Electrochemical DNA sensor detection device and detection method thereof

Abstract

The invention relates to an electrochemical DNA sensor detection device based on a micro-fluidic chip technology. The device comprises a micro-fluidic chip, a heating module, an external pump thrust device, a control module and an electrochemical signal detection module. The micro-fluidic chip is provided with a reaction cavity, an exonuclease storage part, an s-probe storage part, an electrode detection area and a waste liquid storage area. Through accurate temperature control and liquid flow control, the LAMP reaction and the probe hybridization reaction are automatically completed in the micro-fluidic chip, and then DNA signal measurement is performed through the electrochemical signal detection module. The device has the advantages of simplicity in operation, rapidness in detection and suitability for common environments.

Description

Technical Field

[0001] The present invention relates to the field of electrochemical detection technology, and in particular to an electrochemical DNA sensor detection device based on microfluidic chip technology and a detection method thereof, which is mainly used for nucleic acid amplification reaction and electrochemical signal detection. The device can perform rapid, accurate and portable DNA detection in a normal environment. Background Art

[0002] DNA testing has a wide range of applications in biomedicine, environmental monitoring, food safety and other fields. Traditional DNA testing methods rely on complex laboratory equipment and time-consuming operating procedures, usually requiring professional technical operators and a strictly controlled experimental environment. Especially in the electrochemical LAMP (loop-mediated isothermal amplification) reaction, the conventional detection process involves multiple steps, including LAMP product preparation, enzyme inactivation, exonuclease treatment, nucleic acid hybridization and electrochemical signal measurement. This process requires a high-temperature metal bath, precise addition of multiple reagents and time control. The operating steps are cumbersome and are easily affected by manual operation errors, which limits its wide application.

[0003] At present, electrochemical LAMP detection technology is mainly used in laboratory environments. The operation steps are lengthy and costly, making it difficult to achieve fast and convenient on-site detection needs. With the development of microfluidic chip technology, portable, economical, and automated detection devices have gradually become a research hotspot. Microfluidic chips have become an ideal carrier for integrated DNA detection systems due to their advantages such as miniaturization, rapid response, and low reagent consumption. However, existing microfluidic electrochemical detection systems still have certain limitations, such as insufficient detection sensitivity, low degree of automation in the operation process, and complex chip manufacturing processes.

[0004] Therefore, designing an electrochemical DNA sensor detection device based on a microfluidic chip can not only realize automated and integrated operation, but also perform efficient and rapid DNA detection under ordinary conditions without the need for professional operators. It has important technical innovation and practical application value. Summary of the invention

[0005] The technical problem to be solved by the present invention is: in order to solve the problems that the existing electrochemical LAMP reaction detection and signal measurement process is complicated, time-consuming and requires a professional laboratory environment and technicians, the present invention provides an electrochemical DNA sensor detection device based on microfluidic chip technology, which realizes automated operation and simplifies the steps by integrating heating, reaction control and electrochemical signal detection modules, and can quickly complete the detection under normal environment, avoiding errors caused by repeated operations, saving manpower and time.

[0006] The technical solution adopted by the present invention to solve the technical problem is: an electrochemical DNA sensor detection device, comprising: A microfluidic chip, wherein a plurality of functional areas are provided on the microfluidic chip, and the functional areas are sequentially connected through microfluidic channels, and the functional areas include a reaction chamber, a nuclease storage area, an s-probe storage area, an electrode detection area, and a waste liquid storage area; An electrochemical signal detection module, the module comprising a printed electrode disposed in an electrode detection area, and the printed electrode is used to perform electrochemical signal detection on DNA after hybridization reaction, and the printed electrode is covered with a PDMS film to improve detection performance and protect the electrode; A heating module is arranged in the bottom structure of the microfluidic chip and is used to control the temperature of the LAMP reaction and the inactivation reaction in the reaction chamber. The heating module is also integrated with a temperature sensor device for monitoring and adjusting the reaction temperature; An external pump thrust device is used to control the flow of the reaction liquid between the functional areas of the microfluidic chip, and complete the reaction, mixing and electrochemical signal detection in sequence; A control module, used to control the temperature regulation of the heating module and the operation of the external pump thrust device to achieve automated reaction control; A photosensor for microfluidic control is provided at the bottom of the microfluidic chip. The photosensor is connected to the control module. When liquid flows through the photosensor, the change of light produces refraction. The photosensor recognizes the change signal, and the control module stops the pushing of the external pump thrust device.

[0007] Preferably, the microfluidic chip consists of three layers: upper, middle and lower. The above layers are made by hot pressing, and the chip material is PMMA (polymethyl methacrylate). Before hot pressing, the s-probe is dripped on the filter membrane of the bottom plate in advance, and then integrated into the microfluidic chip by hot pressing technology after it dries and solidifies, and the nucleic acid exonuclease reagent is integrated into the microfluidic chip by freeze-drying technology.

[0008] Preferably, the microfluidic chip is provided with an upper plate for sealing the printed electrodes and the waste liquid storage area, and the upper plate is bonded to the chip body through a pressure-sensitive film to ensure sealing performance and prevent external contamination.

[0009] Preferably, the control module is also used to receive monitoring data from a temperature sensing device to achieve precise control of the LAMP reaction and inactivation reaction temperatures to ensure optimal reaction conditions.

[0010] Preferably, the electrochemical signal detection module further includes a data processing unit for receiving the electrochemical signal detected by the printed electrode, and processing and outputting the signal.

[0011] On the other hand, the present application also provides an electrochemical DNA detection method, using the electrochemical DNA sensor detection device according to the present invention, the method comprising the following steps: Adding LAMP reaction reagents into the reaction chamber; The heating module was controlled to heat at 65°C for 25 minutes, and then heated to 85°C for 7 minutes to complete the LAMP reaction and the inactivation enzyme reaction; The LAMP reaction product was pushed to the exonuclease storage area by an external pump thrust device, and the reaction was carried out at 37°C for 40 minutes; The treated product is then pushed to the s-probe storage area for mixing through an external pump thrust device, and then pushed to the electrode detection area; Electrochemical signal measurement was performed after hybridization at 42°C for 1 hour, and the obtained signal was processed by an electrochemical signal detection module.

[0012] Preferably, the material of the microfluidic chip is PMMA, the chip is processed by CNC (computer numerical control) or laser cutting, and is cleaned by ultrasonic cleaning technology after processing to ensure the cleanliness of the chip and the reliability of the reaction.

[0013] Preferably, the external pump thrust device can accurately control the flow and residence time of the sample between the reaction chamber, the exonuclease storage area, the s-probe storage area, the electrode detection area and the detection port through a program to ensure the full progress of each step of the reaction.

[0014] The beneficial effect of the present invention is that it provides an electrochemical DNA sensor detection device based on microfluidic chip technology, which can realize integrated and automated operation without the need for professional technicians to operate in a complex laboratory environment. The device reduces the operating steps and shortens the detection time through precise temperature control and liquid flow control, and effectively reduces experimental errors. The device is small in size, easy to carry, suitable for a variety of environments, has the advantages of high efficiency, convenience, and economy, and is widely used in fields related to gene detection. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0016] Figure 1 It is a schematic structural diagram of the optimal embodiment of the electrochemical DNA sensor detection device of the present invention.

[0017] Figure 2 It is an exploded view of the best embodiment of the electrochemical DNA sensor detection device of the present invention.

[0018] In the figure, 1, reaction chamber, 2, nuclease storage, 3, s-probe storage, 4, electrode detection area, 5, PDMS film, 6, sample injection port, 7, microfluidic chip, 8, detection port, 9, waste liquid storage area, 10, bottom plate, 11, middle plate, 12, top plate, 13, first upper plate, 14, second upper plate, 15, third upper plate, 16, heating plate. DETAILED DESCRIPTION

[0019] Embodiments of the present invention are described in detail below, examples of which 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 only used to explain the present invention, and cannot be understood as limiting the present invention.

[0020] 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 directly connected or indirectly connected 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.

[0021] Combined with Figure 1 and attached Figure 2 The embodiments of the present invention describe in detail the structure and working principle of the electrochemical DNA sensor detection device based on microfluidic chip technology. The embodiments are only some embodiments of the present invention. Based on this, those skilled in the art can make other forms of changes without paying creative labor, and these changes should also be included in the protection scope of the present invention.

[0022] 1. Structural composition As attached Figure 1 and attached Figure 2 As shown, the electrochemical DNA sensor detection device mainly includes a microfluidic chip, a heating module, an external pump thrust device, an electrochemical signal detection module and a control module.

[0023] Microfluidic chip7 The microfluidic chip 7 is composed of three layers: a top plate 12, a middle plate 11 and a bottom plate 10, and specifically includes: The bottom plate 10, the middle plate 11 and the top plate 12 are made by hot pressing and are made of PMMA material to ensure the stability and sealing of the chip. Before hot pressing, the s-probe solution is dripped onto the filter membrane of the bottom plate 10 and dried naturally to form a solid state. Then, the cured s-probe is integrated into the microfluidic chip by hot pressing technology to ensure that it can be effectively released during detection.

[0024] Exonuclease III is prepared into freeze-dried microspheres by freeze-drying technology and added to the reaction chamber of the microfluidic chip. During the reaction, the control module controls the external pump thrust device to mix the reaction liquid with the freeze-dried microspheres to achieve a rapid reaction.

[0025] The bottom heating module and temperature sensor are integrated into the bottom plate 10 of the chip to provide temperature control. The heating module includes three heating plates 16 for providing the required heating; The first upper plate 13, the second upper plate 14 and the third upper plate 15 are used to seal the printed electrodes and the waste liquid storage area 9 to ensure that the reaction area inside the chip is not polluted by the outside world.

[0026] Functional areas and microfluidic channels As attached Figure 1 As shown, a plurality of functional areas are arranged in the microfluidic chip, and the functional areas are sequentially connected through microfluidic channels, specifically including: Reaction chamber 1: used for LAMP reaction, accommodating samples to be tested and performing amplification reaction.

[0027] Exonuclease storage 2: used to store exonuclease and process the reaction products in subsequent steps.

[0028] s-probe storage 3: used for hybridization reaction with the probe to ensure that the DNA sample is bound to the probe and prepare for electrochemical signal measurement.

[0029] Electrode detection area 4: A printed electrode is provided to detect the DNA signal after the hybridization reaction and perform electrochemical signal detection. The surface of the printed electrode is covered with a PDMS film 5 to improve the stability and sensitivity of the signal detection. The electrode detection area 4 is also connected to a detection port 8. The reaction product is hybridized at 42°C for 1 hour on the electrode detection area 4, and finally the electrochemical signal is measured by the detection port 8.

[0030] Waste liquid storage area 9: used to collect waste liquid generated during the experiment to ensure the cleanliness of the chip and the integrity of the reaction process.

[0031] Liquid flow path The liquid flow path starts from the sample injection port 6, passes through the reaction chamber 1, the exonuclease storage 2, the s-probe storage 3, and finally reaches the electrode detection area 4, the detection port 8 and the waste liquid storage area 9. The flow of liquid between different functional areas is controlled by an external pump thrust device to ensure that each step is completed in a predetermined order and under predetermined conditions.

[0032] 2. Heating module and temperature control The heating module is integrated in the bottom plate 10, and specifically provides temperature control for the reaction chamber 1 through the heating plate 16. The temperature sensor is embedded in the bottom plate 10, and is used to monitor the temperature of the reaction chamber 1 in real time and transmit the temperature data to the control module.

[0033] The LAMP reaction requires precise temperature control. The reaction is first maintained at 65°C for 25 minutes, then raised to 85°C for 7 minutes to inactivate the enzyme. The entire process is automatically regulated by the control module.

[0034] 3. Electrochemical signal detection module The electrochemical signal detection module is located in the electrode detection area 4 and mainly includes printed electrodes. Figure 1 and attached Figure 2 As shown, the printed electrode is used to capture the electrochemical signal generated by the DNA hybridization reaction. The surface of the printed electrode is covered with a PDMS film 5 to improve the sensitivity and accuracy of the detection. The electrochemical signal is transmitted to an external data processing unit through the printed electrode for amplification, analysis and output.

[0035] 4. Control module The control module is integrated in the microfluidic chip, and automatically adjusts the working state of the heating module by receiving feedback signals from the temperature sensor. In addition, the control module also controls the external pump thrust device to ensure that the liquid flows in the microfluidic channel according to the predetermined steps and flow rate, ensuring the automation and accuracy of the detection process. A photosensor is integrated at the bottom of the microfluidic chip. The photosensor monitors the refraction changes of light when the liquid flows. The photosensor is connected to the control module. When the light changes, the sensor recognizes the signal and stops the external pump thrust device from pushing, thereby achieving precise flow control.

[0036] 5. Specific operation steps Sample injection: The DNA sample to be detected is injected through the sample injection port 6 and flows into the reaction chamber 1 .

[0037] LAMP reaction: The reaction chamber 1 was heated by the heating module and maintained at 65°C for 25 minutes for LAMP reaction, and then heated at 85°C for 7 minutes for enzyme inactivation.

[0038] Exonuclease treatment: The LAMP reaction product was pushed to the exonuclease storage 2 by an external pump thrust device and subjected to exonuclease treatment at 37°C for 40 minutes.

[0039] Probe hybridization: The processed products are pushed to the s-probe storage 3 for mixing.

[0040] Electrochemical signal detection: The sample flows into the electrode detection area 4 for hybridization, and the hybridization reaction lasts for 1 hour at 42°C. After the hybridization reaction is completed, the sample flows into the detection port 8, and the electrochemical signal is detected through the printed electrode. The detection signal is amplified, analyzed and the result is output by the external data processing unit.

[0041] 6. Overall beneficial effects By combining microfluidic chip technology, heating module and external pump thrust device, the present invention provides an integrated and automated electrochemical DNA detection device. The device can complete rapid detection under ordinary conditions, reduce dependence on the experimental environment, and reduce operational complexity. In addition, through precise temperature control and liquid flow control, the repeatability and reliability of the experiment are improved.

[0042] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0043] Based on the above ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above description. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. An electrochemical DNA sensor detection device, characterized in that: include: A microfluidic chip, wherein a plurality of functional areas are provided on the microfluidic chip, and the functional areas are sequentially connected through microfluidic channels, and the functional areas include a reaction chamber, a nuclease storage area, an s-probe storage area, an electrode detection area, and a waste liquid storage area; An electrochemical signal detection module, the module comprising a printed electrode disposed in an electrode detection area, and the printed electrode is used to perform electrochemical signal detection on DNA after hybridization reaction, and the printed electrode is covered with a PDMS film to improve detection performance and protect the electrode; A heating module is arranged in the bottom structure of the microfluidic chip and is used to control the temperature of the LAMP reaction and the inactivation reaction in the reaction chamber. The heating module is also integrated with a temperature sensor device for monitoring and adjusting the reaction temperature; An external pump thrust device is used to control the flow of the reaction liquid between the functional areas of the microfluidic chip, and complete the reaction, mixing and electrochemical signal detection in sequence; A control module, used to control the temperature regulation of the heating module and the operation of the external pump thrust device to achieve automated reaction control; A photosensor for microfluidic control is provided at the bottom of the microfluidic chip. The photosensor is connected to the control module. When liquid flows through the photosensor, the change of light produces refraction. The photosensor recognizes the change signal, and the control module stops the pushing of the external pump thrust device.

2. The electrochemical DNA sensor detection device according to claim 1, characterized in that: The microfluidic chip consists of three layers: upper, middle and lower. The above layers are made by hot pressing. PMMA (polymethyl methacrylate) is selected as the chip material. Before hot pressing, s-probe is dripped on the filter membrane of the bottom plate in advance, and then integrated into the microfluidic chip by hot pressing technology after it dries and solidifies. The nuclease reagent is integrated into the microfluidic chip by freeze-drying technology.

3. The electrochemical DNA sensor detection device according to claim 2, characterized in that: The microfluidic chip is provided with an upper plate for sealing the printed electrodes and the waste liquid storage area, and the upper plate is bonded to the chip body through a pressure-sensitive film to ensure sealing performance and prevent external contamination.

4. The electrochemical DNA sensor detection device according to claim 1, characterized in that: The control module is also used to receive monitoring data from the temperature sensing device to achieve precise control of the LAMP reaction and inactivation reaction temperatures to ensure optimal reaction conditions.

5. The electrochemical DNA sensor detection device according to claim 1, characterized in that: The electrochemical signal detection module also includes a data processing unit for receiving the electrochemical signal detected by the printed electrode, and processing and outputting the signal.

6. An electrochemical DNA detection method, characterized in that: Using the electrochemical DNA sensor detection device according to claim 1, the method comprises the following steps: Adding LAMP reaction reagents into the reaction chamber; The heating module was controlled to heat at 65°C for 25 minutes, and then heated to 85°C for 7 minutes to complete the LAMP reaction and the inactivation enzyme reaction; The LAMP reaction product was pushed to the exonuclease storage area by an external pump thrust device, and the reaction was carried out at 37°C for 40 minutes; The treated product is then pushed to the s-probe storage area for mixing through an external pump thrust device, and then pushed to the electrode detection area; Electrochemical signal measurement was performed after hybridization at 42°C for 1 hour, and the obtained signal was processed by an electrochemical signal detection module.

7. The electrochemical DNA detection method according to claim 6, characterized in that: The material of the microfluidic chip is PMMA. The chip is processed by CNC (computer numerical control) or laser cutting, and is cleaned by ultrasonic cleaning technology after processing to ensure the cleanliness of the chip and the reliability of the reaction.

8. The electrochemical DNA detection method according to claim 6, characterized in that: The external pump thrust device can accurately control the flow and residence time of the sample between the reaction chamber, the exonuclease storage area, the s-probe storage area and the electrode detection area through a program, ensuring that each step of the reaction is fully carried out.

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