PCR detection device based on ultrasonic chip and resistance method

Abstract

A disclosed PCR detection apparatus based on an ultrasonic chip and a resistance method comprises a silicon-based chip and an MEMS ultrasonic module, the silicon-based chip comprises a microfluidic chip, an electrode, a first membrane, a first cavity and a silicon substrate, the lower surface of the microfluidic chip is fixedly provided with the electrode, the lower surface of the electrode is fixedly provided with the first membrane, and the first membrane is fixedly provided with the second cavity. A silicon substrate is fixedly installed on the lower surface of the first film. According to the PCR detection device based on the ultrasonic chip and the resistance method, ultrasonic waves are emitted based on the structural mode of the silicon chip, the device is small and simple, the ultrasonic module is combined with the micro-fluidic chip, the requirements for miniaturization and rapidness are met, the requirement for high-precision performance can be met while POCT miniaturization, rapidness and convenience are met, and the device is suitable for popularization and application. The MEMS ultrasonic nucleic acid splitting (extraction) technology realizes the miniaturization of a nucleic acid extraction module, so that the nucleic acid extraction process can be carried out in a smaller space, the size of equipment is greatly reduced, and the cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of variable temperature fluorescent microelectronics, and in particular to a PCR detection device based on an ultrasonic chip and a resistance method. Background Art

[0002] At present, the nucleic acid extraction methods mainly include the following methods: 1. Alkaline lysis method: Alkaline lysis method is based on the difference between DNA denaturation and renaturation to achieve separation purpose 2. Boiling method: During boiling, the DNA in the sample is released by the action of nucleic acid lysis buffer. After centrifugation, impurities are removed and the supernatant can be used for PCR amplification.

[0003] 3. Concentrated salt method: RNA and DNA are separated by taking advantage of their different solubility in the electrolyte solution. The commonly used method is to extract with 1M sodium chloride. The obtained DNP mucus is shaken with chloroform containing a small amount of octanol to emulsify it, and then centrifuged to remove the protein. At this time, the protein gel remains between the aqueous phase and the chloroform phase, while the DNA is located in the upper aqueous phase. The DNA sodium salt can be precipitated with 2 times the volume of 95% ethanol.

[0004] 4. Phenol Extraction: Phenol acts as a protein denaturant and also inhibits DNase degradation. When the homogenate is treated with phenol, the bonds between the protein and DNA are broken. The protein molecules also contain numerous polar groups on their surfaces that are similarly soluble in phenol. Protein molecules dissolve in the phenol phase, while DNA dissolves in the aqueous phase. After centrifugation, the aqueous phase is removed and the process is repeated several times. The aqueous phase containing the DNA is then combined. Taking advantage of the alcohol-insolubility of nucleic acids, the DNA is precipitated with ethanol. At this point, the DNA is a very viscous substance that can be gently rolled into a ball on a glass for extraction. This method preserves the extracted DNA in its native state.

[0005] 5. Water Extraction: Utilizing the water-soluble nature of nucleic acids, tissue cells are disrupted and RNA is removed using a low-salt solution. The precipitate is then dissolved in water to fully dissolve the DNA. After centrifugation, the supernatant is collected. Solid sodium chloride is added to the supernatant to adjust the concentration to 2.6M. Two volumes of 95% ethanol are added and immediately stirred. The sample is then washed with 66%, 80%, and 95% ethanol, followed by acetone, and finally air-dried to obtain the DNA sample. This method produces DNA with a high protein content and is generally not used. To remove protein, this method can be modified by adding SDS during the extraction process.

[0006] 6. Anionic detergent method: Using detergents such as SDS or sodium dibenzoate to denature proteins allows DNA to be directly extracted from biological materials. Since DNA and proteins in cells are often bound by electrostatic attraction or coordination bonds, and since anionic detergents can disrupt these bonds, they are often used to extract DNA.

[0007] 7. Magnetic bead method: generally used for automated extraction. Biomagnetic beads are a new type of functional solid carrier with active groups coated on its surface. They can couple with a variety of biologically active substances. They have the characteristics of liquid fluidity and solid magnetic materials. They can be directed and concentrated under the action of an external magnetic field. When the external magnetic field is removed, they can be evenly dispersed in the liquid with a little shaking or suction, making the separation of solid and liquid phases very quick and convenient. Targeted substances with high purity can be obtained through simple elution.

[0008] Due to its convenience and cost-effectiveness, magnetic bead methods are currently used for the majority of nucleic acid extraction in the market, with the exception of point-of-care (POCT) products. Magnetic bead methods can be further categorized based on their principles: ultrasonic extraction, magnetic rod extraction, and external magnetic extraction. Magnetic rod and external magnetic extraction remain the mainstream options for nucleic acid extraction. Ultrasonic extraction is currently under research, with only a limited number of methods currently in the research phase. All three methods are currently in the standalone or automated instrumentation phase and are not used in POCT settings.

[0009] An ultrasonic vibration micromixer is used in a magnetic bead-based nucleic acid extraction microfluidic system to mix the magnetic bead biomixture. By leveraging the concentrated energy and high frequency of ultrasonic vibration, the efficiency of magnetic bead mixing is improved, mixing time is reduced, and nucleic acid extraction efficiency is accelerated. Comparative experimental results demonstrate the role of acoustic vibration in promoting the movement and mixing of magnetic beads in the magnetic bead biomixture.

[0010] Due to its convenience and cost-effectiveness, magnetic bead-based methods are currently the most widely used for nucleic acid testing in the market, with the exception of point-of-care (POCT) products. Traditional magnetic bead-based methods often require separate instruments, making them unsuitable for POCT applications. This approach, however, hinders rapid detection and the ability to provide results quickly. To simplify PCR systems and provide more portable and cost-effective applications, electrochemical techniques are being applied to PCR systems. EIS-based PCR technology is gradually entering the market. As the name suggests, it utilizes the electrochemical impedance spectroscopy (EIS) principle, measuring the impedance changes within a microchamber after different PCR cycles to determine reaction completion. The detection electrodes in this type of PCR system are in direct contact with the sample, eliminating the need for fixed pretreatment or complex external optical instrumentation. By applying a signal of a specific frequency and amplitude to the electrode tip, the real or imaginary impedance values ​​within different PCR cycles are measured, enabling the determination of reaction positivity.

[0011] Existing electrochemical detection of amplicons usually uses voltammetry to measure sample impedance. In order to enhance the signal for the desired detection, a pre-labeling process is usually used, which results in additional intercalants or requires additional fixation pretreatment to improve the signal-to-noise ratio. Traditional PCR relies on fluorescent labeling and optical detection, and the equipment is complex and costly, making it difficult to apply in resource-limited scenarios. Sample pretreatment (such as cell lysis and nucleic acid extraction) typically requires steps such as centrifugation and chemical reagents, which is time-consuming and difficult to integrate with PCR. Although electrical impedance analysis can indirectly detect nucleic acid concentration through changes in solution conductivity, existing technologies do not combine ultrasonic extraction with microfluidic PCR and lack sensitivity. To address these issues, we propose a PCR detection device based on an ultrasonic chip and electrical impedance analysis. Summary of the Invention

[0012] The main purpose of the present invention is to provide a PCR detection device based on an ultrasonic chip and a resistance method, which can effectively solve the problems in the background technology.

[0013] To achieve the above object, the technical solution adopted by the present invention is: A PCR detection device based on an ultrasonic chip and a resistance method includes a silicon-based chip and a MEMS ultrasonic module. The silicon-based chip includes a microfluidic chip, an electrode, a membrane No. 1, a cavity No. 1 and a silicon substrate. The electrode is fixedly mounted on the lower surface of the microfluidic chip, the membrane No. 1 is fixedly mounted on the lower surface of the electrode, the silicon substrate is fixedly mounted on the lower surface of the membrane No. 1, and the cavity No. 1 is opened in the middle of the upper surface of the silicon substrate.

[0014] Preferably, the MEMS ultrasonic module includes a top electrode, a piezoelectric layer, a bottom electrode, a No. 2 membrane, a No. 2 cavity, a base and a driving power supply. The piezoelectric layer is fixedly installed on the lower surface of the top electrode, the bottom electrode is fixedly installed on the lower surface of the piezoelectric layer, the base is fixedly installed on the peripheral position of the lower surface of the bottom electrode, the No. 2 membrane is fixedly installed on the lower surface of the bottom electrode inside the base, and the No. 2 cavity is provided under the No. 2 membrane.

[0015] Preferably, the top electrode and the bottom electrode are electrically connected to the positive and negative output electrodes of the driving power supply, respectively.

[0016] Preferably, the MEMS ultrasonic module is integrated on a silicon-based chip.

[0017] Compared with the prior art, the present invention has the following beneficial effects: In this invention, an ultrasound module is integrated onto a silicon-based chip. Piezoelectric MEMS ultrasound is used for nucleic acid purification and extraction. The silicon chip-based structure emits ultrasound waves in a compact and simple manner. Combining the ultrasound module with a microfluidic chip achieves miniaturization and rapidity, meeting the demands for miniaturization, rapidity, and convenience in point-of-care (POCT) while also achieving high-precision performance. MEMS ultrasonic nucleic acid lysis (extraction) technology miniaturizes the nucleic acid extraction module, allowing the extraction process to be performed in a smaller space, significantly reducing the size and cost of the equipment. Precisely controlling the frequency and intensity of the MEMS ultrasound waves achieves efficient and uniform disruption of cell walls and membranes to release nucleic acids, improving extraction efficiency. Specially designed sample processing channels and isolation structures are proposed to effectively reduce the risk of cross-contamination, ensuring the independence and safety of each sample. Compared to traditional nucleic acid extraction techniques, this technology offers significant improvements in efficient disruption, cross-contamination control, and rapidity and simplicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the overall structure of a PCR detection device based on an ultrasonic chip and a resistance method according to the present invention; Figure 2 This is a schematic diagram of a MEMS ultrasonic module of a PCR detection device based on an ultrasonic chip and a resistance method according to the present invention; Figure 3 This is an example diagram of a microfluidic chip of a PCR detection device based on an ultrasonic chip and a resistance method according to the present invention.

[0019] In the figure: 1. Microfluidic chip; 2. Electrode; 3. Membrane No. 1; 4. Cavity No. 1; 5. Silicon substrate; 6. Silicon-based chip; 7. MEMS ultrasonic module; 71. Top electrode; 72. Piezoelectric layer; 73. Bottom electrode; 74. Membrane No. 2; 75. Cavity No. 2; 76. Base; 77. Driving power supply. DETAILED DESCRIPTION

[0020] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below in conjunction with specific implementation methods.

[0021] like Figure 1-3 As shown, a PCR detection device based on an ultrasonic chip and a resistance method includes a silicon-based chip 6 and a MEMS ultrasonic module 7. The silicon-based chip 6 includes a microfluidic chip 1, an electrode 2, a first membrane 3, a first cavity 4, and a silicon substrate 5. The electrode 2 is fixedly mounted on the lower surface of the microfluidic chip 1, the first membrane 3 is fixedly mounted on the lower surface of the electrode 2, the silicon substrate 5 is fixedly mounted on the lower surface of the first membrane 3, and the silicon substrate 5 has a first cavity 4 defined in the middle of the upper surface of the silicon substrate 5. Specifically, the MEMS ultrasonic module 7 is combined with the microfluidic chip 1 to achieve the requirements of miniaturization and rapidity, meeting the requirements of miniaturization, rapidity, and convenience of POCT while achieving high-precision performance. MEMS ultrasonic nucleic acid lysis (extraction) technology realizes the miniaturization of the nucleic acid extraction module, allowing the nucleic acid extraction process to be carried out in a smaller space, greatly reducing the size of the equipment and reducing costs. It is proposed to achieve efficient and uniform fragmentation of cell walls and cell membranes to release nucleic acids and improve the efficiency of nucleic acid extraction by precisely controlling the frequency and intensity of MEMS ultrasound. It is proposed to effectively reduce the risk of cross-contamination by designing special sample processing channels and isolation structures to ensure the independence and safety of each sample. Compared with traditional nucleic acid extraction technology, this technology has significant improvements in efficient fragmentation, cross-contamination control, and rapidity and simplicity.

[0022] The MEMS ultrasonic module 7 includes a top electrode 71, a piezoelectric layer 72, a bottom electrode 73, a second membrane 74, a second cavity 75, a base 76, and a driving power supply 77. The piezoelectric layer 72 is fixedly mounted on the lower surface of the top electrode 71, the bottom electrode 73 is fixedly mounted on the lower surface of the piezoelectric layer 72, the base 76 is fixedly mounted on the peripheral position of the lower surface of the bottom electrode 73, the second membrane 74 is fixedly mounted on the lower surface of the bottom electrode 73 and located inside the base 76, and the second cavity 75 is provided below the second membrane 74. Specifically, the structure of the silicon-based chip 6 emits ultrasound, which is small and simple.

[0023] The top electrode 71 and the bottom electrode 73 are electrically connected to the positive and negative output electrodes of the driving power supply 77 respectively; The MEMS ultrasonic module 7 is integrated on the silicon-based chip 6; Specifically, the MEMS ultrasonic module 7 is integrated on the silicon-based chip 6, and the piezoelectric MEMS ultrasonic is used for nucleic acid purification and nucleic acid extraction.

[0024] It should be noted that this invention is a PCR detection device based on an ultrasonic chip and electrical impedance detection. It integrates sample lysis, nucleic acid amplification, and label-free detection through microfluidic technology, making it suitable for on-site rapid molecular diagnosis. The chip integrates ultrasonic lysis, microfluidic PCR temperature control, and electrical impedance detection.

[0025] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A PCR detection device based on an ultrasonic chip and a resistance method, characterized by: The invention comprises a silicon-based chip (6) and a MEMS ultrasonic module (7), wherein the silicon-based chip (6) comprises a microfluidic chip (1), an electrode (2), a membrane (3), a cavity (4) and a silicon substrate (5), wherein the electrode (2) is fixedly mounted on the lower surface of the microfluidic chip (1), the membrane (3) is fixedly mounted on the lower surface of the electrode (2), the silicon substrate (5) is fixedly mounted on the lower surface of the membrane (3), and the cavity (4) is opened in the middle of the upper surface of the silicon substrate (5).

2. The PCR detection device based on an ultrasonic chip and an electrical resistance method according to claim 1, characterized in that: The MEMS ultrasonic module (7) includes a top electrode (71), a piezoelectric layer (72), a bottom electrode (73), a second membrane (74), a second cavity (75), a base (76) and a driving power supply (77), wherein the piezoelectric layer (72) is fixedly mounted on the lower surface of the top electrode (71), the bottom electrode (73) is fixedly mounted on the lower surface of the piezoelectric layer (72), the base (76) is fixedly mounted on the peripheral position of the lower surface of the bottom electrode (73), the second membrane (74) is fixedly mounted on the inner side of the base (76) on the lower surface of the bottom electrode (73), and the second cavity (75) is provided below the second membrane (74).

3. The PCR detection device based on an ultrasonic chip and an electrical resistance method according to claim 2, characterized in that: The top electrode (71) and the bottom electrode (73) are electrically connected to the positive and negative output electrodes of the driving power supply (77), respectively.

4. The PCR detection device based on an ultrasonic chip and an electrical resistance method according to claim 3, characterized in that: The MEMS ultrasonic module (7) is integrated on a silicon-based chip (6).

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