Gene chip, method for preparing the same, and method for detecting a gene

Abstract

The application provides a gene chip and a preparation method and a gene detection method thereof, and relates to the technical field of gene chips.The gene chip comprises a substrate and an optical resonant cavity arranged on the substrate, wherein the optical resonant cavity is a concave micro-nano structure with an opening facing the upper side of the substrate; the bottom surface and the side wall of the optical resonant cavity are made of a high-refractive-index material, and a gene probe is fixed on the bottom surface and / or the side wall of the optical resonant cavity.The application develops a gene chip which has a low-cost preparation process and the feasibility of portable detection.

Description

Technical Field

[0001] This invention relates to the field of gene chip technology, and in particular to a gene chip and its preparation method and gene detection method. Background Technology

[0002] Gene chips are miniaturized biodetection devices that immobilize a large number of gene probes with known sequences in an array on the surface of a solid substrate. Based on the principle of complementary base pairing between nucleic acid chains, this technology performs parallel detection of target nucleic acid molecules in the sample through hybridization reactions. It features high throughput and strong parallelism and has been widely applied in fields such as gene expression profiling, disease diagnosis, drug screening, gene sequencing, and forensic identification.

[0003] Traditional gene chip detection methods primarily employ fluorescent labeling. During chip fabrication, gene probe molecules are labeled with fluorescent dyes. During detection, after the sample to be tested is hybridized with the chip, a laser of a specific wavelength is used to excite the fluorescent groups, and the fluorescence signal is collected using a fluorescence microscope or a dedicated scanner. The target molecule sequence is then determined based on the fluorescence distribution generated by the binding of the target molecule to the probe array on the gene chip. This fluorescent labeling-based gene chip technology requires the modification of nucleotides with fluorescent proteins during fabrication, which increases manufacturing complexity and cost, and also affects the lifespan of the gene chip. Furthermore, the detection process requires a strong light source to excite the fluorescent probe molecules, further increasing the complexity of the detection system and impacting its convenience and market penetration.

[0004] Therefore, how to develop a gene chip that combines low-cost preparation process with portable detection feasibility has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] This invention provides a gene chip and its preparation method and gene detection method, which can provide a gene chip that has both low-cost preparation process and portable detection feasibility.

[0006] This invention provides a gene chip, comprising: A substrate and an optical resonant cavity disposed on the substrate, wherein the optical resonant cavity is a concave micro / nano structure with its opening facing upwards from the substrate; The bottom surface and sidewalls of the optical resonant cavity are made of a high refractive index material, and gene probes are fixed on the bottom surface and / or sidewalls of the optical resonant cavity.

[0007] According to a gene chip provided by the present invention, the optical resonant cavity is a Mie Void optical resonant cavity, wherein the in-plane dimensions of the Mie Void optical resonant cavity are 200nm-2000nm and the depth is 50nm-1000nm.

[0008] According to a gene chip provided by the present invention, the optical resonant cavity comprises multiple such cavities arranged in an array on the substrate, with an array period of 300nm-100μm.

[0009] According to a gene chip provided by the present invention, the bottom surface of the optical resonant cavity is further provided with a protruding structure, the refractive index of the protruding structure is lower than the refractive index of the high refractive index material, and the height of the protruding structure is less than the depth of the optical resonant cavity.

[0010] According to a gene chip provided by the present invention, a biocompatible material layer is disposed between the bottom surface and / or sidewall of the optical resonant cavity and the gene probe, wherein the thickness of the biocompatible material layer is less than 20 nm.

[0011] According to a gene chip provided by the present invention, the refractive index of the high refractive index material is >2.5, and the high refractive index material is selected from at least one of the following materials: monocrystalline silicon, polycrystalline silicon, amorphous silicon, monocrystalline germanium, polycrystalline germanium, amorphous germanium, gallium arsenide.

[0012] According to a gene chip provided by the present invention, the gene probe is any one of deoxyribonucleic acid (DNA), deoxyribonucleic acid (RNA), and oligonucleotide sequences.

[0013] According to a gene chip provided by the present invention, the cross-sectional shape of the optical resonant cavity in the substrate normal direction is circular, square, rectangular or elliptical.

[0014] This invention also provides a gene detection method, comprising: Provide a gene chip as described in any of the preceding claims, wherein a gene probe of a known sequence is fixed within the optical resonant cavity of the gene chip; The sample to be tested is introduced into the gene chip so that the molecules to be tested in the sample come into contact with the gene probe. The color information of the scattered light generated by the optical resonant cavity on the gene chip under white light illumination is collected; Based on the change in the color of the scattered light relative to the initial color of the scattered light in the optical resonant cavity before the reaction, it is determined whether a specific binding reaction has occurred in the optical resonant cavity, so as to determine the target molecule sequence information in the sample to be detected.

[0015] According to a gene detection method provided by the present invention, the scattered light color information is acquired by a color image sensor.

[0016] According to a gene detection method provided by the present invention, the step of determining whether a specific binding reaction has occurred in the optical resonant cavity based on the change in the color information of the scattered light relative to the initial scattered light color of the optical resonant cavity before the reaction, in order to determine the target molecule sequence information in the sample to be detected, includes: The color information of the scattered light is compared with the color of the initial scattered light from the optical resonant cavity before the reaction using colorimetry. When the color change exceeds a preset threshold, it is determined that a specific binding reaction has occurred in the optical resonant cavity; Based on the gene probe with a known sequence fixed in the optical resonant cavity, the target molecular sequence information in the sample to be tested is determined.

[0017] This invention also provides a method for preparing a gene chip, comprising: Provide a base; Based on the refractive index of the substrate, the substrate is processed to form an optical resonant cavity; the optical resonant cavity is a concave micro / nano structure with its opening facing upwards from the substrate, and the bottom surface and sidewalls of the optical resonant cavity are made of a high refractive index material; Gene probes are fixed on the bottom surface and / or sidewalls of the optical resonant cavity.

[0018] According to a method for preparing a gene chip provided by the present invention, the step of processing the substrate based on the refractive index of the substrate to form an optical resonant cavity includes: When the substrate is a high refractive index material, an upward-opening concave micro / nano structure is formed in the substrate to obtain the optical resonant cavity; or, When the substrate is a low-refractive-index material, an upward-opening concave micro / nano structure is formed on the substrate, and then a high-refractive-index material coating is formed on the surface of the concave micro / nano structure to obtain the optical resonant cavity; or, When the substrate is a low refractive index material, a high refractive index material coating is formed on the substrate, and then an upward-opening concave micro / nano structure is formed in the high refractive index material coating to obtain the optical resonant cavity; Wherein, the refractive index of the low refractive index material is ≤1.5.

[0019] According to a method for preparing a gene chip provided by the present invention, the step of forming an upward-opening concave micro / nano structure on the substrate includes: A photoresist layer is formed on the substrate; A pattern is defined in the photoresist layer using a photolithography process; The photoresist is removed by a development process to form a patterned photoresist mask. Using the patterned photoresist mask as an etching barrier layer, the exposed substrate is etched to transfer the pattern into the substrate, forming the concave micro / nano structure.

[0020] According to a method for preparing a gene chip provided by the present invention, the step of fixing gene probes on the bottom surface and / or sidewalls of the optical resonant cavity includes: Nucleotide monomers with photosensitive protecting groups are sequentially introduced in gas phase; Gene probes with pre-defined sequences are synthesized layer by layer by spatially selective ultraviolet light irradiation using maskless lithography controlled by digital micromirror devices.

[0021] This invention provides a gene chip, its preparation method, and a gene detection method. The gene chip includes a substrate and an optical resonant cavity disposed on the substrate. The optical resonant cavity is a concave micro / nano structure with its opening facing upwards from the substrate. Its bottom surface and sidewalls are made of a high-refractive-index material, and gene probes are fixed on its bottom surface and / or sidewalls. Because the bottom surface and sidewalls of the optical resonant cavity are made of a high-refractive-index material, when white light is incident, the light can form effective resonance enhancement within the cavity, generating a scattered spectral signal that can be recognized by conventional imaging systems. Therefore, no specific wavelength laser excitation source is required during the detection process; only a white light source and basic imaging device are needed to acquire the signal. This characteristic reduces the complexity and cost of the supporting detection equipment, freeing gene chip detection from dependence on professional fluorescence scanners and providing a structural basis for the realization of portable gene detection systems. Simultaneously, the gene probes are directly fixed to the bottom surface and / or sidewalls of the optical resonant cavity, enabling the gene chip to specifically capture complementary nucleic acid molecules in the sample to be detected. Since the detection signal originates from changes in the optical properties of the optical resonant cavity, rather than the fluorescence emission of the probe molecules, there is no need to label the gene probes with fluorescent dyes. This eliminates the fluorescent modification step required in traditional methods, reduces raw material costs and process complexity, and avoids the negative impact of fluorescent labeling on probe molecule activity and chip storage stability. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the structure of a traditional gene chip.

[0024] Figure 2 This is a schematic diagram illustrating the principle of traditional gene chip detection using fluorescence detection.

[0025] Figure 3 This is one of the structural schematic diagrams of the gene chip provided by the present invention.

[0026] Figure 4 This is the second schematic diagram of the structure of the gene chip provided by the present invention.

[0027] Figure 5 This is a schematic flowchart of the gene detection method provided by the present invention.

[0028] Figure 6 This is a schematic diagram illustrating the principle of the gene detection method provided by the present invention.

[0029] Figure 7 This is a schematic flowchart of the gene chip preparation method provided by the present invention.

[0030] Figure 8 This is one of the schematic diagrams of the gene chip preparation method provided by the present invention.

[0031] Figure 9 This is the second schematic diagram of the gene chip preparation method provided by the present invention.

[0032] Figure 10 This is a schematic diagram of the system for immobilizing gene probes during the gene chip fabrication process provided by the present invention.

[0033] Figure label: 01. Base; 02. Gene probe; 10. Substrate; 20. Optical resonant cavity; 30. High refractive index material coating; 40. Protruding structure; 51. Photochemical reactor; 52. Gene chip synthesis mask; 53. Ultraviolet light source; 54. Gene chip synthesis stage; 55. Carrier gas input port; 56. Cleaning gas input port; 57. Gas output port. Detailed Implementation

[0034] To facilitate understanding of the technical solution of this invention, a brief explanation of traditional gene chips and their detection principles will be given below.

[0035] Traditional gene chips, such as Figure 1 As shown, it is a fabrication of micro- and nano-sized units on a flat substrate, each unit containing the same gene probe.

[0036] Traditional gene chip detection methods primarily employ fluorescent labeling detection schemes. For example... Figure 2As shown, during the chip fabrication process, gene probe molecules immobilized on the substrate need to be labeled with fluorescent dyes. During detection, the target molecule sequence in the sample to be detected is hybridized with the chip: when the target molecule sequence and the probe molecule on the chip are complementary, a double-stranded structure is formed. At this point, under the excitation of a laser of a specific wavelength, the hybridization site generates a first characteristic fluorescent signal (e.g., ...). Figure 2 (As shown by fluorescence λ0); when the target molecule sequence and the probe molecule are not paired, stable hybridization cannot be formed, and the corresponding probe site does not produce a fluorescence signal or produces background fluorescence of different wavelengths (e.g., λ0). Figure 2 The fluorescence emission λ1 is shown in the figure. The above fluorescence signals are acquired by fluorescence microscopy or a dedicated scanner. The sequence information of the target molecule can be determined based on the distribution and intensity of the fluorescence signals.

[0037] Traditional fluorescently labeled gene chip technologies require modification of nucleotides with fluorescent proteins during fabrication, increasing manufacturing complexity and cost, and affecting the chip's lifespan. Furthermore, the detection process demands a strong light source to excite the probe fluorescent molecules, further complicating the detection system and impacting its convenience and market penetration. Therefore, this invention develops a gene chip that combines a low-cost fabrication process with portable detection feasibility.

[0038] The gene chip of the present invention is described in detail below. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0039] The terms “center,” “longitudinal,” “lateral,” “length,” “width,” “thickness,” “upper,” “lower,” “front,” “rear,” “left,” “right,” “vertical,” “horizontal,” “top,” “bottom,” “inner,” “outer,” “clockwise,” “counterclockwise,” “axial,” “radial,” and “circumferential” indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the present invention and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting the present invention.

[0040] It should also be noted that, in the description of the embodiments of the present invention, unless otherwise stated, "a plurality of" means two or more. Furthermore, unless otherwise explicitly specified and limited, the terms "installed," "connected," and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0041] The following is combined with Figures 3 to 4 A gene chip according to an embodiment of the present invention.

[0042] Figure 3 This is one of the structural schematic diagrams of the gene chip provided by the present invention. For example... Figure 3 As shown, the gene chip includes a substrate 10 and an optical resonant cavity 20 disposed on the substrate 10. The optical resonant cavity 20 is a concave micro / nano structure with its opening facing upwards from the substrate.

[0043] The bottom surface and sidewalls of the optical resonant cavity 20 are made of a high refractive index material, and gene probes are fixed on the bottom surface and / or sidewalls of the optical resonant cavity 20.

[0044] The substrate refers to the solid support material used to support the optical resonant cavity.

[0045] An optical resonant cavity is a microstructure that enables light of a specific wavelength to resonate and enhance within it.

[0046] High refractive index materials refer to materials with a refractive index significantly higher than that of ordinary media (such as air, water, and glass). They are usually materials with a refractive index greater than 2.5, such as silicon (Si), germanium (Ge), and gallium arsenide (GaAs).

[0047] Gene probes are DNA or RNA fragments with known base sequences that can specifically recognize and bind to target nucleic acid molecules through the principle of complementary base pairing.

[0048] The gene chip in this embodiment of the invention includes a substrate 10, the surface of which needs to be flat to facilitate subsequent micro / nano fabrication. The substrate 10 can be made of a low-refractive-index material, such as glass or quartz, or a high-refractive-index material, such as monocrystalline silicon, polycrystalline silicon, or amorphous silicon. An optical resonant cavity 20 is disposed on the substrate 10, which is a concave micro / nano structure with its opening facing upwards from the substrate. The optical resonant cavity 20 may include one or more.

[0049] The bottom surface and sidewalls of the optical resonant cavity 20 are made of a high refractive index material.

[0050] In one embodiment, when the substrate itself is a high refractive index material, such as silicon, a semiconductor manufacturing process can be used to directly form an upward-facing concave micro / nano structure on the substrate through a process of spin coating, photolithography, development, and etching. In this case, the bottom surface and sidewalls of the concave micro / nano structure are the silicon material itself, which meets the requirement of using a high refractive index material.

[0051] In another embodiment, when the substrate is a low refractive index material, such as glass, a semiconductor manufacturing process can be used to form an upward-facing concave micro / nano structure on the substrate through a process of homogenization, photolithography, development, and etching. Then, a high refractive index material coating 30 is deposited on the surface of the concave micro / nano structure to obtain an optical resonant cavity.

[0052] In another embodiment, when the substrate is a low refractive index material, such as glass, it is necessary to first deposit a high refractive index material coating 30, such as amorphous silicon, on the glass substrate, and then etch an upward-facing concave micro / nano structure in the coating, so that the bottom surface and sidewalls of the concave micro / nano structure are made of the amorphous silicon thin film.

[0053] Gene probes with known sequences are fixed on the bottom surface and / or sidewalls of the optical resonant cavity. These gene probes are used to specifically hybridize with target molecular sequences in the sample to be tested, thereby identifying target molecules complementary to the probe.

[0054] It should be understood that when there are multiple optical resonant cavities, different sequences of gene probes can be immobilized in different optical resonant cavities.

[0055] This invention provides a gene chip comprising a substrate and an optical resonant cavity disposed on the substrate. The optical resonant cavity is a concave micro / nano structure with its opening facing upwards from the substrate. Its bottom surface and sidewalls are made of a high-refractive-index material, and gene probes are fixed on its bottom surface and / or sidewalls. Because the bottom surface and sidewalls of the optical resonant cavity are made of a high-refractive-index material, when white light is incident, the light can form effective resonance enhancement within the cavity, generating a scattered spectral signal that can be recognized by conventional imaging systems. Therefore, no specific wavelength laser excitation source is required during detection; only a white light source and basic imaging device are needed to acquire the signal. This characteristic reduces the complexity and cost of the supporting detection equipment, freeing gene chip detection from dependence on professional fluorescence scanners and providing a structural basis for the realization of portable gene detection systems. Simultaneously, the gene probes are directly fixed to the bottom surface and / or sidewalls of the optical resonant cavity, enabling the gene chip to specifically capture complementary nucleic acid molecules in the sample to be detected. Since the detection signal originates from changes in the optical properties of the optical resonant cavity, rather than the fluorescence emission of the probe molecules, there is no need to label the gene probes with fluorescent dyes. This eliminates the fluorescent modification step required in traditional methods, reduces raw material costs and process complexity, and avoids the negative impact of fluorescent labeling on probe molecule activity and chip storage stability.

[0056] Based on any of the above embodiments, the optical resonator is a Mie Void optical resonator, and the in-plane dimensions of the Mie Void optical resonator are 200nm-2000nm, and the depth is 50nm-1000nm.

[0057] The Mie Void optical resonator is an open-type optical resonator that operates based on the Mie scattering principle. Its geometric dimensions are on the same order of magnitude as the wavelength of visible light, causing a significant Mie scattering effect when incident white light interacts with the concave micro / nano structure, thus forming an optical resonance.

[0058] In-plane dimensions refer to the characteristic dimensions of the openings in a concave micro / nano structure, such as the diameter of a circular opening or the side length of a square opening. Depth refers to the vertical distance from the opening plane to the bottom of the cavity.

[0059] Specifically, such as Figure 3 As shown, the in-plane dimensions d of the Mie Void optical resonator are 200nm-2000nm, and the depth t is 50nm-1000nm.

[0060] The gene chip provided in this invention ensures that the Mie Void optical resonator can undergo Mie scattering and resonance with visible light by limiting the size of the Mie Void optical resonator within the aforementioned range. This size range allows the optical resonator to generate a distinguishable color scattering signal under standard white light illumination, while ensuring sufficient space inside the cavity to accommodate gene probes and target molecules.

[0061] Based on any of the above embodiments, the optical resonant cavity includes multiple resonators arranged in an array on the substrate, with an array period of 300nm-100μm.

[0062] In this embodiment of the invention, the optical resonant cavities include multiple cavities arranged in an array on a substrate. The array arrangement refers to the arrangement of these optical resonant cavities in a regular one-dimensional linear array or two-dimensional grid pattern, for example, arranged into a rectangular grid with fixed row and column spacing.

[0063] The center distance between adjacent optical resonators, i.e., the array period, is between 300 nm and 100 μm. Preferably, the array period is 10 μm, which ensures no optical crosstalk between adjacent optical resonators while allowing a sufficient number of resonators to be integrated within a unit area. Taking a 10 mm × 10 mm gene chip as an example, if the array period is 10 μm, approximately one million independent optical resonator units can be integrated.

[0064] When the array period is less than 300 nm, the optical crosstalk between adjacent optical resonant cavities is enhanced, affecting the detection accuracy; when the array period is greater than 100 μm, the number of probes per unit area is significantly reduced, and the detection throughput of the gene chip decreases.

[0065] This invention provides a gene chip that achieves high-throughput parallel detection capabilities by arranging multiple optical resonant cavities in an array on a substrate. Simultaneously, the defined array period ensures the independence of optical signals between each resonant cavity, avoiding mutual interference and guaranteeing the accuracy of the detection results.

[0066] Based on any of the above embodiments, the bottom surface of the optical resonant cavity is further provided with a protruding structure, the refractive index of the protruding structure is lower than the refractive index of the high refractive index material, and the height of the protruding structure is less than the depth of the optical resonant cavity.

[0067] Figure 4 This is the second schematic diagram of the gene chip structure provided by the present invention, as shown below. Figure 4 As shown, a protruding structure 40 is also provided on the bottom surface of the optical resonant cavity.

[0068] The refractive index of the protrusion structure 40 is lower than that of the high-refractive-index material that makes up the bottom and sidewalls of the optical resonant cavity 20. For example, the cavity wall material is high-refractive-index amorphous silicon with a refractive index of about 3.5, while the protrusion structure can be made of silicon dioxide with a lower refractive index of about 1.5.

[0069] The height of the protrusion 40 is less than the depth t of the optical resonant cavity 20. That is, the top surface of the protrusion is lower than the opening plane of the optical resonant cavity 20, thus forming a three-dimensional space with a central protrusion inside the resonant cavity. The gene probe can be fixed to the top surface and sidewalls of the protrusion 40, as well as the bottom surface and sidewalls of the original optical resonant cavity 20.

[0070] This invention provides a gene chip that increases the surface area available for fixing gene probes by setting protrusions with a refractive index lower than that of the cavity wall material on the bottom surface of the optical resonant cavity, thereby enabling the fixing of a larger number of probes. Simultaneously, the protrusions elevate the fixing position of some gene probes from the bottom surface of the cavity to a position closer to the concentrated light field region inside the cavity. This allows the same number of binding events to cause a more significant shift in the resonant wavelength, thereby enhancing the refractive index change effect caused by the binding of the target molecule and improving detection sensitivity.

[0071] Based on any of the above embodiments, a biocompatible material layer is disposed between the bottom surface and / or sidewall of the optical resonant cavity and the gene probe, and the thickness of the biocompatible material layer is less than 20 nm.

[0072] The biocompatible material layer is an intermediate layer material located between the cavity wall (including the bottom surface and / or sidewalls) of the optical resonant cavity and the gene probe. This material does not cause non-specific adsorption or loss of biological activity upon contact with biomolecules and provides stable chemical binding sites. The biocompatible material layer is selected from at least one of the following materials: gold (Au), silane coupling agents, streptavidin, and polylysine. The silane coupling agent may include, but is not limited to, APTES (3-aminopropyltriethoxysilane) and AEAPTES (γ-aminoethyl-aminopropyltriethoxysilane).

[0073] In this embodiment of the invention, a biocompatible material layer is first formed on the bottom surface and / or sidewalls of the optical resonant cavity before immobilizing the gene probe, thereby achieving efficient immobilization of the gene probe. The thickness of this biocompatible material layer is controlled to be below 20 nm to avoid significant impact on the optical performance of the optical resonant cavity.

[0074] In one specific embodiment, a 10 nm thick gold film is deposited on the surface of the optical resonant cavity using electron beam evaporation. The thickness of the gold film is controlled to be below 20 nm to ensure that it does not significantly alter the optical characteristics of the resonant cavity. Furthermore, before depositing the gold film, a 2 nm thick chromium film is deposited as an adhesion layer to enhance the bonding force between the gold film and the substrate material.

[0075] In another specific embodiment, a monolayer, such as an APTES monolayer, is formed on the surface of the cavity through a liquid-phase or gas-phase self-assembly process.

[0076] The gene chip provided in this embodiment of the invention enhances the bonding force between the gene probe and the cavity surface and improves the stability of gene probe fixation by setting a biocompatible material layer with a thickness of less than 20 nm between the bottom surface and / or sidewall of the optical resonance and the gene probe.

[0077] Based on any of the above embodiments, the refractive index of the high refractive index material is >2.5, and the high refractive index material is selected from at least one of the following materials: monocrystalline silicon, polycrystalline silicon, amorphous silicon, monocrystalline germanium, polycrystalline germanium, amorphous germanium, gallium arsenide.

[0078] The high-refractive-index material has a refractive index greater than 2.5, measured at a visible light wavelength of 550 nm. When materials with a refractive index lower than 2.5 are used in conjunction with the structure of this invention, their ability to confine the light field is insufficient, making it difficult to form an optical resonant cavity with a high quality factor, resulting in insignificant changes in the color of the scattered light.

[0079] Specifically, the high refractive index material is selected from at least one of the following materials: monocrystalline silicon, polycrystalline silicon, amorphous silicon, monocrystalline germanium, polycrystalline germanium, amorphous germanium, and gallium arsenide. Among these, monocrystalline silicon has a refractive index of approximately 3.5, polycrystalline silicon has a refractive index of approximately 3.4-3.6, amorphous silicon has a refractive index of approximately 3.5, monocrystalline germanium has a refractive index of approximately 4.0, polycrystalline germanium has a refractive index of approximately 4.0, amorphous germanium has a refractive index of approximately 4.0, and gallium arsenide has a refractive index of approximately 3.8.

[0080] This invention provides a gene chip that, by limiting the refractive index of a high-refractive-index material to greater than 2.5, ensures sufficient refractive index contrast between the optical resonant cavity wall and the internal medium, thereby effectively confining light within the cavity to form resonance. The selected materials are all mature materials used in the semiconductor industry, possessing good processing compatibility and consistency, which helps to achieve low-cost and large-scale manufacturing of this invention.

[0081] Based on any of the above embodiments, the gene probe is any one of deoxyribonucleic acid (DNA), deoxyribonucleic acid (RNA), and oligonucleotide sequences.

[0082] In this embodiment of the invention, the gene probe is any one of deoxyribonucleic acid (DNA), RNA (ribonucleic acid), or an oligonucleotide sequence. DNA probes, due to their high chemical stability, are suitable for detecting DNA target molecules or detecting RNA via reverse transcription. RNA probes are suitable for detecting RNA viruses or analyzing gene expression. Oligonucleotide probes (typically referring to single-stranded nucleic acids less than 50 bases in length) have the advantages of low synthesis cost, flexible sequence design, and high hybridization specificity.

[0083] The present invention provides a gene chip that is not limited to a single type of probe. It can select DNA probes, RNA probes or oligonucleotide probes according to the specific detection target, thus expanding the application scenarios of gene chips and enabling them to cover multiple fields such as genomics, transcriptomics, and pathogen detection.

[0084] Based on any of the above embodiments, the cross-sectional shape of the optical resonant cavity in the substrate normal direction is circular, square, rectangular or elliptical.

[0085] The shape of the cross-section from the base upwards, that is, the shape of the opening observed from above the gene chip, can be flexibly selected as circular, square, rectangular or elliptical according to actual needs.

[0086] Furthermore, the following is combined with Figures 5 to 6This invention describes a gene detection method according to embodiments of the present invention. Obviously, the described embodiments are only a portion, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0087] Figure 5 This is a flowchart illustrating the gene detection method provided by the present invention, as shown below. Figure 5 As shown, the gene detection method includes steps S110, S120 and S130.

[0088] Step S110: Provide a gene chip as described above, wherein a gene probe with a known sequence is fixed in the optical resonant cavity of the gene chip.

[0089] A gene chip as described above is provided, wherein gene probes with known sequences are immobilized in each optical resonant cavity. It should be understood that gene probes with the same or different sequences may be immobilized in different optical resonant cavities.

[0090] Furthermore, before use, the gene chip can be illuminated with white light, and the initial scattered light color of each optical resonant cavity can be recorded by a color image sensor as a reference color.

[0091] Step S120: The sample to be tested is introduced into the gene chip, so that the molecule to be tested in the sample comes into contact with the gene probe.

[0092] The sample to be tested refers to a biological sample that contains or may contain the target molecule to be tested, such as blood, saliva, tissue homogenate, cell lysate, PCR (Polymerase Chain Reaction) amplification products, etc.

[0093] The sample to be tested is introduced onto the surface of the gene chip, so that the sample solution covers the gene chip and comes into contact with the gene probe in the optical resonant cavity. It is then incubated at a suitable temperature for a certain period of time, such as 30 minutes to 2 hours at 42℃-65℃, so that the molecules to be tested in the sample undergo a specific hybridization reaction with the complementary gene probe.

[0094] Step S130: Collect the color information of the scattered light generated by the optical resonant cavity on the gene chip under white light illumination.

[0095] The gene chip after the reaction was illuminated with white light, and the color information of the scattered light generated by the optical resonant cavity on the gene chip was collected.

[0096] Step S140: Based on the change in the color of the scattered light relative to the initial scattered light color of the optical resonant cavity before the reaction, determine whether a specific binding reaction has occurred in the optical resonant cavity, so as to determine the target molecule sequence information in the sample to be detected.

[0097] The collected scattered light color information is compared with the initial scattered light color recorded before the reaction. If the color of an optical resonator undergoes a detectable change, such as changing from green to yellow or red, it indicates that a specific binding reaction has occurred within that optical resonator; conversely, if the color does not change, it indicates that no specific binding reaction has occurred.

[0098] Based on the known gene probe corresponding to the optical resonant cavity whose color changes, the target molecule sequence information contained in the sample to be tested can be deduced.

[0099] Furthermore, combined Figure 6 The principle of the gene detection method according to embodiments of the present invention will be briefly explained. Because the optical resonant cavity uses a bottom and sidewalls made of a high-refractive-index material, it will resonate and scatter light of specific wavelengths under white light illumination, exhibiting a characteristic color. This characteristic color is sensitive to changes in the refractive index within the cavity.

[0100] like Figure 6 As shown, before the hybridization reaction, as Figure 6 In (a), different optical resonant cavities exhibit different resonant scattering wavelengths due to differences in probe sequences or initial states within the cavities. Specifically, the resonant scattering wavelength of the resonant cavity containing the probe that matches the sequence of the target molecule is λ1, while the resonant scattering wavelength of the resonant cavity containing the probe that does not match the sequence of the target molecule is λ2 (λ2≠λ1).

[0101] When the target molecule sequence in the sample to be tested hybridizes with the matching probe fixed in the resonant cavity, such as Figure 6 In (b), the refractive index within the resonant cavity changes, causing its resonant scattering wavelength to shift from λ1 to λ1+Δλ. For the originally mismatched probe, since no hybridization occurs, its resonant scattering wavelength remains unchanged at λ2.

[0102] By acquiring the color information of scattered light from each optical resonator before and after hybridization using a color image sensor, and determining whether a specific binding reaction occurs in each optical resonator based on whether the resonant scattering wavelength changes from λ1 to λ1+Δλ (i.e., whether the color shifts), the target molecule sequence information in the sample to be tested can be determined.

[0103] This invention provides a gene detection method that utilizes the sensitivity of the scattered color of an optical resonant cavity to changes in the refractive index of the cavity medium to achieve label-free detection of gene hybridization events using white light illumination. This detection process eliminates the need for fluorescent labeling of gene probes or samples, and also eliminates the need for a specific wavelength excitation source. Detection results are obtained simply by recording color changes before and after the hybridization reaction using conventional color imaging equipment. Compared to existing fluorescent labeling detection methods, this invention simplifies the detection process and equipment, reduces detection costs, and provides a feasible solution for portable, low-cost gene detection.

[0104] Based on any of the above embodiments, the scattered light color information is acquired by a color image sensor.

[0105] In this embodiment of the invention, the color information of the scattered light is acquired by a color image sensor.

[0106] Among them, the color image sensor can be a standalone color CCD (Charge Coupled Device) camera, a color CMOS (Complementary Metal Oxide Semiconductor) camera, or a color camera integrated into a portable device (such as a smartphone).

[0107] This invention provides a gene detection method that uses a color image sensor to collect scattered light color information, enabling objective recording of color changes before and after hybridization. This provides a data foundation for subsequent colorimetric comparisons and reduces the reliance on specialized instruments. In particular, when a smartphone camera is used as the acquisition device, the cost of the detection device is significantly reduced, and it becomes portable, providing a technological basis for the application of gene chips in rapid on-site detection and personalized medicine.

[0108] Based on any of the above embodiments, step S140 includes: step S141, step S142 and step S143.

[0109] Step S141: Perform a colorimetric comparison between the scattered light color information and the initial scattered light color of the optical resonant cavity before the reaction.

[0110] The color information of the scattered light from each optical resonator collected after the reaction is compared with the color of the initial scattered light before the reaction using colorimetry.

[0111] Specifically, the RGB (Red-Green-Blue) values ​​of the scattered light from each optical resonator collected after the reaction are compared with the reference RGB values ​​of the same optical resonator recorded before the reaction.

[0112] In one embodiment, the comparison method can be to calculate the change in each of the three RGB channels.

[0113] In another implementation, the color difference value can also be calculated using a different color space. This other color space can be Lab (including lightness L). and chromaticity a b ) color space, HSV (Hue, Saturation, Value) color space.

[0114] Step S142: When the color change exceeds a preset threshold, it is determined that a specific binding reaction has occurred in the optical resonant cavity.

[0115] A preset threshold for color change is set, which can be determined through a negative control experiment. For example, a blank sample containing no target molecules is used for detection, and the average and standard deviation of all optical resonator color change values ​​are calculated. The preset threshold is then set to the average plus three or six times the standard deviation.

[0116] When the color change value of an optical resonator exceeds the preset threshold, it is determined that a specific binding reaction has occurred in the optical resonator; when the color change value of an optical resonator does not exceed the preset threshold, it is determined that no specific binding reaction has occurred in the optical resonator.

[0117] Step S143: Determine the target molecular sequence information in the sample to be tested based on the gene probe with a known sequence fixed in the optical resonant cavity.

[0118] Based on the position coordinates of the optical resonator determined to have undergone a binding reaction on the gene chip, the gene probe with a known sequence fixed at that pre-recorded position is located, thereby determining the presence of a target molecular sequence complementary to the gene probe in the sample to be tested, and thus obtaining the target molecular sequence information.

[0119] The gene detection method provided in this invention achieves objective and quantitative determination of hybridization events by comparing the color information of scattered light before and after the reaction and setting a preset threshold.

[0120] Furthermore, the following is combined with Figures 7 to 10 This invention describes a method for preparing a gene chip according to embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0121] Figure 7 This is a schematic flowchart of the gene chip preparation method provided by the present invention, as shown below. Figure 7 As shown, the preparation method of the gene chip includes steps S210, S220 and S230.

[0122] Step S210, providing a substrate.

[0123] The substrate material can be a low-refractive-index material, such as glass or quartz, or a high-refractive-index material, such as monocrystalline silicon, polycrystalline silicon, or amorphous silicon. The choice of substrate depends on the application requirements and cost considerations of the final gene chip.

[0124] Step S220: Based on the refractive index of the substrate, the substrate is processed to form an optical resonant cavity; the optical resonant cavity is a concave micro / nano structure with its opening facing upwards from the substrate, and the bottom surface and sidewalls of the optical resonant cavity are made of a high refractive index material.

[0125] Based on the refractive index of the substrate, the substrate is processed to form an optical resonant cavity. The optical resonant cavity is a concave micro / nano structure with its opening facing upwards from the substrate, and its bottom surface and sidewalls are made of a high refractive index material.

[0126] In one embodiment, when the substrate is a high refractive index material, an upward-opening concave micro / nano structure is formed in the substrate to obtain an optical resonant cavity.

[0127] In another embodiment, when the substrate is a low refractive index material, an upward-opening concave micro / nano structure is formed on the substrate, and a high refractive index material coating is formed on the surface of the concave micro / nano structure to obtain an optical resonant cavity.

[0128] In another embodiment, when the substrate is a low refractive index material, a high refractive index material coating is formed on the substrate, and then an upward-opening concave micro / nano structure is formed in the high refractive index material coating to obtain an optical resonant cavity.

[0129] Among them, the refractive index of low-refractive-index materials is ≤1.5.

[0130] For specific implementation details, please refer to the following examples, which will not be elaborated here.

[0131] Step S230: Fix the gene probe on the bottom surface and / or sidewall of the optical resonant cavity.

[0132] Gene probes are fixed on the bottom surface and / or sidewalls of the formed optical resonant cavity.

[0133] Gene probe immobilization can be performed using electrochemical or photochemical methods. Taking the photochemical method as an example, nucleotide monomers with photosensitive protecting groups are used to synthesize probe molecules with a predetermined sequence layer by layer through spatially selective irradiation. Specific implementation methods are described in the following examples and will not be repeated here.

[0134] This invention provides a gene detection method that uses a substrate-based refractive index-selective processing technique to form an optical resonant cavity, and then immobilizes gene probes within the cavity, thus achieving complete gene chip fabrication. In this fabrication process, fluorescent labeling of the gene probes is unnecessary, reducing fabrication costs.

[0135] Based on any of the above embodiments, step S120 includes: step S121, or step S122, or step S123.

[0136] Step S121: When the substrate is a high refractive index material, an upward-opening concave micro / nano structure is formed in the substrate to obtain the optical resonant cavity.

[0137] When the substrate is a high refractive index material, such as single-crystal silicon with a refractive index > 2.5, an upward-opening concave micro / nano structure is directly formed in the substrate, and this concave structure itself constitutes an optical resonant cavity.

[0138] In another embodiment, when the substrate is a low refractive index material, such as glass, it is necessary to first deposit a high refractive index material coating 30, such as amorphous silicon, on the glass substrate, and then etch an upward-facing concave micro / nano structure in the coating, so that the bottom surface and sidewalls of the concave micro / nano structure are made of the amorphous silicon thin film.

[0139] Step S122: When the substrate is a low refractive index material, a concave micro / nano structure with an upward opening is formed on the substrate, and a high refractive index material coating is formed on the surface of the concave micro / nano structure to obtain the optical resonant cavity.

[0140] When the substrate is a low refractive index material, such as glass with a refractive index ≤ 1.5, Figure 8 As shown, a concave micro / nano structure with an upward opening is first formed on a substrate using semiconductor manufacturing processes such as photoresist coating, photolithography, development, and etching. Then, a high-refractive-index material coating is formed on the surface of this concave micro / nano structure using a conformal deposition process. This coating uniformly covers the bottom surface and sidewalls of the concave structure, thereby forming an optical resonant cavity.

[0141] Step S123: When the substrate is a low refractive index material, a high refractive index material coating is formed on the substrate, and then an upward-opening concave micro / nano structure is formed in the high refractive index material coating to obtain the optical resonant cavity.

[0142] When the substrate is a low refractive index material, such as glass with a refractive index ≤ 1.5, as Figure 9 As shown, a high refractive index material coating is first formed on a flat substrate using a conformal deposition process. Then, using semiconductor manufacturing processes, a concave micro / nano structure with an upward opening is formed in the high refractive index material coating through spin coating, photolithography, development, and etching. This concave structure constitutes an optical resonant cavity.

[0143] The gene detection method provided in this embodiment of the invention provides three processing routes for substrates with different refractive indices, enabling the gene chip of this embodiment of the invention to adapt to a variety of material systems.

[0144] Based on any of the above embodiments, the step "forming an upward-opening concave micro / nano structure on the substrate" includes: steps S1211, S1212, S1213, and S1214.

[0145] Step S1211: A photoresist layer is formed on the substrate.

[0146] A photoresist layer is formed on the substrate surface using a spin-coating process. The type (positive or negative) and thickness of the photoresist are selected based on the required etching depth and are not specifically limited here.

[0147] Step S1212: Define a pattern in the photoresist layer using a photolithography process.

[0148] The photoresist layer is irradiated by photolithography, and a pre-defined concave micro / nano structure pattern is defined on the photoresist layer using a photomask or a maskless photolithography system.

[0149] In one embodiment, ultraviolet lithography is used for exposure, and the photomask is designed with a transparent pattern in the shape of a circle, square, rectangle, or ellipse. After exposure, the irradiated area of ​​the photoresist undergoes a photochemical reaction and becomes soluble in the developing solution.

[0150] Step S1213: Remove the photoresist through a development process to form a patterned photoresist mask.

[0151] The photoresist in the exposed area (for positive photoresist) or unexposed area (for negative photoresist) is removed by developing a developer, thereby forming patterned openings in the photoresist layer to expose the underlying substrate or coating material, thus forming an imaged photoresist mask.

[0152] Step S1214: Using the patterned photoresist mask as an etching barrier layer, the exposed substrate is etched to transfer the pattern into the substrate, forming the concave micro / nano structure.

[0153] Using a patterned photoresist mask as an etching barrier layer, dry or wet etching processes are employed to etch the exposed substrate or coating material, removing the exposed material and transferring the pattern on the photoresist into the substrate to form a concave micro / nano structure. The etching depth is controlled by the etching time.

[0154] Furthermore, after etching is complete, the residual photoresist is removed.

[0155] The gene detection method provided in this invention achieves high-precision and high-reproducibility fabrication of concave micro / nano structures through the above-mentioned standard semiconductor process.

[0156] Based on any of the above embodiments, step S230 includes: step S231 and step S232.

[0157] Step S231: Nucleotide monomers with photosensitive protecting groups are sequentially introduced in gas phase.

[0158] Step S232: Gene probes with preset sequences are synthesized layer by layer by performing spatially selective ultraviolet light irradiation through maskless photolithography controlled by digital micromirror devices.

[0159] In this embodiment of the invention, photochemical method is used as an example to immobilize gene probes, and the preparation system is as follows: Figure 10 As shown. The fabrication system comprises three parts: the first part is an ultraviolet light source 53; the second part is a photochemical reactor 51 in which the photochemical reaction occurs and a substrate 10 with an optical resonant cavity 20 disposed therein; the third part is a gene chip synthesis mask 52 for implementing and controlling the spatial distribution of the photochemical reaction, wherein the control of the photochemical reaction is exemplified by maskless photolithography controlled by a microarray.

[0160] Considering that if the reaction molecules and cleaning reagents in the photochemical reactor 51 are liquid, it would be difficult for them to form the required DNA / RNA / oligonucleotide sequence molecules at the bottom of the concave micro / nano structure. Therefore, this embodiment of the invention provides a carrier gas input port 55, a cleaning gas input port 56, and a gas output port 57 on the photochemical reactor 51. The carrier gas input port 55 is used to carry gaseous reaction molecules into the reactor, the cleaning gas input port 56 is used to input cleaning gas, and the gas output port 57 is used to vent the gas from the reactor.

[0161] First, a cleaning gas (such as high-purity nitrogen) is introduced into the photochemical reactor 51 through a pipe and a cleaning gas inlet port 56 to purge and clean the substrate 10 and the optical resonant cavity 20, removing impurities physically adsorbed on the surface. The cleaning gas is discharged through the gas outlet port 57. Subsequently, surface pretreatment molecules (such as gaseous APTES) are introduced into the reactor through a carrier gas inlet port 55, where they are adsorbed and reacted on the bottom surface and / or sidewalls of the optical resonant cavity 20 to form a layer of active groups. Unreacted pretreatment molecules are discharged through the gas outlet port 57.

[0162] After surface pretreatment, the first sequence molecule (i.e., a nucleotide monomer with a photosensitive protecting group) is introduced into the photochemical reactor 51 via carrier gas (argon) through carrier gas input port 55, according to the preset DNA molecule sequence. This nucleotide monomer molecule diffuses into the bottom of the optical resonant cavity 20 under gas phase conditions and is adsorbed and fixed to the bottom surface and / or sidewalls of the cavity through the chemical action of surface-active groups. After adsorption is complete, excess nucleotide monomer vapor is vented through gas output port 57.

[0163] A microarray controlled by a DMD (Digital Micromirror Device) emits ultraviolet light from a light source 53, selectively irradiating the optical resonant cavity 20 where the probe to be synthesized. The ultraviolet wavelength and irradiation time can be selected according to the type of photosensitive protecting group, and are not specifically limited here. The inert modified molecules (i.e., photosensitive protecting groups) at the ends of nucleotide monomers in the irradiated area are photolyzed and removed, exposing the reactive sites; the protecting groups on the nucleotide monomers in the unirradiated area remain unchanged. After irradiation, the ultraviolet light of the DMD is turned off.

[0164] A cleaning gas (such as high-purity nitrogen) is introduced through the cleaning gas inlet port 56 to purge the surface of the photochemical reactor 51 and the substrate 10, removing byproducts and unreacted monomer molecules from the photolysis reaction. The cleaning gas and byproducts are then discharged through the gas outlet port 57.

[0165] After cleaning, a second-base nucleotide monomer (with a photosensitive protecting group) is introduced through carrier gas input port 55 and diffuses to the bottom of optical resonant cavity 20 under gas phase conditions. This nucleotide monomer only couples with the ends of the nucleotide chains whose protecting groups were removed in the previous step, thereby extending the probe chain. Unreacted nucleotide monomers are vented through gas output port 57.

[0166] Repeat the above cyclic process, adding one base in each cycle. It should be understood that the DMD will generate different illumination patterns according to the required probe sequence, thereby enabling the parallel synthesis of gene probes with different sequences in different optical resonant cavities 20.

[0167] After all bases have been added, a deprotection gas is introduced through the carrier gas input port 55 to remove the remaining protecting groups on the nucleotides, thereby fully activating the probe molecules and obtaining a biologically active gene probe.

[0168] Through the above steps, the desired gene probe sequence can be fixed on the bottom surface and / or sidewall of the optical resonant cavity 20 of the substrate 10.

[0169] This invention provides a gene detection method that uses a gas-phase method to immobilize gene probes, solving the problems of poor wetting and residue in micro / nano concave structures caused by liquid-phase methods. Gas molecules can enter the bottom of sub-micron scale optical resonant cavities without hindrance, ensuring high efficiency and uniformity of probe synthesis within each cavity. Combined with DMD maskless lithography, tens of thousands or even millions of different gene probe sequences can be synthesized in parallel on a single gene chip, significantly reducing manufacturing costs and time.

[0170] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gene chip, characterized in that, include: A substrate and an optical resonant cavity disposed on the substrate, wherein the optical resonant cavity is a concave micro / nano structure with its opening facing upwards from the substrate; The bottom surface and sidewalls of the optical resonant cavity are made of a high refractive index material, and gene probes are fixed on the bottom surface and / or sidewalls of the optical resonant cavity.

2. The gene chip according to claim 1, characterized in that, The optical resonant cavity is a Mie Void optical resonant cavity, with an in-plane dimension of 200nm-2000nm and a depth of 50nm-1000nm.

3. The gene chip according to claim 1, characterized in that, The optical resonant cavity comprises multiple resonators arranged in an array on the substrate, with an array period of 300nm-100μm.

4. The gene chip according to claim 1, characterized in that, The bottom surface of the optical resonant cavity is also provided with a protruding structure. The refractive index of the protruding structure is lower than that of the high refractive index material, and the height of the protruding structure is less than the depth of the optical resonant cavity.

5. The gene chip according to claim 1, characterized in that, A biocompatible material layer is disposed between the bottom surface and / or sidewall of the optical resonant cavity and the gene probe, and the thickness of the biocompatible material layer is less than 20 nm.

6. The gene chip according to any one of claims 1 to 5, characterized in that, The high refractive index material has a refractive index > 2.5, and the high refractive index material is selected from at least one of the following materials: monocrystalline silicon, polycrystalline silicon, amorphous silicon, monocrystalline germanium, polycrystalline germanium, amorphous germanium, gallium arsenide.

7. The gene chip according to any one of claims 1 to 5, characterized in that, The gene probe is any one of deoxyribonucleic acid (DNA), deoxyribonucleic acid (RNA), and oligonucleotide sequences.

8. The gene chip according to any one of claims 1 to 5, characterized in that, The cross-sectional shape of the optical resonant cavity in the normal direction of the substrate is circular, square, rectangular or elliptical.

9. A gene detection method, characterized in that, include: A gene chip as described in any one of claims 1 to 8 is provided, wherein a gene probe of a known sequence is fixed in the optical resonant cavity of the gene chip; The sample to be tested is introduced into the gene chip so that the molecules to be tested in the sample come into contact with the gene probe. The color information of the scattered light generated by the optical resonant cavity on the gene chip under white light illumination is collected; Based on the change in the color of the scattered light relative to the initial scattered light color of the optical resonant cavity before the reaction, it is determined whether a specific binding reaction has occurred in the optical resonant cavity, so as to determine the target molecule sequence information in the sample to be detected.

10. The gene detection method according to claim 9, characterized in that, The color information of the scattered light is acquired by a color image sensor.

11. The gene detection method according to claim 9 or 10, characterized in that, The step of determining whether a specific binding reaction has occurred within the optical resonant cavity based on the change in the scattered light color information relative to the initial scattered light color of the optical resonant cavity before the reaction, in order to determine the target molecule sequence information in the sample to be detected, includes: The color information of the scattered light is compared with the color of the initial scattered light from the optical resonant cavity before the reaction using colorimetry. When the color change exceeds a preset threshold, it is determined that a specific binding reaction has occurred in the optical resonant cavity; Based on the gene probe with a known sequence fixed in the optical resonant cavity, the target molecular sequence information in the sample to be tested is determined.

12. A method for preparing a gene chip, characterized in that, include: Provide a base; Based on the refractive index of the substrate, the substrate is processed to form an optical resonant cavity; The optical resonant cavity is a concave micro / nano structure with its opening facing upwards from the substrate, and the bottom surface and sidewalls of the optical resonant cavity are made of a high refractive index material; Gene probes are fixed on the bottom surface and / or sidewalls of the optical resonant cavity.

13. The preparation method according to claim 12, characterized in that, The process of treating the substrate based on its refractive index to form an optical resonant cavity includes: When the substrate is a high refractive index material, an upward-opening concave micro / nano structure is formed in the substrate to obtain the optical resonant cavity; or, When the substrate is a low-refractive-index material, an upward-opening concave micro / nano structure is formed on the substrate, and then a high-refractive-index material coating is formed on the surface of the concave micro / nano structure to obtain the optical resonant cavity; or, When the substrate is a low refractive index material, a high refractive index material coating is formed on the substrate, and then an upward-opening concave micro / nano structure is formed in the high refractive index material coating to obtain the optical resonant cavity; Wherein, the refractive index of the low refractive index material is ≤1.

5.

14. The method for preparing a gene chip according to claim 13, characterized in that, The formation of an upward-opening concave micro / nano structure on the substrate includes: A photoresist layer is formed on the substrate; A pattern is defined in the photoresist layer using a photolithography process; The photoresist is removed by a development process to form a patterned photoresist mask. Using the patterned photoresist mask as an etching barrier layer, the exposed substrate is etched to transfer the pattern into the substrate, forming the concave micro / nano structure.

15. The preparation method according to any one of claims 12 to 14, characterized in that, The fixation of the gene probe on the bottom surface and / or sidewalls of the optical resonant cavity includes: Nucleotide monomers with photosensitive protecting groups are sequentially introduced in gas phase; Gene probes with pre-defined sequences are synthesized layer by layer by spatially selective ultraviolet light irradiation using maskless lithography controlled by digital micromirror devices.

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