A method for interfering with DNA recognition based on ultrasonic driving device

Abstract

This invention relates to the field of biosensors and microfluidics, and particularly to a method for treating interfering DNA based on an ultrasonic-driven device. The method includes an ultrasonic-driven device comprising a piezoelectric substrate, Rayleigh wave drive, microfluidic reaction, and drive control module. First, a nucleic acid hybridization system is constructed to achieve initial binding and recognition of interfering DNA. Then, Rayleigh waves are excited to generate physical shearing force. Based on the difference in binding force between the interfering DNA and the target DNA and probe, precise mechanical feature recognition is achieved. By adjusting the radio frequency signal parameters to make the shearing force between the two types of binding forces, the interfering DNA is dissociated and expelled, completing the selective removal process.

Description

Technical Field

[0001] This invention relates to the field of biosensors and microfluidics, specifically to a method for processing DNA recognition interference based on an ultrasonic-driven device. Background Technology

[0002] Nucleic acid hybridization-based biosensors, such as DNA chips and surface plasmon resonance spectrometers, are core tools for gene detection and disease diagnosis. They capture complementary target nucleic acid chains using immobilized probe nucleic acid chains and achieve quantitative analysis of the target DNA through hybridization signals. However, in real-world complex biological samples, interfering DNAs such as mismatched DNA, truncated DNA, and non-specifically adsorbed nucleic acids are commonly present. These substances compete with the target DNA for probe binding sites, causing not only non-specific binding of interfering DNA to the probe but also increased background signal, decreased sensitivity, and even false positive results, affecting the effective identification of the target DNA.

[0003] Existing methods for treating interfering DNA mainly involve precise temperature control, repeated washing with formamide denaturing agents, and the addition of blocking agents. However, these methods are difficult to effectively identify highly homologous nucleic acid sequences, easily damage the perfectly complementary double strands of the target DNA-probe, and are cumbersome and time-consuming. They cannot achieve in-situ, real-time identification and removal of interfering DNA, making them unsuitable for the online detection requirements of microfluidic chips and limiting the application effect of biosensors in complex sample analysis. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for identifying and processing interfering DNA based on an ultrasound-driven device. This method utilizes Rayleigh wave-induced physical shearing force to identify the mechanical characteristics of interfering DNA and target DNA. By precisely controlling the shearing force to match the binding force between the interfering DNA and the probe, the method completes the dissociation and expulsion of interfering DNA without damaging the perfectly complementary double strands of the target DNA-probe pair. This solves the problems of existing methods, such as inaccurate identification of interfering DNA, poor processing results, easy damage to the target double strand, and cumbersome operation.

[0005] To achieve the above objectives, this invention provides a method for processing interference in DNA recognition based on an ultrasound-driven device. The ultrasound-driven device includes a piezoelectric substrate module, a Rayleigh wave driving module, a microfluidic reaction module, and a driving control module. The Rayleigh wave driving module is fabricated on the surface of the piezoelectric substrate module. The microfluidic reaction module is bonded above the piezoelectric substrate module and covers the acoustic propagation path of the Rayleigh wave driving module. The driving control module is electrically connected to the Rayleigh wave driving module. The processing method includes the following steps: S1. Construct an interference DNA recognition and processing system using an ultrasonic-driven device. Place a bio-substrate with immobilized probe DNA in the DNA probe binding region of a microfluidic reaction module. Inject a mixed solution of target DNA and interference DNA into the microfluidic reaction module to establish a nucleic acid hybridization reaction system. S2. Perform nucleic acid hybridization reaction. Under normal temperature and light-proof conditions, the probe DNA fully binds to the target DNA and interfering DNA to form complementary double strands of target DNA-probe and non-complementary double strands of interfering DNA-probe, thus completing the initial binding and recognition of interfering DNA. S3. Start the Rayleigh wave excitation program of the ultrasonic drive device. Apply a radio frequency drive signal to the Rayleigh wave drive module through the drive control module. The piezoelectric substrate module converts electrical energy into Rayleigh waves and propagates along the surface. After being enhanced by reflection by the reflective grating, a localized strong sound field is formed. S4. Rayleigh waves are used to identify the mechanical characteristics of interfering DNA. Rayleigh waves induce an acoustic flow effect at the solid-liquid interface to generate physical shear force. Based on the difference in binding force between interfering DNA and probe and target DNA and probe, the mechanical characteristics of interfering DNA and target DNA are distinguished and identified through physical shear force. S5. Selective removal of interfering DNA by regulating physical shearing force: The radio frequency signal parameters are adjusted by the drive control module to make the physical shearing force between the interfering DNA-probe binding force and the target DNA-probe binding force, thereby dissociating the interfering DNA from the probe and using acoustic flow to drive the dissociated interfering DNA away from the DNA probe binding area. S6. After completing the identification and processing of interfering DNA, shut down the drive control module and perform subsequent detection and analysis on the purified target DNA-probe system.

[0006] Preferably, the piezoelectric substrate module of the ultrasonic driving device is a 128°YX tangential lithium niobate piezoelectric crystal, and the Rayleigh wave driving module includes an input interdigital transducer and a reflective grating. The input interdigital transducer is a standard single-finger structure with 25 pairs of interdigits and a designed center frequency of 39MHz. The reflective grating is located on the acoustic propagation side of the input interdigital transducer and has 439 grid pairs.

[0007] Preferably, the electrodes of the Rayleigh wave driving module adopt a multilayer metal film structure, consisting of a 10nm thick chromium layer and a 300nm thick aluminum layer from bottom to top. The chromium layer is an adhesion layer, and the aluminum layer is a conductive layer, ensuring the bonding force between the electrode and the piezoelectric substrate and the acoustic wave excitation efficiency at high frequencies.

[0008] Preferably, the ultrasonic driving device has an upper, middle and lower sandwich structure, with the Rayleigh wave driving module located in the upper layer, the microfluidic reaction module being a closed microfluidic channel made of PDMS material located in the middle layer, and the biological substrate of the DNA probe binding area located in the lower layer, thereby realizing the physical separation of the driving component and the biosensing component.

[0009] Preferably, the adjustable radio frequency signal parameters of the drive control module include frequency, power, pulse duration and duty cycle, and the center frequency of the output radio frequency signal matches the design center frequency of the input interdigital transducer, which is 39MHz.

[0010] Preferably, in step S4, the mechanical feature identification is based on the binding force of the interfering DNA probe being 5-10 pN and the binding force of the target DNA probe being 20-30 pN. The mechanical action of physical shear force is used to achieve accurate differentiation and identification of the two binding systems.

[0011] Preferably, in step S5, adjusting the radio frequency signal parameters through the drive control module specifically involves adjusting the power of the radio frequency signal to precisely control the physical shearing force within 5-20 pN, ensuring that only the non-complementary double strands of the interfering DNA probe are dissociated, without damaging the perfectly complementary double strands of the target DNA probe.

[0012] Preferably, in step S5, the interfering DNA includes single nucleotide polymorphism mismatched DNA, truncated DNA, and non-specifically adsorbed nucleic acid molecules. During the removal process, the dissociated interfering DNA is discharged through the sample outlet of the microfluidic reaction module via acoustic flow, achieving in-situ, reagent-free removal.

[0013] Compared with the closest existing technology, the present invention has the following advantages: A system for identifying and processing interfering DNA was constructed using an ultrasonic drive device. First, preliminary binding and identification of interfering DNA was achieved through nucleic acid hybridization. Then, Rayleigh wave-induced physical shearing force was used to precisely identify the mechanical characteristics of the interfering DNA and the target DNA, forming a dual identification system of "binding identification + mechanical characteristic identification." This significantly improves the accuracy of interfering DNA identification and effectively distinguishes highly homologous nucleic acid sequences. A 128°YX tangential lithium niobate piezoelectric crystal and a Rayleigh wave drive module with a center frequency of 39MHz were used to achieve efficient conversion of electrical energy to acoustic energy. The thickness of the Rayleigh wave-induced acoustic flow boundary layer is matched with the scale of the DNA molecule and the binding interface, which can efficiently transfer momentum to the surface-bound DNA molecules, optimizing the mechanical characteristic identification and shearing processing effects. Precise control of the physical shearing force was achieved by adjusting the radio frequency signal power, ensuring that the shearing force is between the probe binding force of the interfering and target DNA. This ensures that the target DNA is removed while simultaneously achieving dissociation and removal of the interfering DNA. The integrity of the A-probe double strand is ensured, solving the problem of damage to the target double strand in traditional methods. The entire identification and processing is based on physical shear force, requiring no chemical denaturants or blocking agents. It is simple to operate and fast, and can complete in-situ, real-time identification and removal at room temperature, adapting to the online detection requirements of microfluidic chips. The ultrasonic drive device adopts a sandwich structure to physically separate the drive component from the biosensor component. The drive module is reusable, reducing detection and processing costs. Furthermore, the PDMS microfluidic channel can block heat from the drive, maintaining a stable temperature in the reaction zone and ensuring the accuracy of interfering DNA identification and processing. It achieves integrated processing of interfering DNA from identification to removal, with an interfering DNA removal rate of over 95% and a target DNA retention rate of over 98%. It can significantly reduce background signals, improve the signal-to-noise ratio and detection sensitivity of biosensors, and effectively avoid false positive results. It is suitable for high-resolution genotyping and nucleic acid detection analysis of complex biological samples. Attached Figure Description

[0014] Figure 1 This is a flowchart of a method for processing DNA recognition interference based on an ultrasound-driven device provided by the present invention; Figure 2 This is a CAD design drawing of a Rayleigh wave driver based on an ultrasonic drive device for interfering with DNA recognition processing, provided by the present invention. Detailed Implementation

[0015] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0017] This invention provides a method for processing DNA recognition interference based on an ultrasound-driven device, such as... Figure 1 As shown, a dual recognition and selective removal process for interfering DNA is achieved using an ultrasonic driving device. This ultrasonic driving device includes a piezoelectric substrate module, a Rayleigh wave driving module, a microfluidic reaction module, and a driving control module. The piezoelectric substrate module is a 128° YX tangential lithium niobate piezoelectric crystal, such as... Figure 2 As shown, the Rayleigh wave driving module consists of 25 pairs of interdigital input transducers and 439 pairs of reflective grids, with a designed center frequency of 39MHz. The microfluidic reaction module is a closed microfluidic channel made of PDMS material, and the driving control module is an RF signal generator with adjustable RF parameters. The following details the specific implementation steps. Example

[0018] In this embodiment, the interfering DNA is single nucleotide polymorphism mismatched DNA and truncated DNA, and the target DNA is full-length DNA that is completely complementary to the probe DNA. The specific identification and processing steps are as follows: S1. Construction of an interference DNA recognition and processing system: A 128°YX tangential lithium niobate piezoelectric crystal was selected as the piezoelectric substrate. A Rayleigh wave driving module consisting of 25 pairs of standard single-finger interdigital transducers and 439 pairs of reflective gratings was prepared on its surface. The electrodes adopted a 10nmCr / 300nmAl multilayer metal film structure. A microfluidic reaction module made of PDMS material was bonded to the piezoelectric substrate to ensure that the microfluidic channel covered the Rayleigh wave acoustic propagation path. A bio-substrate with probe DNA fixed was placed in the DNA probe binding area of ​​the microfluidic reaction module. A mixed solution of target DNA and interference DNA was injected into the microfluidic channel through the injection port to establish a nucleic acid hybridization reaction system. The device as a whole has an upper, middle and lower sandwich structure, with the Rayleigh wave driving module on top, the microfluidic reaction module in the middle and the bio-substrate at the bottom.

[0019] S2. Nucleic acid hybridization and preliminary binding recognition: Seal the inlet of the microfluidic reaction module and place the device in a room temperature, light-proof environment for 15 minutes to allow the probe DNA to fully undergo nucleic acid hybridization with the target DNA and interfering DNA in the mixed solution. The probe DNA and target DNA form perfectly complementary double strands, while the probe DNA and interfering DNA form non-complementary double strands. The preliminary binding recognition of the interfering DNA is completed through the principle of complementary base pairing.

[0020] S3. Start the Rayleigh wave excitation program: Connect the RF signal generator as the drive control module to the input interdigital transducer of the Rayleigh wave drive module. Start the drive control module, set the center frequency of the output RF signal to 39MHz, and apply the RF signal to the input interdigital transducer. The 128°YX tangential lithium niobate piezoelectric substrate efficiently converts electrical energy into Rayleigh waves. The Rayleigh waves propagate along the surface of the piezoelectric substrate towards the direction of the reflective grating. After being reflected by 439 pairs of reflective gratings, they are transmitted back to the DNA probe binding area of ​​the microfluidic reaction module. They are superimposed with the original propagating Rayleigh waves to form a high-Q acoustic resonant cavity, forming a localized strong sound field in the working area. The sound field intensity is 8 times higher than that of the design without reflective gratings.

[0021] S4. Mechanical Feature Identification of Interfering DNA: Rayleigh waves leak energy at the solid-liquid interface between the piezoelectric substrate and the microfluidic, inducing a strong nonlinear acoustic flow effect, which forms a physical shear force acting on the DNA-probe binding interface. It is known that the binding force of interfering DNA-probe is 5-10 pN, and the binding force of target DNA-probe is 20-30 pN. By utilizing the mechanical effect of physical shear force and based on the difference in binding force between the two binding systems, the mechanical features of interfering DNA and target DNA can be accurately distinguished and identified, and the target site of the interfering DNA to be treated can be clearly identified.

[0022] S5. Selective Removal of Interfering DNA: By gradually adjusting the power of the radio frequency signal through the drive control module, the physical shearing force is precisely controlled at 10pN. This shearing force is greater than the binding force of the interfering DNA-probe but less than the binding force of the target DNA-probe. Only the non-complementary double strands of the interfering DNA and the probe are dissociated, while the perfectly complementary double strands of the target DNA-probe remain intact. After dissociation, the interfering DNA is rapidly driven away from the DNA probe binding area by the acoustic flow and discharged through the sample outlet of the microfluidic reaction module, realizing in-situ and selective removal of interfering DNA. The radio frequency signal is continuously applied for 30 seconds to ensure complete removal of the interfering DNA.

[0023] S6. Complete identification and subsequent detection: After the interfering DNA is removed, the drive control module is turned off. At this time, the DNA probe binding region retains only the target DNA-probe complementary double strand, completing the integrated processing of interfering DNA from identification to removal. Hybridization signal detection and quantitative analysis can be performed directly on the purified system to achieve accurate detection of the target DNA.

[0024] In this embodiment, after processing by this method, the accuracy rate of interfering DNA identification reaches over 99%, the removal rate reaches 96%, the retention rate of target DNA reaches 98.5%, the detection signal-to-noise ratio of the biosensor is improved by more than 12 times, effectively avoiding the generation of false positive results and significantly improving detection accuracy.

[0025] Those skilled in the art will understand that embodiments of the present invention can be provided as methods, systems, or computer program products. Therefore, the present invention can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, the present invention can take the form of a computer program product embodied on one or more computer-usable storage media (including, but not limited to, disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0026] This invention is described with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of this application. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, special-purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, generate instructions for implementing the flowchart illustrations and / or block diagrams. Figure 1 One or more processes and / or boxes Figure 1 A device that provides the functions specified in one or more boxes.

[0027] These computer program instructions may also be stored in a computer-readable storage medium that can direct a computer or other programmable data processing device to function in a particular manner, such that the instructions stored in the computer-readable storage medium produce an article of manufacture including instruction means, which are implemented in a process Figure 1 One or more processes and / or boxes Figure 1 The function specified in one or more boxes.

[0028] These computer program instructions may also be loaded onto a computer or other programmable data processing equipment to cause a series of operational steps to be performed on the computer or other programmable equipment to produce a computer-implemented process, thereby providing instructions that execute on the computer or other programmable equipment for implementing the process. Figure 1 One or more processes and / or boxes Figure 1 The steps of the function specified in one or more boxes.

[0029] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for processing DNA recognition interference based on an ultrasound-driven device, characterized in that, The ultrasonic driving device includes a piezoelectric substrate module, a Rayleigh wave driving module, a microfluidic reaction module, and a driving control module. The Rayleigh wave driving module is fabricated on the surface of the piezoelectric substrate module. The microfluidic reaction module is bonded above the piezoelectric substrate module and covers the acoustic propagation path of the Rayleigh wave driving module. The driving control module is electrically connected to the Rayleigh wave driving module. The processing method includes the following steps: S1. Construct an interference DNA recognition and processing system using an ultrasonic-driven device. Place a bio-substrate with immobilized probe DNA in the DNA probe binding region of a microfluidic reaction module. Inject a mixed solution of target DNA and interference DNA into the microfluidic reaction module to establish a nucleic acid hybridization reaction system. S2. Perform nucleic acid hybridization reaction. Under normal temperature and light-proof conditions, the probe DNA fully binds to the target DNA and interfering DNA to form complementary double strands of target DNA-probe and non-complementary double strands of interfering DNA-probe, thus completing the initial binding and recognition of interfering DNA. S3. Start the Rayleigh wave excitation program of the ultrasonic drive device. Apply a radio frequency drive signal to the Rayleigh wave drive module through the drive control module. The piezoelectric substrate module converts electrical energy into Rayleigh waves and propagates along the surface. After being enhanced by reflection by the reflective grating, a localized strong sound field is formed. S4. Rayleigh waves are used to identify the mechanical characteristics of interfering DNA. Rayleigh waves induce an acoustic flow effect at the solid-liquid interface to generate physical shear force. Based on the difference in binding force between interfering DNA and probe and target DNA and probe, the mechanical characteristics of interfering DNA and target DNA are distinguished and identified through physical shear force. S5. Selective removal of interfering DNA by regulating physical shearing force: The radio frequency signal parameters are adjusted by the drive control module to make the physical shearing force between the interfering DNA-probe binding force and the target DNA-probe binding force, thereby dissociating the interfering DNA from the probe and using acoustic flow to drive the dissociated interfering DNA away from the DNA probe binding area. S6. After completing the identification and processing of interfering DNA, shut down the drive control module and perform subsequent detection and analysis on the purified target DNA-probe system.

2. The method for processing DNA recognition interference based on an ultrasound-driven device according to claim 1, characterized in that, The piezoelectric substrate module is a 128°YX tangential lithium niobate piezoelectric crystal. The Rayleigh wave driving module includes an input interdigital transducer and a reflective grating. The input interdigital transducer is a standard single-finger structure with 25 pairs of interdigits and a designed center frequency of 39MHz. The reflective grating is located on the acoustic propagation side of the input interdigital transducer and has 439 grid pairs.

3. The method for processing DNA recognition interference based on an ultrasound-driven device according to claim 2, characterized in that, The electrodes of the Rayleigh wave driving module adopt a multilayer metal film structure, consisting of a 10nm thick chromium layer and a 300nm thick aluminum layer from bottom to top. The chromium layer is an adhesion layer, and the aluminum layer is a conductive layer.

4. The method for processing DNA recognition interference based on an ultrasound-driven device according to claim 1, characterized in that, The ultrasonic driving device has an upper, middle and lower sandwich structure. The Rayleigh wave driving module is located in the upper layer, the microfluidic reaction module is a closed microfluidic channel made of PDMS material and is located in the middle layer, and the biological substrate with DNA probe is located in the lower layer and corresponds to the binding area of ​​the DNA probe.

5. The method for processing DNA recognition interference based on an ultrasound-driven device according to claim 1, characterized in that, The drive control module is a radio frequency signal generator. The adjustable radio frequency signal parameters include frequency, power, pulse duration and duty cycle. The center frequency of its output radio frequency signal matches the design center frequency of the input interdigital transducer, which is 39MHz.

6. The method for processing DNA recognition interference based on an ultrasound-driven device according to claim 1, characterized in that, In step S4, the binding force for mechanical feature recognition is based on the following: the binding force of the interfering DNA probe is 5-10 pN, and the binding force of the target DNA probe is 20-30 pN. The mechanical action of physical shear force is used to achieve accurate differentiation and recognition of the two binding systems.

7. The method for processing DNA recognition interference based on an ultrasound-driven device according to claim 6, characterized in that, In step S5, adjusting the radio frequency signal parameters through the drive control module specifically involves adjusting the power of the radio frequency signal to precisely control the physical shear force within 5-20 pN.

8. The method for processing DNA recognition interference based on an ultrasound-driven device according to claim 1, characterized in that, In step S2, the nucleic acid hybridization reaction is allowed to stand for 10-20 minutes to allow the probe DNA to fully bind with the target DNA and interfering DNA, ensuring the accuracy of the initial binding and identification.

9. The method for processing DNA recognition interference based on an ultrasound-driven device according to claim 1, characterized in that, In step S5, the radio frequency signal is continuously applied for 20-60 seconds to ensure that the dissociated interfering DNA is completely removed from the DNA probe binding area, and the interfering DNA is discharged through the sample outlet of the microfluidic reaction module.

10. A method for processing DNA recognition interference based on an ultrasound-driven device according to any one of claims 1-9, characterized in that, The interfering DNA includes single nucleotide polymorphism mismatched DNA, truncated DNA, and non-specifically adsorbed nucleic acid molecules. The entire identification and processing process is in situ and real-time, and no chemical reagents need to be added. The target DNA retention rate is ≥98%, and the interfering DNA removal rate is ≥95%.

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