A biosensor and a preparation method and application thereof

Abstract

The application relates to the technical field of biosensors, and specifically discloses a biosensor and a preparation method and application thereof, wherein the biosensor comprises a substrate, a DNA origami structure adsorbed on the substrate, and a CRISPR recognition unit adsorbed on the DNA origami structure; the substrate comprises a double-layered corner graphene layer and a gold nano array layer which are arranged in a stack; the DNA origami structure is a tetrahedron structure constructed by eight DNA single chains shown in nucleic acid sequences such as SEQ ID No. 1-8 through a DNA origami technology; and the CRIPSR recognition unit is specifically combined with a nucleic acid sequence to be detected. The biosensor can recognize low-abundance nucleic acid targets in complex biological samples by combining a double-layered corner graphene, a gold nano array, a CRISPR-Cas unit and a DNA origami unit, and high-specificity detection of single nucleotide polymorphism in ultra-low-concentration samples is realized.

Description

Technical Field

[0001] This application relates to the field of biosensor technology, and in particular to a biosensor, its preparation method, and its application. Background Technology

[0002] Circulating tumor DNA (ctDNA) refers to somatic DNA from tumor cells that is shed or released into the human circulatory system after apoptosis. It is characterized by high sensitivity, high specificity, and a short half-life, making it a distinctive tumor biomarker. The main methods for detecting single nucleotide polymorphisms (SNPs) in ctDNA are polymerase chain reaction (PCR) based methods, including next-generation sequencing (NGS), droplet digital PCR (ddPCR), and amplified difficult-to-resolve mutation systems (ARMS). However, these methods are time-consuming and cannot identify all SNPs.

[0003] With increasing attention being paid to ctDNA, other detection technologies have been developed, including isothermal amplification, microfluidics, and biosensors. Surface plasmon resonance (SPR) biosensors offer several advantages, including tagless operation, fast real-time response, and high-throughput testing. However, current SPR biosensors still face many challenges, making it difficult to achieve highly specific detection of SNPs in ultra-low concentration samples. Therefore, existing technologies require further improvement. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this application is to provide a biosensor, its preparation method and application, which aims to solve the problems of low sensitivity and poor specificity of the prior art in detecting single nucleotide polymorphisms in ultra-low concentration samples.

[0005] The technical solution of this application is as follows:

[0006] A first aspect of this application provides a biosensor comprising: a substrate including a stacked bilayer corner graphene layer and a gold nanoarray layer; a DNA origami structure adsorbed on the gold nanoarray layer; and a CRISPR recognition unit adsorbed on the DNA origami structure; wherein the DNA origami structure is a tetrahedral structure constructed by DNA origami technology from eight single strands of DNA with nucleic acid sequences as shown in SEQ ID No. 1-8; and the CRISPR recognition unit specifically binds to the nucleic acid sequence to be detected.

[0007] Preferably, the torsion angle θ of the two graphene layers in the double-layered corner graphene layer satisfies 5°≤θ<30°.

[0008] Preferably, the substrate further includes a substrate layer comprising a silicon dioxide layer and a silicon layer stacked sequentially, wherein the silicon dioxide layer is located on the side of the bilayer corner graphene layer opposite to the gold nanoarray layer.

[0009] A second aspect of this application provides a method for preparing a biosensor according to the first aspect of this application, comprising the steps of: S1, preparing a substrate; S2, preparing a DNA origami structure; S3, preparing a CRISPR recognition unit; and S4, combining the DNA origami structure and the CRISPR recognition unit with the substrate to obtain a biosensor.

[0010] Preferably, the method for preparing the substrate includes the following steps:

[0011] S11. Spin-coating polymethyl methacrylate (PMMA) onto a chemical vapor deposition-grown graphene single crystal grown on copper foil to obtain a first substrate precursor; S12. Cutting the first substrate precursor using a femtosecond laser and etching the copper foil therein with an etching solution to obtain a second substrate precursor with parallel crystal phases; S13. Transferring the second substrate precursor to the center of a silicon dioxide / silicon substrate and removing the polymethyl methacrylate using an acetone solution to obtain a third substrate precursor; S14. Transferring the second substrate precursor to an attached polymethyl methacrylate... A fourth substrate precursor is obtained on a glass slide containing methylsiloxane; S15, the third substrate precursor is placed on a high-precision rotating platform and rotated by a torsion angle θ. The fourth substrate precursor is then attached to the third substrate precursor by a 3D rotating platform to obtain a fifth substrate precursor; S16, the fifth substrate precursor is etched using a plasma lithography machine to remove polymethyl methacrylate from the fifth substrate. Subsequently, a chromium metal layer and a gold metal layer are deposited sequentially on the fifth substrate precursor using a vapor deposition apparatus to obtain the substrate.

[0012] Preferably, the torsion angle θ satisfies 5°≤θ<30°.

[0013] Preferably, the method for preparing the DNA origami structure includes the following steps: adding a solution containing eight single-stranded DNA molecules with nucleic acid sequences as shown in SEQ ID No. 1-8 to a Tris-EDTA buffer solution for mixing, followed by denaturation and annealing treatment to form a DNA origami structure with a tetrahedral structure.

[0014] Preferably, the denaturation treatment temperature is 95°C, and the remodeling treatment temperature is 0–4°C.

[0015] Preferably, the specific steps for binding the DNA origami structure and CRISPR recognition unit to the substrate include: S41, rinsing the substrate with TM buffer and drying it with nitrogen; S42, dropping the DNA origami structure onto the substrate, and then rinsing the substrate with TM buffer; S43, dropping the CRISPR recognition unit onto the product of S42, coating it with 6-mercapto-1-hexanol, then dropping a gold nanoparticle solution, and finally rinsing with TM buffer and drying it with nitrogen to obtain the biosensor.

[0016] A third aspect of this application provides an application of the biosensor of the first aspect of this application in the preparation of gene diagnostic products.

[0017] Compared with the prior art, this application has the following advantages:

[0018] (1) This application uses bilayer twisted graphene (tBLG) to precisely match the absorption characteristics of the van Hoff singularity (VHS) in tBLG with the resonance peak of the heterojunction structure between the gold nanoarray layers on the surface of tBLG, thereby significantly enhancing the photocurrent of the biosensor and amplifying the signal.

[0019] (2) The DNA fold structure binds to gold nanoparticles (AuNPs) in the gold nanoarray layer, causing the DNA fold structure to adsorb onto the surface of the gold nanoarray layer, thus maintaining the precise distance between each AuNP in the gold nanoarray layer. The presence of AuNPs can significantly change the resonance peak position of the heterojunction structure of the gold nanoarray, disrupting its coupling mode with tBLG, resulting in a reduction in photocurrent signal. The CRISPR recognition unit ensures the single-base recognition capability of the biosensor, and then the cleaving capability of the CRISPR recognition unit cuts the DNA origami structure to release the encapsulated AuNPs. As the external dielectric environment recovers, the coupling mode is regenerated. That is, by controlling the coupling mode formed between tBLG and the heterojunction structure of the gold nanoarray layer, the photocurrent of the biosensor is changed, and different DNA sequences are responded to, thus completing the detection. Combined with the precise manipulation of the DNA origami structure at the nanoscale, the biosensor can achieve detection in the attomole (amol) range.

[0020] (3) The biosensor of this application combines tBLG, gold nanoarray, CRISPR recognition unit and DNA origami structure to identify low-abundance nucleic acid targets in complex biological samples, achieving highly specific detection of SNPs in ultra-low concentration samples. The biosensor of this application provides a new paradigm for DNA detection in the two-dimensional limit through light-matter interaction, laying the foundation for the development of high-sensitivity molecular detection and nonlinear electro-optic modulation. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0022] Figure 1 This is a schematic diagram of the structure of the biosensor provided in the embodiments of this application;

[0023] Figure 2 A schematic flowchart of the substrate preparation method provided in the embodiments of this application;

[0024] Figure 3 The sensitivity detection diagram of the biosensor provided in the embodiments of this application. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings and examples. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of this application. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0026] It should be noted that if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on enabling those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed in this application.

[0027] In recent years, cancer deaths have risen so dramatically that by the end of 2020, it was considered the leading cause of death globally, with nearly 10 million deaths recorded. The success of cancer treatment largely depends on the stage at which it is detected, making early diagnosis crucial for effective treatment.

[0028] ctDNA refers to the somatic DNA of tumor cells released into the circulatory system after shedding or apoptosis, and is a characteristic tumor biomarker. ctDNA detection can detect traces of tumors in the blood. ctDNA is cell-free extracellular DNA found in bodily fluids such as blood, synovial fluid, and cerebrospinal fluid. It is mainly composed of single-stranded or double-stranded DNA, as well as mixtures of single-stranded or double-stranded DNA, existing in both DNA-protein complexes and free DNA forms. It is a tumor marker with broad application prospects, high sensitivity, and high specificity, applicable to various types of tumors. Compared to protein markers, ctDNA rarely produces false positives because it originates from genomic mutations in tumor cells. Furthermore, ctDNA has a short half-life, accurately reflecting the current state of the tumor. Single nucleotide polymorphisms (SNPs) refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. Disease diagnosis, customized treatment, and precision medicine require the ability to elucidate gene mutations and variations at the molecular, single-base level. Therefore, detecting SNPs in ctDNA is an ideal detection method.

[0029] The primary methods for detecting single nucleotide polymorphisms (SNPs) in ctDNA are polymerase chain reaction (PCR)-based methods, including next-generation sequencing (NGS), droplet digital PCR (ddPCR), and amplified mutagenesis systems (ARMS). However, these methods are time-consuming and cannot identify all SNPs. With increasing focus on ctDNA, other detection technologies have been developed, including isothermal amplification, microfluidics, and biosensors. Surface plasmon resonance (SPR) biosensors offer advantages such as tagless operation, rapid real-time response, and high-throughput testing. However, current SPR biosensors still face many challenges, making it difficult to achieve highly specific detection of SNPs in ultra-low concentration samples.

[0030] On the other hand, graphene has attracted widespread attention due to its outstanding characteristics such as electrochemical properties, adsorption properties, mechanical strength, and flexibility, making it a candidate material for biosensors. Twisted bilayer graphene (tBLG) leads to enhanced light absorption and Raman resonance. Integration with optical nanoantennas enhances the light-matter interaction in two-dimensional materials, and the detection sensitivity of sensors can be effectively improved through the rational construction of tBLG / nanoantenna structures. However, graphene-based biosensors also face many challenges, such as the need to enhance the signal-to-noise ratio (SNR) to identify low-abundance nucleic acid targets in complex biological samples. CRISPR-Cas technology is a revolutionary gene-editing technology that can distinguish nucleic acid sequences at the single-base level, and subsequent trans-cutting can be used for various gene detection methods. However, traditional single-stranded DNA (ssDNA) probes immobilized on the chip surface hinder probe-target interactions due to two main problems: entanglement between adjacent probes (uncontrolled distance between gold particles) and entanglement between probes located on the surface (uncontrolled distance between gold particles and the surface), both of which result in invalid probes occupying the chip surface. It is evident that existing biosensors still have many problems, making it difficult to achieve highly specific detection of SNPs in ultra-low concentration samples.

[0031] Based on this, a first aspect of the embodiments of this application provides a biosensor, including:

[0032] The substrate, along with the DNA origami structure and CRISPR recognition unit adsorbed onto it, are described. The substrate comprises a stacked bilayer of corner-turned graphene layers and a gold nanoarray layer. The DNA origami structure is a tetrahedral structure constructed from eight single-stranded DNA molecules, the sequences of which are shown in SEQ ID No. 1-8. The CRISPR recognition unit is configured to specifically bind to a particular nucleic acid sequence to achieve its recognition function.

[0033] In some embodiments, the 5' end of SEQ ID No. 1, SEQ ID No. 3, and SEQ ID No. 5 is also connected to a globular group -SH-C6-. The addition of the globular group can improve the adsorption of the DNA origami structure with the gold nanoparticles in the gold nanoarray.

[0034] In some embodiments, the torsion angle θ of the bilayer torsion graphene metal layer in the bilayer torsion graphene structure substrate satisfies 5° ≤ θ < 30°. For example, the torsion angle θ is an angle less than 30°, such as 5°, 7°, 9°, 9.4°, 10°, 13.2°, 15°, 20°, 22°, 25°, 26°, 27°, 28°, 29°, or 29.9°.

[0035] In some embodiments, the substrate includes a substrate layer comprising a silicon dioxide layer and a silicon layer stacked sequentially, the silicon dioxide layer being located on the side of the bilayer corner graphene layer facing away from the gold nanoarray layer.

[0036] The CRISPR recognition unit includes a CRISPR-A sequence and a crRNA sequence. The CRISPR-A sequence adsorbs onto the DNA origami structure, and the crRNA is used to specifically aggregate the nucleic acid to be tested. In some embodiments, the CRISPR-A sequence is shown in SEQ ID No. 9, and the crRNA sequence is shown in SEQ ID No. 10. It is used to recognize T-DNA obtained by reverse transcription of miRNA21.

[0037] Furthermore, the working principle of biosensors is as follows: Figure 1 As shown, in bilayer twisted graphene (tBLG), the Dirac cones of each monolayer intersect to form saddle points, leading to van Hoff singularities (VHS) in the density of states (DOS), thereby enhancing light absorption and Raman resonance. Creating high local density of states (LDOS) near the interface between nanostructures and two-dimensional materials is considered to induce coupling modes in light-matter interactions. Since the formation of these coupling modes requires precise alignment between structural resonances and material band gaps, primers from external reactors may disrupt these modes, thus presenting a novel biosensing mechanism.

[0038] By using bilayer twisted graphene (tBLG), the absorption characteristics of the van Hoff singularity (VHS) in tBLG are precisely matched with the resonance peak of the heterojunction structure located between the tBLG surface and the gold nanoarray layer, which significantly enhances the photocurrent of the biosensor and thus amplifies the signal.

[0039] Furthermore, DNA origami technology involves the self-assembly of two-dimensional or three-dimensional structures, eliminating the persistent problems of entanglement and surface aberrations in traditional ssDNA probes and improving sensor sensitivity. Gold nanoparticles (AuNPs) with tetrahedral structures are combined with DNA origami structures and fixed on the surface of a gold nanodisk to maintain precise distances between individual AuNPs within the gold nanodisk layer. The presence of AuNPs significantly alters the resonance peak positions of the heterojunction structure of the gold nanodisk, disrupting its coupling mode with tBLG and leading to a reduction in photocurrent signal. A CRISPR recognition unit ensures the biosensor's single-base recognition capability. The CRISPR recognition unit then cleaves the DNA fold structure to release the encapsulated AuNPs, and the coupling mode is regenerated as the external dielectric environment recovers. In other words, by controlling the coupling mode formed between tBLG and the heterojunction structure of the gold nanodisk layer, the photocurrent of the biosensor is altered, enabling responses to different DNA sequences and achieving detection. By combining precise manipulation of the nanoscale DNA origami structure and the ultrathin structure of tBLG, the biosensor can achieve detection in the attomole (amol) range.

[0040] The biosensor of the first aspect of this application combines tBLG, gold nanoarray, CRISPR recognition unit and DNA origami structure to identify low-abundance nucleic acid targets in complex biological samples, thereby achieving highly specific detection of SNPs in ultra-low concentration samples.

[0041] A second aspect of this application provides a method for preparing a biosensor according to the first aspect of this application, comprising the steps of:

[0042] S1. Prepare the substrate;

[0043] S2. Prepare DNA origami structures;

[0044] S3. Fabricate the CRISPR recognition unit;

[0045] S4. Combine the DNA origami structure and CRISPR recognition unit with the substrate to prepare a biosensor.

[0046] In some implementations, please refer to Figure 2 The substrate preparation method includes the following steps:

[0047] S11. Spin-coating polymethyl methacrylate (PMMA) onto a graphene single crystal grown by chemical vapor deposition on a copper foil to obtain the first substrate precursor.

[0048] S12. Use a femtosecond laser to cut the first substrate precursor and use an etching solution to etch the copper foil therein to obtain a second substrate precursor with parallel crystal phases.

[0049] S13. Transfer the second substrate precursor to the center of the silicon dioxide / silicon substrate layer, and remove the PMMA layer using acetone solution to obtain the third substrate precursor.

[0050] S14. The second substrate precursor is transferred to a glass slide with polydimethylsiloxane (PDMS) attached to obtain the fourth substrate precursor.

[0051] S15. Place the third substrate precursor on a high-precision rotating platform, rotate it by a torsion angle θ, and then attach the fourth substrate precursor to the third substrate precursor using a 3D rotating platform to obtain the fifth substrate precursor.

[0052] S16. Use an electro-lithography (EBL) machine to etch the fifth substrate precursor to remove PMMA from the fifth substrate. Then use a vapor deposition machine to deposit a chromium metal layer and a gold metal layer onto the fifth substrate precursor in sequence to obtain the substrate.

[0053] In some embodiments, the specific steps of spin coating in S11 are as follows: first spin coating PMMA at 750 rpm for 10 s, then spin coating PMMA at 2000 rpm for 60 s, followed by heating at 100°C for 60 s.

[0054] In some embodiments, the etching solution includes hydrochloric acid, hydrogen peroxide, and water, with a volume ratio of hydrochloric acid, hydrogen peroxide, and water of 1:1:8.

[0055] In some implementations, the torsion angle θ in S14 satisfies 5° ≤ θ < 30°. For example, the torsion angle is an angle less than 30°, such as 5°, 7°, 9°, 9.4°, 10°, 13.2°, 15°, 20°, 22°, 25°, 26°, 27°, 28°, 29°, or 29.9°.

[0056] In some embodiments, the thickness of the chromium metal layer deposited by vapor deposition is 5 nm, and the thickness of the gold metal layer deposited by vapor deposition is 45 nm.

[0057] In some embodiments, the method for preparing DNA origami structures includes the following steps:

[0058] S21. The eight nucleotide sequences (as shown in SEQ ID No. 1-8) for constructing the DNA origami structure are added to Tris-EDTA (TE) buffer solution and mixed for denaturation and annealing to form a DNA origami structure with a tetrahedral structure.

[0059] In some implementations, the denaturation temperature is 95°C, the denaturation time is 10 min, and the annealing temperature is 0–4°C. For example, annealing can be performed directly on ice.

[0060] In some embodiments, the method for fabricating the CRISPR recognition unit includes the following steps:

[0061] S31. Prepare the CRISPR-A sequence into a solution to obtain the CRISPR recognition unit.

[0062] In some implementations, the specific steps of S4 include:

[0063] S41. Rinse the substrate with TM buffer and dry it with nitrogen gas.

[0064] S42. Drop the DNA origami structure onto the substrate and rinse with TM buffer for 5 minutes after 30 minutes.

[0065] S43. The CRISPR recognition unit is dropped onto the product from S42. After 30 min, 6-mercapto-1-hexanol is coated, followed by the addition of a gold nanoparticle solution after 15 min. After waiting 30 min, the mixture is rinsed with TM buffer and dried with nitrogen gas to obtain the biosensor. The TM buffer contains Tris-HCl and MgSO4. 6-Mercapto-1-hexanol (MCH) can seal the surface of the gold nanoarray, preventing adsorption of other substances during subsequent preparation or reactions. The added gold nanoparticle solution binds to the PloyA structure at the top of the DNA origami structure, disrupting the localization of the light field of the gold nanostructure.

[0066] After matching the absorption peak of the twisted graphene with the plasmon resonance peak, a light source with the matching absorption peak is selected to illuminate the sensor. DNA strands that can be cleaved by the Cas12a protein are then bound to DNA origami attached to the surface of the nanoarray, with the other end connected to gold nanoparticles (AuNPs). Due to the alteration of the external environment caused by the AuNPs, the resonance peak of the gold nanoarray shifts, resulting in a mismatch between the electromagnetic field localization of the nanoarray and the twisted graphene, leading to a decrease in photoelectric conversion efficiency and photocurrent. Upon detection of the target DNA, the Cas12a protein, guided by crRNA, cleaves the DNA strand, causing the AuNPs to detach from the sensor surface. This results in a significant change in the near-field electromagnetic field intensity, causing large fluctuations in the photocurrent value. By calculating the change in current value before and after cleavage, the target virus can be identified and quantitatively analyzed, thus enabling the sensor to recognize the target DNA.

[0067] A third aspect of this application provides an application of the biosensor of the first aspect of this application in the preparation of gene diagnostic products. The diagnostic products can be used to diagnose whether a patient has a certain type of cancer or carries a certain virus.

[0068] In some implementations, when using diagnostic products to detect whether a patient has a certain type of cancer, only a blood sample needs to be taken. The miRNA in the sampled blood is reverse transcribed to create T-DNA. A crRNA is designed based on the biomarker of the cancer to be detected. The T-DNA, Cas12a protein reaction solution (which is commercially available and provides suitable reaction conditions for Cas12a protein), Cas12 protein, and crRNA are mixed and added to a sensor. The sensor is then placed in a flow cell and incubated with a CRISPR reaction at 37°C for 30 minutes. The sensor is then rinsed with TM buffer, and proteinase K is added for another 10 minutes to remove non-specific adsorption. Finally, the sensor is rinsed with TM buffer. The change in photocurrent before and after sample addition can be used to identify specific cancers.

[0069] In some implementations, the amount of Cas12a protein added is 20 μL, the amount of protein reaction solution added is 20 μL, the amount of crRNA added is 10 μL, the amount of T-DNA added is 10 μL, the total volume of the detection solution system is 200 μL, and ultrapure water is used to make up the volume.

[0070] In some implementations, when using diagnostic products to detect whether a patient has a certain type of cancer, only a blood sample needs to be taken. A crRNA marker designed based on the virus to be detected is then used. The sampled blood, Cas12a protein reaction solution, Cas12 protein, and crRNA are mixed and added to a sensor. The sensor is then placed in a flow cell and incubated with a CRISPR reaction at 37°C for 30 minutes. The sensor is then rinsed with TM buffer, and proteinase K is added for another 10 minutes to remove non-specific adsorption. Finally, the sensor is rinsed with TM buffer. The change in photocurrent before and after sample addition can be used to diagnose specific cancers.

[0071] In some implementations, the amount of Cas12a protein added is 20 μL, the amount of protein reaction solution added is 20 μL, the amount of crRNA added is 10 μL, the amount of sampled blood added is 10 μL, the total volume of the detection system is 200 μL, and ultrapure water is used to make up to the final volume.

[0072] The following specific examples will provide further details.

[0073] Example 1: Fabrication of a biosensor

[0074] The specific preparation method includes the following steps:

[0075] (1) Polymethyl methacrylate (PMMA) was spin-coated onto a single crystal of graphene (Copper foil / Gr) grown on a copper foil by chemical vapor deposition. The spin-coating was first performed at 750 rpm for 10 s, then at 2000 rpm for 60 s, followed by heating at 100 °C for 60 s to obtain PMMA / graphene / copper foil (Copper foil / Gr / PMMA). The PMMA / graphene / copper foil in (1) was cut using a femtosecond laser with a wavelength of 800 nm. The copper foil was etched using an etchant with a volume ratio of HCl:H2O2:H2O of 1:1:8 to obtain two PMMA / graphene (PMMA / Gr) pieces with parallel crystal phases. One of the PMMA / graphene pieces was transferred to the center of a silicon dioxide / silicon substrate to obtain Si / SiO2 / Gr / PMMA. The PMMA coating was removed using an acetone solution to obtain Si / SiO2 / Gr. A Si / SiO2 / Gr substrate was placed on a high-precision rotating platform as the bottom layer of the tBLG. Another PMMA / Gr substrate was then transferred onto a glass carrier with attached polydimethylsiloxane (PDMS) to obtain a glass carrier / PDMS / PMMA / graphene substrate (Glass / PDMS / PMMA / Gr). The bottom graphene was rotated 9.4° on the silicon dioxide / silicon substrate using the rotating platform, and the top graphene was adhered to the bottom graphene using a 3D translation platform (E1-G, Meta) to obtain Si / SiO2 / tBLG / PMMA / Glass. The substrate was then immersed in pure acetone solution for 30 min and thermally annealed at 340 °C under vacuum for 3 h to remove residual PMMA, yielding Si / SiO2 / tBLG. PMMA was spin-coated onto Si / SiO2 / tBLG at 750 rpm for 10 s, followed by 4000 rpm for 60 s, and then heated at 100°C for 60 s to obtain Si / SiO2 / tBLG / PMMA. PMMA was etched using an electroplating (EBL) machine, and after development and fixing, the exposed PMMA was removed. 5 nm of chromium and 45 nm of gold were deposited using a deposition apparatus, and excess metal was removed using acetone to obtain Si / SiO2 / tBLG / Au-nanodisk.

[0076] (2) Eight solutions containing single-stranded DNA (sequences shown in SEQ ID No. 1-8, where the 5' end of single-stranded DNA of types 1, 3, and 5 includes a globular group -SH-C6-) were centrifuged at 7000×g for 5 min at 4 °C, then diluted to 100 μM with Tris-EDTA (TE) buffer and stored at -20 to -80 °C. 2 μL of each prepared solution was mixed with 184 μL of Tris-magnesium sulfate (TM) buffer to bring the final concentration of each single-stranded DNA to 1 μM. The prepared solution was heated to 95 °C and held for 10 min, then rapidly cooled on ice to obtain the DNA origami structure for later use.

[0077] (3) The Si / SiO2 / tBLG / Au-nanodisk prepared in (1) was washed with TM buffer and dried with nitrogen. The DNA origami structure in (2) was incubated for 30 min, followed by washing with TM buffer for 5 min. Then, a solution containing CRISPR-A (sequence shown in SEQ ID No. 9) was added and incubated for 30 min. Then, 6-mercapto-1-hexanol was added for MCH blocking treatment for 15 min. Finally, a gold nanoparticle (Au-NP) solution was added and incubated for 30 min.

[0078] (4) The product in (3) is washed and dried with nitrogen to obtain a biosensor with the structure Si / SiO2 / tBLG / Au-nanodisk / DNA origami / AuNP.

[0079] Example 2: Sensitivity Detection of Biosensors

[0080] To characterize the sensitivity of the biosensor, the photocurrent values ​​generated before and after different concentrations (100 pmol-10 amol) of target DNA (T-DNA, sequence as shown in SEQ No ID.11) were added to the biosensor prepared in Example 1 were measured under optimal conditions.

[0081] Electrical measurements were performed using a CIQTEK Melab instrument with a lock-in amplifier, while photoelectric measurements were performed using a scanning photocurrent microscope. A laser source with adjustable output frequencies at wavelengths of 405 nm, 532 nm, 660 nm, and 785 nm was used. The output frequency of the light source was set to 1 kHz, and the laser light was focused onto the device through a ×100 objective lens to form a 1 μm spot. The device was connected to a preamplifier and an ITECH IT2805 voltage source to provide bias voltage, and the photocurrent was measured using a lock-in amplifier. During the scanning of the laser spot on the instrument, the induced photocurrent and beam position were simultaneously recorded and displayed with the aid of a computer. All electrical and photoelectric measurements were performed at room temperature.

[0082] T-DNA (sequence shown in SEQ ID No. 11) was generated by reverse transcription from miRNA-21, and the crRNA sequence is shown in SEQ ID No. 10. A mixture of crRNA and T-DNA was added to the biosensor and incubated with a CRISPR reaction at 37°C. Subsequently, the signal threshold was assessed using the synthesized target DNA template. The threshold was calculated according to the guidelines of the International Union of Pure and Applied Chemistry (IUPAC) and included the sum of blank measurements and three times their standard deviation. The test results are as follows... Figure 3 As shown, from Figure 3 As can be seen, the LOD value is significantly reduced, which may be attributed to the high sensitivity of the coupling mode between the Au-nanodisk structural resonance and the bandgap alignment of the tBLG material to the external environment, as well as the highly versatile control of 3D spatial structures through DNA origami. This further demonstrates that the biosensor prepared in Example 1 can achieve highly specific detection of target SNPs at ultra-low concentrations (below 1 fM). Compared with traditional CRISPR detection technologies, such as CRISPR-SHERLOCK, its detection limit is enhanced by more than four orders of magnitude.

[0083] In summary, the biosensor of this application, by combining tBLG, gold nanoarrays, CRISPR recognition units, and DNA origami structures, can identify low-abundance nucleic acid targets in complex biological samples, achieving highly specific detection of SNPs in ultra-low concentration samples. This sensor, based on light-matter interaction, provides a new paradigm for DNA detection in the two-dimensional limit, laying the foundation for the development of highly sensitive molecular detection and nonlinear electro-optic modulation.

[0084] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A biosensor, characterized in that, The biosensor includes: The substrate includes a substrate layer and a stacked bilayer corner graphene layer and a gold nanoarray layer; the substrate layer includes a silicon dioxide layer and a silicon layer stacked sequentially, the silicon dioxide layer being located on the side of the bilayer corner graphene layer opposite to the gold nanoarray layer; DNA origami structure adsorbed on the gold nanoarray layer; CRISPR recognition units adsorbed onto the DNA origami structure; The DNA origami structure is a tetrahedral structure constructed by DNA origami technology from eight single strands of DNA with nucleic acid sequences as shown in SEQ ID No. 1-8. The CRISPR recognition unit specifically binds to the nucleic acid to be detected; The torsion angle θ of the two graphene layers in the double-layered corner graphene layer is 9.4°.

2. A method for preparing the biosensor according to claim 1, characterized in that, Including the following steps: S1. Prepare the substrate; S2. Prepare DNA origami structures; S3. Fabricate the CRISPR recognition unit; S4. Combine the DNA origami structure and CRISPR recognition unit with the substrate to prepare a biosensor.

3. The preparation method according to claim 2, characterized in that, The method for preparing the substrate includes the following steps: S11. Spin-coating polymethyl methacrylate onto graphene grown on copper foil using chemical vapor deposition to obtain the first substrate precursor PMMA / graphene / copper foil. S12. The first substrate precursor is cut using a femtosecond laser, and the copper foil therein is etched using an etching solution to obtain a second substrate precursor PMMA / graphene with parallel crystal phases. S13. The second substrate precursor is transferred to the center of the substrate layer, and polymethyl methacrylate is removed using acetone solution to obtain the third substrate precursor Si / SiO2 / graphene. S14. The second substrate precursor is transferred to a glass slide with polydimethylsiloxane attached to obtain the fourth substrate precursor glass slide / PDMS / PMMA / graphene. S15. Place the third substrate precursor on a high-precision rotating platform and rotate it by a torsion angle θ. Then, attach the fourth substrate precursor to the third substrate precursor using a 3D rotating platform to obtain the fifth substrate precursor Si / SiO2 / tBLG / PMMA / glass substrate. S16. The fifth substrate precursor is immersed in acetone solution to remove PMMA, resulting in Si / SiO2 / tBLG; PMMA is spin-coated onto Si / SiO2 / tBLG to obtain Si / SiO2 / tBLG / PMMA; Si / SiO2 / tBLG / PMMA is etched using a plasma lithography machine to remove polymethyl methacrylate, and then a chromium metal layer and a gold metal layer are sequentially deposited using a vapor deposition machine to obtain the substrate.

4. The preparation method according to claim 2, characterized in that, The method for preparing the DNA origami structure includes the following steps: A solution containing eight single-stranded DNA molecules with nucleic acid sequences as shown in SEQ ID No. 1-8 was added to a Tris-EDTA buffer solution and mixed, followed by denaturation and annealing to form a DNA origami structure with a tetrahedral structure.

5. The preparation method according to claim 4, characterized in that, The denaturation treatment temperature is 95℃, and the remodeling treatment temperature is 0~4℃.

6. The preparation method according to claim 2, characterized in that, The specific steps for integrating the DNA origami structure and CRISPR recognition unit with the substrate include: S41. Rinse the substrate with TM buffer and dry it with nitrogen gas. S42. Drop the DNA origami structure onto the substrate, then rinse the substrate with TM buffer; S43. The CRISPR recognition unit is dropped onto the product of S42, coated with 6-mercapto-1-hexanol, followed by the addition of a gold nanoparticle solution. Finally, the product is rinsed with TM buffer and dried with nitrogen to obtain the biosensor.

7. The application of the biosensor according to claim 1 in the preparation of gene diagnostic products.

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