Rapid nucleic acid extraction device based on ultrasonic technology

Abstract

The invention discloses a rapid nucleic acid extraction device based on an ultrasonic technology, and relates to the technical field of nucleic acid extraction, the rapid nucleic acid extraction device comprises an ultrasonic module and a microfluidic chip, the microfluidic chip is installed at the top end of the ultrasonic module, the ultrasonic module further comprises a base, a cavity is formed in the middle of the base, a bottom electrode is deposited on the upper end face of the base, and the bottom electrode is arranged in the cavity. A piezoelectric layer is deposited on the bottom electrode, a top electrode is deposited on the upper end face of the piezoelectric layer, and the micro-fluidic chip is installed on the upper end face of the top electrode, the ultrasonic module and the micro-fluidic chip are combined, the requirements for miniaturization and rapidness are met, the requirement for high-precision performance can be met while the POCT miniaturization, rapidness and convenience are met, and the cost is reduced. Ultrasonic waves are emitted based on a structural mode of the silicon chip, the device is small and simple, the miniaturization of the nucleic acid extraction module is realized, 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 invention relates to the technical field of nucleic acid extraction, in particular to a nucleic acid rapid extraction device based on ultrasonic technology. Background Art

[0002] Nucleic acid extraction is the process of separating DNA or RNA from biological samples. This process usually includes several key steps, the purpose of which is to release nucleic acids from cells or tissues and remove proteins, lipids and other impurities. The nucleic acid extraction methods we currently use mainly include the following methods, including:

[0003] 1. Alkaline lysis method: Alkaline lysis method is based on the difference between DNA denaturation and renaturation to achieve separation purpose;

[0004] 2. Boiling method: During boiling, the DNA in the sample is released by the action of nucleic acid lysis solution, and the impurities are removed after centrifugation. The supernatant can be used for PCR amplification;

[0005] 3. Concentrated salt method: RNA and DNA are separated by different solubility in the electrolyte solution. The commonly used method is to extract with 1M sodium chloride, shake the obtained DNP mucus with chloroform containing a small amount of octanol to emulsify it, and then centrifuge to remove the protein. At this time, the protein gel stays between the water phase and the chloroform phase, while the DNA is located in the upper water phase. DNA sodium salt can be precipitated with 2 volumes of 95% ethanol;

[0006] 4. Phenol extraction method: Phenol is used as a protein denaturant and inhibits the degradation of DNase. When the homogenate is treated with phenol, the bond between protein and DNA has been broken, and the surface of the protein molecule contains many polar groups that are similar to phenol and are soluble in water. Protein molecules are soluble in the phenol phase, while DNA is soluble in the water phase. After centrifugation and stratification, the water layer is taken out, and the operation is repeated many times. The water phase containing DNA is then combined, and the DNA is precipitated with ethanol using the property that nucleic acid is insoluble in alcohol. At this time, the DNA is a very viscous substance and can be rolled into a ball with glass and taken out. The characteristic of this method is that it keeps the extracted DNA in its natural state;

[0007] 5. Water extraction method: Using the property of nucleic acid being soluble in water, the tissue cells are broken, and RNA is removed with a low-salt solution. Then the precipitate is dissolved in water to fully dissolve the DNA in water, and the supernatant is collected after centrifugation. Solid sodium chloride is added to the supernatant to adjust to 2.6M. 95% ethanol is added in twice the volume, and it is immediately stirred out by stirring. Then it is washed with 66%, 80% and 95% ethanol and acetone respectively, and finally dried in the air to obtain a DNA sample. The protein content in the DNA extracted by this method is relatively high, so it is generally not used. This method can be improved to remove protein by adding SDS during the extraction process;

[0008] 6. Anionic detergent method: Use detergents such as SDS or sodium dibenzoate to denature proteins, and DNA can be directly extracted from biological materials. Since DNA and proteins in cells are often bound by electrostatic attraction or coordination bonds, anionic detergents are often used to extract DNA because they can destroy such valence bonds.

[0009] 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 be coupled 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 vibration 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.

[0010] Due to the convenience and cost-effectiveness of the magnetic bead method, most nucleic acid tests on the market, except for POCT products, use the magnetic bead method. In traditional detection technologies, the detection of the magnetic bead method often uses a separate instrument and cannot be applied to POCT products, so rapid detection cannot be achieved and test results cannot be provided in a short time. Summary of the invention

[0011] The purpose of the present invention is to provide a nucleic acid rapid extraction device based on ultrasonic technology to solve the problems raised in the above background technology.

[0012] To achieve the above-mentioned purpose, the present invention provides the following technical solutions: a rapid nucleic acid extraction device based on ultrasonic technology, comprising an ultrasonic module and a microfluidic chip, wherein the microfluidic chip is installed at the top of the ultrasonic module, the ultrasonic module also comprises a base, a cavity is arranged in the middle of the base, a bottom electrode is deposited on the upper end surface of the base, a piezoelectric layer is deposited on the bottom electrode, a top electrode is deposited on the upper end surface of the piezoelectric layer, and the microfluidic chip is installed on the upper end surface of the top electrode.

[0013] Preferably, a diaphragm is connected to the bottom of the bottom electrode at the position of the cavity, and the diaphragm is embedded in the cavity.

[0014] Preferably, a protrusion is provided on the upper end surface of the top electrode, a groove is provided at the bottom of the microfluidic chip, and the protrusion is embedded in the groove.

[0015] Preferably, the sample channel of the microfluidic chip is arranged on the left side, obliquely downwardly connected to the magnetic bead channel, and the magnetic bead channel is connected to the ultrasonic lysis zone, and a magnetic bead fixation zone is arranged on the right side of the ultrasonic lysis zone, the ultrasonic lysis zone and the magnetic bead fixation zone are connected to each other through a meandering channel, and the connection with the magnetic bead fixation zone is upwardly diverted and respectively connected to the oil raw material zone, the buffer zone, the eluent zone and the washing liquid zone, and the sample extraction and collection zone and the waste liquid zone are connected to the right side of the magnetic bead fixation zone.

[0016] Preferably, the upper end surface of the top electrode is coated with a layer of silver paint.

[0017] Preferably, the left and right end surfaces of the cavity are both inclined outwards.

[0018] Preferably, the upper end surface of the top electrode and the bottom of the microfluidic chip are cleaned using a dust-free cloth soaked in isopropyl alcohol.

[0019] Preferably, the base is made of silicon-based material.

[0020] Preferably, the top electrode and the bottom electrode are connected to an external power source.

[0021] In summary, the beneficial effects of the present invention are:

[0022] The present invention combines an ultrasonic module with a microfluidic chip to achieve miniaturization and rapidity, meet the requirements of miniaturization, rapidity and convenience of POCT, and achieve high-precision performance. The structure mode based on the silicon chip emits ultrasound, which is small and simple, and realizes the miniaturization of the nucleic acid extraction module, so that the nucleic acid extraction process can 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 extraction efficiency of nucleic acids by precisely controlling the frequency and intensity of MEMS ultrasound. It is proposed to effectively reduce the risk of cross contamination by designing a special sample processing channel and isolation structure 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1This is a schematic diagram of the structure of a rapid nucleic acid extraction device based on ultrasonic technology of the present invention;

[0025] Figure 2 This is a schematic diagram of the structure of a MEMS ultrasonic module of a nucleic acid rapid extraction device based on ultrasonic technology of the present invention;

[0026] Figure 3 It is a schematic structural diagram of the microfluidic chip and top electrode installation of a nucleic acid rapid extraction device based on ultrasound technology of the present invention;

[0027] Figure 4 This is a schematic diagram of microfluidic chip sampling of a nucleic acid rapid extraction device based on ultrasonic technology of the present invention.

[0028] The markings in the accompanying drawings are described as follows: top electrode 1; piezoelectric layer 2; bottom electrode 3; diaphragm 4; cavity 5; base 6; microfluidic chip 7; protrusion 8; groove 9; driving power supply 10; silver paint 11; washing area 12; sample channel 71; magnetic bead channel 72; ultrasonic lysis area 73; magnetic bead fixing area 74; sample extraction and collection area 75; waste liquid area 76; oil raw material area 77; buffer area 78; eluent area 79. DETAILED DESCRIPTION

[0029] The present invention will now be further described in detail in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. These drawings are all simplified schematic diagrams, which only illustrate the basic structure of the present invention in a schematic way, and therefore they only show the structures related to the present invention.

[0030] To facilitate understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Several embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0031] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0032] Any feature disclosed in this specification (including any additional claims, abstract and drawings), unless otherwise stated, may be replaced by other equivalent or alternative features with similar purposes. That is, unless otherwise stated, each feature is only an example of a series of equivalent or similar features.

[0033] In the present invention, unless otherwise clearly defined and specified, the terms "installed", "connected", "connected", "fixed" and the like 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, a direct connection, or an indirect connection through an intermediate medium, it can be the internal connection of at least two elements or the interaction relationship between at least two elements, unless otherwise clearly defined. 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.

[0034] Combine the following Figure 1-3 The present invention is described in detail. For the convenience of description, the directions mentioned below are defined as follows: the up, down, left, right, front, and back directions mentioned below are the same as Figure 1 The view direction is consistent in front, back, left, right, up and down directions. Figure 1 The directions shown are consistent with the front, back, left, right, up and down directions of the device of the present invention when viewed from the front.

[0035] See also Figure 1-3 , an embodiment provided by the present invention: a rapid nucleic acid extraction device based on ultrasonic technology, comprising an ultrasonic module and a microfluidic chip 7, wherein the ultrasonic module is a MEMS piezoelectric ultrasonic module, comprising a base 6, the base 6 is a silicon-based material, a cavity 5 is arranged in the middle of the base 6, a bottom electrode 3 is deposited on the upper end surface of the base 6, a piezoelectric layer 2 is deposited on the bottom electrode 3, a top electrode 1 is deposited on the upper end surface of the piezoelectric layer 2, and a diaphragm 4 is connected to the bottom of the bottom electrode 3 at the position of the cavity 5, the diaphragm 4 is embedded in the cavity 5, and the diaphragm 4 vibrates downwardly to generate ultrasound. It should be noted that the left and right side end surfaces of the cavity 5 are both inclined outward, so that the cavity 5 is trapezoidal, and the stability and service life of the entire base 6 are longer, and the top electrode 1 and the bottom electrode 3 are connected to an external power supply, and the ultrasonic frequency and intensity can be controlled by controlling the voltage at both ends of the electrodes, and finally the microfluidic chip 7 is installed on the upper end surface of the top electrode 1;

[0036] The material of the piezoelectric layer 2 may be lead zirconate titanate (PZT), aluminum nitride (AlN), etc.

[0037] The sample channel 71 of the microfluidic chip 7 is arranged on the left side, and is connected with the magnetic bead channel 72 obliquely downward, and the magnetic bead channel 72 is connected with the ultrasonic lysis area 73. A magnetic bead fixing area 74 is arranged on the right side of the ultrasonic lysis area 73. The ultrasonic lysis area 73 and the magnetic bead fixing area 74 are connected with each other through a circular channel, and the connection with the magnetic bead fixing area 74 is split upward, and is respectively connected with the oil raw material area 77, the buffer area 78, the eluent area 79 and the washing liquid area. The sample extraction and collection area 75 and the waste liquid area 76 are connected to the right side of the magnetic bead fixing area 74;

[0038] When extracting nucleic acid, the sample to be extracted and the lysis solution are added from the sample channel 71, and the magnetic beads enter from the magnetic bead channel 72 and enter the ultrasonic lysis area 73 together with the sample. The chemical functional groups on the surface of the magnetic beads can specifically bind to the nucleic acid. Once bound, the magnetic beads and the nucleic acid bound thereto can be separated from the liquid by applying an external magnetic field. Finally, the magnetic beads enter the magnetic bead fixing area 74 together with the oil, nucleic acid extraction BUFFER, eluent and washing solution. The magnetic beads are moved to the washing area under the action of the magnetic field, and the unnecessary substances are removed by multiple washings, thereby eliminating the lysis process. Proteins, salts and other impurities produced in the elution process. Elution is the process of separating nucleic acids from magnetic beads. It is usually achieved using a mild buffer. The eluted nucleic acids can be directly used for subsequent PCR amplification or other molecular biological analysis. Finally, the purified nucleic acids in the eluent are collected and enter the sample extraction collection area 75 for further research, while the waste liquid enters the waste liquid area 76. The entire process is completed in an ultrasonic environment, using the advantages of concentrated energy and high frequency of ultrasonic vibration to improve the efficiency of magnetic bead mixing, reduce mixing time and thus speed up nucleic acid extraction efficiency. The comparative experimental results show the role of acoustic vibration in promoting the movement and mixing of magnetic beads in the magnetic bead biological mixture;

[0039] By precisely controlling the frequency and intensity of MEMS ultrasound, the cell walls and cell membranes can be efficiently and evenly broken to release nucleic acids, thus improving the efficiency of nucleic acid extraction. It is proposed that the risk of cross-contamination can be effectively reduced 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 significantly improved in terms of efficient fragmentation, cross-contamination control, and rapidity and simplicity.

[0040] It should be noted that, in this embodiment, the ultrasonic lysis area 73 and the magnetic bead fixing area 74 are both arranged in the shape of water droplets.

[0041] In addition, in one embodiment, when the microfluidic chip 7 is installed on the top electrode 1, in order to further improve the stability of the chip, a protrusion 8 is provided on the upper end surface of the top electrode 1, and a groove 9 is provided at the bottom of the microfluidic chip 7, and the protrusion 8 is embedded in the groove 9. At the same time, during the installation process, the top electrode 1 and the bottom of the microfluidic chip 7 are first wiped with a dust-free cloth soaked in isopropyl alcohol, and then a layer of silver paint is coated on the upper end surface of the top electrode 1. When the microfluidic chip 7 is installed on the upper end surface of the top electrode 1, the protrusion 8 is embedded in the groove 9. At the same time, the surrounding silver paint will penetrate into the groove 9 along with the extrusion to form a more stable connection, while improving the contact performance, which can ensure the transmission performance of sound waves.

[0042] When nucleic acid extraction is required, the frequency and intensity of the MEMS ultrasound are first controlled so that the ultrasound penetrates the micro-control flow chip 7. When the sample for nucleic acid extraction is added to the micro-control flow chip 7, when the sample and the magnetic beads enter the ultrasonic lysis area 73 together, under the action of ultrasound, the cell wall and cell membrane are efficiently and evenly broken to release the nucleic acid, thereby improving the extraction efficiency of the nucleic acid. Finally, the sample enters the magnetic bead fixing area 74 together with the oil, nucleic acid extraction BUFFER, eluent and washing liquid. The magnetic beads are moved to the washing area under the action of the magnetic field, and unnecessary substances are removed by multiple washings, and proteins, salts and other impurities generated during the lysis process are removed. Elution is the process of separating the nucleic acid from the magnetic beads, which is usually achieved using a mild buffer. The eluted nucleic acid can be directly used for subsequent PCR amplification or other molecular biological analysis. Finally, the purified nucleic acid in the eluent is collected and enters the sample extraction collection area 75 for further research, and the waste liquid enters the waste liquid area 76.

[0043] In summary, the present invention integrates the ultrasonic module on the silicon-based chip, and the piezoelectric MEMS ultrasound is used for nucleic acid purification and nucleic acid extraction. The structure mode based on the silicon chip emits ultrasound, which is small and simple. The ultrasonic module is combined with the microfluidic chip to achieve miniaturization and rapidity requirements, meet the miniaturization, rapidity and convenience of POCT, and can achieve high-precision performance requirements. MEMS ultrasonic nucleic acid lysis (extraction) technology realizes the miniaturization of the nucleic acid extraction module, so that the nucleic acid extraction process can 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 crushing of cell walls and cell membranes to release nucleic acids and improve the extraction efficiency of nucleic acids 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 crushing, cross contamination control, and rapidity and simplicity.

[0044] The above is only a specific implementation of the invention, but the protection scope of the invention is not limited to it. Any changes or substitutions that are not conceived through creative work should be included in the protection scope of the invention. Therefore, the protection scope of the invention should be based on the protection scope defined in the claims.

Claims

1. A rapid nucleic acid extraction device based on ultrasonic technology, characterized in that: The invention comprises an ultrasonic module and a microfluidic chip (7), wherein the microfluidic chip (7) is mounted on the top of the ultrasonic module, and the ultrasonic module further comprises a base (6), wherein a cavity (5) is arranged in the middle of the base (6), a bottom electrode (3) is deposited on the upper end surface of the base (6), a piezoelectric layer (2) is deposited on the bottom electrode (3), a top electrode (1) is deposited on the upper end surface of the piezoelectric layer (2), and the microfluidic chip (7) is mounted on the upper end surface of the top electrode (1).

2. The nucleic acid rapid extraction device based on ultrasonic technology according to claim 1, characterized in that: The bottom of the bottom electrode (3) is connected to a diaphragm (4) at the position of the cavity (5), and the diaphragm (4) is embedded in the cavity (5).

3. The nucleic acid rapid extraction device based on ultrasonic technology according to claim 2, characterized in that: The upper end surface of the top electrode (1) is provided with a protrusion (8), the bottom of the microfluidic chip (7) is provided with a groove (9), and the protrusion (8) is embedded in the groove (9).

4. The nucleic acid rapid extraction device based on ultrasonic technology according to claim 3, characterized in that: The sample channel (71) of the microfluidic chip (7) is arranged on the left side and is connected to the magnetic bead channel (72) at an angle downward, and the magnetic bead channel (72) is connected to the ultrasonic lysis zone (73). A magnetic bead fixing zone (74) is arranged on the right side of the ultrasonic lysis zone (73). The ultrasonic lysis zone (73) and the magnetic bead fixing zone (74) are connected to each other through a circular channel, and the connection with the magnetic bead fixing zone (74) is split upward and connected to the oil raw material zone (77), the buffer zone (78), the eluent zone (79) and the washing liquid zone respectively. The right side of the magnetic bead fixing zone (74) is connected to a sample extraction and collection zone (75) and a waste liquid zone (76).

5. The nucleic acid rapid extraction device based on ultrasonic technology according to claim 4, characterized in that: The upper end surface of the top electrode (1) is coated with a layer of silver paint.

6. The nucleic acid rapid extraction device based on ultrasonic technology according to claim 5, characterized in that: The left and right end surfaces of the cavity (5) are both inclined outwards.

7. The nucleic acid rapid extraction device based on ultrasonic technology according to claim 6, characterized in that: The upper end surface of the top electrode (1) and the bottom of the microfluidic chip (7) are cleaned using a dust-free cloth soaked in isopropyl alcohol.

8. The nucleic acid rapid extraction device based on ultrasonic technology according to claim 7, characterized in that: The base (6) is made of silicon-based material.

9. The nucleic acid rapid extraction device based on ultrasonic technology according to claim 8, characterized in that: The top electrode (1) and the bottom electrode (3) are connected to an external power source.

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