a container lid 4, which cooperates with the reaction container 3 to form a sealed space; and a scale marking, which cooperates with the microfluidic structure 1 and the reaction container 3, for naked-eye reading; such as Figure 2As shown on the left, when the device is placed upright, the pressure difference between the reaction vessel 3 and the microfluidic structure 1 is zero, and at this time, liquid 6 will not enter the microfluidic structure 1; Figure 2 As shown on the right, when the device is inverted, the upper chamber of the reaction vessel 3 is upside down. The liquid 6 in the reaction vessel 3 is concentrated in the upper chamber of the reaction vessel 3 under the action of gravity, sealing the microfluidic structure 1. When the reaction medium reacts chemically with the target, the gas generated increases the air pressure 7 in the reaction vessel 3, causing the liquid in the vessel 3 to be pushed into the microfluidic structure 1 under the pressure difference. The length of the liquid column in the microfluidic structure 1 is positively correlated with the concentration of the target, so the rapid quantitative detection of the target molecule concentration can be achieved by reading the length of the liquid column in the microfluidic structure 1 with the naked eye.

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

[0066] like Figure 1 As shown, in some embodiments, the detection device includes a graduated partition 2 disposed between the microfluidic structure 1 and the reaction vessel 3, the surface of which is provided with graduated markings. The inlet 5 of the microfluidic structure 1 is aligned and communicates with the orifice of the reaction vessel 3 and the orifice of the graduated partition 2.

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

[0068] like Figure 1 As shown, in some embodiments, the reaction vessel 3 is a disposable semi-micro reaction dish, the container lid 4 is a rubber stopper that matches the reaction dish, and the container lid 4 can achieve a complete seal with the reaction vessel 3.

[0069] In some embodiments, the reaction medium may be a hydrogen peroxide solution containing a colored dye, and the target to be tested may be selected from substances with hydrogen peroxide catalytic activity or their conjugates. The substances with hydrogen peroxide catalytic activity include, but are not limited to, catalase, platinum nanoparticles (PtNP), and other compounds or materials with similar catalytic activity. The conjugates include antibody- or nucleic acid aptamer-labeled catalase / nanozymes, used to achieve the determination of biomolecules such as nucleic acids and proteins through the specific recognition of the target by the conjugate molecules. The conjugates can achieve highly specific detection of specific biomolecules by forming a sandwich complex similar to the "magnetic bead-capture antibody-target protein-catalase-labeled antibody" in CLIA methods.

[0070] This invention provides a visual quantitative detection device for biomarkers. A microfluidic structure is connected to the upper part of a reaction vessel, using the length of the liquid column in the channel as a reading indicator, cleverly creating a naked-eye readable detection device. The upper part of the vessel contains a certain volume of air, while the lower part contains a mixture of hydrogen peroxide (which may contain colored dye) and the target substance. When the device is placed upright, the internal and external pressure differences are zero because the microfluidic structure connects the inside and outside of the vessel. When the device is inverted, the liquid inside concentrates at the bottom of the vessel due to gravity (air concentrates at the top), sealing the channel. Since there is no pressure difference between the inside and outside of the reaction vessel, the liquid will not enter the microfluidic structure. As the reaction proceeds, the target substance with hydrogen peroxide catalytic activity (such as catalase, platinum nanoparticles (PtNP), or their conjugates) catalyzes the decomposition of hydrogen peroxide in the vessel, producing water and oxygen. The continuously generated oxygen accumulates in the vessel, causing an increase in internal pressure. Ultimately, the liquid in the vessel is pushed into the microfluidic structure under the pressure difference. Within the same time frame, the length of the liquid column in the flow channel is positively correlated with the concentration of the target substance. By visually measuring the length of the liquid column in the microchannel structure and then basing the readings on the correlation between the liquid column length and the target concentration, rapid quantitative detection of the target molecule concentration can be achieved. This invention can be used for the detection of active substances that generate catalytic gases.

[0071] This detection device can be used not only for the direct detection of substances with hydrogen peroxide catalytic activity (such as catalase, nanozymes, etc.), but also for the determination of conjugates of the above substances (such as antibody / nucleic acid aptamer-labeled catalase / nanozymes). Through the specific recognition of the target by the conjugate molecules, the determination of biomolecules such as nucleic acids and proteins can be achieved. This device is suitable not only for reaction systems that catalyze the decomposition of hydrogen peroxide to produce oxygen, but also for any reaction system that utilizes a liquid reaction to produce gas, resulting in a change in pressure inside and outside the container. This invention has many advantages, including no instrument required, naked-eye reading, high sensitivity, quantitative determination, rapid detection, simple operation, and low cost. It also has strong scalability and is expected to be used in many fields such as chemical analysis and biological target detection, possessing broad application value.

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

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

[0074] S2. Prepare 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 reaction medium and target to be tested.

[0075] S3. Configure a container lid to fit the reaction vessel and form a sealed space;

[0076] S4. Create scale markings that fit the microfluidic structure and reaction vessel for naked-eye reading; wherein, the relationship between the liquid column length in the microfluidic structure and the concentration of the target to be measured is calibrated, so that the target molecule concentration can be rapidly quantitatively detected by reading the liquid column length in the microfluidic structure with the naked eye.

[0077] The following describes specific embodiments of the present invention.

[0078] Structural Design of This Detection Device

[0079] This detection device mainly consists of four parts: a microfluidic structure 1, a graduated partition 2, a reaction vessel 3, and a vessel lid. Its main structure is as follows: Figure 1 As shown. This detection device uses a reaction vessel (including a lid) as its main body, with a small hole on one side of the upper part of the reaction vessel. The above-mentioned side of the reaction vessel is attached to an opaque partition with a small hole and graduations at the corresponding position. This graduation partition (with distance or concentration graduations printed on its surface) is then attached to a polydimethylsiloxane (PDMS) microchannel structure. The small hole in the reaction vessel, the small hole in the graduation partition, and the inlet 5 of the PDMS microchannel structure are aligned, so that the interior of the assembled reaction vessel is connected to the external atmosphere through the small hole in the graduation partition and the microchannel structure.

[0080] The following optimizations can be made to the structures mentioned above:

[0081] ① The microchannel structure described above is designed in an S-shape to maximize the channel length, but it can also be designed in other shapes. The channel can use different widths / heights (or combinations 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 detection sensitivity, while increasing the channel width / height can improve the detection range.

[0082] ②The microchannel structure described above is made of PDMS material, but other materials can also be used instead.

[0083] ③ The above-mentioned reaction vessel is a disposable semi-micro reaction dish, and the container lid is a rubber stopper that matches the reaction dish, ensuring a complete seal between the container lid and the reaction vessel. The above-mentioned reaction vessel and container lid can also be replaced with other functional devices of different materials, sizes, or shapes.

[0084] ④ The above-mentioned graduated partition pattern can also be printed directly on the side of the reaction vessel, thus omitting the graduated partition.

[0085] Operating method of this detection device

[0086] A suitable volume of hydrogen peroxide solution containing colored dye and a certain volume of catalase test solution of unknown concentration are mixed and added to the reaction vessel. The liquid level in the reaction vessel should not exceed the height of the orifice. The vessel is then covered. Since the air inside and outside the reaction vessel is connected, the pressure difference between the inside and outside is zero. The detection device is then inverted. Due to gravity, the liquid in the reaction vessel will concentrate at the bottom, sealing the flow channel. The catalase in the solution catalyzes the decomposition of H2O2 in the reaction vessel, producing water and oxygen. The generated oxygen accumulates in the sealed container, increasing the internal pressure. Ultimately, the liquid in the reaction vessel is pushed into the microchannel structure from the inlet by the pressure difference. Because the pushed-out liquid contains colored dye, the length of the liquid column in the flow channel can be directly observed with the naked eye. Within the same time frame, the length of the liquid column in the flow channel is positively correlated with the catalase concentration. By establishing a fitting relationship between the liquid column length and the catalase concentration, rapid quantitative detection of the target molecule can be achieved. The operation method is as follows: Figure 2 As shown. Figure 2 The device shown is a perspective view and does not include the graduated dividers.

[0087] The following optimizations can be made to the components mentioned above:

[0088] ① The analyte (catalase) can be replaced with other substances that have catalase catalytic decomposition activity (such as platinum nanoparticles) to achieve the concentration determination of the substance.

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

[0090] ③The above reaction system for catalytic decomposition of hydrogen peroxide to produce oxygen can be replaced with any other reaction system that uses liquid reaction to produce gas, resulting in pressure changes inside and outside the container.

[0091] Example

[0092] The invention describes the fabrication of a detection device and the use of this device to determine the concentration of catalase solution of unknown concentration. The specific implementation is as follows:

[0093] ①Preparation of this detection device:

[0094] 1. Fabrication of the microchannel structure: 2 mL of SU-8 photoresist was dropped onto a 3-inch silicon wafer and uniformly dispersed on the wafer surface using a homogenizer. The homogenized silicon wafer was placed in a dryer and dried for 7 minutes (65℃) and 30 minutes (95℃), respectively, with an expected lithography height of 170 μm. The lithography machine was turned on, and a photomask was placed on the silicon wafer (the width of the first half of the channel was designed to be 0.3 mm, and the width of the second half of the channel was designed to be 0.6 mm), exposed at 2.14V for 35 seconds. Then, the silicon wafer was placed in the dryer and dried for 5 minutes (65℃) and 12 minutes (95℃), respectively. The dried silicon wafer was immersed in the developer for 12 minutes, and then rinsed with ethanol and developer alternately three times. The cleaned silicon wafer was placed in the dryer and dried for 10 minutes (150℃), completing the fabrication of the channel template. The flow channel template was placed in a circular petri dish. 15g of PDMS component 1 and 1.5g of component 2 (crosslinking agent component) were weighed and stirred thoroughly with a glass rod before being poured onto a silicon wafer template. After the bubbles evaporated, the material was heated on a 65°C hot plate for 3 hours until the PDMS solidified. The template was then peeled off the silicon wafer template (molding) and cut into 40mm × 12mm PDMS flow channel templates. A small hole was drilled at the PDMS flow channel inlet corresponding to a PDMS film of the same size. The template and the PDMS flow channel were then placed in a plasma chamber for 5 minutes. After bonding the PDMS flow channel to the PDMS film, the mixture was heated at 65°C for 2 hours to obtain a complete microchannel structure. The overall PDMS flow channel preparation process is as follows: Figure 3 As shown, the microchannel structure is as follows Figure 4 As shown.

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

[0096] 2. Preparation of the reaction vessel: A disposable semi-micro reaction vessel is used as the reaction vessel, and the vessel lid is a rubber stopper that matches the reaction vessel. A small hole is drilled at the corresponding position at the inlet of the microfluidic structure, thus completing the preparation of the reaction vessel.

[0097] 3. Overall Assembly of the Detection Device: Print scale markings on waterproof paper. For easy reading, divide the length of each channel into 5 equal parts and use small black dots as scale markings. Define each part as one relative length unit (au). Make a small hole at the corresponding position of the scale partition and the microchannel structure inlet. Use double-sided tape to attach the microchannel structure, scale partition, and reaction vessel together in sequence. Align the small holes in the reaction vessel, scale partition, and PDMS microchannel structure inlet so that the interior of the assembled reaction vessel can communicate with the outside through the small holes in the scale partition and the microchannel structure. This completes the overall assembly of the detection device.

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

[0099] 1. Construction of calibration curves: First, using phosphate-buffered saline (PBS) as the buffer, a series of catalase solutions with known concentrations (0, 2, 4, 6, 8, and 10 μg / mL) were prepared. One mL of each of these different concentrations of catalase solution was mixed with 1 mL of 20 mM H₂O₂ solution containing dark ink and added to the assembled reaction vessel. The vessel was then sealed. The reaction apparatus was quickly inverted and reacted at 25°C for 5 minutes. The length of liquid expelled from the flow channel was then read. Each sample was tested three times, and the mean was taken. A concentration-length fitting curve was established based on the relationship between the concentration of catalase solution and the length reading for the known concentration gradients. The positive correlation satisfies a four-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 R0 is 3.26. 2 =0.9938. The fitted calibration curve is as follows: Figure 5 As shown. Finally, based on the fitted curve, the length reading is converted into the catalase solution concentration, and a graduated diaphragm with direct markings for the corresponding concentration readings is made, assembling the device into the detection apparatus for testing samples.

[0100] 2. Determination of catalase samples of unknown concentration: Using PBS as buffer, mix 1 mL of the sample of unknown concentration with 1 mL of H2O2 solution (20 mM) containing dark ink, and add the mixture to the assembled reaction apparatus with concentration markings. Seal the reaction container by closing the lid. Then, quickly invert the reaction apparatus and react at 25°C for 5 minutes. Read the concentration reading directly with the naked eye at the position of the displaced liquid in the flow channel (see physical sample). Figure 6 As shown in the figure, this is the concentration of catalase in the sample to be tested. Figure 6A photograph of the detection device used for catalase assay is shown, with scale length marking 9 and scale number marking 10.

[0101] Analysis of the advantages of this testing device:

[0102] This detection device can rapidly determine catalase concentration within 5 minutes. It achieves a low-cost, simple, instrument-free, highly sensitive, naked-eye quantitative, and rapid catalase concentration determination. Furthermore, this method has extremely high scalability. By replacing catalase with a conjugate of a catalase-active substance and a target-recognition substance (such as antibody / nucleic acid aptamer-labeled catalase / platinum nanoparticles), the specific recognition of target molecules by the conjugate enables the determination of biomolecules such as proteins and nucleic acids. This device is applicable not only to reaction systems that catalyze the decomposition of hydrogen peroxide to produce oxygen, but also to any reaction system that utilizes a liquid reaction to produce gas, resulting in a change in pressure inside and outside the container. It holds promise for applications in chemical analysis, biological target detection, and many other fields, possessing broad 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 qualitatively detect target molecules. This invention utilizes the correspondence between the length of the ejected liquid column and the concentration of the analyte to achieve quantitative detection of target molecules. The quantitative results have important practical significance for the determination of many targets, and therefore quantitative detection has a wider range of application prospects.

[0105] (2) High sensitivity detection: The reaction process of this invention is carried out in solution, which has better reaction kinetic conditions than immunochromatography, making the reaction process faster and more thorough; on the other hand, due to the small cross-sectional area of ​​the microchannel structure, even a small amount of gas generated in the reaction container can push out a long liquid column, which is beneficial for high sensitivity detection of low concentration samples.

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

[0107] (4) No instruments required, low cost: This method requires no instruments or equipment, involves few types of raw materials with low prices (such as disposable reaction vessels and hydrogen peroxide solution), and has a simple preparation process. The overall detection cost is significantly lower than that of traditional detection technologies.

[0108] (5) Strong application scalability: By replacing catalase with a conjugate of catalase-active substances and target recognition substances (such as antibody / nucleic acid aptamer-labeled catalase / platinum nanoparticles), the conjugate molecule can achieve specific target recognition, thereby enabling the determination of biomolecules 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 utilizes liquid reactions to generate gas, resulting in pressure changes inside and outside the container, demonstrating extremely strong application scalability.

[0109] The above description provides a further detailed explanation of the present invention in conjunction with specific / preferred embodiments, and it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various substitutions or modifications can be made to these described embodiments without departing from the concept of the present invention, and all such substitutions or modifications should be considered within the scope of protection of the present invention. In the description of this specification, the reference to terms such as "an embodiment," "some embodiments," "preferred embodiment," "example," "specific example," or "some examples," etc., indicates that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any suitable manner in one or more embodiments or examples. Without contradiction, those skilled in the art can combine and integrate 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 visualizing quantitative detection device, characterized by, The device comprises: a microfluidic structure made of transparent material for liquid flow and liquid column length measurement; a reaction container, the upper chamber of which is in communication with the upper inlet of the microfluidic structure, and the reaction container is used to load a liquid mixture of reaction medium and target to be detected; a container cover cooperating with the reaction container to form a sealed space; a scale mark cooperating with the microfluidic structure and the reaction container for naked eye reading; the upper inlet of the microfluidic structure is in communication with the small hole of the scale spacer and the small hole of the reaction container, and when the device is placed in the normal direction, the inside of the reaction container is in communication with the atmosphere outside the device through the small hole of the scale spacer and the microfluidic structure; when the device is placed in the normal direction, the air pressure difference between the inside of the reaction container and the inside of the microfluidic structure is zero, and at this time the liquid will not enter the microfluidic structure; when the device is placed upside down, the upper chamber of the reaction container is inverted below, the liquid in the reaction container is concentrated in the upper chamber of the reaction container under the action of gravity, the microfluidic structure is closed, and the gas generated by the chemical reaction of the reaction medium and the target to be detected increases the air pressure in the reaction container, so that the liquid in the container is pushed into the microfluidic structure under the driving of the air pressure difference; wherein the liquid column length in the microfluidic 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 naked eye reading of the liquid column length in the microfluidic structure.

2. The biomarker visualizing quantitative detection device according to claim 1, wherein, The microfluidic structure is made of polydimethylsiloxane.

3. The biomarker visualization and quantitative detection device of claim 1, wherein, The microfluidic structure is designed in S shape.

4. The biomarker visualizing quantitative test device according to any one of claims 1 to 3, wherein The device comprises a scale spacer arranged between the microfluidic structure and the reaction container, the surface of the scale spacer is provided with a scale mark, or the scale mark is directly printed on the side of the reaction container.

5. The biomarker visualizing quantitative test device according to any one of claims 1 to 3, wherein The reaction container is a disposable semi-micro reaction dish, the container cover is a reaction dish matched rubber plug, and the container cover can realize complete sealing with the reaction container.

6. The biomarker visualizing quantitative test device according to any one of claims 1 to 3, wherein The reaction medium is a hydrogen peroxide solution containing a color dye, and the target to be detected is selected from substances with hydrogen peroxide catalytic activity or their conjugates, wherein the substances with hydrogen peroxide catalytic activity include catalase and platinum nanoparticles; the conjugates include antibody or nucleic acid aptamer labeled catalase / nanoplasma, which is used for the determination of biomolecules by specific recognition of the target by the conjugated molecules.

7. A method for preparing a biomarker visualizing quantitative detection device according to any one of claims 1 to 6, characterized in that, The device comprises the following steps: S1, preparing 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 in communication with the upper inlet of the microfluidic structure, and the reaction container is used to load a mixture of reaction medium and target to be detected; S3, configuring a container cover cooperating with the reaction container to form a sealed space; S4, making scale mark cooperating with micro-channel structure and reaction container for naked eye reading; the upper inlet of the micro-channel structure is communicated with the small hole of the reaction container and the small hole of the scale layer, when the device is placed in the right direction, the inside of the reaction container is communicated with the atmosphere outside the device through the small hole of the scale layer and the micro-channel structure; wherein the relationship between the liquid column length in the micro-channel structure and the concentration of the target to be detected is calibrated, so that the concentration of the target molecule can be quickly quantitatively detected by reading the liquid column length in the micro-channel structure with naked eye.

8. The method for preparing the biomarker visualizing and quantifying test device according to claim 7, wherein, Step S1 includes: S1.1, coating photoresist on a silicon wafer and performing exposure and development to form a flow channel template; S1.2, copying the flow channel template using PDMS material to form a PDMS flow channel; S1.3, post-processing the PDMS flow channel, including punching the inlet and plasma treatment, and then adhering with a PDMS film to obtain a complete micro-channel structure.

9. The method for preparing the biomarker visualizing and quantifying test device according to claim 7 or 8, wherein, In step S2, the reaction container is a disposable semi-micro reaction dish, and the container cover is a reaction dish matched rubber plug. A small hole is punched at the corresponding position of the inlet of the reaction container and the micro-channel structure to complete the preparation of the reaction container.

10. The method for preparing the biomarker visualizing and quantifying test device according to any one of claims 7 to 8, wherein, In step S4, the scale mark corresponding to the concentration reading is made by establishing the fitting relationship between the liquid column length and the target concentration.

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