A DNA origami microarray biochip based on plasmon surface lattice resonance

Abstract

The present invention relates to the technical field of biochips and provides a DNA origami microarray biochip based on plasmon surface lattice resonance, comprising a chip substrate and arrayed DNA origami-plasmonic nanostructure-biomolecular probe units. A preparation method thereof comprises the following steps: extending a class A single strand from the DNA origami surface for grabbing and connecting plasmonic nanoparticles and biomolecular probes; then modifying the plasmonic nanoparticles and biomolecular probes with a class B single strand DNA that base-pairs complementary with the class A single strand; constructing hydrophilic array-arranged points on the substrate surface; connecting DNA origami to each point to form a microarray; and assembling biomolecular probes and plasmonic nanoparticles on the DNA origami through base-pairing complementary to form a plasmon-biomolecular probe microarray. The biochip can be customized for the detection of multiple or single pathogens, and the manufacturing method is economical and efficient, meeting personalized medical needs.

Description

Technical Field

[0001] The present invention relates to the technical field of biochips, and in particular to a DNA origami microarray biochip based on plasmon surface lattice resonance, and a preparation method and application thereof. Background Art

[0002] Biochips are a technology that enables high-throughput biomolecule detection and analysis by immobilizing a large number of molecular probes on a small solid substrate. They are used in areas such as drug action research, infectious disease detection, cancer diagnosis, drug screening, and food safety monitoring. Optical sensing biochips designed using optical sensing mechanisms such as surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) enable rapid response and real-time monitoring. SPR-based optical sensing enables label-free detection, eliminating the need for fluorescent or radioactive labeling of biomolecules. This reduces sample preparation steps, lowers costs, and avoids changes in the structure and function of biomolecules caused by labeling. Consequently, the role of SPR in label-free bioassays has attracted considerable attention in recent years.

[0003] The SPR effect is a key optical property of plasmonic nanomaterials. The principle of label-free detection based on SPR is that when a light source and the on-chip plasmon nanomaterial undergo surface plasmon resonance, a strong resonance peak is generated in the extinction spectrum. When biomolecules interact with molecules near the plasmonic nanomaterial (e.g., antibodies and antigens), the SPR characteristics change, shifting the plasmon resonance wavelength and phase.

[0004] When plasmonic nanostructures are arrayed at micron-nanoscale spacing, their arrangement forms a lattice-like structure, resulting in plasmon surface lattice resonance (SLR). Compared to the SPR effect, SLR produces a plasmon resonance peak with a very narrow bandwidth (2 nm). Due to the narrowband nature of SLR, it has a higher sensitivity for detecting interactions with surrounding molecules.

[0005] Existing label-free detection biochips based on the SLR effect are mainly based on simple plasmon nanoparticle arrays. The attached adsorbed biomolecular probes are usually randomly distributed, resulting in unstable detection efficiency, low device consistency and standardization. In addition, the accuracy of plasmon nanostructures is usually 50 nm or above, which limits the dynamic detection range of light sensing. Summary of the Invention

[0006] To achieve the goal of fabricating low-cost, high-precision, highly sensitive, and customizable label-free detection biochips, the present invention aims to provide a DNA origami microarray biochip based on plasmon surface lattice resonance and its preparation method. Based on the DNA origami microarray, this invention proposes a cross-scale fabrication process from the core unit of the "DNA origami-plasmonic nanostructure-biomolecular probe" assembly with sub-10 nm precision to micron-scale arrays. Leveraging the SLR effect of the plasmonic nanoarray and the interaction between the integrated biomolecular probe (such as a specific antibody) and the target analyte, real-time, label-free, highly sensitive detection is achieved.

[0007] Compared to existing technologies, the DNA origami self-assembly technique used to construct DNA origami microarray biochips allows for the self-assembly of plasmonic nanostructures—biomolecular probe units—with a precision of less than 10 nm. Each array unit can be customized with a variety of biomolecular probes (such as various specific antibodies). This high-precision DNA origami microarray biochip fabrication method is cost-effective and efficient, improves the biochemical properties of label-free detection biochips, and can meet personalized medical needs.

[0008] The embodiments of the present invention are achieved through the following technical solutions:

[0009] A DNA origami microarray biochip based on plasmon surface lattice resonance has an overall structure consisting of two parts: a chip substrate and an array of DNA origami-plasmon nanostructure-biomolecule probe units. The chip substrate is made of SiO2 with a refractive index between 1.45 and 1.5.

[0010] Furthermore, the chip substrate is periodically patterned using nanosphere etching technology, with a period of 100-1000 nm and a number of periods of the order of 100-10,000, arranged in one of a hexagonal array and a square array.

[0011] Furthermore, the DNA origami is folded by a long DNA single strand (such as a long scaffolding strand, such as the M13mp18 phage single strand) and several short DNA single strands (such as staple short strands) through base complementary pairing; its shape is one of a triangle, a quadrilateral, a hexagon, an octagon or a circle; the size of the DNA origami is between 50-300 nm.

[0012] Furthermore, the plasmonic nanostructure is a single plasmonic nanoparticle and / or a plurality of plasmonic nanoparticles.

[0013] Furthermore, the plasmon nanoparticles are one of metal nanoparticles, hybrid metal nanoparticles, and hybrid metal-dielectric nanoparticles, and their shapes are one of spheres, rods, cubes, stars, disks, and core-shell spherical structures.

[0014] A method for preparing a DNA origami microarray biochip based on plasmon surface lattice resonance comprises the following steps:

[0015] Step 1: Design DNA origami with a type-a single strand extending from its surface for grabbing and connecting one or more plasmonic nanoparticles and biomolecular probes;

[0016] Step 2: Modifying plasmonic nanoparticles and biomolecular probes with type B single-stranded DNA that is complementary to type A single-stranded bases;

[0017] Step 3: The substrate is patterned by nanosphere etching technology and surface oxygen plasma treatment to form hydrophilic array points;

[0018] Step 4: DNA origami is connected to each spot to form a microarray; specifically, the spots are connected by Mg 2+ Connecting DNA origami with hydroxyl groups to form DNA origami arrays;

[0019] Step 5: Through complementary base pairing, the self-assembled biomolecular probes and plasmonic nanoparticles are precisely and controllably positioned on the DNA origami to form a plasmon-biomolecular probe microarray, where both the biomolecular probes and plasmonic nanoparticles can be of multiple types and are combined into units in a designed manner.

[0020] More specifically, the preparation method is as follows:

[0021] S1. Mix long DNA strands (scaffold strands, such as M13mp18 phage strands) with short DNA strands (staple strands), plasmonic nanoparticle grabber strands, and biomolecule grabber strands at a molar ratio of 1:15-25:15-25:15-25. Heat to 70-90°C in a nucleic acid amplifier for 5-10 minutes, then anneal from 75°C to 25-30°C. Purify by gel electrophoresis in 0.5× TAE-MgSO4 buffer. 2+ ;

[0022] S2. Using a SiO2 substrate, treated with O2 plasma, nanospheres (e.g., polystyrene spheres) with a diameter of 500 nm are deposited on the substrate to form a hexagonally close-packed monolayer / multilayer of nanospheres. The substrate is then treated with a hexamethyldisilazane (HMDS) solution in a vacuum desiccator to add hydrophobic trimethylsilyl groups to the surface. Finally, the substrate is ultrasonically treated in water to remove the nanospheres from its surface. Hydrophilic silanol groups are then generated at the circular array sites using O2 plasma treatment. A DNA origami solution is dripped onto the etched array and incubated in a moist container for one hour to allow the DNA origami to attach to the hydrophilic sites. Excess DNA origami is then removed by multiple washes with a 0.07% Tween-20 buffer (Tris, MgCl2).

[0023] S3. Plasmonic nanoparticles (e.g., gold nanospheres / gold nanorods) and biomolecular probes (e.g., specific IgG antibodies) are modified with thiolated single-stranded DNA (e.g., PolyT, CT-9) and purified by gel electrophoresis in 0.5×TBE buffer. The modified plasmonic nanoparticle solution is added to the buffer on the surface of the substrate, and the temperature is controlled to anneal and incubate overnight under moist conditions. The substrate surface is then washed with a buffer containing MgCl2 to remove excess plasmonic nanoparticles. The modified biomolecular probe (e.g., specific IgG antibody) solution is then added to the buffer on the surface of the substrate, incubated at 37°C for at least 1 hour, and the substrate surface is rinsed with buffer. Because the grabbing strands for nanoparticles and biomolecules are pre-designed at specific locations on the DNA origami surface, the plasmonic nanoparticles and biomolecules modified with complementary single-stranded DNA will effectively connect to specific sites on the DNA origami through base complementary pairing, thus forming a "DNA origami-plasmonic nanostructure-biomolecular probe" composite structural unit.

[0024] In another aspect, the present invention also provides an application of the aforementioned biochip for biomolecule detection. Specifically, light emitted by a light source penetrates blood droplets and the biochip, and biomolecule detection data can be directly obtained based on the spectrum and resonance peak shift analysis transmitted by the photodetector and processed by a computer. Multiple resonance peak shifts can be used in conjunction to determine biomolecule detection.

[0025] The technical solutions of the embodiments of the present invention have at least the following advantages and beneficial effects:

[0026] 1. The present invention uses DNA origami self-assembly technology to construct microarray units and biomolecular probes, so that the biochip can be customized for the detection of multiple or one pathogen.

[0027] 2. The present invention manufactures array patterns by etching nanospheres, further realizing plasmon-bioprobe microarrays, which has low cost, high scalability, and high device consistency and standardization of the chip.

[0028] 3. The biological probe of the present invention has a high degree of integration, and more than a million molecular probes are highly and orderly integrated within the millimeter-scale range. Only 50 μL of blood sample is needed to detect the target object.

[0029] 4. The present invention is based on the SLR effect, has a high Q value optical sensing signal, and can achieve ultra-low concentration detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 Schematic diagram of the DNA origami microarray biochip and the "DNA origami-plasmonic nanostructure-biomolecular probe" unit provided in Example 1 of the present invention;

[0032] Figure 2 Schematic diagram of realizing a DNA origami array using a polystyrene sphere-etched substrate, as provided in Example 1 of the present invention;

[0033] Figure 3 This is a schematic diagram of the principle of the biochip provided in Example 1 of the present invention for performing biomolecule detection based on the SLR effect;

[0034] Figure 4 This is the simulated light transmittance of the plasmon nanostructure array used in Example 1 of the present invention. DETAILED DESCRIPTION

[0035] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0036] Example 1

[0037] This embodiment provides a DNA origami microarray biochip based on plasmon surface lattice resonance, such as Figure 1 As shown, Figure 1The basic structure of the biochip of the present invention is shown. This embodiment uses hexagonal DNA origami with a diameter of about 240 nm to construct two C6 rotationally symmetric plasmonic nanostructure units, one of which is a chiral assembly such as Figure 1 (1) shows that one is an achiral assembly such as Figure 1 (2) and connected to specific IgG antibodies in the gap.

[0038] The method for preparing the biochip of this embodiment includes the following steps:

[0039] S1. The M13mp18 phage single strand was mixed with the designed staple short strand, plasmonic nanoparticle grabber strand, and biomolecule grabber strand at a molar ratio of 1:20:20:20. The mixture was heated to 80°C in a nucleic acid amplifier for 5 minutes, then annealed from 75°C to 27°C. Finally, the mixture was purified by gel electrophoresis in a 0.5× TAE-Mg buffer. 2+ .

[0040] S2. Figure 2 This paper demonstrates a process for etching a substrate using polystyrene spheres (PS spheres) and attaching DNA origami. Using a SiO2 substrate, treated with O2 plasma, PS spheres with a diameter of 500 nm are deposited onto the substrate, forming hexagonally close-packed monolayers or multilayers of nanospheres. The substrate is then treated with a hexamethyldisilazane (HMDS) solution in a vacuum desiccator to add hydrophobic trimethylsilyl groups to the surface. Finally, the substrate is sonicated in water to remove the PS spheres from the surface. O2 plasma treatment then creates hydrophilic silanol groups on the circular array sites. A DNA origami solution is then dropped onto the etched array and incubated in a humidified container for one hour to allow the DNA origami to attach to the hydrophilic sites. Excess DNA origami is then removed by washing five times with a 0.07% Tween-20 buffer (Tris, MgCl2). Following this step, the DNA origami array can be characterized using atomic force microscope (AFM).

[0041] S3. Gold nanospheres / gold nanorods and specific IgG antibodies are modified with thiol-single-stranded DNA (such as PolyT or CT-9) and purified by gel electrophoresis in a 0.5× TBE buffer. The modified plasmonic nanoparticle solution is added to the buffer on the substrate surface. After annealing and incubation overnight under humid conditions, the substrate surface is washed with a buffer containing MgCl₂ to remove excess plasmonic nanoparticles. The modified IgG antibody solution is then added to the buffer on the substrate surface and incubated at 37°C for at least 1 hour. After rinsing the substrate surface with the buffer, an array of DNA origami-plasmonic nanostructure-biomolecular probe composite structural units is formed on the substrate surface, resulting in the biochip.

[0042] Experimental example

[0043] This experimental example provides a schematic diagram of the principle of biochip prepared in the embodiment for biomolecule detection based on SLR effect; see Figure 3 ,Depend on Figure 3 It can be seen that when specific antibody molecules near the surface of plasmon nanoparticles bind to antigens in the test blood, causing a change in the refractive index, it will cause a shift in the plasmon resonance wavelength.

[0044] Figure 4 The present invention demonstrates the resonance peak spectra generated by SPR and SLR. Because the plasmonic nanostructure includes both spherical and rod-shaped nanoparticles, it exhibits two SPR resonance peaks, generating two simultaneous SLR resonance peaks. The narrow resonance band of SLR can significantly improve detection limits and sensitivity. By transmitting light from a light source through blood droplets and biochips, biomolecule detection data can be directly obtained based on the spectrum and resonance peak shift analysis transmitted by the photodetector and on the PC. Multiple resonance peak shifts can be used synergistically as a basis for determining biomolecule detection.

[0045] The above embodiment is only one of the embodiments of the present invention. The DNA origami used includes but is not limited to four shapes: triangle, quadrilateral, octagon, and circle. The plasmonic nanostructures prepared include but are not limited to chiral and achiral array assemblies formed by six types of nanoparticles: spherical, rod-shaped, cubic, star-shaped, disk-shaped, and core-shell spherical structures. Any modifications made to the main design of the present invention that are meaningless and that solve the technical problems that are still consistent with the present invention should be included in the scope of protection of the present invention.

Claims

1. A DNA origami microarray biochip based on plasmon surface lattice resonance, characterized in that: include: DNA origami and plasmonic nanostructures arranged on substrates and arrays - biomolecular probe units; The substrate surface has a plurality of spots arranged in a hydrophilic array, and DNA origami with hydroxyl groups are connected to the spots to form a DNA origami array; The plasmonic nanostructure and the biomolecular probe are modified by thiol-single-stranded DNA and connected to the DNA origami according to the principle of complementary base pairing; The method for preparing the biochip comprises the following steps: S1. Mix the long DNA strands with the short DNA strands, plasmonic nanoparticle grabber strands, and biomolecular probe grabber strands. Heat the mixture in a nucleic acid amplifier for a period of time, anneal it, and then purify it. S2. Nanospheres are deposited on the substrate surface using a nanosphere etching technique. The substrate surface is then treated to be hydrophobic, causing the nanospheres to fall off, forming circular array sites. Hydrophilic sites are then generated on the circular array sites using oxygen plasma treatment. A DNA origami solution is dripped onto the etched hydrophilic sites, incubated for a period of time to allow the DNA origami to attach to the hydrophilic sites, and then washed with a buffer solution to form a DNA origami array. S3. Plasmonic nanoparticles and biomolecular probes are modified with thiolated single-stranded DNA, the modified plasmonic nanoparticle solution is added to the buffer solution on the substrate surface in S2, the temperature is controlled under wet conditions, the substrate surface is annealed and incubated overnight, and then the substrate surface is washed with a buffer solution containing magnesium ions; the modified biomolecular probe solution is then added to the buffer solution on the substrate surface, incubated for a period of time, and the substrate surface is washed with the buffer solution for a period of time, thereby forming an arrayed DNA origami-plasmonic nanostructure-biomolecular probe composite structural unit on the substrate surface; and the biochip is obtained.

2. The DNA origami microarray biochip based on plasmon surface lattice resonance according to claim 1, characterized in that: The substrate is periodically patterned using nanosphere etching technology, with a period of 100-1000 nm and a number of periods of the order of 100-10,000, and is arranged in one of a hexagonal array and a square array.

3. The DNA origami microarray biochip based on plasmon surface lattice resonance according to claim 2, characterized in that: The substrate is made of SiO2, and its refractive index is between 1.45 and 1.

5.

4. The DNA origami microarray biochip based on plasmon surface lattice resonance according to claim 1, characterized in that: The DNA origami is formed by folding a long DNA single strand and several short DNA single strands through base complementary pairing; its shape is one of a triangle, a quadrilateral, a hexagon, an octagon or a circle; and the size of the DNA origami is between 50-300 nm.

5. The DNA origami microarray biochip based on plasmon surface lattice resonance according to claim 1, characterized in that: The plasmon nanostructure is a single plasmon nanoparticle and / or a plurality of plasmon nanoparticles.

6. The DNA origami microarray biochip based on plasmon surface lattice resonance according to claim 5, characterized in that: The plasmon nanoparticles are one of metal nanoparticles, hybrid metal nanoparticles, and hybrid metal-dielectric nanoparticles, and their shapes are one of spheres, rods, cubes, stars, disks, and core-shell spherical structures.

7. The DNA origami microarray biochip based on plasmon surface lattice resonance according to claim 1, characterized in that: In S1, the long DNA single strand, the short DNA single strand, the plasmon nanoparticle grabbing strand, and the biomolecule probe grabbing strand are mixed in a molar ratio of 1:15-25:15-25:15-25.

8. The DNA origami microarray biochip based on plasmon surface lattice resonance according to claim 1, characterized in that: When the substrate surface is subjected to hydrophobic treatment, it is treated with a hexamethyldisilazane solution to add hydrophobic trimethylsilyl groups to the substrate surface.

9. Use of the DNA origami microarray biochip based on plasmon surface lattice resonance according to any one of claims 1 to 8 in biomolecule detection.

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