Bacterial lysis and nucleic acid extraction integrated device based on surface acoustic waves
By using surface acoustic wave and microfluidic chip technology in the integrated device of bacterial lysis and nucleic acid extraction, efficient bacterial lysis and rapid nucleic acid extraction are achieved, the problem of inhibitor introduction is solved, and the sensitivity and reliability of detection are improved.
Patent Information
- Application Number
- CN202510147895.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-11
- Publication Date
- 2025-06-13
AI Technical Summary
The prior art is difficult to avoid the introduction of inhibitors during bacterial lysis and nucleic acid extraction, resulting in low sensitivity of nucleic acid detection and high false negative rate.
A integrated device for bacterial lysis and nucleic acid extraction based on surface acoustic waves is adopted. The device includes a piezoelectric substrate, an interdigital transducer and a microfluidic chip. The acoustic microfluidic effect is used to achieve bacterial lysis, and nucleic acid extraction is achieved through electrostatic adsorption of the microcolumn array, avoiding the use of inhibitors.
It realizes efficient bacterial cleavage and rapid nucleic acid extraction, reduces the introduction of impurities, improves the sensitivity and reliability of nucleic acid detection, and avoids potential amplification inhibition.
Smart Images
Figure CN120137744A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of nucleic acid extraction, and particularly relates to an integrated device for bacterial lysis and nucleic acid extraction based on surface acoustic waves. Background Art
[0002] In recent years, under the significant demand for nucleic acid detection in biomedicine, biological sample preparation has helped nucleic acid analysis become the cornerstone of clinical applications. The progress of nucleic acid extraction mainly stems from improving the yield of the isolated nucleic acid, minimizing the amount of impurities co-extracted with the nucleic acid, reducing sample preparation time and sample volume. The most commonly used cell lysis methods include physical methods and chemical methods. The latest research on cell lysis focuses on the development of new lysis agents and microfluidic chips capable of achieving high lysis efficiency for POCT.
[0003] With the continuous development of microfluidic chip technology, it has gradually become possible to use microfluidic chips to provide a simple, fast and practical lysis method for cell lysis. In traditional chemical cell lysis methods, reagents such as bioenzymes, lysis solutions, and surfactants are generally used, which can easily destroy the cell membrane and release the contents between cells. However, the remaining chemical reagents are difficult to remove completely, affecting subsequent extraction and amplification. Enzyme detection methods (such as polymerase chain reaction and sequencing) are highly sensitive to inhibitors, usually resulting in low sensitivity and false negatives. Therefore, sample preparation methods must remove inhibitors to ensure accurate and reproducible results. And some physical methods such as electrochemical lysis also require lysis reagents to improve the lysis rate during lysis. In contrast, surface acoustic wave cell lysis devices have the advantages of high lysis efficiency, short cycle, and non-contact, and have broad application prospects in bacterial lysis technology. However, on the one hand, bacteria are more difficult to lyse than cells, and on the other hand, the extraction and purification of nucleic acids after lysis are often interfered by inhibitors. Therefore, there is an urgent need for a lysis device that can achieve efficient bacterial lysis without introducing inhibitors. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide an integrated device for bacterial lysis and nucleic acid extraction based on surface acoustic waves, which realizes the integration of bacterial lysis and nucleic acid extraction and can better avoid potential amplification inhibition.
[0005] The present invention provides an integrated device for bacterial lysis and nucleic acid extraction based on surface acoustic waves, including a piezoelectric substrate and an interdigital transducer disposed on the piezoelectric substrate; on the piezoelectric substrate, at the same plane as the interdigital electrode, a microfluidic chip is further provided opposite to the propagation direction of the surface acoustic wave of the interdigital transducer; a microcolumn array for colliding with bacteria and enriching nucleic acids is provided in the microfluidic chip.
[0006] Preferably, the piezoelectric substrate is a lithium niobate wafer cut in the 128°-Y direction.
[0007] Preferably, the interdigital transducer includes interdigital electrodes capable of generating surface acoustic waves and connected floating electrodes capable of reducing clutter reflection.
[0008] Preferably, the surface acoustic wave generated by the interdigital transducer is a Rayleigh wave. When the Rayleigh wave enters the microfluidic chip, an acoustic streaming effect is generated.
[0009] Preferably, the interdigital transducer is a unidirectional interdigital transducer.
[0010] Preferably, the number of the interdigital transducers is single or multiple; the number of interdigital pairs is 25 to 45; the interdigital width is 10 to 100 microns; and the aperture width is 4 to 8 mm.
[0011] Preferably, the surface of the microcolumn array is modified with chitosan to capture nucleic acids through electrostatic interaction.
[0012] Preferably, the number of the microcolumn arrays is 20 to 40; the diameter of the microcolumns is 0.5 to 10 microns; and the height of the microcolumns is 0.5 to 20 microns.
[0013] Preferably, a perforated glass slide is provided above the microfluidic chip. The perforated glass slide is an ordinary cover glass with the same size as the microfluidic chip.
[0014] Preferably, the microfluidic chip further includes a bacterial lysis chamber, a liquid injection port, a waste liquid chamber, a collection chamber, and a serpentine liquid channel facing the direction in which the surface acoustic wave generated by the interdigital transducer propagates.
[0015] Preferably, the upper side of the microcolumn array is connected to the bacterial lysis chamber and is an integral structure.
[0016] Preferably, the two side outlets of the serpentine liquid channel are respectively connected to the liquid injection port, the bacterial lysis chamber, the waste liquid chamber, and the collection chamber.
[0017] Preferably, the top layer of the bacterial lysis chamber is closed, and the size of the lysis chamber can be changed according to the aperture width of the interdigital transducer or the amount of the lysis sample.
[0018] Preferably, the sizes of the waste liquid chamber and the collection chamber can be changed according to the amount of the lysis sample. The waste liquid chamber and the collection chamber are hollow and not closed.
[0019] Preferably, the perforated positions of the perforated glass slide correspond to the liquid injection port, the waste liquid chamber, and the collection chamber.
[0020] Preferably, the punched glass slide is further provided with a one-way valve inlet and a sealing cover. The position of the one-way valve inlet corresponds to the liquid injection port, and the position of the sealing cover corresponds to the collection chamber. The material is made of polypropylene, and the size can be adjusted according to the diameters of the liquid injection port, the waste liquid chamber, and the collection chamber.
[0021] The present invention utilizes the acoustic streaming effect of surface acoustic waves to drive the bacterial solution and the bacteria in the solution to move rapidly in the microfluidic chip. During the rapid movement, the dynamic bacteria and the surface of the static micro-column array collide with each other due to different speeds, resulting in bacterial lysis. At the same time, during the rapid movement, the electrostatic interaction of the micro-column array modified with chitosan is utilized, and the dynamic nucleic acid and the surface of the static micro-column array collide with each other due to different speeds, thereby capturing the nucleic acid.
[0022] Beneficial effects
[0023] (1) The present invention utilizes the acoustic streaming effect of the interdigital transducer to assist in lysing bacteria in the micro-column array in the bacterial lysis chamber with the help of a microfluidic chip. Specifically, the bacterial solution to be lysed is injected from the liquid injection port of the microfluidic chip, flows into the bacterial lysis chamber through the serpentine liquid channel, and under the surface acoustic wave generated by the interdigital transducer, the bacteria collide with the sound wave and the micro-columns to achieve bacterial lysis. During the entire lysis process, except for the direct action of the surface acoustic wave, there is no contact with any foreign objects, avoiding the introduction of impurities.
[0024] (2) The interdigital electrode for generating surface acoustic waves in the present invention contains a connected floating electrode, which reduces the loss of acoustic wave energy in the same direction and improves the lysis efficiency. The entire lysis process can complete bacterial lysis in only one minute, and the lysis rate is fast.
[0025] (3) During the bacterial lysis process of the present invention, under the acoustic streaming effect of the surface acoustic wave, the dynamic bacteria and the static micro-column array collide with each other in the microfluidic chip with a micro-column array, improving the lysis efficiency.
[0026] (4) The micro-column array in the lysis chamber of the microfluidic chip of the present invention is modified with chitosan, and nucleic acid extraction is realized through the action of electrostatic adsorption and hydrogen bonds, achieving the integration of bacterial lysis and nucleic acid extraction.
[0027] (5) During the process of nucleic acid adsorption and desorption by the micro-column array in the lysis chamber of the present invention, with the participation and oscillation of the surface acoustic wave, the nucleic acid capture and desorption efficiency are significantly improved, realizing the rapid extraction of nucleic acid.
[0028] (6) For bacterial lysis, nucleic acid enrichment, and purification in the present invention, nucleic acid is adsorbed in an acidic solution and released in an alkaline solution through electrostatic adsorption and hydrogen bonds. Nucleic acid extraction from bacteria can be achieved without introducing organic reagents, well avoiding potential amplification inhibition. Description of the Drawings
[0029] Figure 1 is a schematic structural diagram of the device of the present invention; wherein, 1 - piezoelectric substrate; 2 - microfluidic chip; 3 - interdigital transducer; 12 - punched glass slide; 13 - one-way valve inlet; 14 - sealing cover;
[0030] Figure 2 is a flowchart of the use of the device of the present invention. Detailed implementation manners
[0031] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.
[0032] Embodiment 1
[0033] As Figure 1 shown, this embodiment provides an integrated device for bacterial lysis and nucleic acid extraction based on surface acoustic waves, including a piezoelectric substrate and an interdigital transducer disposed on the piezoelectric substrate; on the piezoelectric substrate, at the same plane as the interdigital electrode, a microfluidic chip is further provided opposite to the propagation direction of the surface acoustic wave of the interdigital transducer; a microcolumn array for colliding with bacteria and enriching nucleic acids is provided in the microfluidic chip.
[0034] Preferably, the piezoelectric substrate is a lithium niobate wafer cut in the 128°-Y direction.
[0035] Preferably, the interdigital transducer includes interdigital electrodes capable of generating surface acoustic waves and a connected floating electrode capable of reducing clutter reflection.
[0036] Preferably, the surface acoustic wave generated by the interdigital transducer is a Rayleigh wave. After the Rayleigh wave enters the microfluidic chip, an acoustic streaming effect is generated.
[0037] Preferably, the interdigital transducer is a unidirectional interdigital transducer.
[0038] Preferably, the number of the interdigital transducers is single or multiple; the number of interdigital fingers is 25 to 45; the width of the interdigital fingers is 10 to 100 microns; the aperture width is 4 to 8 mm.
[0039] Preferably, the surface of the microcolumn array is modified with chitosan to capture nucleic acids by electrostatic interaction.
[0040] Preferably, the number of the microcolumn arrays is 20 to 40; the diameter of the microcolumns is 0.5 to 10 microns; the height of the microcolumns is 0.5 to 20 microns.
[0041] Preferably, a perforated glass slide is provided above the microfluidic chip. The perforated glass slide is an ordinary cover glass, and its size is equal to that of the microfluidic chip.
[0042] Preferably, the material of the microfluidic chip is PDMS.
[0043] Preferably, the microfluidic chip further includes a bacterial lysis chamber, a liquid injection port, a waste liquid chamber, a collection chamber, and a serpentine liquid channel that are directly opposite to the direction in which the surface acoustic wave generated by the interdigital transducer propagates.
[0044] Preferably, the upper side of the microcolumn array is connected to the bacterial lysis chamber and is an integral structure.
[0045] Preferably, the two side outlets of the serpentine liquid channel are respectively connected to the liquid injection port, the bacterial lysis chamber, the waste liquid chamber, and the collection chamber.
[0046] Preferably, the diameter of the serpentine liquid channel is 1 to 1.5 millimeters.
[0047] Preferably, the top layer of the bacterial lysis chamber is closed, and the size of the lysis chamber can be changed according to the aperture width of the interdigital transducer or the amount of the lysis sample. In this embodiment, the length × width × height of the bacterial lysis chamber = 6 mm × 4 mm × 4 mm.
[0048] Preferably, the sizes of the waste liquid chamber and the collection chamber can be changed according to the amount of the lysis sample. The waste liquid chamber and the collection chamber are hollow and not closed. In this embodiment, the diameter × height of the waste liquid chamber and the collection chamber = 5 mm × 4 mm.
[0049] Preferably, the microfluidic chip is parallel to the fingers of the interdigital transducer and is 1 to 4 millimeters apart.
[0050] Preferably, the perforated positions of the perforated glass slide correspond to the liquid injection port, the waste liquid chamber, and the collection chamber.
[0051] Preferably, the perforated glass slide is further provided with a one-way valve inlet and a sealing cover. The position of the one-way valve inlet corresponds to the liquid injection port, the position of the sealing cover corresponds to the collection chamber, the material is made of polypropylene, and the size can be adjusted according to the diameters of the liquid injection port, the waste liquid chamber, and the collection chamber.
[0052] As Figure 2 shown, this embodiment also provides a method for using an integrated device for bacterial lysis and nucleic acid extraction based on surface acoustic waves, including the following steps:
[0053] (1) Inject the bacterial solution to be lysed through the liquid injection port, flow it into the bacterial lysis chamber through the serpentine liquid channel, and the bacteria collide and lyse with the micro-columns under the surface acoustic wave generated by the interdigital transducer to obtain the nucleic acid lysate. The entire lysis process only takes one minute to complete bacterial lysis, with a fast lysis rate and no contact at the same time, avoiding the introduction of impurities.
[0054] (2) Adjust the solution to an acidic condition (pH = 4 - 6) before injecting the bacterial solution. The nucleic acid lysate formed after bacterial lysis repeatedly collides with the chitosan-modified micro-column array under the surface acoustic wave, prompting the chitosan of the micro-column array to rapidly adsorb nucleic acids under acidic conditions. After the lysis is completed, seal the collection chamber, inject gas through the liquid injection port, and drain the bacterial lysate in the bacterial lysis chamber into the waste liquid chamber. After taking out the bacterial lysate, inject the nucleic acid elution buffer under an alkaline condition (pH = 8 - 10) through the liquid injection port. After flowing into the bacterial lysis chamber, under the oscillation of the surface acoustic wave generated by the interdigital transducer, the nucleic acids quickly dissolve into the nucleic acid elution buffer. After the elution is completed, seal the waste liquid chamber, inject gas through the liquid injection port, and drain the nucleic acid elution buffer solution into the collection chamber. Take out the liquid to obtain the nucleic acid solution. The entire elution process only takes a few minutes to complete. The enrichment and collection of nucleic acids thus achieve nucleic acid extraction without introducing organic reagents, which can better avoid potential amplification inhibition.
Claims
1. An integrated device for bacterial lysis and nucleic acid extraction based on surface acoustic waves, characterized in that: It includes a piezoelectric substrate and an interdigital transducer arranged on the piezoelectric substrate; on the piezoelectric substrate, at the same plane as the interdigital electrodes, a microfluidic chip is arranged opposite to the outgoing direction of the surface acoustic wave of the interdigital transducer; the microfluidic chip is provided with a microcolumn array for colliding with bacteria and enriching nucleic acids.
2. The device according to claim 1, characterized in that: The piezoelectric substrate is a lithium niobate wafer cut in the 128°-Y direction.
3. The device according to claim 1, characterized in that: The interdigital transducer comprises interdigital electrodes capable of generating surface acoustic waves and interconnected suspension electrodes capable of reducing clutter reflection.
4. The device according to claim 1, characterized in that: The number of the interdigital transducers is single or multiple; the number of interdigital pairs is 25 to 45; the width of the interdigital fingers is 10 to 100 microns; and the aperture width is 4 to 8 millimeters.
5. The device according to claim 1, characterized in that: The surface of the micro-column array is modified by chitosan, and nucleic acids are captured through electrostatic action.
6. The device according to claim 1, characterized in that: The number of the micro-column array is 20 to 40; the diameter of the micro-column is 0.5 to 10 microns; and the height of the micro-column is 0.5 to 20 microns.
7. The device according to claim 1, characterized in that: A perforated glass slide is arranged above the microfluidic chip.
8. The device according to claim 1, characterized in that: The microfluidic chip also includes a bacteria lysis chamber, a liquid injection port, a waste liquid chamber, a collection chamber and a serpentine liquid channel which are opposite to the outgoing direction of the surface acoustic wave generated by the interdigital transducer.
9. The device according to claim 8, characterized in that: The upper side of the micro-column array is connected to the bacteria lysis chamber.
10. The device according to claim 8, characterized in that: The outlets on both sides of the serpentine liquid channel are respectively connected to the liquid injection port, the bacteria lysis chamber, the waste liquid chamber, and the collection chamber.