Single-molecule fluorescence excitation device and gene sequencing chip composed of the same

Abstract

The present invention provides a single-molecule fluorescence excitation device and a gene sequencing chip composed of the same, the single-molecule fluorescence excitation device includes a ring and eleven micropillars located in the ring, the refractive index of the ring is higher than the refractive index of the eleven micropillars, the eleven micropillars are respectively the first to the eleventh micropillars, the refractive index of the first to the fifth micropillars is the same as the refractive index of the seventh to the eleventh micropillars, the first to the fifth micropillars and the eleventh to the seventh micropillars are respectively arranged in order of refractive index from the edge of the ring to the center, and the sixth micropillar is located at the center of the ring; the ring and the first micropillar, the second micropillar and the ring and the tenth micropillar and the eleventh micropillar respectively enclose a holding space, and a medium with the same refractive index as the ring is respectively embedded in the two holding spaces. The present invention can effectively avoid the problem of low loading rate of DNA molecules in nanopores, and at the same time, the metasurface lens can realize wavelength selection and multi-focus focusing, further improving the integration of the chip system.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid sequencing, and in particular to a single-molecule fluorescence excitation device and a gene sequencing chip composed of the same. Background Art

[0002] Gene sequencing is a new genetic testing technology that can analyze and determine the complete genome sequence from blood or saliva, predicting the susceptibility of various diseases and identifying individual behavioral characteristics and rationale for these behaviors. Gene sequencing technology can pinpoint individual pathogenic genes, enabling early prevention and treatment. The first-generation Sanger sequencing method, which uses multiple primer sets, can only detect consensus sequences of common mutations and is unable to detect mutations at the level of individual viral molecules.

[0003] Although the second-generation gene sequencing technology can obtain a large amount of sequencing data cheaply, its read length limitation makes it impossible to simply and intuitively see the structural changes across the entire genome. A large number of sequencing fragments need to be correctly spliced ​​to construct the genome map, which cannot meet the needs of functional genomics research such as gene recognition, identification, cloning, gene structure, function and their interrelationships, gene expression regulation, identification of protein structure, function and interaction.

[0004] Currently, the mainstream third-generation long-read PCR-free sequencers include PacBio's single-molecule fluorescence technology and ONT protein nanopore technology. Single-molecule fluorescence sequencing uses fluorescent labeling to sequence single DNA molecules simultaneously by synthesis. Typical third-generation gene sequencing technologies also include FRET and transmission electron microscopy sequencing. PacBio's single-molecule fluorescence technology is accurate, rapid, and stable, but its drawback is its high price. ONT protein nanopore technology offers portability and ultra-long reads, but its accuracy needs further improvement. Founded in 2004, PacBio's products primarily utilize zero-mode waveguides for single-molecule real-time sequencing. Through continuous updates and iterations, it has become the only product in the world capable of single-molecule fluorescence sequencing with long reads and high accuracy, but it is expensive. Patents cover all aspects of sequencing chips, supporting reagents, and system integration, resulting in a technological monopoly that is difficult to overcome using mature technologies. The Sequel system, launched in 2015, surpasses the RS II in key performance areas such as read length and data throughput. In particular, it boasts a sevenfold increase in the number of ZMW pores, resulting in a sevenfold increase in throughput compared to the RS II within the same runtime. However, PacBio's SMRT chip still has obvious shortcomings. Low throughput is a common problem of third-generation technology. In addition, the PacBio system also has the following problems:

[0005] (1) The loading efficiency of DNA molecules in the nanopore is low;

[0006] (2) Limited by the statistical law of Poisson distribution, the diffusion-based loading method of nucleic acid fragment-enzyme mixture will lead to low utilization of zero-mode waveguide sequencing chips. Summary of the Invention

[0007] The purpose of the present invention is to overcome the shortcomings of existing technologies and propose a single-molecule fluorescence excitation device and a gene sequencing chip composed of it. It can integrate beam splitting and localized light field. By controlling the longitudinal distribution of the light field, it can simultaneously excite each unit under parallel light incidence conditions and significantly reduce the fluorescence background of non-detected molecules, meeting the requirements of high signal-to-noise ratio and high-throughput sequencing. Compared with the current PacBio zero-mode waveguide chip, it is expected to reduce production costs and may be reused.

[0008] To achieve the above objectives, the present invention adopts the following specific technical solutions:

[0009] The single-molecule fluorescence excitation device provided by the present invention includes a circular ring and eleven micropillars located within the circular ring. The refractive index of the circular ring is higher than the refractive index of the eleven micropillars. The eleven micropillars are respectively first to eleventh micropillars, the refractive index of the first to fifth micropillars is the same as the refractive index of the seventh to eleventh micropillars, the first to fifth micropillars and the eleventh to seventh micropillars are arranged progressively from the edge to the center of the circular ring in ascending order of refractive index, and the sixth micropillar is located at the center of the circular ring. The circular ring and the first and second micropillars, as well as the circular ring and the tenth and eleventh micropillars, respectively, form a receiving space, and a medium with the same refractive index as the circular ring is embedded in each of the two receiving spaces.

[0010] Preferably, the calculation formulas for the second to fifth microcolumns are: Among them, g represents the gradient change index, N represents the total number of layers, and m represents the mth layer.

[0011] The gene sequencing chip provided by the present invention includes a detector, a transmission layer located above the detector, a metasurface lens located above the transmission layer, a waveguide structure located above the metasurface lens, and the above-mentioned single-molecule fluorescence excitation device located above the waveguide structure.

[0012] Preferably, the metasurface lens includes a substrate and a dielectric layer prepared on the substrate, and a unit structure array is photoetched on the dielectric layer, which is used to focus incident light of different wavelengths within the incident light band to different focal positions on the same focal plane, and the distance between two adjacent focal points is greater than 2μm.

[0013] Preferably, the numerical aperture of the metasurface lens is greater than 0.4.

[0014] Preferably, the substrate is made of silicon dioxide material, and the dielectric layer is made of titanium dioxide material, gallium nitride material or hafnium dioxide material.

[0015] Preferably, the shape of the unit structures in the unit structure array is rectangular, triangular, cylindrical or elliptical.

[0016] The present invention can achieve the following technical effects: integrating beam splitting with light field localization, and by controlling the longitudinal distribution of the light field, simultaneously exciting each unit under the condition of parallel light incidence and significantly reducing the fluorescence background of non-detected molecules, meeting the requirements of high signal-to-noise ratio and high-throughput sequencing. Compared with the current PacBio zero-mode waveguide chip, it is expected to reduce production costs and may be reused. It can not only avoid the blockade of existing patented technologies such as PacBio, but more importantly, the use of super-resolution nano-light localization technology effectively avoids the problem of low DNA molecule loading rate in the nanopore. At the same time, the metasurface lens can achieve wavelength selection and multi-focal focusing, further improving the integration of the chip system. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 2 is a schematic structural diagram of a single-molecule fluorescence excitation device provided according to an embodiment of the present invention;

[0018] Figure 2 2. It is a schematic diagram of a transverse electric field distribution curve of a single-molecule fluorescence excitation device provided in an embodiment of the present invention;

[0019] Figure 3 This is a schematic diagram of the structure of a gene sequencing chip provided according to an embodiment of the present invention;

[0020] Figure 4 3 is a schematic structural diagram of a metasurface lens provided according to an embodiment of the present invention.

[0021] The reference numerals therein include: single-molecule fluorescence excitation device 1, waveguide structure 2, metasurface lens 3, substrate 31, unit structure 32, transmission layer 4, detector 5, fluorescence signal 6, and focal plane 7. DETAILED DESCRIPTION

[0022] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, identical modules are denoted by identical reference numerals. In the case of identical reference numerals, their names and functions are also identical. Therefore, their detailed description will not be repeated.

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation of the present invention.

[0024] This invention innovatively uses a photonic nanojet (PNJ) to excite fluorescence. Due to factors such as the diffraction limit, the current FWHM that super-resolution PNJs can achieve is around 100 nanometers. To address the need for localized nanoscale high-energy light in single-molecule detection, this invention combines the introduction of a high-refractive-index medium with micropillars arranged with a refractive-index gradient. This unique micropillar structure, with an internal micropillar with a horizontally gradient refractive index distribution, an external high-refractive-index ring, and high-refractive-index medium embedded at both ends, produces a PNJ with a narrower beamwidth. The advantages of this structure are:

[0025] First, the use of high-refractive-index media can increase the numerical aperture of the structure, which manifests itself in the frequency domain as the spectrum of the structure can be extended to higher frequency components, and in the spatial domain as a stronger lateral compression capability of the PNJ;

[0026] Second, the use of an external high-refractive-index ring can increase the energy flux distribution along the arc side at both ends of the micropillar (in previous designs, the energy flux distributed on both sides of the micropillar was not large, and few people paid attention to this point), which helps to further compress the PNJ beam width;

[0027] Third, the high-refractive-index embedding bodies at both ends are used to regulate the energy flux distribution of the PNJ so that it is not compressed inside, that is, to guide the PNJ to emit.

[0028] In summary, the unique structure we proposed can achieve high-intensity PNJ with a beam width within a hundred nanometers, which can effectively excite single-molecule fluorescence.

[0029] Figure 1 The structure of a single-molecule fluorescence excitation device provided according to an embodiment of the present invention is shown.

[0030] like Figure 1 As shown, the single-molecule fluorescence excitation device provided by the embodiment of the present invention includes a high-refractive-index ring and eleven low-refractive-index microcolumns located inside the ring, and the refractive index of the ring is higher than that of the eleven microcolumns.

[0031] The eleven microcolumns are the first to the eleventh microcolumns, the refractive indexes of the first to the eleventh microcolumns are n1-n11 respectively, and the refractive index of the ring is n 12 Figure 1 The refractive index n1-n 11 The first to eleventh microcolumns are designated by the refractive index n 12 Refers to a ring.

[0032] Among the eleven microcolumns, the refractive index n6 of the sixth microcolumn is the largest, the refractive index n1 of the first microcolumn to the refractive index n5 of the fifth microcolumn increases in sequence, the refractive index n7 of the seventh microcolumn to the refractive index n1 of the eleventh microcolumn increases in sequence, and the refractive index n 11 The refractive index n1 of the first microcolumn decreases in sequence, and the refractive index n1 of the eleventh microcolumn is 11The refractive index of the second microcolumn is n2, which is the same as that of the tenth microcolumn. 10 The refractive index n3 of the third microcolumn is the same as the refractive index of n9 of the ninth microcolumn, the refractive index n4 of the fourth microcolumn is the same as the refractive index of n8 of the eighth microcolumn, and the refractive index n5 of the fifth microcolumn is the same as the refractive index of n7 of the seventh microcolumn.

[0033] The sixth microcolumn is located at the center of the ring, and the first to fifth microcolumns and the seventh to eleventh microcolumns are symmetrically distributed with the sixth microcolumn as the axis. The first to fifth microcolumns and the eleventh to seventh microcolumns are arranged progressively from the edge of the ring to the center in order of refractive index from small to large.

[0034] With the sixth microcolumn as the innermost layer and the first microcolumn and the eleventh microcolumn as the outermost layer, the refractive index increases gradually from the outermost layer to the innermost layer.

[0035] In a specific example of the present invention, the refractive index of the first microcolumn n1=1.46, the refractive index of the sixth microcolumn n6=1.72, then n2-n5, n7-n 10 The calculation formula is:

[0036]

[0037] Among them, g represents the gradient change index, N represents the total number of layers, and m represents the mth layer.

[0038] Therefore, n2=n 10 =1.4653, n3=n9=1.4720, n4=n8=1.4811, n5=n7=1.4960, the width of each microcolumn except the sixth microcolumn is 0.5 μm, and the width of the sixth microcolumn is 1 μm.

[0039] The refractive index n of the ring 12 =2.6μm, the ring width is 0.1μm, the ring is Figure 1 The a area in .

[0040] from Figure 1 It can be seen that the upper and lower sides of the third to ninth micropillars are in contact with the ring respectively, while the upper sides of the first, second, tenth and eleventh micropillars do not touch the ring. Therefore, an accommodating space is formed between the first, second and third micropillars and the ring, and between the ninth, tenth and eleventh micropillars and the ring, that is, Figure 1 The two b areas in .

[0041] In the two b regions, the same refractive index as the ring (also n 12=2.6), h = 0.25 μm, h refers to the vertical height of the bottom of region b relative to the center of the sphere (0, 0), that is, the height relative to the line y = 0.

[0042] When 500nm parallel light is incident vertically along the negative x-axis, the lateral full width at half maximum is about 87.47nm, and the light intensity is about 45 times that of the incident light. The lateral electric field distribution on the surface is as follows: Figure 2 shown.

[0043] The present invention forms a microsphere by combining a circular ring with eleven micropillars and a medium embedded in region b. Microspheres with a refractive index greater than 2 face the problem of light spots being compressed within the microsphere. This problem is also addressed by adding a high-refractive-index circular ring to the exterior of the micropillars. However, embedding a high-refractive-index medium in region b increases the energy flux distribution and optical vortex distribution on both sides, modulating the light spot output and forming a nanojet with an extremely narrow beamwidth (extremely small FWHM), thus resolving this problem.

[0044] The present invention provides a new microsphere design concept, namely, a high-refractive-index ring on the outside, with high-refractive-index media embedded at both ends to adjust the energy flux distribution and emit a light spot, further breaking through the diffraction limit and solving the problem of microspheres with a refractive index greater than 2, where the light spot is compressed inside the microsphere. Its advantages are:

[0045] 1. The beam width of 87nm is very good (the FWHM is much narrower, further breaking the diffraction limit);

[0046] 2. This result (FWHM) broke through to below 100nm, breaking a limit.

[0047] The above content describes in detail the structure of the single-molecule fluorescence excitation device provided in an embodiment of the present invention. Corresponding to the single-molecule fluorescence excitation device, an embodiment of the present invention also provides a gene sequencing chip composed of the single-molecule fluorescence excitation device.

[0048] Figure 3 The structure of a gene sequencing chip provided according to an embodiment of the present invention is shown.

[0049] like Figure 3 As shown, the gene sequencing chip provided by an embodiment of the present invention includes a single-molecule fluorescence excitation device 1, a waveguide structure 2, a metasurface lens 3, a transmission layer 4 and a detector 5 prepared in sequence from top to bottom. The waveguide structure 2 is prepared on the substrate of the metasurface lens 3 by a sputtering process or an atomic deposition process, the single-molecule fluorescence excitation device 1 is prepared on the waveguide structure 2 by a sputtering process or an atomic deposition process, the transmission layer 4 is prepared on the unit structure array of the metasurface lens 3 by a sputtering process or an atomic deposition process, and the detector 5 is prepared on the transmission layer 4.

[0050] The waveguide structure 2 is used to transmit the excitation light to the single-molecule fluorescence excitation device 1 to excite the fluorescence signal. The waveguide structure 2 includes a core layer and a cladding layer. The cladding layer is wrapped around the outside of the core layer. The refractive index of the core layer is higher than the refractive index of the cladding layer.

[0051] The single-molecule fluorescence excitation device 1 is used to excite nucleic acid molecules through excitation light, so that the nucleic acid molecules emit fluorescence signals.

[0052] The metasurface lens 3 is used to collect the fluorescence signal excited by the single-molecule fluorescence excitation device 1 , and the fluorescence signals of different wavelengths are focused to different focal positions on the same focal plane.

[0053] The transmission layer 4 is used to ensure a certain distance between the metasurface lens 3 and the detector 5, ensuring that the fluorescent signal collected by the metasurface lens 3 is focused on the detector 5. The transmission layer 4 can be made of materials such as silicon dioxide, hafnium dioxide or titanium dioxide.

[0054] The detector 5 is located on the focal plane of the metasurface lens 3 and is used to receive the fluorescence signals of various wavelengths focused by the metasurface lens 3 .

[0055] To ensure that different fluorescence signals are effectively distinguished by the detector 5 , the distance between two adjacent focal points of the metasurface lens 3 is greater than 2 μm.

[0056] The working principle of the gene sequencing chip is as follows: the PNJ generated by the single-molecule fluorescence excitation device 1 is used to excite the nucleic acid molecules to emit fluorescence. The excitation light is transmitted along the waveguide structure 2. The fluorescence signal is collected by the metasurface lens 3 located below the waveguide structure 2 and focused on the detector 5 through the transmission layer 4.

[0057] Because fluorescence signals have low intensity and a large divergence angle, a metasurface lens with a large numerical aperture is required to collect them. To distinguish between different fluorescence signals, fluorescence signals of different wavelengths must be focused at different focal points. Based on these two requirements, a metasurface lens must have both a large numerical aperture and multiple focal points.

[0058] Based on the characteristics of the metasurface lens, the specific design of the metasurface lens is as follows:

[0059] 1. A large numerical aperture metasurface lens with a numerical aperture greater than 0.4 is selected to collect the fluorescence signal to meet the requirements of large fluorescence signal divergence angle and low signal intensity.

[0060] The diameter and focal length of the metasurface lens are selected based on the numerical aperture. For example, if the diameter of the metasurface lens is 10 μm and the focal length is 12 μm, the numerical aperture is set to ensure that the fluorescence signal can be effectively collected.

[0061] 2. Design the size and shape of the unit structure to correspond to fluorescent signals of different wavelengths so that fluorescent signals of different wavelengths are focused at different focal positions on the same focal plane.

[0062] More specifically, the phase distribution of the metasurface lens is designed according to the focal positions corresponding to different fluorescence signal wavelengths; and the unit structure is arranged according to the phase distribution to achieve the purpose of focusing at different positions on the same focal plane under different fluorescence signal wavelengths.

[0063] Figure 4 The structure of the metasurface lens according to an embodiment of the present invention is shown.

[0064] like Figure 4 As shown, the metasurface lens includes a substrate 31 and a unit structure 32. The number of unit structures 32 is multiple, forming a unit structure array. The unit structure 32 is formed by photolithography of a dielectric layer. Specifically, a dielectric layer is prepared on the substrate 31, and the dielectric layer is patterned by electron beam lithography technology to form a unit structure array composed of multiple unit structures 32.

[0065] Fluorescence signals 6 of different wavelengths are focused onto different focal positions on the same focal plane 7 through their corresponding unit structures 32. The metasurface lens can achieve the fluorescence signal focusing function of a Fresnel lens. Compared to a Fresnel lens, the metasurface lens has a simpler structure, lower complexity, simpler manufacturing process, and lower production cost.

[0066] In an example of the present invention, the substrate 31 may be made of silicon dioxide or other materials, and the dielectric layer may be made of titanium dioxide, gallium nitride, or hafnium dioxide or other materials.

[0067] One unit structure 32 can focus a fluorescence signal of one wavelength at one focal point, and the unit structure array can focus fluorescence signals of different wavelengths at different focal positions on the same focal plane.

[0068] Taking a silicon dioxide substrate and a titanium dioxide medium as an example, the wavelength center peaks of 532nm, 578nm, 645nm, and 721nm are selected as the operating wavelengths. Based on these four operating wavelengths, four unit structures that can respond to them are designed. For example, the unit structure corresponding to the wavelength of 532nm is rectangular, the unit structure corresponding to the wavelength of 532nm is cylindrical, the unit structure corresponding to the wavelength of 645nm is elliptical, and the unit structure corresponding to the wavelength of 645nm is triangular. Then, according to the focal positions corresponding to the wavelengths of 532nm, 578nm, 645nm, and 721nm, the four unit structures of rectangle, cylinder, elliptical, and triangle are arranged to realize the focusing function of the metasurface lens.

[0069] The present invention innovatively introduces a polarization-independent multi-wavelength multi-focus metasurface lens. According to the off-axis focusing pb phase formula f represents the focal length, (x f ,y f ) represents the focal position of different wavelengths, (x l ,y l ) represents the position of any unit on the hypersurface, and the calculated θ ideal (x l ,y l ) is the ideal phase distribution of the metasurface. Since the ideal phases of different wavelengths are different, a constant phase C(λ) related to the wavelength is introduced here. i ) is used to characterize the difference between the ideal phase of different wavelengths and the transmission phase fit of the unit structure. Finally, an optimization algorithm is used to match the transmission phase with the ideal phase of different wavelengths. This metasurface not only focuses light waves of different wavelengths to different positions, but also has no polarization-selective properties for the incident light waves. Because this metasurface lens is polarization-free, it can be used to split and converge light waves of different wavelengths, achieving both a collection and a splitting effect.

[0070] The designed metasurface lens has the characteristics of large numerical aperture, no requirements for the incident polarization of light, and different focal distribution positions according to different wavelengths. It can replace the usual filters and optical convergence imaging functions, meet the needs of effective collection of excited fluorescence signals in the process of single-molecule fluorescence gene sequencing, ensure a high signal-to-noise ratio, and distinguish the focal positions at different wavelengths, reducing the complexity of the chip, making the entire sequencing system more integrated and the production more digital, while also improving the reliability of molecular manipulation. It is a solution with high application value for third-generation and fourth-generation gene sequencing technologies.

[0071] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0072] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

[0073] The above specific embodiments of the present invention do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A single-molecule fluorescence excitation device, characterized in that: It includes a ring and eleven microcolumns located in the ring, and the refractive index of the ring is higher than the refractive index of the eleven microcolumns; wherein, The eleven micropillars are respectively the first to the eleventh micropillars, the refractive index of the first to the fifth micropillars is the same as the refractive index of the seventh to the eleventh micropillars, the first to the fifth micropillars and the eleventh to the seventh micropillars are arranged in ascending order of refractive index from the edge to the center of the ring, and the sixth micropillar is located at the center of the ring; The ring and the first and second micropillars, as well as the ring and the tenth and eleventh micropillars, respectively form accommodation spaces. A medium with the same refractive index as the ring is embedded in the two accommodation spaces. A microsphere is formed by the ring and the eleven micropillars and the medium embedded in the accommodation spaces.

2. The single-molecule fluorescence excitation device according to claim 1, wherein The calculation formula for the second to fifth microcolumns is: ; Where g represents the gradient change index, N represents the total number of layers, and m represents the mth layer.

3. A gene sequencing chip comprising a detector, a transmission layer located above the detector, a metasurface lens located above the transmission layer, and a waveguide structure located above the metasurface lens, characterized in that: It also includes the single-molecule fluorescence excitation device as claimed in claim 1 or 2, which is located above the waveguide structure.

4. The gene sequencing chip according to claim 3, wherein: The metasurface lens includes a substrate and a dielectric layer prepared on the substrate. A unit structure array is photoetched on the dielectric layer, which is used to focus incident light of different wavelengths within the incident light band onto different focal positions on the same focal plane, and the distance between two adjacent focal points is greater than 2μm.

5. The metasurface lens according to claim 4, wherein: The numerical aperture of the metasurface lens is greater than 0.

4.

6. The metasurface lens according to claim 4, wherein: The substrate is made of silicon dioxide material, and the dielectric layer is made of titanium dioxide material, gallium nitride material or hafnium dioxide material.

7. The metasurface lens according to claim 4, wherein: The shape of the unit structures in the unit structure array is rectangular, triangular, cylindrical or elliptical.

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