A LAMP detection chip and detection method based on capillary force self-drive

Abstract

The present invention provides a LAMP detection chip and detection method based on capillary force self-drive, wherein the detection chip comprises a base plate, an upper cover plate and a blood filter membrane. A sample loading bump is provided on one side of the base plate, a drainage notch connected to the main pipeline is provided, deep sidewall grooves are provided on both sides of the main pipeline, and a reaction chamber is provided. The upper cover plate is fixed to the base plate by hot pressing, and a sample loading window, a flow control port and a packaging port are provided. The blood filter membrane is laid on the sample loading window and pressed by a gasket. The chip accurately controls the plasma flow rate through capillary force self-drive and deep sidewall grooves, thereby realizing the integration of plasma separation and LAMP reaction. The chip does not require external equipment and professional operators, is easy to operate, and has a high degree of automation, which significantly improves the detection efficiency and accuracy, reduces errors, steps and costs, and is suitable for convenient detection under ordinary conditions.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a LAMP detection chip and a detection method based on capillary force self-drive. Background Art

[0002] Whole-blood LAMP (loop-mediated isothermal amplification) reaction detection technology, as an efficient and sensitive nucleic acid amplification technology, is widely used in the field of molecular diagnosis. Traditional whole-blood LAMP reaction detection usually needs to be performed in a laboratory environment and relies on professional technical operators, specific external equipment and cumbersome operating steps. Specifically, existing detection methods usually first require the use of dedicated equipment to separate the plasma and blood cells in the whole blood sample. The plasma is then added to the PCR tube along with the premix and primers for reaction. After the reaction is completed, the PCR tube is removed and the reaction results are observed. The entire process is not only time-consuming (usually 2-3 hours) but also requires operation in a specific experimental environment. The operator must have certain professional skills and is prone to human error.

[0003] Furthermore, the plasma separation step in traditional methods relies on specialized equipment, such as centrifuges, which are large, expensive, and complex to operate, requiring considerable time to process blood samples. These factors hinder widespread application of existing methods in resource-limited settings, particularly in settings without access to laboratory equipment or specialized operators, resulting in low test efficiency and complex operations.

[0004] Therefore, there is an urgent need for a more convenient, rapid, and economical detection technology that can perform whole-blood LAMP reaction testing in ordinary environments, even without specialized equipment and technicians. This technology should integrate plasma separation and LAMP reaction detection, automate each step, reduce human error, improve detection efficiency, and be low-cost. Summary of the Invention

[0005] The technical problem to be solved by the present invention is: to solve the problems of cumbersome operation, the need for specific experimental equipment and professional operators, and errors in the experimental process in the existing whole blood LAMP reaction detection method. In the traditional method, whole blood separation and reaction detection processes are separated, and usually rely on complex external equipment, resulting in long detection time, high cost and complicated operation. In order to improve detection efficiency, reduce experimental errors, and enable the technology to be implemented under ordinary conditions, the present invention provides a LAMP detection chip based on capillary force self-drive, which integrates whole blood separation and LAMP reaction detection, and accurately controls the plasma flow rate through capillary force self-drive and side wall deep groove structure, thereby realizing an efficient, automated, external equipment-free and simple detection process.

[0006] The technical solution adopted by the present invention to solve the technical problem is: a LAMP detection chip based on capillary force self-drive, comprising a bottom plate, an upper cover plate and a blood filter membrane;

[0007] A sample loading convex block is provided on one side of the head of the bottom plate, and a drainage notch connected to the main pipe is provided on the sample loading convex block. Deep sidewall grooves are provided on both sides of the main pipe, and a reaction chamber is also provided on the main pipe.

[0008] The upper cover is placed on the bottom plate and fixed to each other by heat pressing. The upper cover is provided with a sample loading window corresponding to the sample loading protrusion. The blood filter membrane is laid on the sample loading window and pressed tightly by a gasket.

[0009] The upper cover plate is located above the reaction chamber and is further provided with a packaging port, and the upper cover plate is further provided with a flow control port adjacent to the packaging port;

[0010] During testing, the capillary force is used to drive the plasma flow through the synergistic effect of the main channel and the deep grooves on the side walls.

[0011] Preferably, the sidewall deep groove has a depth of 0.8-1.2 mm, a width of 700-900 μm, and a length of 20-30 mm.

[0012] Preferably, a heating plate is provided at the bottom of the base plate;

[0013] A photosensor is provided above the flow control port for monitoring the flow of plasma and generating an optical signal to control the heating plate.

[0014] Preferably, a spectral sensor is provided above the packaging port for detecting the product after the LAMP reaction and judging the positive or negative nature based on the color change.

[0015] Preferably, the upper cover plate is located above the main pipeline and is further provided with a waste liquid outlet for discharging waste liquid generated during the experiment.

[0016] A method for manufacturing a LAMP detection chip based on capillary force self-drive comprises the following steps:

[0017] S1. Prepare the base plate and upper cover plate, and assemble them together by hot pressing to form the main structure of the chip. When preparing the base plate made of polymethyl methacrylate (PMMA), avoid the rest of the main pipeline and cover the non-treated area of the base plate with transparent tape. Then, perform plasma hydrophilic treatment on the main pipeline at a power of 55-65% for 4-6 minutes. After the treatment, remove the transparent tape. Set the sidewall groove to a depth of 0.8-1.2 mm, a width of 700-900 μm, and a length of 20-30 mm.

[0018] S2. Laying a blood filter membrane at the sample loading window and pressing it tightly with a gasket to separate plasma from whole blood;

[0019] S3. Install a heating plate at the bottom of the base plate to heat the liquid in the reaction chamber;

[0020] S4. Installing a photosensor above the flow control port to monitor the plasma flow and generate an optical signal to control the heating plate;

[0021] S5. Install a spectral sensor above the packaging port to detect the product after the LAMP reaction and determine the positive or negative status based on the color change;

[0022] S6. Add the freeze-dried premix and primers into the reaction chamber through the packaging port, cover with sealing film, and complete the chip packaging.

[0023] A detection method for a LAMP detection chip comprises the following steps:

[0024] L100, filtering the whole blood sample through the blood filter membrane of the sample loading window, and allowing the separated plasma to enter the main pipeline after the hydrophilic treatment through the drainage gap;

[0025] L200, through the capillary resistance of the deep grooves on both sides of the main pipe, the plasma flow rate is adjusted to 0.0041-0.0043m / s, so that the plasma flows along the main pipe to the reaction chamber;

[0026] L300, when the front end of the plasma touches the photosensor at the fluid control port, the photosensor detects the fluid refractive index change signal and activates the bottom plate heating plate;

[0027] L400 and heating plate control the reaction chamber temperature at 65±2℃ for 25 minutes to perform LAMP amplification reaction;

[0028] L500. After the reaction is completed, the color change of the reaction product is detected by the spectral sensor to determine whether the test result is positive or negative.

[0029] Preferably, the flow rate control in step L200 includes:

[0030] Capillary resistance is generated by deep grooves on the side walls with a depth of 0.8-1.2 mm and a width of 700-900 μm, reducing the plasma flow rate.

[0031] The present invention significantly improves detection efficiency and accuracy by integrating plasma separation and LAMP reaction detection on a microfluidic chip, and utilizing capillary self-drive and deep sidewall grooves to precisely control plasma flow rate. The chip requires no external equipment or specialized operators, is easy to operate, and offers a high degree of automation. This reduces experimental errors and operational steps, saving time, manpower, and material resources, lowering costs, and making it suitable for convenient testing in common environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention will be further described below with reference to the accompanying drawings and examples.

[0033] Figure 1 It is a structural schematic diagram of the optimal embodiment of the plasma separation LAMP detection chip based on capillary force self-drive of the present invention.

[0034] Figure 2 This is a schematic diagram of the explosion structure of the optimal embodiment of the plasma separation LAMP detection chip based on capillary force self-drive of the present invention.

[0035] In the figure: 1. Sample addition block, 2. Drainage notch, 3. Main pipeline, 4. Side wall deep groove, 5. Flow control port, 6. Packaging port, 7. Reaction chamber, 8. Waste liquid port, 9. Bottom plate, 10. Upper cover plate, 11. Blood filter membrane, 12. Gasket, 13. Heating plate. DETAILED DESCRIPTION

[0036] The following describes embodiments of the present invention in detail. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended only to explain the present invention and are not to be construed as limiting the present invention.

[0037] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "axial", "radial", "circumferential" and the like to indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0038] In addition, the terms "first", "second", etc. are used for descriptive purposes only and are not to be understood as indicating or implying relative importance. In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "connected" and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. In addition, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0039] like Figure 1-2 As shown, the present invention provides a plasma separation LAMP detection chip based on capillary force self-drive. This chip can efficiently complete functions such as whole blood separation, LAMP reaction detection, and flow rate control in a microfluidic environment, greatly simplifying the cumbersome operation process in existing laboratories. Specifically, the chip of the present invention integrates plasma separation and LAMP reaction detection on a single chip, and uses capillary force self-drive and deep sidewall grooves to control plasma flow rate, reducing dependence on external equipment while achieving rapid and accurate detection.

[0040] The chip includes a base plate, an upper cover plate, and a blood filter membrane. Its specific structure is as follows:

[0041] A sample loading bump 1 is provided on one side of the head of the bottom plate 9, and the sample loading bump 1 is provided with a drainage notch 2 connected to the main pipeline 3. The whole blood sample is introduced through the sample loading window and flows into the main pipeline 3 through the drainage notch 2 for subsequent processing. Side wall deep grooves 4 are provided on both sides of the main pipeline 3. The depth of the side wall deep grooves 4 is 0.8-1.2mm, the width is 700-900μm, and the length is 20-30mm. The structure of the side wall deep grooves 4 can accurately control the plasma flow rate. By adjusting the flow rate, the plasma flow rate is controlled within the range of 0.0041-0.0043m / s. Through this precise flow rate control, it is ensured that the plasma can flow smoothly into the reaction chamber 7, avoiding the use of external equipment. A reaction chamber 7 is also provided on the main pipeline 3, and the reaction chamber 7 is used to receive filtered plasma. When the plasma flows through the reaction chamber 7, it mixes with the frozen pre-mixed solution and primers placed in the reaction chamber 7 in advance to complete the LAMP reaction. The upper cover plate 10 is placed on the bottom plate 9 by hot pressing and fixed by hot pressing. The upper cover plate 10 is provided with a sample loading window corresponding to the sample loading protrusion 1. The blood filter membrane 11 is laid on the sample loading window and pressed by the gasket 12 to ensure that the whole blood sample can be effectively filtered through the blood filter membrane to separate the plasma. The upper cover plate 10 is also provided with a packaging port 6 located above the reaction chamber 7. The packaging port 6 is used to add the freeze-dried premix and primers to the reaction chamber 7. A flow control port 5 is provided on the upper cover plate 10 adjacent to the packaging port 6, which is used to monitor the flow of plasma and control the heating of the heating plate 13 through optical signals. A heating plate 13 is provided at the bottom of the bottom plate 9, which is responsible for heating the liquid in the reaction chamber during the LAMP reaction. The heating plate 13 is controlled by a photosensor. When the photosensor detects the flow of plasma, it generates a refractive index change signal, thereby activating the heating plate 13, so that the reaction temperature is maintained at 65±2°C for 25 minutes to implement the LAMP reaction. A photosensor, located above the flow control port 5, monitors plasma flow and generates optical signals, ensuring precise control of the reaction process. A spectral sensor is located above the packaging port 6 of the upper cover plate 10. This spectral sensor detects the products of the LAMP reaction and determines their positive or negative status based on color changes, ensuring the accuracy of the test results. The upper cover plate 10 also features a waste liquid port 8, located above the main pipeline 3, for draining waste liquid generated during the experiment, ensuring smooth fluid flow within the chip and unaffected by waste liquid accumulation.

[0042] The plasma separation LAMP detection chip of the present invention is mainly composed of a base plate 9, an upper cover plate 10, a blood filter membrane 11 and a heating plate 13. A sample loading bump 1 is provided at the head of the base plate 9, which is connected to the main pipeline 3 through a drainage notch 2. Deep sidewall grooves 4 are provided on both sides of the main pipeline for accurately controlling the plasma flow rate. The upper cover plate 10 of the chip is combined with the base plate 9 by hot pressing, and a sample loading window, a flow control port 5 and a packaging port 6 are provided. The plasma is filtered at the sample loading window through the blood filter membrane 11, and the filtered plasma flows along the main pipeline under the action of capillary force, passes through the flow rate control of the deep sidewall groove 4, and enters the reaction chamber 7 to mix with the freeze-dried premix and primer.

[0043] Compared with existing technologies, traditional plasma separation and LAMP reactions typically require separate equipment, and each step involves complex manual operations and reliance on external equipment. This invention integrates these two steps into a microfluidic chip, enabling automated and precise detection. This not only avoids the use of external equipment but also makes the detection process more convenient and efficient.

[0044] During plasma separation and flow, the design of the deep sidewall groove structure plays a key role in plasma flow rate. Experiments have shown that a deep sidewall groove design with a depth of 1mm, a width of 800μm, and a length of 25mm can effectively reduce the plasma flow rate to 0.0041-0.0043m / s, compared to the flow rate of 0.009-0.011m / s without the deep sidewall groove, a 42% reduction. This structure not only ensures smoother plasma flow, but also avoids the use of external thrust equipment, reducing experimental complexity.

[0045] Unlike traditional methods that rely on external pumps for flow rate control, this invention achieves precise flow rate control through capillary self-drive and fluidic design. The device is also more portable, less expensive, and easier to operate. This innovative design allows the chip to be used in ordinary environments, making it particularly suitable for rapid on-site testing or resource-constrained settings.

[0046] The chip manufacturing method of the present invention is achieved by the following steps:

[0047] S1. Preparation of the base plate and upper cover: First, prepare the base plate and upper cover, and assemble them together using hot pressing to form the main structure of the chip. When preparing the polymethyl methacrylate (PMMA) base plate, use transparent tape to cover the non-treated area of the base plate to prevent the area outside the main pipe from being treated. Then, use plasma hydrophilic treatment to treat the main pipe, with the treatment power set to 60% and the treatment time set to 5 minutes to ensure that the surface of the main pipe has excellent hydrophilicity and promote the flow of plasma;

[0048] S2. Install the blood filter membrane: Place the blood filter membrane over the sample loading window and press it tightly with the gasket. The purpose of the blood filter membrane is to separate the plasma from the whole blood and ensure that the blood cells and plasma are effectively separated. The gasket is designed to ensure that the blood filter membrane is flat and well sealed.

[0049] S3. Installing the heating plate: Install the heating plate on the bottom of the base plate to heat the liquid in the reaction chamber during the LAMP reaction. The temperature control accuracy of the heating plate is crucial for the LAMP reaction, so the present invention has designed a precise heating system to ensure that the temperature is controlled within the range of 65±2℃.

[0050] S4. Installing a photosensor: Install a photosensor above the flow control port to monitor the flow of plasma in real time and activate the heating plate based on light changes. The photosensor determines whether the plasma flow is normal by detecting changes in the refractive index during the flow of plasma and activates the heating system.

[0051] S5. Install the spectral sensor: Install a spectral sensor above the package port to detect the product after the LAMP reaction. The spectral sensor can determine the positive or negative nature of the LAMP reaction based on the color change of the reaction product, ensuring the accuracy of the test results.

[0052] S6. Packaging of premix and primers: Add the freeze-dried premix and primers into the reaction chamber through the packaging port, and then cover with sealing film to complete the chip packaging.

[0053] Based on the design of the above chip, the detection method of the present invention is implemented by the following steps:

[0054] L100, sample addition: First, add the whole blood sample through the sample addition window, and after filtering through the blood filter membrane, the separated plasma enters the main pipeline through the drainage gap;

[0055] L200, Flow Rate Control: The plasma flows through the deep grooves on the sidewalls, and the flow rate is precisely controlled within the range of 0.0041-0.0043 m / s. The deep grooves on the sidewalls are 1 mm deep, 800 μm wide, and 25 mm long. This design significantly reduces the plasma flow rate from 0.009-0.011 m / s without the deeper grooves, making the flow rate control more stable.

[0056] L300, photosensor activates the heating plate: When the front end of the plasma passes through the flow control port, the photosensor detects the change in the optical signal and immediately activates the heating plate. The photosensor determines the flow condition by detecting the change in the refractive index of the fluid, ensuring that the heating plate is heated synchronously with the flow of plasma;

[0057] L400, heating reaction: The heating plate heats the liquid in the reaction chamber to 65°C and maintains it for 25 minutes to perform the LAMP amplification reaction;

[0058] L500, result determination: After the reaction is completed, the reaction product is detected by a spectral sensor to detect the color change of the reaction product and determine its positive or negative nature.

[0059] The technical effects of the present invention are embodied in the following aspects:

[0060] Efficient detection: Compared with traditional laboratory methods, this invention integrates plasma separation and LAMP reaction into a microfluidic chip, which greatly reduces the detection time. The entire detection process only takes 40 minutes, greatly improving the detection efficiency.

[0061] Precise flow rate control: Through the design of deep grooves on the side walls, the present invention effectively controls the plasma flow rate, making it more stable, avoiding the use of external thrust equipment, saving equipment costs and improving flow control accuracy.

[0062] Automation and convenience: The chip design eliminates the need for professional operators and external equipment during the entire detection process, making it easy to operate, reducing human errors and ensuring detection accuracy.

[0063] Low cost and high stability: The chip provided by the present invention has a simple structure, which reduces dependence on complex equipment, and the materials and processes used have low costs, are suitable for use in ordinary environments, and have broad market prospects.

[0064] Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0065] With the above-described preferred embodiments of the present invention as a guide, and with reference to the above description, relevant personnel are fully capable of making various changes and modifications without departing from the technical scope of this invention. The technical scope of this invention is not limited to the contents of the specification and must be determined according to the scope of the claims.

Claims

1. A capillary force self-driven LAMP detection chip, characterized in that: It includes a bottom plate, an upper cover plate and a blood filter membrane; A sample loading convex block is provided on one side of the head of the bottom plate, and a drainage notch connected to the main pipe is provided on the sample loading convex block. Deep sidewall grooves are provided on both sides of the main pipe, and a reaction chamber is also provided on the main pipe. The upper cover is placed on the bottom plate and fixed to each other by heat pressing. The upper cover is provided with a sample loading window corresponding to the sample loading protrusion. The blood filter membrane is laid on the sample loading window and pressed tightly by a gasket. The upper cover plate is located above the reaction chamber and is further provided with a packaging port, and the upper cover plate is further provided with a flow control port adjacent to the packaging port; During testing, the capillary force is used to push the plasma flow through the synergistic effect of the main channel and the deep grooves on the side walls; The sidewall deep groove has a depth of 0.8-1.2 mm, a width of 700-900 μm, and a length of 20-30 mm; A heating plate is provided at the bottom of the base plate; A photosensor is provided above the flow control port for monitoring the flow of plasma and generating an optical signal to control the heating plate; A spectrum sensor is provided above the packaging port for detecting the product after the LAMP reaction and judging the positive or negative according to the color change.

2. The capillary force self-driven LAMP detection chip according to claim 1, characterized in that: The upper cover plate is located above the main pipeline and is also provided with a waste liquid outlet for discharging waste liquid generated during the experiment.

3. A method for manufacturing a LAMP detection chip based on capillary force self-drive, characterized in that: The following steps are involved: S1. Prepare the base plate and upper cover plate as described in claim 1 or 2, and assemble them together by hot pressing to form the main structure of the chip; wherein, when preparing the base plate made of polymethyl methacrylate (PMMA), avoid the rest of the main pipeline and cover the non-treated area of the base plate with transparent tape, then perform plasma hydrophilic treatment on the main pipeline, set the treatment power to 55-65%, and the treatment time to 4-6 minutes. After the treatment, remove the transparent tape; set the sidewall deep groove to a depth of 0.8-1.2 mm, a width of 700-900 μm, and a length of 20-30 mm; S2. Laying a blood filter membrane at the sample loading window and pressing it tightly with a gasket to separate plasma from whole blood; S3. Install a heating plate at the bottom of the base plate to heat the liquid in the reaction chamber; S4. Installing a photosensor above the flow control port to monitor the plasma flow and generate an optical signal to control the heating plate; S5. Install a spectral sensor above the packaging port to detect the product after the LAMP reaction and determine the positive or negative status based on the color change; S6. Add the freeze-dried premix and primers into the reaction chamber through the packaging port, cover with sealing film, and complete the chip packaging.

4. A detection method based on the LAMP detection chip according to claim 1 or 2, characterized in that: The following steps are involved: L100, filtering the whole blood sample through the blood filter membrane of the sample loading window, and allowing the separated plasma to enter the main pipeline after the hydrophilic treatment through the drainage gap; L200, through the capillary resistance of the deep grooves on both sides of the main pipe, the plasma flow rate is adjusted to 0.0041-0.0043m / s, so that the plasma flows along the main pipe to the reaction chamber; L300, when the front end of the plasma touches the photosensor at the fluid control port, the photosensor detects the fluid refractive index change signal and activates the bottom plate heating plate; L400 and heating plate control the reaction chamber temperature at 65±2℃ for 25 minutes to perform LAMP amplification reaction; L500. After the reaction is completed, the color change of the reaction product is detected by the spectral sensor to determine whether the test result is positive or negative.

5. The detection method according to claim 4, characterized in that The flow rate control in step L200 includes: Capillary resistance is generated by deep grooves on the side walls with a depth of 0.8-1.2 mm and a width of 700-900 μm, reducing the plasma flow rate.

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