Method for enhancing DNA specific binding based on acoustic driving
By capturing bubbles in microchannels and using acoustic drive to generate microfluidic phenomena, the problem of low DNA-specific binding efficiency in the prior art is solved, and a significant enhancement of DNA-specific binding is achieved, which is suitable for the field of micromolecular biology.
Patent Information
- Application Number
- CN202311167481.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-12
- Publication Date
- 2025-03-14
AI Technical Summary
Acoustic drives are not seen in the prior art for promoting DNA-specific binding, resulting in low DNA-specific binding efficiency.
By capturing bubbles in microchannels and oscillating the bubbles with acoustic drive, a microfluidic phenomenon is generated, thereby increasing the number of collisions of DNA strands and enhancing DNA-specific binding.
It effectively increases the number of collisions of complementary DNA strands, significantly enhances the specific binding effect of DNA, and has a simple structure and low cost, which is suitable for the field of micromolecular biology.
Smart Images
Figure CN119614678A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the fields of microfluidics and biomedicine, and in particular to a method for enhancing DNA specific binding based on acoustic driving. Background Art
[0002] Over the past two decades, microfluidics have become increasingly popular in a variety of applications, such as biomedical analysis, drug delivery, chemical synthesis enzyme reactions, etc. The promise of microfluidic systems lies in their ability to perform high-resolution reactions and analyses in a time- and cost-effective manner using minute amounts of reagents. These microfluidic systems are generally capable of rapidly and efficiently mixing different solutions at the microscale. To date, many micromixing methods have been demonstrated, such as chaotic advection, hydrodynamic focusing, electrokinetically driven mixing, and thermally and optically induced mixing. Among these methods, acoustic-based mixers have attracted great interest due to their non-invasiveness and simple, inexpensive fabrication procedures.
[0003] Acoustically driven micromixers employ bulk acoustic waves (BAW), surface acoustic waves, and acoustically vibrating microstructures to transfer acoustic energy into a fluid medium and induce mixing. Among the latter, bubbles have attracted great interest due to their strong localized microflows, low power consumption, and simple and inexpensive fabrication procedures, and they do not require interdigitated electrode deposition or expensive piezoelectric substrates. Bubble-based mechanisms are increasingly being applied to applications such as DNA hybridization, enzyme reaction analysis, cell separation, and bacterial aggregation.
[0004] In the DNA molecular structure, due to the difference in base sequences, two single-stranded DNAs with complementary bases will specifically bind based on base pairing. However, in the prior art, there is no report on using acoustic actuation to promote specific DNA binding. Therefore, it is of great significance to develop a method for enhancing specific DNA binding using acoustic actuation. Summary of the invention
[0005] In view of the above shortcomings of the prior art, the present invention provides a method for enhancing DNA specific binding based on acoustic drive, by capturing bubbles in the pre-designed grooves on the side walls of the microchannel. When acoustic drive is applied, the membranes of these captured bubbles begin to oscillate, and the bubble oscillation leads to microflow phenomenon - strong pressure and velocity fluctuations in the liquid, resulting in rapid and uniform mixing of the fluid, thereby increasing the number of collisions between the two complementary DNA chains and enhancing the specific binding of DNA. Finally, the effect of DNA specific binding is determined by measuring the SERS signal intensity. This is specifically achieved through the following technologies:
[0006] The invention provides a method for enhancing DNA specific binding based on acoustic drive, which applies ultrasonic resonance to a microchannel and passes a single-stranded DNA into the microchannel.
[0007] Furthermore, the above method specifically uses a signal generator to generate an electrical signal, and controls the piezoelectric ceramic sheet to convert the electrical signal into an ultrasonic signal; the voltage of the signal generator is 1-50V, and the resonant frequency is 5-60kHz.
[0008] Furthermore, the above method specifically includes:
[0009] (1) preparing a microchannel with a groove on the side wall, bonding the microchannel to a glass slide, and pasting a piezoelectric ceramic sheet on the glass slide;
[0010] (2) Covering the microchannel with a monolayer of metal nanoparticles, and then introducing a single-stranded DNA into the microchannel; turning on a signal generator, connecting the electrodes of the signal generator to the electrodes of the piezoelectric ceramic sheet, introducing a SERS probe complementary to the single-stranded DNA, and determining the specific binding of the DNA by measuring the SERS signal.
[0011] After a lot of creative research, the applicant found that the specific binding effect of DNA will be significantly enhanced due to the microfluidic phenomenon, which will produce rapid and uniform mixing of fluids, enhance the collision of two complementary DNA single strands, and achieve the purpose of DNA specific binding. In the above specific steps, the DNA single strand to be paired and combined is introduced into the microchannel structure, and an electrical signal is generated after the signal generator is turned on. The piezoelectric ceramic sheet connected to the signal generator will convert the generated electrical signal into an ultrasonic signal and act on the microchannel structure. When the DNA single strand passes through the microchannel structure, microbubbles will be formed and resonance will be generated under the action of the ultrasonic signal. At this time, the grooves on the side walls of the microchannel will capture the bubbles, effectively enhancing the collision between the two complementary DNA chains, thereby enhancing the specific binding of DNA.
[0012] More preferably, in the above step (2), the amount of DNA single strand introduced is 20-40 μL, the introduction speed is 0.5-2 μL / min, and the concentration is 50-200 uM; the amount of SERS probe introduced is 20-40 μL, the introduction speed is 0.5-2 μL / min, and the concentration is 50-200 uM.
[0013] More preferably, in the above step (2), a plurality of grooves are provided at equal intervals on both sides of the microchannel, and the grooves on both sides of the microchannel are arranged in a staggered manner; the piezoelectric ceramic sheet is close to the microchannel, and the material of the piezoelectric ceramic sheet is PZT. The use of PZT can make the experimental operation easier, and the cost of PZT is relatively low, and the ultrasonic frequency is relatively low, which is in kHz.
[0014] More preferably, in the above step (2), the method for covering the microchannel with a single layer of metal nanoparticles is: first, 20-40 μL of a PDDA solution with a mass fraction of 1-3% is introduced into the microchannel at a rate of 0.5-1 μL / min, and the solution is rinsed with deionized water at a flow rate of 2-3 μL / min for 3-4 min, and then 20-40 μL of a metal nanoparticle solution is introduced at a rate of 0.5-2 μL / min, and the solution is rinsed with deionized water at a flow rate of 2-3 μL / min for 3-4 min.
[0015] Specifically, the above microchannel structure preferably uses PDMS microchannels, i.e., PDMS chips. The microfluidic chip prepared with PDMS is variable, can have several grooves, and has advantages such as low price compared to conventional silicon chips. The PDMS microchannel is manufactured by conventional soft lithography and mold replication technology.
[0016] Furthermore, the metal nanoparticle solution is one of a silver nanoparticle solution or a gold nanoparticle solution with a concentration of 30-80 nM.
[0017] Compared with the prior art, the present invention is beneficial in that:
[0018] 1. The method for enhancing DNA specific binding based on acoustic drive provided by the present invention can effectively increase the number of collisions between two complementary DNA chains, thereby achieving the effect of enhancing DNA specific binding. The device has a simple structure and low cost, and can be widely used in the field of micromolecular biology.
[0019] 2. This method achieves contactless and non-destructive control and mixing effects. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 1 is a top view of a PDMS microchannel in a specific embodiment, in which 1 is an injection port, 2 is an outflow port, 3 is a rectangular groove, and 4 is a PDMS microchannel;
[0021] Figure 2 4 is a front cross-sectional view of a PDMS microchannel, a glass slide, and a piezoelectric ceramic sheet in a specific embodiment, in which 4 is a PDMS microchannel, 5 is a glass slide, and 6 is a piezoelectric ceramic sheet;
[0022] Figure 3 is a Raman spectrum diagram measured after acoustic driving processing in a specific implementation manner;
[0023] Figure 4 It is a Raman spectrum diagram measured without acoustic driving processing in a specific implementation manner. DETAILED DESCRIPTION
[0024] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0025] In this embodiment, a signal generator is used to generate an electrical signal, and a piezoelectric ceramic piece is controlled to convert the electrical signal into an ultrasonic signal. The ultrasonic resonance acts on a microchannel, and a single-stranded DNA to be paired and combined is introduced into the microchannel structure. When the single-stranded DNA passes through the microchannel structure, microbubbles are formed and resonance is generated under the action of the ultrasonic signal. At this time, the grooves on the side walls of the microchannel will capture the bubbles, effectively enhancing the collision between the two complementary DNA chains, thereby enhancing the specific binding of the DNA.
[0026] The method for enhancing DNA specific binding disclosed in the present invention comprises the following steps: (1) preparing a microchannel, wherein a plurality of grooves are provided at equal intervals on both sides of the microchannel, and the grooves on both sides of the microchannel are arranged in a staggered manner; then the microchannel is bonded to a glass slide, and a PZT piezoelectric ceramic sheet is bonded to a portion of the glass slide close to the microchannel; and (2) firstly introducing 20-40 μL of a PDDA solution with a mass fraction of 1-3% into the microchannel, and rinsing the solution with deionized water at a flow rate of 2-3 μL / min for 3-4 minutes, and then introducing 20-40 μL of a silver nanoparticle solution or a gold nanoparticle solution, and rinsing the solution with deionized water at a flow rate of 2-3 μL / min for 3-4 minutes. Then, 20-40 μL of a single-stranded DNA with a concentration of 50-200 μM is introduced into the microchannel at a speed of 0.5-2 μL / min; the signal generator is turned on, and its voltage range is set to 1-50 V and the resonant frequency range is set to 5-60 kHz. The electrode of the signal generator is connected to the electrode of the piezoelectric ceramic sheet, and 20-40 μL of a SERS probe with a concentration of 50-200 μM complementary to the above-mentioned single-stranded DNA is introduced at a speed of 0.5-2 μL / min, and the specific binding of DNA is determined by measuring the SERS signal.
[0027] The above-defined range parameters and material selections can achieve the effect of enhancing DNA specific binding. In order to simplify the research process, the specific steps of this implementation are as follows:
[0028] Taking the sequences of DNA-1 and DNA-2 as examples, the amino acid sequences of DNA-1 and DNA-2 are shown in Table 1 below:
[0029] Table 1 Amino acid sequences of DNA-1 and DNA-2
[0030]
[0031] 1. Preparation of polydimethylsiloxane (PDMS) microchannels
[0032] The PDMS microchannel was prepared by using standard soft lithography and mold replication technology (for specific steps, see the prior art). In order to compare the effects of DNA specific binding after acoustic driving treatment and without acoustic driving treatment, two microchannels with the same structure were set on PDMS. The PDMS microchannel was provided with an injection port and an outflow port, and a rectangular groove was provided on the side wall. The specific structure is as follows Figure 1 As shown, the sidewalls of the PDMS microchannel contain rectangular grooves arranged in a staggered pattern with equal spacing to capture bubbles.
[0033] 2. Preparation of acoustic drive device
[0034] The PDMS microchannel was ultrasonically cleaned in anhydrous ethanol for 30 minutes, then rinsed with ethanol and dried with nitrogen. The glass slide was ultrasonically cleaned with piranha wash for 30 minutes, then repeatedly rinsed with deionized water and dried with nitrogen. The rinsed PDMS microchannel was bonded to the top of the glass slide, and then a piezoelectric ceramic sheet made of lead zirconate titanate (PZT) was bonded to the carrier and close to the PDMS microchannel. The positional relationship between the PDMS microchannel, the glass slide and the piezoelectric ceramic sheet is shown in the figure. Figure 2 shown.
[0035] 3. Preparation of DNA-modified silver nanoparticles
[0036] 22 mg of silver nitrate was dissolved in 122.5 mL of deionized water and stirred and heated to boiling. 25 mg of sodium citrate was dissolved in 2.5 mL of deionized water and added to the boiling silver nitrate solution. After heating for 45 minutes, the reaction was stopped and cooled naturally to obtain a silver nanoparticle solution. The silver nanoparticle solution was concentrated 5-10 times, 90 ul of the concentrated silver nanoparticle solution was mixed with 5 ul of a 100 mM DNA-2 solution (from Shanghai Shenggong Biotechnology Co., Ltd.), and then 8 ul of a 10 mM DTNB solution (purchased from Sigma Aldrich) was added and mixed; 900 ul of n-butanol was added and hand-shaken for 30 seconds; then 200 ul of TBE solution was added and hand-shaken for 30 seconds; the reaction solution was centrifuged (5000 r / min, 5 min), and the resulting precipitate was redispersed in 90 ul of deionized water to obtain DNA-modified silver nanoparticles and label the SERS probe molecules.
[0037] 4. Covering of single layer of metal nanoparticles in PDMS microchannels
[0038] 30 μL of 1% PDDA solution (purchased from Sigma Aldrich) was introduced into two identical PDMS microchannels at a rate of 0.5 μL / min, and rinsed with deionized water at a rate of 2 μL / min for 3 minutes. Subsequently, 30 μL of silver nanoparticle solution was introduced at a rate of 0.5 μL / min, and rinsed with deionized water at a rate of 2 μL / min for 3 minutes to obtain silver nanoparticles distributed in a monolayer in the PDMS microchannel.
[0039] 5. Determination of DNA-specific binding effect
[0040] 30 μL of single-stranded DNA-1 (10uM) and salmon sperm DNA (0.1mg / ml) were introduced into the inlet of the two identical PDMS microchannels at a speed of 0.5 μL / min, and the signal generator was turned on at the same time. The electrodes of the signal generator were connected to the electrodes of the piezoelectric ceramic sheet, the voltage of the signal generator was set to 20V and the resonant frequency was adjusted to 29kHz. 30 μL of a SERS probe synthesized from a single-stranded DNA-2 complementary to DNA-1 was introduced into one of the PDMS microchannels at a speed of 0.5 μL / min, that is, it was acoustically driven.
[0041] The signal generator was turned off, and 30 μL of a SERS probe synthesized from a single-stranded DNA-2 complementary to DNA-1 was introduced into another PDMS microchannel at a rate of 0.5 μL / min, ie, without acoustic actuation treatment.
[0042] 6. Test results
[0043] The SERS signals of the two PDMS microchannels were measured to determine whether the specific binding of DNA was enhanced under acoustic driving. The SERS signals after and without acoustic driving treatment were measured as follows: Figure 3-4 As shown. It can be seen that the channel under acoustic drive ( Figure 3 ) is stronger than that without acoustic driving treatment, which means that the method for enhancing DNA specific binding based on acoustic driving provided by the present invention can effectively enhance specific binding and can be widely used in the field of micromolecular biology.
[0044] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.
Claims
1. A method for enhancing DNA specific binding based on acoustic drive, characterized in that: Ultrasonic resonance is applied to the microchannel to pass the DNA single strand into the microchannel.
2. The method for enhancing DNA specific binding according to claim 1, characterized in that: A signal generator is used to generate an electrical signal, and the piezoelectric ceramic piece is controlled to convert the electrical signal into the ultrasonic signal.
3. The method for enhancing DNA specific binding according to claim 2, characterized in that: The voltage of the signal generator is 1-50V, and the resonant frequency is 5-60kHz.
4. The method for enhancing DNA specific binding according to claim 3, characterized in that: The specific steps include: (1) preparing a microchannel with a groove on the side wall, bonding the microchannel to a glass slide, and pasting a piezoelectric ceramic sheet on the glass slide; (2) Covering the microchannel with a monolayer of metal nanoparticles, and then introducing a single-stranded DNA into the microchannel; turning on a signal generator, connecting the electrodes of the signal generator to the electrodes of the piezoelectric ceramic sheet, introducing a SERS probe complementary to the single-stranded DNA, and determining the specific binding of the DNA by measuring the SERS signal.
5. The method for enhancing DNA specific binding according to claim 4, characterized in that: In step (2), the amount of the single-stranded DNA introduced is 20-40 μL, the introduction speed is 0.5-2 μL / min, and the concentration is 50-200 μM.
6. The method for enhancing DNA specific binding according to claim 4, characterized in that: In step (2), the amount of the SERS probe introduced is 20-40 μL, the speed of introduction is 0.5-2 μL / min, and the concentration is 50-200 μM.
7. The method for enhancing DNA specific binding according to claim 4, characterized in that: In step (2), the piezoelectric ceramic sheet is close to the microchannel.
8. The method for enhancing DNA specific binding according to claim 4, characterized in that: In step (2), a plurality of grooves are provided at equal intervals on both sides of the microchannel, and the grooves on both sides of the microchannel are arranged in a staggered manner.
9. The method for enhancing DNA specific binding according to claim 4, characterized in that: In step (2), the method for covering the microchannel with a single layer of metal nanoparticles is: firstly pass 20-40 μL of a PDDA solution with a mass fraction of 1-3% into the microchannel for flushing, and then pass 20-40 μL of a metal nanoparticle solution for flushing.
10. The method for enhancing DNA specific binding according to any one of claims 1 to 9, characterized in that: The metal nanoparticle solution is a silver nanoparticle solution or a gold nanoparticle solution.