LAMP (loop-mediated isothermal amplification) detection chip and detection method based on capillary force self-driving

Abstract

The invention provides an LAMP (loop-mediated isothermal amplification) detection chip based on capillary force self-driving and a detection method. The detection chip comprises a bottom plate, an upper cover plate and a blood filtering film, one side of the bottom plate is provided with a sample adding convex block and a drainage notch connected with the main pipeline, and two sides of the main pipeline are provided with side wall deep grooves and reaction cavities. The upper cover plate is fixed on the bottom plate in a hot pressing manner and is provided with a sample adding window, a flow control port and a packaging port. And the blood filtering membrane is laid on the sample adding window and is pressed by a gasket. The chip accurately controls the flow rate of plasma through capillary force self-driving and deep grooves in the side wall, and integration of plasma separation and LAMP reaction is achieved. The chip does not need external equipment and professional operators, is simple and convenient to operate and high in automation degree, remarkably improves the detection efficiency and precision, reduces errors, steps and cost, and is suitable for convenient detection in a common environment.

Description

Technical Field

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

[0002] The 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 carried out in a laboratory environment and relies on professional technical operators, specific external equipment and cumbersome operation steps. Specifically, existing detection methods usually first need to separate plasma from blood cells in a whole blood sample through special equipment, and then add the plasma together with the premix and primers into a PCR tube for reaction. After the reaction ends, take out the PCR tube and observe the reaction result. The whole process not only takes a long time (usually 2 - 3 hours), but also needs to be operated in a specific experimental environment. The operator must have certain professional skills and is easily affected by human errors.

[0003] In addition, the plasma separation step in traditional methods relies on special equipment such as centrifuges. These devices are large in volume, expensive, and have complex operations, and it takes a certain amount of time to complete the processing of blood samples. Due to these factors, existing methods are difficult to be widely applied in many resource-limited environments, especially in scenarios without laboratory equipment or professional operators, where the detection efficiency is low and the operation is complex.

[0004] Therefore, there is an urgent need for a more convenient, fast and economical detection technology that can complete the whole blood LAMP reaction detection in a normal environment, even without professional equipment and technical personnel. This technology should have the functions of integrating plasma separation and LAMP reaction detection, be able to automate each step, reduce human errors, improve detection efficiency, and have a lower 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 the error problems in the experimental process in the existing whole blood LAMP reaction detection method. In traditional methods, the whole blood separation and reaction detection processes are separated and usually rely on complex external equipment, resulting in long detection time, high cost and complex operation. In order to improve detection efficiency, reduce experimental errors, and enable this technology to be realized in a normal environment, the present invention provides a LAMP detection chip based on capillary force self-driving, which integrates whole blood separation and LAMP reaction detection, and precisely controls the plasma flow rate through capillary force self-driving and sidewall 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 its technical problems is: a LAMP detection chip based on capillary force self-driving, including a bottom plate, an upper cover plate and a blood filtration membrane; A sample-adding bump is arranged on one side of the head of the bottom plate. The sample-adding bump is provided with a drainage notch connected to the main pipeline. Side wall deep grooves are arranged on both sides of the main pipeline, and a reaction chamber is also arranged on the main pipeline; The upper cover plate is covered on the bottom plate and fixed to each other by a hot pressing method. The upper cover plate is provided with a sample-adding window corresponding to the sample-adding bump. The blood filtration membrane is laid on the sample-adding window and pressed tightly by a gasket; The upper cover plate is also provided with a packaging port above the reaction chamber, and a fluid control port adjacent to the packaging port is also arranged on the upper cover plate; During detection, through the synergistic effect of the main pipeline and the side wall deep grooves, capillary force is used to push the plasma to flow.

[0007] Preferably, the depth of the side wall deep groove is 0.8-1.2 mm, the width is 700-900 μm, and the length is 20-30 mm.

[0008] Preferably, a heating sheet is arranged at the bottom of the bottom plate; A photosensitive sensor is arranged above the fluid control port for monitoring the plasma flow and generating an optical signal to control the heating sheet.

[0009] Preferably, a spectral sensor is arranged above the packaging port for detecting the product after the LAMP reaction and judging positive or negative according to the color change.

[0010] Preferably, a waste liquid port is also arranged above the main pipeline of the upper cover plate for discharging the waste liquid generated during the experiment.

[0011] A manufacturing method of a LAMP detection chip based on capillary force self-driving includes the following steps: S1. Prepare the bottom plate and the upper cover plate described above, and assemble them together by a hot pressing method to form the main structure of the chip; among them, when preparing the bottom plate made of polymethyl methacrylate (PMMA), avoid the rest of the main pipeline, cover the non-treated area of the bottom plate with transparent tape, then perform plasma hydrophilic treatment on the main pipeline, set the treatment power to 55-65%, the treatment time to 4-6 minutes, and tear off the transparent tape after the treatment; set the depth of the side wall deep groove to 0.8-1.2 mm, the width to 700-900 μm, and the length to 20-30 mm; S2. Lay the blood filtration membrane at the sample-adding window and press it tightly by a gasket for separating the plasma in whole blood; S3. Install a heating sheet at the bottom of the bottom plate for heating the liquid in the reaction chamber; S4. Install a photosensitive sensor above the flow control port to monitor the plasma flow and generate an optical signal to control the heating sheet. S5. Install a spectral sensor above the encapsulation port to detect the product after the LAMP reaction and determine the positive or negative result based on the color change. S6. Add the freeze-dried premix and primers into the reaction chamber through the encapsulation port, cover the sealing film, and complete the encapsulation of the chip.

[0012] A detection method for a LAMP detection chip includes the following steps: L100. After filtering the whole blood sample through the blood filtration membrane of the sample loading window, make the separated plasma enter the hydrophilized main pipeline through the drainage notch. L200. Regulate the plasma flow rate to 0.0041 - 0.0043 m / s through the capillary resistance of the deep grooves on both side walls of the main pipeline, and make the plasma flow along the main pipeline towards the reaction chamber. L300. When the front end of the plasma touches the photosensitive sensor at the flow control port, the photosensitive sensor detects the fluid refractive index change signal and activates the bottom heating sheet. L400. The heating sheet controls the temperature of the reaction chamber at 65 ± 2 °C and maintains it for 25 minutes to perform the LAMP amplification reaction. L500. After the reaction ends, detect the color change of the reaction product through the spectral sensor to determine the positive or negative result of the detection.

[0013] Preferably, the flow rate control in step L200 includes: Generate capillary resistance through the side wall deep grooves with a depth of 0.8 - 1.2 mm and a width of 700 - 900 μm to reduce the plasma flow rate.

[0014] The beneficial effects of the present invention are that by integrating plasma separation and LAMP reaction detection on a microfluidic chip and using capillary force self-driving and side wall deep groove structure to precisely control the plasma flow rate, the detection efficiency and accuracy are significantly improved. This chip does not require external equipment and professional operators, is easy to operate, has a high degree of automation, reduces experimental errors and operation steps, saves time, manpower and material resources, reduces costs, and is suitable for convenient detection in ordinary environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will be further described below in conjunction with the drawings and embodiments.

[0016] Figure 1 It is a schematic structural diagram of the optimal embodiment of the plasma separation LAMP detection chip based on capillary force self-driving of the present invention.

[0017] Figure 2It is an exploded structural schematic diagram of the optimal embodiment of the plasma separation LAMP detection chip based on capillary force self-driving of the present invention.

[0018] In the figure: 1 sample loading bump, 2 drainage notch, 3 main pipeline, 4 side wall deep groove, 5 flow control port, 6 encapsulation port, 7 reaction chamber, 8 waste liquid port, 9 bottom plate, 10 upper cover plate, 11 blood filtration membrane, 12 gasket, 13 heating sheet. Detailed implementation manners

[0019] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention and should not be construed as a limitation of the present invention.

[0020] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "axial", "radial", "circumferential", etc. indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings. It is 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 thus should not be construed as a limitation of the present invention.

[0021] In addition, the terms "first", "second", etc. are only used for descriptive purposes and should not be construed as indicating or implying relative importance. In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, the terms "connected" and "connected" should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection, or an integral connection; it may be a mechanical connection or an electrical connection; it may be directly connected or indirectly connected through an intermediate medium. For those of ordinary skill in the art, 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, the meaning of "a plurality" is two or more.

[0022] Such as Figure 1-2As shown in the figure, the present invention provides a plasma separation LAMP detection chip based on capillary force self-driving. 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 one chip, and uses capillary force self-driving and sidewall deep groove structure to control the plasma flow rate, reducing the dependence on external equipment and achieving rapid and accurate detection.

[0023] The chip includes a bottom plate, an upper cover plate, and a blood filtration membrane, and its specific structure is as follows: On one side of the head of the bottom plate 9, there is a sample-adding bump 1, and the sample-adding bump 1 is provided with a drainage notch 2 connected to the main pipeline 3. The whole blood sample is introduced through the sample-adding window, flows into the main pipeline 3 through the drainage notch 2 for subsequent processing. On both sides of the main pipeline 3, there are sidewall deep grooves 4. The depth of the sidewall deep grooves 4 is 0.8 - 1.2 mm, the width is 700 - 900 μm, and the length is 20 - 30 mm. The structure of the sidewall deep grooves 4 can precisely control the plasma flow rate. By adjusting the flow rate, the plasma flow rate is controlled within the range of 0.0041 - 0.0043 m / s. Through this precise flow rate control, it is ensured that the plasma can smoothly flow into the reaction chamber 7, avoiding the use of external equipment. There is also a reaction chamber 7 opened on the main pipeline 3, and the reaction chamber 7 is used to receive the filtered plasma. When the plasma flows through the reaction chamber 7, it is mixed with the freeze-dried premixed solution and primers pre-placed in the reaction chamber 7 to complete the LAMP reaction. The upper cover plate 10 is covered on the bottom plate 9 by hot pressing and fixed by hot pressing. The upper cover plate 10 is provided with a sample-adding window corresponding to the sample-adding bump 1, and the blood-filtering membrane 11 is laid on the sample-adding window and pressed tightly by the gasket 12 to ensure that the whole blood sample can be effectively filtered through the blood-filtering membrane to separate the plasma. The upper cover plate 10 is also provided with a packaging port 6 above the reaction chamber 7, and the packaging port 6 is used to add the freeze-dried premixed solution and primers into the reaction chamber 7. There is a flow control port 5 opened on the upper cover plate 10 adjacent to the packaging port 6, which is used to monitor the plasma flow and control the heating of the heating sheet 13 through an optical signal. The bottom of the bottom plate 9 is provided with a heating sheet 13, which is responsible for heating the liquid in the reaction chamber during the LAMP reaction. The heating sheet 13 is controlled by a photosensitive sensor. When the photosensitive sensor detects the plasma flow, a refractive index change signal is generated, thereby starting the heating sheet 13 to keep the reaction temperature at 65 ± 2 °C for 25 minutes to implement the LAMP reaction. The photosensitive sensor is located above the flow control port 5 and is used to monitor the plasma flow condition and generate an optical signal to ensure the precise control of the reaction process. Above the packaging port 6 of the upper cover plate 10, there is a spectral sensor, which is used to detect the product after the LAMP reaction and judge the positive and negative according to the color change to ensure the accuracy of the detection result. The upper cover plate 10 is also provided with a waste liquid port 8 above the main pipeline 3, which is used to discharge the waste liquid generated during the experiment to ensure the smooth flow of the fluid in the chip and is not affected by the accumulation of waste liquid.

[0024] The plasma separation LAMP detection chip of the present invention mainly consists of a bottom plate 9, an upper cover plate 10, a blood filtration membrane 11, a heating sheet 13, etc. A sample-adding bump 1 is provided at the head of the bottom plate 9, which is connected to the main pipeline 3 through a drainage notch 2. Side wall deep grooves 4 are provided on both sides of the main pipeline to precisely control the plasma flow rate. The upper cover plate 10 of the chip is combined with the bottom plate 9 by a hot pressing method, and a sample-adding window, a flow control port 5 and a packaging port 6 are provided. Through the blood filtration membrane 11, the plasma is filtered at the sample-adding window, and the filtered plasma flows along the main pipeline under the action of capillary force. After the flow rate is controlled by the side wall deep grooves 4, it enters the reaction chamber 7 and is mixed with the freeze-dried premixed solution and primers.

[0025] Compared with the prior art, traditional plasma separation and LAMP reactions usually need to be carried out in different devices respectively, and each step involves complex manual operations and dependence on external devices. The present invention integrates these two steps into a microfluidic chip, realizing automated and precise detection. It not only avoids the use of external devices, but also makes the detection process more convenient and efficient.

[0026] During the plasma separation and flow process, the design of the side wall deep groove structure plays a key role in the plasma flow rate. Experiments show that by adopting a side wall deep groove design with a depth of 1 mm, a width of 800 μm, and a length of 25 mm, the plasma flow rate can be effectively reduced to 0.0041 - 0.0043 m / s. Compared with the flow rate of 0.009 - 0.011 m / s without side wall deep grooves, the flow rate is reduced by 42%. This structure not only makes the plasma flow more stable, but also avoids the use of external thrust devices and reduces the complexity of the experiment.

[0027] Different from the traditional method that relies on an external pump for flow rate control, the present invention realizes precise flow rate control through capillary force self-driving and flow control design, and the device is more portable, has a lower cost, and is easier to operate. This innovative design enables the chip to be used in ordinary environments, especially suitable for on-site rapid detection or resource-constrained environments.

[0028] The chip manufacturing method of the present invention is realized through the following steps: S1. Preparation of the bottom plate and the upper cover plate: First, prepare the bottom plate and the upper cover plate, and assemble them together by a hot pressing method to form the main structure of the chip. When preparing the bottom plate made of polymethyl methacrylate (PMMA) material, cover the non-treated area of the bottom plate with transparent tape to avoid the area outside the main pipeline being treated. Then, use the plasma hydrophilic treatment method to treat the main pipeline, set the treatment power to 60%, and the treatment time to 5 minutes to ensure that the surface of the main pipeline has excellent hydrophilicity and promotes the flow of plasma; S2. Installation of the blood filtration membrane: Lay the blood filtration membrane at the sample addition window and press it tightly with a gasket. The purpose of the blood filtration membrane is to separate the plasma in whole blood and ensure effective separation of blood cells and plasma. The design of the gasket can ensure that the blood filtration membrane is flat and well-sealed; S3. Installation of the heating element: Install the heating element at 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 element is crucial for the LAMP reaction. Therefore, the present invention designs a precise heating system to ensure that the temperature is controlled within the range of 65 ± 2°C; S4. Installation of the photosensitive sensor: Install the photosensitive sensor above the fluid control port to monitor the flow of plasma in real time and activate the heating element according to the change in light. The photosensitive sensor determines whether the plasma flow is normal by detecting the change in refractive index during the plasma flow and starts the heating system; S5. Installation of the spectral sensor: Install the spectral sensor above the encapsulation port to detect the product after the LAMP reaction. The spectral sensor can determine the positive or negative result of the LAMP reaction according to the color change of the reaction product to ensure the accuracy of the detection result; S6. Encapsulation of the premix and primers: Add the lyophilized premix and primers into the reaction chamber through the encapsulation port, and then cover the sealing film to complete the encapsulation of the chip.

[0029] Based on the above chip design, the detection method of the present invention is realized through the following steps: L100. Sample addition: First, add the whole blood sample through the sample addition window, and after filtration by the blood filtration membrane, the separated plasma enters the main pipeline through the drainage notch; L200. Flow rate control: The plasma passes through the side wall deep groove, and the flow rate is precisely controlled within the range of 0.0041 - 0.0043 m / s. The depth of the side wall deep groove is 1 mm, the width is 800 μm, and the length is 25 mm. Such a design significantly reduces the plasma flow rate compared to 0.009 - 0.011 m / s without the deep groove, and the flow rate control is more stable; L300. The photosensitive sensor activates the heating element: When the front end of the plasma passes through the fluid control port, the photosensitive sensor monitors the change in the optical signal and immediately activates the heating element. The photosensitive sensor determines the flow situation by detecting the change in refractive index of the fluid to ensure that the heating of the heating element is synchronized with the plasma flow; L400. Heating reaction: The heating element heats the liquid in the reaction chamber to 65°C and maintains it for 25 minutes to perform the LAMP amplification reaction; L500. Result determination: After the reaction ends, detect the reaction product through the spectral sensor, detect the color change of the reaction product and determine its positive or negative result.

[0030] The technical effects of the present invention are reflected in the following aspects: Efficient Detection: Compared with traditional laboratory methods, the present invention integrates plasma separation and LAMP reaction in a microfluidic chip, greatly reducing the detection time. The entire detection process only takes 40 minutes, significantly improving the detection efficiency.

[0031] Precise Flow Rate Control: Through the design of sidewall deep grooves, the present invention effectively controls the plasma flow rate, making it more stable, avoiding the use of external thrust devices, saving equipment costs and improving flow control accuracy.

[0032] Automation and Convenience: The chip design enables the entire detection process to be carried out without professional operators and external equipment. The operation is simple, reducing human errors and ensuring the accuracy of detection.

[0033] Low Cost and High Stability: The chip provided by the present invention has a simple structure, reducing the dependence on complex equipment. Moreover, the materials and processes used have low costs and are suitable for application in ordinary environments, with broad market prospects.

[0034] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0035] Taking the above-mentioned ideal embodiment of the present invention as an inspiration, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in the specification, and its technical scope 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 arranged on one side of the head of the bottom plate, and a drainage notch connected to the main pipeline is provided on the sample loading convex block, and deep side wall grooves are arranged on both sides of the main pipeline, and a reaction chamber is also provided on the main pipeline; The upper cover plate is placed on the bottom plate and fixed to each other by heat pressing. The upper cover plate 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 by a gasket. The upper cover plate is located above the reaction chamber and is provided with a packaging port, and the upper cover plate is also provided with a flow control port adjacent to the packaging port; 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.

2. The capillary force self-driven LAMP detection chip according to claim 1, characterized in that: The side wall deep groove has a depth of 0.8-1.2 mm, a width of 700-900 μm, and a length of 20-30 mm.

3. The capillary force self-driven LAMP detection chip according to claim 2, characterized in that: A heating plate is provided at the bottom of the bottom plate; A photosensitive sensor is arranged above the flow control port to monitor the flow of plasma and generate an optical signal to control the heating plate.

4. The capillary force self-driven LAMP detection chip according to claim 3, characterized in that: A spectral sensor is arranged above the packaging port to detect the product after the LAMP reaction and judge the positive and negative properties according to the color change.

5. The capillary force self-driven LAMP detection chip according to claim 4, 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.

6. A method for manufacturing a LAMP detection chip based on capillary force self-driving, characterized in that: The following steps are involved: S1. Prepare the bottom plate and the upper cover plate as described in any one of claims 1 to 5, and assemble them together by hot pressing to form the main structure of the chip; wherein, when preparing the bottom plate made of polymethyl methacrylate (PMMA), avoid the rest of the main pipeline, cover the non-treated area of ​​the bottom plate with transparent tape, and then perform plasma hydrophilic treatment on the main pipeline, the treatment power is set to 55-65%, the treatment time is 4-6 minutes, and the transparent tape is torn off after the treatment; set the depth of the side wall deep groove to 0.8-1.2mm, the width to 700-900μm, and the length to 20-30mm; 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, installing a heating plate at the bottom of the bottom plate to heat the liquid in the reaction chamber; S4, installing a photosensor above the flow control port to monitor the flow of plasma 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 and negative properties based on the color change; S6. Add the freeze-dried premix and primers into the reaction chamber through the packaging port, cover with a sealing film, and complete the packaging of the chip.

7. A detection method based on the LAMP detection chip according to any one of claims 1 to 5, 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, the plasma flow rate is regulated to 0.0041-0.0043 m / s by the capillary resistance of the deep grooves on both sides of the main pipeline, so that the plasma flows along the main pipeline to the reaction chamber; L300, when the front end of the plasma touches the photosensor at the flow control port, the photosensor detects the fluid refractive index change signal and activates the bottom plate heating plate; L400, heating plate to control the temperature of the reaction chamber at 65 ± 2 ° C, maintain for 25 minutes to implement LAMP amplification reaction; L500. After the reaction is completed, the color change of the reaction product is detected by the spectral sensor to determine the positive or negative nature of the detection result.

8. The detection method according to claim 7, 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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