Gel self-interference based nucleic acid detection method and microfluidic detection platform

Abstract

The application provides a nucleic acid detection method based on gel self-interference and a microfluidic detection platform. The method comprises the following steps: after adding a to-be-detected sample, the to-be-detected sample is sequentially mixed with a lysis solution, magnetic beads, a magnetic bead cleaning solution and a nucleic acid elution solution through a digital microfluidic chip to extract sample nucleic acid droplets; the sample nucleic acid droplets are mixed with first-round RPA reagents and second-round RPA reagents through the digital microfluidic chip to perform two rounds of RPA amplification, and sample product droplets are obtained; the sample product droplets are mixed with CRISPR reagents through the digital microfluidic chip, and are moved to a solid-phase gel for sequence recognition and cleavage reaction, and solid-phase gel after cleavage reaction is obtained; and self-interference signal detection of the solid-phase gel in the solution after cleavage reaction is performed through an optical detection system, and a nucleic acid detection result of whether the to-be-detected sample contains a to-be-detected nucleic acid sequence is output. The method realizes high-specificity, fully-automatic and non-labeled PRA detection.

Description

Technical Field

[0001] This application relates to the field of nucleic acid detection technology, and in particular to a nucleic acid detection method based on gel self-interference and a microfluidic detection platform. Background Technology

[0002] Nucleic acid detection is a method that analyzes biological samples (such as blood samples) under laboratory conditions to determine the presence of specific nucleic acid sequences carrying the target nucleic acid. Currently, mainstream RPA nucleic acid detection technologies primarily use fluorescent probes or lateral flow test strips to characterize amplicones. For example, using exo fluorescent probes to detect amplicones can detect as few as 7.74 copies of RNA molecules in 1 μL of template solution; alternatively, RPA amplification products labeled with FAM and biotin are bound to anti-FAM gold particles, and then the biotin ligand on the lateral flow test strip is used to detect the amplification products with the anti-FAM probe, achieving a detection limit of 1.0 copies / μL. However, current RPA nucleic acid detection technologies mostly use fluorescence or other labels to read out amplicones, increasing detection costs and the complexity of the detection process. Highly integrated label-free RPA amplicon detection requires further development. Summary of the Invention

[0003] This application provides a nucleic acid detection method based on gel self-interference and a microfluidic detection platform, realizing a highly specific, fully automated, label-free PRA detection general method. The technical solution is as follows:

[0004] According to one aspect of this application, a nucleic acid detection method based on gel self-interference is provided. The method is applied to a microfluidic detection platform, which includes a digital microfluidic chip and an optical detection system. The digital microfluidic chip includes an upper chip and a lower chip. A solid-phase gel is embedded in the upper chip, and the lower chip contains lysis buffer and magnetic beads, magnetic bead cleaning solution, nucleic acid elution buffer, first-round RPA reagent, second-round RPA reagent, and CRISPR reagent. The digital microfluidic chip is placed on the stage of the optical detection system. The method includes:

[0005] After the sample to be tested is added to the lower chip, the sample to be tested is mixed sequentially with the lysis buffer and magnetic beads, the magnetic bead cleaning solution and the nucleic acid elution solution through the digital microfluidic chip to extract sample nucleic acid droplets from the sample to be tested;

[0006] The sample nucleic acid droplets are mixed with the first round RPA reagent and the second round RPA reagent using the digital microfluidic chip to perform two rounds of RPA amplification, resulting in sample product droplets containing nucleic acid amplification products from both rounds.

[0007] The sample product droplets are mixed with the CRISPR reagent using the digital microfluidic chip and moved to the solid gel for sequence recognition and cleavage reaction to obtain the cleavage reaction solid gel.

[0008] The optical detection system performs self-interference signal detection on the solid gel in the solution after the cutting reaction to determine and output the nucleic acid detection result of whether the sample contains the nucleic acid sequence to be tested.

[0009] According to another aspect of this application, a microfluidic detection platform is provided, the microfluidic detection platform including a digital microfluidic chip and an optical detection system, the digital microfluidic chip including an upper chip and a lower chip, the upper chip embedding a solid-phase gel, the lower chip storing lysis buffer and magnetic beads, magnetic bead cleaning solution, nucleic acid elution buffer, first-round RPA reagent, second-round RPA reagent and CRISPR reagent, the digital microfluidic chip being placed on the stage of the optical detection system;

[0010] The digital microfluidic chip is used to add the sample to be tested to the lower chip, and then mix the sample to be tested sequentially with the lysis buffer and magnetic beads, the magnetic bead cleaning solution and the nucleic acid elution solution to extract sample nucleic acid droplets from the sample to be tested.

[0011] The digital microfluidic chip is also used to mix the sample nucleic acid droplets with the first round RPA reagent and the second round RPA reagent to perform two rounds of RPA amplification, thereby obtaining sample product droplets containing the nucleic acid amplification products of the two rounds.

[0012] The digital microfluidic chip is also used to mix the sample product droplets with the CRISPR reagent and move them to the solid gel for sequence recognition and cleavage reaction to obtain the solid gel after cleavage reaction.

[0013] The optical detection system is used to perform self-interference signal detection on the solid gel after the cutting reaction, and to determine and output the nucleic acid detection result of whether the sample contains the nucleic acid sequence to be tested.

[0014] According to one aspect of this application, a nucleic acid detection device is provided, comprising: a processor and a memory storing a program, the program including instructions that, when executed by the processor, cause the processor to perform the gel self-interference-based nucleic acid detection method as described above.

[0015] According to another aspect of this application, a non-transitory computer-readable storage medium is provided storing computer instructions for causing the computer to perform the gel self-interference-based nucleic acid detection method as described above.

[0016] According to another aspect of this application, a computer program product is provided, comprising computer instructions stored in a computer-readable storage medium. A processor of a nucleic acid detection device reads the computer instructions from the computer-readable storage medium and executes the computer instructions, causing the computer device to perform the aforementioned gel-based self-interference nucleic acid detection method.

[0017] The beneficial effects of the technical solutions provided in this application include at least the following:

[0018] By integrating an optical detection system and a digital microfluidic chip, a solid-phase gel is embedded within the digital microfluidic chip as a sensing unit for the nucleic acid sequence to be tested. This allows for exponential amplification of the nucleic acid sequence to be tested through nested RPA amplification during the nucleic acid detection process. Furthermore, by combining this with Cas12a cleavage of the solid-phase gel, the presence of the target nucleic acid sequence in the sample is determined by detecting the self-interference signal of the solid-phase gel. This achieves both highly sensitive nucleic acid detection and a highly specific label-free RPA detection method. In addition, the integration of the optical detection system and the digital microfluidic chip has led to the construction of a new automated microfluidic detection platform, further enhancing the automation and integration of the nucleic acid detection process. Attached Figure Description

[0019] Further details, features, and advantages of this application are disclosed in the following description of exemplary embodiments in conjunction with the accompanying drawings, in which:

[0020] Figure 1 A flowchart of a nucleic acid detection method based on gel self-interference according to an exemplary embodiment of this application is shown;

[0021] Figure 2 This is a construction process for a solid-phase gel provided in an exemplary embodiment of this application;

[0022] Figure 3 A flowchart of another nucleic acid detection method based on gel self-interference according to an exemplary embodiment of this application is shown;

[0023] Figure 4 This is a schematic diagram of the structure of a digital microfluidic chip provided in an exemplary embodiment of this application;

[0024] Figure 5 This is a schematic diagram of the structure of an optical detection system provided in an exemplary embodiment of this application;

[0025] Figure 6 This is an optical schematic diagram of gel self-interference provided in an exemplary embodiment of this application;

[0026] Figure 7This is a schematic diagram of the structure of a microfluidic detection platform provided in an embodiment of this application. Detailed Implementation

[0027] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While some embodiments of this application are shown in the drawings, it should be understood that this application can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of this application. It should be understood that the drawings and embodiments of this application are for illustrative purposes only and are not intended to limit the scope of protection of this application.

[0028] It should be understood that the steps described in the method embodiments of this application may be performed in different orders and / or in parallel. Furthermore, the method embodiments may include additional steps and / or omit the steps shown. The scope of this application is not limited in this respect.

[0029] The term "comprising" and its variations as used herein are open-ended, meaning "including but not limited to". The term "based on" means "at least partially based on". The term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments". Definitions of other terms will be given in the following description. It should be noted that the concepts of "first", "second", etc., mentioned in this application are only used to distinguish different devices, modules, or units, and are not intended to limit the order of functions performed by these devices, modules, or units or their interdependencies. It should be noted that the modifications "a" and "a plurality" mentioned in this application are illustrative and not restrictive, and those skilled in the art should understand that unless explicitly indicated in the context, they should be understood as "one or more". The names of messages or information exchanged between multiple devices in the embodiments of this application are for illustrative purposes only and are not intended to limit the scope of these messages or information.

[0030] The present invention will now be described with reference to the accompanying drawings. The technical solutions provided by the embodiments of the present invention will be explained in detail through specific examples and application scenarios.

[0031] Currently, mainstream RPA (Recombinase Polymerase Amplification) nucleic acid detection technologies primarily employ fluorescent probes or lateral flow test strips to characterize amplicones. For example, using exo fluorescent probes to detect amplicones can detect as few as 7.74 copies of RNA (ribonucleic acid) molecules in 1 μL of template solution. Alternatively, RPA amplification products labeled with FAM (carboxyfluorescein) and biotin are bound to anti-FAM gold particles, and then the amplification products are detected using biotin ligands on lateral flow test strips along with anti-FAM probes, achieving a detection limit of 1.0 copies / μL. However, current RPA nucleic acid detection technologies mostly use fluorescence or other labels to read out amplicones, increasing detection costs and the complexity of the detection process. Highly integrated label-free RPA amplicon detection requires further development.

[0032] To achieve highly sensitive, highly specific, and highly integrated label-free RPA nucleic acid detection, this application provides a microfluidic detection platform integrating interferometric sensing with RPA nucleic acid detection. The detection of amplified products is achieved through interferometric sensing, eliminating the need for other markers. Please refer to... Figure 1 This document illustrates a flowchart of a gel self-interference-based nucleic acid detection method according to an exemplary embodiment of this application. The method is described using an application to a microfluidic detection platform as an example. Figure 1 As shown, the method includes:

[0033] Step 101: After adding the sample to be tested to the lower chip, the sample to be tested is mixed with lysis buffer and magnetic beads, magnetic bead cleaning solution and nucleic acid elution solution in sequence through a digital microfluidic chip to extract sample nucleic acid droplets from the sample to be tested.

[0034] The microfluidic detection platform provided in this application includes a digital microfluidic chip and an optical detection system. The digital microfluidic chip includes an upper chip (also referred to as an upper electrode) and a lower chip (also referred to as a lower electrode). A solid-phase gel is embedded in the upper chip and contacts the elongated control electrodes on both sides of the lower chip via conductive tape. The gel surface of the solid-phase gel is suspended above the lower electrode. When a droplet passes through the lower electrode, the solid-phase gel contacts the droplet. The lower chip includes a storage electrode, a sample loading electrode, a waste liquid storage electrode, and an electrode array for droplet manipulation. The storage electrode is used to store lysis buffer, magnetic beads, magnetic bead washing solution, nucleic acid elution buffer, first-round RPA reagent, second-round RPA reagent, and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) reagent. When the digital microfluidic chip is placed on the stage of the optical detection system, the optical acquisition module of the optical detection system can acquire the interference signal of the solid-phase gel through the lower electrode.

[0035] For example, the upper-level chip in a digital microfluidic chip can be made of ITO (Indium Tin Oxide) coated glass, and the lower-level chip can be made of ITO electrodes to form liquid storage electrodes, sample loading electrodes, waste liquid storage electrodes and droplet manipulation electrode arrays.

[0036] Unlike related technologies that use fluorescence or other labels to read the nucleic acid sequence to be tested, this application uses a solid-phase gel (also known as a solid-phase DNA gel) as the sensing unit for the nucleic acid sequence to be tested, and characterizes the detection of the nucleic acid sequence to be tested by the change in the thickness of the solid-phase gel. Figure 2 The diagram illustrates a construction process for a solid-phase gel provided in an exemplary embodiment of this application. Figure 2 As shown, firstly, primer 1 (…) is used in the liquid phase… Figure 2 The short chain 1) in the synthesis of the cyclic template ( Figure 2 The long chain 2) forms a circular template precursor. In the T4 enzyme ( Figure 2 Under the action of step 3), the annular template precursor is connected into a ring. Then, the annular template is added to a silicon wafer modified with solid-phase primer 1. Figure 2 The surface of primer 1 (4) is used in the formation of the circular template. Since the 3' end of primer 1 is modified with a C3 spacer, it does not compete with the solid-phase primers. The immobilized primer 1 binds to the circular template and, under the action of the RCA (Rolling Circle Amplification) reaction, forms a long-chain amplification primer (…). Figure 2 (5) Afterwards, the silicon wafer is immersed in a solution containing primer 2 ( Figure 2 6) Primer 3 Figure 2 In the RCA reaction system (7) of the formula, primer 2 binds to the long single-stranded DNA formed by long-chain amplification primer 5 at multiple sites, and amplifies it again to form a double-stranded product with single-stranded branching structures. These branching structures repeat the circular template sequence, and under the action of primer 3, they are further amplified, causing the single-stranded branching structures to form double strands with secondary single-stranded branching structures. This process alternates, and the DNA (Deoxyribonucleic Acid) molecules on the silicon wafer continuously increase. Each round of RCA generates a single-stranded template for the next round of RPA, ultimately forming a solid-phase DNA gel. Figure 2 9) Figure 2 The number 8 in the diagram represents the RCA reaction system containing elements 6 and 7. It should be noted that the thickness of the resulting solid-phase gel should be no less than 20 nm. This ensures that the thickness change of the solid-phase gel after CRISPR cleavage is sufficiently significant compared to the sub-nanometer sensitivity of optical interference, thereby guaranteeing the signal-to-noise ratio of nucleic acid detection.

[0037] In one possible implementation, after constructing the solid-phase gel, it is embedded in the upper layer of a digital microfluidic chip. Lysis buffer and magnetic beads, magnetic bead cleaning solution, nucleic acid elution solution, first-round RPA reagent, second-round RPA reagent, and CRISPR reagent are added to the reservoir electrode of the lower-level chip of the digital microfluidic chip, respectively. After adding the sample to be tested to the sample loading electrode, the subsequent nucleic acid amplification and nucleic acid detection process begins. First, the sample to be tested is sequentially mixed with lysis buffer and magnetic beads, magnetic bead cleaning solution, and nucleic acid elution solution using the digital microfluidic chip to extract nucleic acid from the sample, resulting in a sample nucleic acid droplet containing the sample nucleic acid.

[0038] For example, the sample to be tested can be a blood sample or other biological products from which nucleic acids can be extracted.

[0039] Step 102: The sample nucleic acid droplets are mixed with the first and second RPA reagents using a digital microfluidic chip to perform two rounds of RPA amplification, resulting in sample product droplets containing the nucleic acid amplification products from both rounds.

[0040] To achieve highly sensitive detection of the nucleic acid sequence to be tested, this application utilizes nested RPA amplification to achieve exponential amplification of the nucleic acid sequence. Specifically, after extracting the nucleic acid from the blood sample, a digital microfluidic chip is used to mix the sample nucleic acid droplets with first-round and second-round RPA reagents respectively to perform two rounds of RPA amplification, resulting in sample product droplets containing amplification products from both rounds. These two rounds of RPA amplification can amplify a sufficient concentration of recognition products for subsequent nucleic acid detection.

[0041] Step 103: The sample product droplets are mixed with CRISPR reagents using a digital microfluidic chip and moved to the solid gel for sequence recognition and cleavage reaction to obtain the solid gel after cleavage reaction.

[0042] The principle of nucleic acid detection in this application is as follows: The self-interference thickness of a solid-phase DNA gel is changed through CRISPR / Cas12a protein. First, the specific nucleic acid fragment of the target sample is identified, and the crRNA recognition sequence is determined according to the technical requirements of the Cas12a protein. Then, the target nucleic acid sequence is exponentially amplified through two nested rounds of RPA amplification. The products of the two rounds of nucleic acid amplification contain the crRNA recognition region of the CRISPR system. When the two rounds of RPA amplification produce a sufficient concentration of crRNA recognition products, the Cas12a protein in the CRISPR system can highly specifically recognize the target nucleic acid sequence, activating the molecular mechanism of DNA cleavage and cleaving the solid-phase DNA gel on the silicon wafer. After the solid DNA gel molecules are cleaved, the physical thickness decreases, resulting in a decrease in the self-interference phase of the solid-phase DNA gel. This allows the detection of the self-interference signal of the solid-phase DNA gel to determine whether the sample contains the target nucleic acid sequence.

[0043] Based on this nucleic acid detection principle, after obtaining sample product droplets through two rounds of RPA amplification, the sample product droplets are further mixed with CRISPR reagents using a digital microfluidic chip. crRNA recognizes specific sequences, and the mixed droplets are transferred to a solid gel for sequence recognition and cleavage reaction. After incubation, the droplets are transferred to a waste liquid electrode, and the solid gel after the cleavage reaction is detected to determine whether the nucleic acid sequence to be tested is present.

[0044] Step 104: The solid-phase gel in the solution after the cutting reaction is subjected to self-interference signal detection by an optical detection system to determine and output the nucleic acid detection result of whether the sample contains the nucleic acid sequence to be tested.

[0045] In one possible implementation, a digital microfluidic chip is placed on a stage. A light source in the optical detection system illuminates the surface of the solid-phase DNA gel within the digital microfluidic chip via a beam splitter and objective lens. The reflected light is focused by an imaging lens onto the end face of a multimode fiber, and then connected to a spectrometer and data analysis equipment to detect the self-interference signal of the solid-phase gel in the solution after the cleavage reaction. Since the self-interference signal is related to the thickness of the solid-phase gel, and whether the thickness of the solid-phase gel changes is related to whether the sample contains the target nucleic acid sequence, based on this correlation, the nucleic acid detection result (whether the sample contains the target nucleic acid sequence) can be determined and output based on the detection result of the self-interference signal.

[0046] In summary, this application provides a nucleic acid detection method based on gel self-interference: by integrating an optical detection system and a digital microfluidic chip, a solid-phase gel is embedded in the digital microfluidic chip as a sensing unit for the nucleic acid sequence to be tested. This allows for exponential amplification of the nucleic acid sequence to be tested through nested RPA amplification during the nucleic acid detection process, and combines this with Cas12a cleavage of the solid-phase gel to determine whether the sample contains the nucleic acid sequence to be tested by detecting the self-interference signal of the solid-phase gel. This achieves both high-sensitivity nucleic acid detection and a highly specific label-free RPA detection method. Furthermore, the integration of the optical detection system and the digital microfluidic chip constructs a new automated microfluidic detection platform, further enhancing the automation and integration of the nucleic acid detection process.

[0047] Digital microfluidic chips use electrowetting to control microdroplets of samples and reagents. By applying potential to electrodes, micro-droplets can be combined, mixed, distributed, and separated on an electrode array coated with a hydrophobic layer, thus automating, miniaturizing, and integrating the detection process.

[0048] Please refer to Figure 3 This document illustrates a flowchart of another gel self-interference-based nucleic acid detection method according to exemplary embodiments of this application. The method is described using an application to a microfluidic detection platform as an example. Figure 3 As shown, the method includes:

[0049] Step 301: After adding the sample to be tested to the lower chip, the sample to be tested and the lysis buffer are mixed with the magnetic beads through the electrode array to release the sample nucleic acid in the sample to be tested, and the sample nucleic acid is enriched on the magnetic beads.

[0050] Step 302: The magnetic beads are cleaned by mixing the magnetic beads with the magnetic bead cleaning solution through the electrode array.

[0051] Step 303: The cleaned magnetic beads are mixed with nucleic acid elution solution using an electrode array to obtain sample nucleic acid droplets.

[0052] Step 304: The first round of RPA reagents is mixed with sample nucleic acid droplets through an electrode array to obtain the first round of amplification products.

[0053] Step 305: The second round of RPA reagent is mixed with the first round of amplification products through an electrode array to obtain a sample product droplet containing the nucleic acid amplification products from both rounds.

[0054] Step 306: The sample product droplets are mixed with CRISPR reagents through an electrode array and moved to the solid gel for sequence recognition and cleavage reaction to obtain the solid gel after cleavage reaction.

[0055] Step 307: Use an optical detection system to detect the self-interference signal of the solid gel in the solution after the cutting reaction to determine whether the thickness of the solid gel has changed.

[0056] In this embodiment, the lower layer (lower electrode plate) of the digital microfluidic chip is fabricated with an ITO electrode, comprising a sample loading electrode, a waste liquid storage electrode, an electrode array, and multiple reservoir electrodes. The number of electrodes in the electrode array can be increased by adding electrodes or solid-phase gels according to the detection throughput. The multiple reservoir electrodes are used to store lysis buffer and magnetic beads, magnetic bead cleaning buffer, nucleic acid elution buffer, first-round RPA reagent, second-round RPA reagent, and CRISPR reagent, respectively. The sample loading electrode stores the sample to be tested. The electrode array is used to control the movement of the sample to be tested, sample nucleic acid droplets, and sample product droplets.

[0057] For example, such as Figure 4 As shown, it is a schematic diagram of the structure of a digital microfluidic chip provided in an exemplary embodiment of this application. Figure 4 As shown, Figure 4 18 is the sample loading electrode, into which blood samples can be added; Figure 4 Electrodes 19, 20, 21, 22, 23, and 24 are six liquid storage electrodes. Electrode 19 can be filled with lysis buffer and magnetic beads, electrode 20 can be filled with magnetic bead cleaning solution, electrode 21 can be filled with nucleic acid elution buffer, electrode 22 can be filled with first-round RPA reagent, electrode 23 can be filled with second-round RPA reagent, and electrode 24 can be filled with CRISPR reagent. Figure 4 25 in the figure represents an embedded gel silicon wafer; Figure 4 26 in the figure is the sample loading control electrode. Figure 4 27 in the diagram is a transparent 4×16 electrode array used for droplet manipulation.

[0058] Optionally, taking blood samples as an example, the chip dielectric layer (thickness 0-15 μm) and hydrophobic layer (thickness 0-15 μm) are optimized to improve droplet manipulation precision and reduce electrode residue, while preventing electrode breakdown caused by high-conductivity blood samples. Furthermore, the electrode array design (4×16) and optimized electrode spacing contribute to precise droplet distribution and separation, ensuring reaction efficiency. By optimizing the droplet distribution, mixing, and magnetic bead separation processes, the sensitivity and stability of nucleic acid extraction and amplification are improved, thereby achieving efficient and automated nucleic acid detection.

[0059] Based on the above description of the structure of the digital microfluidic chip, the droplet control process in the nucleic acid detection test includes the following steps: (1) After adding the sample to be tested to the lower chip, one drop is separated from the sample to be tested, the lysis buffer and the magnetic beads through the electrode array; (2) One drop of sample to be tested and one drop of lysis buffer are mixed with the magnetic beads to release the sample nucleic acid in the sample to be tested through the lysis buffer, and the released sample nucleic acid is incubated and enriched on the magnetic beads; (3) The magnetic beads are fixed by magnetic control, the solution is removed, and the magnetic bead cleaning solution is mixed with the magnetic beads through the electrode array, that is, the magnetic bead cleaning solution is added to the retained magnetic beads to clean the magnetic beads and remove excess solution; (4) After the magnetic beads are cleaned, the magnetic beads are retained, and the magnetic bead cleaning solution is moved to the waste liquid storage electrode through the electrode array; (5) The droplets are separated from the nucleic acid elution buffer through the electrode array, and the droplets of the nucleic acid elution buffer are mixed with the cleaned magnetic beads and incubated to separate the sample nucleic acid enriched on the magnetic beads from the magnetic beads and obtain sample nucleic acid droplets. (6) Remove the sample nucleic acid droplet through the electrode array and mix the first round RPA reagent with the sample nucleic acid droplet for amplification to obtain the first round amplification product. The droplet can be moved to improve the amplification efficiency; (7) Mix the second round RPA reagent with the first round amplification product through the electrode array to obtain a sample product droplet containing the nucleic acid amplification products of both rounds. The amplification efficiency can also be improved by moving the droplet; (8) Mix the sample product droplet with CRISPR reagent through the electrode array, and crRNA recognizes specific sequences; (9) Move the mixed droplet to the solid gel through the electrode array for sequence recognition and cleavage reaction. After incubation, move the droplet to the waste liquid storage electrode to obtain the solid gel after the cleavage reaction; (10) Use an optical detection system to detect the self-interference signal of the solid gel in the solution after the cleavage reaction to determine whether the thickness of the solid gel has changed, so as to obtain the nucleic acid detection result of the nucleic acid sequence to be tested.

[0060] Please refer to Figure 5 This is a schematic diagram of the structure of an optical detection system provided in an exemplary embodiment of this application. Figure 5 As shown, after being collimated by the collimating lens 12, the light source is irradiated onto the surface of the solid gel in the solution by the beam splitter 14 and objective lens 15 of the optical detection system. The solid gel is embedded in the digital microfluidic chip 16. The reflected interference signal is focused by the imaging lens 13 through the beam splitter 14 onto the end face of the multimode fiber 17. The fiber is connected to an external spectrometer and then to a computer for data analysis.

[0061] In order to infer the nucleic acid detection result of the target nucleic acid sequence based on the detection result of the self-interference signal, it is first necessary to analyze the optical principle of solid-phase gel self-interference. For example, please refer to... Figure 6 , Figure 6 This is an optical schematic diagram of a gel self-interference provided in an exemplary embodiment of this application. (See diagram for example.) Figure 6As shown in (a), when unit intensity perpendicularly incident light shines on the silicon wafer, it is reflected by the upper surface of the solid DNA gel 9 and then reflected through the solid DNA gel 9 at the interface between the silica layer 10 and the silicon substrate 11. The two reflected beams are denoted as U1(k) and U2(k), where k is the vacuum wavenumber (since the refractive indices of silica and DNA molecules are not significantly different, reflections between these two layers are often ignored in self-interference sensors). The two reflected beams interfere, and the interference phase can be expressed as: 2n0k(d+δ). Here, n0 represents the average refractive index of silica and the solid DNA gel (≈1.47), d is the thickness of the silica layer, and δ is the thickness of the gel layer of the solid gel.

[0062] When a solid-phase DNA gel reacts in solution, part of the incident light U0(k) is also interfered with by the weakly scattering medium in the solution, such as... Figure 6 As shown in (b) above, the weakly scattering medium is equivalently considered as a spherical particle with radius a and refractive index n, and the distribution of its scattering amplitude f is shown. (q) It can be represented as Where q is the scattered wave vector, which physically represents the vector difference between the scattered wave and the incident wave. Therefore, the scattered wave vector generated by part of the incident light can be expressed as: Because the molecular size of the scattering medium is much smaller than the wavelength, and u can be approximated s (k)≈C k U0(k)(n 2 -1)V, where C k Here are the constants generated in the calculation, and V is the volume of the scattering medium.

[0063] Therefore, due to the presence of a weakly scattering medium in the solution, the objective lens can collect a beam of backscattered light, and the wave vector can be expressed as u3(k)=α k C k U0(k)(n 2 -1)V, where α k This represents the ratio of the objective lens convergence angle to the 4π space. Scattering simultaneously weakens the illumination intensity on the solid gel. When the incident light passes through the solution and illuminates the solid DNA gel, the reflected light U1(k) and U2(k) are scattered again by the medium after passing through the solution, further reducing their amplitude. Let A1(k) and A2(k) be the amplitudes of the two reflected beams collected by the objective lens, respectively, and let A3(k) be the amplitude of the wave vector u3(k). The composite beam consists of the two reflected beams and the scattered light, and can be represented as follows: Where n wLet be the average refractive index of the solution, and z be the axial distance of the scattering medium relative to the upper surface of the gel. The reflection intensity I(k) is the sum of squares of the combined beams. Since the interference term includes the phase difference, the reflection intensity of the self-interference spectrum can be expressed as: I(k)=|U(k)| 2 ≈

[0064] A1(k) 2 +A2(k) 2 +A3(k) 2 +2A2(k)A3(k)cos[2n0k(d+δ)]+2a1(k)A2(k)cos[2n0k(d+δ)+2n w According to common methods of light field reconstruction, theoretically, the multiple interferences with relatively low intensity and the interference between U1(k) and U3(k) are ignored.

[0065] In terms of analytical methods, the interference spectrum exhibits a cosine function distribution along the wavenumber k dimension. The Fast Fourier Transform (FFT) can be used to convert spectral frequency domain information to axial spatial domain information, making cos[2n0k(d+δ)] and cos[2n0k(d+δ)+2n] equal. w The interference information represented by [kz] can be effectively separated, meaning the self-interference signal of the solid-phase gel can be separated from the scattering signal in the solution via FFT. Since the FFT result of the digital spectral signal is discrete, its sensitivity is insufficient to resolve nanometer-scale δ values. This application resolves δ using the phase information of FFT. During the resolution process, the reflectance spectrum R0(k) of a standard silicon wafer with an oxide layer thickness of d is pre-recorded, and the reflectance spectrum of the solid-phase gel in the solution is recorded as R1(k). An FFT is performed on R0(k)-R1(k), because 2n0k(d+δ)≈2n0k d +2n0k c δ(k c (where the spectral average wavenumber is 2n0k) is calculated after FFT. d The phase information of the corresponding peak divided by the coefficient 2n0k c The value of δ can then be obtained.

[0066] Based on the analytical principle of the self-interference signal described above, the change in the thickness (δ) of the solid gel will cause a change in the phase (2n0k(d+δ)) of the self-interference signal. Correspondingly, the change in the phase of the self-interference signal can be detected to determine whether the thickness of the solid gel has changed. In an exemplary example, step 307 may also include steps 307A and 307B.

[0067] Step 307A: The solid-phase gel in the solution after the cutting reaction is subjected to self-interference signal detection by an optical detection system to obtain the target interference signal.

[0068] Step 307B: If the phase of the target interference signal changes, the thickness change of the solid gel is determined by the optical detection system.

[0069] In one possible implementation, a white LED, after being collimated, illuminates the surface of a solid gel in solution through a beam splitter and objective lens of an optical detection system. The reflected interference signal is then focused by an imaging lens onto the end face of a multimode optical fiber. An external spectrometer is connected to the fiber and then to a computer. The target interference signal is separated using FFT, and phase analysis is performed to determine if the phase of the target interference signal has changed. If the phase of the target interference signal has changed, it is determined that the thickness of the solid gel has changed. Conversely, if the phase of the target interference signal has not changed, it is determined that the thickness of the solid gel has not changed.

[0070] Due to the influence of the scattering medium in the solution, the optical detection system initially receives a composite beam, which consists of interference and scattering signals. Therefore, before analyzing the interference signal, it is necessary to separate it. In one possible implementation, after detecting the self-interference signal of the solid-phase gel in the solution after the cutting reaction using the optical detection system and obtaining the composite reflection signal (i.e., the composite beam) within the microfluidic chip, considering that the interference signal and the scattering signal in the solution have different characteristics in the frequency domain, the target interference signal can be separated from the composite reflection signal within the microfluidic chip using a Fast Fourier Transform (FFT). Specifically, through FFT, the spatial domain information of the reflection spectrum can be converted into frequency domain information. The low-frequency part corresponds to the interference signal, and the high-frequency part corresponds to the scattering signal in the solution. By extracting the low-frequency peak from the FFT conversion result, the target interference signal can be extracted.

[0071] Step 308: If the thickness of the solid gel changes, the nucleic acid detection result of the sample containing the nucleic acid sequence to be tested is output through the optical detection system.

[0072] If the sample contains the target nucleic acid sequence, nested RPA amplification can increase the nucleic acid concentration of the target sequence. Then, CRISPR reagent recognition and cleavage can reduce the physical thickness of the solid gel, leading to a decrease in the phase of the self-interference signal. Therefore, by analyzing whether the phase of the self-interference signal changes through an optical detection system, it can be determined whether the thickness of the solid gel has changed (i.e., whether the thickness has decreased). If the thickness of the solid gel changes, it can be confirmed that the sample contains the target nucleic acid sequence, and the corresponding nucleic acid detection result will be output, indicating that the sample contains the target nucleic acid sequence.

[0073] Conversely, if the sample does not contain the target nucleic acid sequence, nested RPA amplification will not increase the nucleic acid concentration of the target sequence, nor will there be subsequent recognition and cleavage reactions. The physical thickness of the solid gel will not change, and the phase of the self-interference signal detected by the optical detection system will not change. Therefore, if the solid gel does not change in thickness, it can be determined that the sample does not contain the target nucleic acid sequence.

[0074] Step 309: If the sample to be tested contains the nucleic acid sequence to be tested, analyze the phase change value of the target interference signal through an optical detection system.

[0075] Step 310: Determine and output the nucleic acid concentration of the nucleic acid sequence to be tested based on the phase change value using an optical detection system.

[0076] In addition to outputting the nucleic acid detection results of the nucleic acid sequence to be tested (i.e. whether the sample to be tested contains the nucleic acid sequence to be tested), after the sample to be tested contains the nucleic acid sequence to be tested, the thickness (δ) of the solid gel and the nucleic acid concentration (C) of the nucleic acid sequence to be tested can also be determined by analyzing the phase change value of the target interference signal.

[0077] In one exemplary example, the method for analyzing the thickness of a solid-phase gel may include the following steps:

[0078] I. Analysis of the interference signal after FFT: After FFT transformation, the frequency domain representation of the interference term is as follows:

[0079] 2n0k0(d+δ)=2n0k0d+2n0k0cδ

[0080] Where: c is the average wavenumber of the spectrum; n0 is the average refractive index of silica and DNA gel; d represents the thickness of the silica layer; δ represents the thickness of the solid gel; and k0 represents the wavenumber corresponding to the low-frequency peak.

[0081] II. Extracting Phase Information: Extract the phase information of the low-frequency peak from the FFT results to obtain the phase of the interference peak. Assuming the phase of this peak is Φ(k0), δ can be calculated using the following steps:

[0082] (1) Calculate the phase information of the interference term: Φ(k0)=2n0k0(d+δ)

[0083] (2) Solving for δ: By comparing with the known standard silicon wafer reflectance spectrum R0(k), we obtain:

[0084]

[0085] Where, Phase of ΔR(k)peak atk0 represents the FFT of the standard silicon wafer reflectance spectrum R0(k)-R1(k) after 2n0k.d Phase information corresponding to the peak.

[0086] The thickness change of the solid-phase gel has a direct linear relationship with the nucleic acid concentration of the target nucleic acid sequence. As the nucleic acid concentration increases, the amplification products increase, leading to an increase in CRISPR / Cas12a reaction products, which in turn causes a change in the thickness of the solid-phase gel. By performing Fourier analysis on the interference signal, the thickness change of the solid-phase gel can be accurately determined, thereby achieving quantitative detection of the nucleic acid concentration of the target nucleic acid sequence. In another exemplary example, the method for determining the nucleic acid concentration of the target nucleic acid sequence may include the following steps:

[0087] I. Analysis of the relationship between solid-phase gel thickness and amplification products: It is assumed that there is a linear relationship between the change in solid-phase gel thickness δ and the nucleic acid concentration c of the target nucleic acid sequence. That is:

[0088] δ=αC+β(1)

[0089] Where: δ represents the thickness change of the solid-phase gel layer, C is the nucleic acid concentration of the nucleic acid sequence to be tested, α is a coefficient related to reaction efficiency and gel properties, representing the proportion of the solid-phase gel thickness change caused by the change in the nucleic acid concentration of the nucleic acid sequence to be tested; β is a constant representing the baseline thickness of the solid-phase gel at zero nucleic acid concentration. In practical applications, β is usually assumed to be 0 because there is no thickness change of the solid-phase gel at zero nucleic acid concentration.

[0090] II. Analysis of the relationship between interference signal and gel thickness: The reflection intensity I(k) is expressed as: I(k)=|U(k)| 2 ≈A1(k) 2 +A2(k) 2 +A3(k) 2 +2A2(k)A3(k)cos(2n0k(d+δ))+2A1(k)A2(k)cos(2n0k(d+δ)+2n w kz), where the effect of the solid gel thickness variation on the interference signal is reflected by the phase term 2n0k(d+δ), where δ in the phase term directly affects the intensity of the interference signal. Correspondingly, by analyzing the phase change of the interference signal, i.e., the effect of the solid gel thickness variation δ on the interference signal, it can be expressed as:

[0091] Δφ=2n0kδ (2)

[0092] Here, Δφ is the phase change caused by the change in gel thickness.

[0093] III. Analysis of the relationship between the thickness of the solid-phase gel and the nucleic acid concentration of the nucleic acid sequence to be tested: Based on formulas (1) and (2), a linear relationship can be obtained - formula (3):

[0094] Δφ=2n0k(αC) (3)

[0095] Simplifying, we get formula (4):

[0096] Δφ=γC (4)

[0097] Wherein, γ=2n0kα is a constant related to the experimental settings (e.g., wavelength λ, refractive index n0, etc.) and the properties of the solid-phase gel.

[0098] IV. Analyzing the quantitative relationship between the concentration of the nucleic acid to be detected and the change in gel thickness (or phase change): By performing linear regression on the experimental data, the value of γ can be further determined, and a specific linear relationship can be obtained. By measuring the reflection signal I(k) or phase change Δφ at different nucleic acid concentrations C, the quantitative relationship between the nucleic acid concentration of the nucleic acid sequence to be detected and the change in the thickness (or phase change) of the solid gel layer can be obtained. Therefore, the final linear relationship formula is shown in formula (5):

[0099]

[0100] Where δ represents the thickness variation of the solid phase gel layer, n0 is the average refractive index of silica and DNA gel, k represents the wavenumber, γ = 2n0kα is a constant value, and C represents the nucleic acid concentration of the nucleic acid sequence to be tested.

[0101] In this embodiment, the detected interference signal is analyzed using an optical detection system. Whether the phase change of the interference signal changes determines whether the thickness of the solid-phase gel changes, and further, whether the sample contains the target nucleic acid sequence is determined based on the change in the thickness of the solid-phase gel. This achieves label-free nucleic acid detection. Furthermore, by analyzing the phase change value, the thickness change of the solid-phase gel and the nucleic acid concentration of the target nucleic acid sequence can be obtained, achieving label-free nucleic acid concentration detection.

[0102] Please refer to Figure 7 This is a schematic diagram of the structure of a microfluidic detection platform provided in an embodiment of this application. For example, as shown... Figure 7 As shown, the microfluidic detection platform 700 includes a digital microfluidic chip 701 and an optical detection system 702. The digital microfluidic chip 701 includes an upper chip and a lower chip. A solid-phase gel is embedded in the upper chip, and the lower chip contains lysis buffer and magnetic beads, magnetic bead cleaning solution, nucleic acid elution buffer, first-round RPA reagent, second-round RPA reagent, and CRISPR reagent. The digital microfluidic chip 701 is placed on the stage of the optical detection system 702.

[0103] The digital microfluidic chip 701 is used to add the sample to be tested to the lower chip, and then mix the sample to be tested sequentially with the lysis buffer and magnetic beads, the magnetic bead cleaning solution and the nucleic acid elution solution to extract sample nucleic acid droplets from the sample to be tested.

[0104] The digital microfluidic chip 701 is also used to mix the sample nucleic acid droplets with the first round RPA reagent and the second round RPA reagent to perform two rounds of RPA amplification, thereby obtaining sample product droplets containing the nucleic acid amplification products of the two rounds.

[0105] The digital microfluidic chip 701 is also used to mix the sample product droplets with the CRISPR reagent and move them to the solid gel for sequence recognition and cleavage reaction to obtain the solid gel after cleavage reaction.

[0106] The optical detection system 702 is used to perform self-interference signal detection on the solid-phase gel in the solution after the cutting reaction, and to determine and output the nucleic acid detection result of whether the sample to be tested contains the nucleic acid sequence to be tested.

[0107] Optionally, the optical detection system 702 is further used for:

[0108] Self-interference signal detection is performed on the solid gel in the solution after the cutting reaction to determine whether the thickness of the solid gel has changed.

[0109] If the thickness of the solid gel changes, the nucleic acid detection result is output, indicating that the sample contains the nucleic acid sequence to be tested.

[0110] Optionally, the optical detection system 702 is further used for:

[0111] The target interference signal was obtained by detecting the self-interference signal of the solid-phase gel in the solution after the cutting reaction.

[0112] If the phase of the target interference signal changes, it is determined that the thickness of the solid gel has changed.

[0113] Optionally, the optical detection system 702 is further used for:

[0114] Self-interference signal detection was performed on the solid-phase gel in the solution after the cutting reaction to obtain the synthetic reflection signal;

[0115] The target interference signal is separated from the synthetic reflection signal by performing a fast Fourier transform on the synthetic reflection signal within the microfluidic chip.

[0116] Optionally, the optical detection system 702 is further used for:

[0117] If the sample to be tested contains the nucleic acid sequence to be tested, analyze the phase change value of the target interference signal;

[0118] Based on the phase change value, the nucleic acid concentration of the nucleic acid sequence to be tested is determined and output.

[0119] Optionally, the lower-layer chip includes a sample loading electrode, a waste liquid storage electrode, an electrode array, and multiple liquid storage electrodes. The multiple liquid storage electrodes are respectively used to store the lysis buffer and magnetic beads, the magnetic bead cleaning solution, the nucleic acid elution buffer, the first round RPA reagent, the second round RPA reagent, and the CRISPR reagent. The sample loading electrode is used to store the sample to be tested. The electrode array is used to control the movement of the sample to be tested, the sample nucleic acid droplets, and the sample product droplets.

[0120] Optionally, the digital microfluidic chip 701 is further used for:

[0121] The sample to be tested and the lysis buffer are mixed with magnetic beads to release the sample nucleic acid in the sample to be tested, and the sample nucleic acid is enriched on the magnetic beads;

[0122] The magnetic beads are mixed with the magnetic bead cleaning solution to clean the magnetic beads.

[0123] The cleaned magnetic beads are mixed with the nucleic acid elution solution to obtain the sample nucleic acid droplets;

[0124] The digital microfluidic chip 701 is also used for:

[0125] The first round of RPA reagent was mixed with the sample nucleic acid droplets to obtain the first round of amplification products;

[0126] The second round of RPA reagent is mixed with the first round of amplification products to obtain the sample product droplet containing the nucleic acid amplification products from both rounds.

[0127] In summary, this application provides a nucleic acid detection method based on gel self-interference: by integrating an optical detection system and a digital microfluidic chip, a solid-phase gel is embedded in the digital microfluidic chip as a sensing unit for the nucleic acid sequence to be tested. This allows for exponential amplification of the nucleic acid sequence to be tested through nested RPA amplification during the nucleic acid detection process, and combines this with Cas12a cleavage of the solid-phase gel to determine whether the sample contains the nucleic acid sequence to be tested by detecting the self-interference signal of the solid-phase gel. This achieves both high-sensitivity nucleic acid detection and a highly specific label-free RPA detection method. Furthermore, the integration of the optical detection system and the digital microfluidic chip constructs a new automated microfluidic detection platform, further enhancing the automation and integration of the nucleic acid detection process.

[0128] An exemplary embodiment of this application also provides a nucleic acid detection device, including: at least one processor; and a memory communicatively connected to the at least one processor. The memory stores a computer program executable by the at least one processor, which, when executed by the at least one processor, causes the nucleic acid detection device to perform a gel self-interference-based nucleic acid detection method according to an embodiment of this application.

[0129] It should be noted that, in addition to processors and memory, this nucleic acid testing device may also include digital microfluidic chips and optical detection systems.

[0130] An exemplary embodiment of this application also provides a non-transitory computer-readable storage medium storing a computer program, wherein the computer program, when executed by a processor of a nucleic acid detection device, is used to cause the computer to perform a gel self-interference-based nucleic acid detection method according to an embodiment of this application.

[0131] An exemplary embodiment of this application also provides a computer program product, including a computer program, wherein, when executed by a computer's processor, the computer program is used to cause the computer to perform a gel-based self-interference nucleic acid detection method according to an embodiment of this application.

Claims

1. A nucleic acid detection method based on gel self-interference, characterized in that, The method is applied to a microfluidic detection platform, which includes a digital microfluidic chip and an optical detection system. The digital microfluidic chip includes an upper chip and a lower chip. A solid-phase gel is embedded in the upper chip. The lower chip includes a sample loading electrode, a waste liquid storage electrode, an electrode array, and multiple liquid storage electrodes. The multiple liquid storage electrodes are respectively used to store lysis buffer and magnetic beads, magnetic bead cleaning buffer, nucleic acid elution buffer, first-round RPA reagent, second-round RPA reagent, and CRISPR reagent. The sample loading electrode is used to store the sample to be tested. The dielectric layer thickness of the digital microfluidic chip is 5 μm. The electrode array is designed as 4×16. The digital microfluidic chip is placed on the stage of the optical detection system. The method includes: After the sample to be tested is added to the lower chip, the sample to be tested is mixed sequentially with the lysis buffer and magnetic beads, the magnetic bead cleaning solution and the nucleic acid elution solution through the digital microfluidic chip to extract sample nucleic acid droplets from the sample to be tested; The sample nucleic acid droplets are mixed with the first and second rounds of RPA reagents using the digital microfluidic chip to perform two rounds of RPA amplification, resulting in sample product droplets containing the nucleic acid amplification products from both rounds. The electrode array is used to control the movement of the sample to be tested, the sample nucleic acid droplets, and the sample product droplets. The step of mixing the sample nucleic acid droplets with the first and second rounds of RPA reagents using the digital microfluidic chip to perform two rounds of RPA amplification, resulting in sample product droplets containing the nucleic acid amplification products from both rounds, includes: The first round of RPA reagent is mixed with the sample nucleic acid droplets through the electrode array to obtain the first round of amplification products; The second round of RPA reagent is mixed with the first round of amplification products through the electrode array to obtain the sample product droplet containing the nucleic acid amplification products of both rounds. The sample product droplets are mixed with the CRISPR reagent using the digital microfluidic chip and moved to the solid gel for sequence recognition and cleavage reaction to obtain the cleavage reaction solid gel. The optical detection system is used to detect the self-interference signal of the solid gel in the solution after the cutting reaction, and the nucleic acid detection result of whether the sample contains the nucleic acid sequence to be tested is determined and output. The step of detecting the self-interference signal of the solid-phase gel in the solution after the cleavage reaction using the optical detection system to determine and output the nucleic acid detection result of whether the sample contains the nucleic acid sequence to be tested includes: The target interference signal is obtained by detecting the self-interference signal of the solid gel in the solution after the cutting reaction using the optical detection system. If the phase of the target interference signal changes, the optical detection system determines that the thickness of the solid gel has changed. If the thickness of the solid gel changes, the optical detection system outputs the nucleic acid detection result, indicating that the sample contains the nucleic acid sequence to be tested.

2. The method according to claim 1, characterized in that, The step of detecting the self-interference signal of the solid-phase gel in the solution after the cutting reaction using the optical detection system to obtain the target interference signal includes: The optical detection system is used to detect the self-interference signal of the solid gel in the solution after the cutting reaction to obtain the synthetic reflection signal. The optical detection system performs a fast Fourier transform on the synthetic reflection signal to separate the target interference signal from the synthetic reflection signal within the microfluidic chip.

3. The method according to claim 1, characterized in that, The method further includes: If the sample to be tested contains the nucleic acid sequence to be tested, the phase change value of the target interference signal is analyzed by the optical detection system; The optical detection system determines and outputs the nucleic acid concentration of the nucleic acid sequence to be tested based on the phase change value.

4. The method according to claim 1, characterized in that, The step of mixing the sample to be tested sequentially with the lysis buffer and magnetic beads, the magnetic bead washing solution, and the nucleic acid elution buffer using the digital microfluidic chip to extract sample nucleic acid droplets from the sample to be tested includes: The sample to be tested and the lysis buffer are mixed with magnetic beads through the electrode array to release the sample nucleic acid in the sample to be tested, and the sample nucleic acid is enriched on the magnetic beads; The magnetic beads are cleaned by mixing the magnetic beads with the magnetic bead cleaning solution using the electrode array. The cleaned magnetic beads are mixed with the nucleic acid elution solution using the electrode array to obtain the sample nucleic acid droplets; 5. A microfluidic detection platform, characterized in that, The microfluidic detection platform includes a digital microfluidic chip and an optical detection system. The digital microfluidic chip includes an upper chip and a lower chip. The upper chip embeds a solid-phase gel. The lower chip includes a sample loading electrode, a waste liquid storage electrode, an electrode array, and multiple liquid storage electrodes. The multiple liquid storage electrodes are used to store lysis buffer and magnetic beads, magnetic bead cleaning buffer, nucleic acid elution buffer, first-round RPA reagent, second-round RPA reagent, and CRISPR reagent, respectively. The sample loading electrode is used to store the sample to be tested. The dielectric layer thickness of the digital microfluidic chip is 5 μm. The electrode array is designed as 4×16. The digital microfluidic chip is placed on the stage of the optical detection system. The digital microfluidic chip is used to add the sample to be tested to the lower chip, and then mix the sample to be tested sequentially with the lysis buffer and magnetic beads, the magnetic bead cleaning solution and the nucleic acid elution solution to extract sample nucleic acid droplets from the sample to be tested. The digital microfluidic chip is also used to mix the sample nucleic acid droplets with the first round RPA reagent and the second round RPA reagent to perform two rounds of RPA amplification, thereby obtaining sample product droplets containing the nucleic acid amplification products of the two rounds; the electrode array is used to control the movement of the sample to be tested, the sample nucleic acid droplets, and the sample product droplets. The digital microfluidic chip is also used to mix the sample product droplets with the CRISPR reagent and move them to the solid gel for sequence recognition and cleavage reaction to obtain the solid gel after cleavage reaction. Specifically, the digital microfluidic chip is used to mix the first round of RPA reagent with the sample nucleic acid droplets through the electrode array to obtain the first round of amplification products; The second round of RPA reagent is mixed with the first round of amplification products through the electrode array to obtain the sample product droplet containing the nucleic acid amplification products of both rounds. The optical detection system is used to perform self-interference signal detection on the solid gel after the cutting reaction, and to determine and output the nucleic acid detection result of whether the sample to be tested contains the nucleic acid sequence to be tested; Specifically, the optical detection system is used to detect the self-interference signal of the solid gel in the solution after the cutting reaction to obtain the target interference signal; if the phase of the target interference signal changes, it is determined that the thickness of the solid gel has changed; if the thickness of the solid gel changes, the nucleic acid detection result of the sample containing the nucleic acid sequence to be tested is output.

6. A nucleic acid testing device, comprising: processor; as well as Stored program memory, The program includes instructions that, when executed by the processor, cause the processor to perform the nucleic acid detection method based on gel self-interference according to any one of claims 1-4.

7. A non-transitory computer-readable storage medium storing computer instructions, wherein, The computer instructions are used to cause the computer to execute the nucleic acid detection method based on gel self-interference according to any one of claims 1-4.

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