CN112779153A - Gene sequencing chip and system - Google Patents
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- CN112779153A CN112779153A CN202011630254.3A CN202011630254A CN112779153A CN 112779153 A CN112779153 A CN 112779153A CN 202011630254 A CN202011630254 A CN 202011630254A CN 112779153 A CN112779153 A CN 112779153A
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- 238000012163 sequencing technique Methods 0.000 title claims abstract description 63
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 45
- 239000013307 optical fiber Substances 0.000 claims abstract description 90
- 238000001514 detection method Methods 0.000 claims abstract description 20
- 238000005253 cladding Methods 0.000 claims abstract description 15
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims abstract description 6
- 102000004190 Enzymes Human genes 0.000 claims abstract description 6
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 6
- 230000005284 excitation Effects 0.000 claims description 36
- 238000003752 polymerase chain reaction Methods 0.000 claims description 7
- 239000010410 layer Substances 0.000 claims description 6
- 239000012792 core layer Substances 0.000 claims description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 4
- 239000002184 metal Substances 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 3
- 238000003491 array Methods 0.000 claims description 2
- 238000003384 imaging method Methods 0.000 abstract description 6
- 230000008878 coupling Effects 0.000 abstract description 3
- 238000010168 coupling process Methods 0.000 abstract description 3
- 238000005859 coupling reaction Methods 0.000 abstract description 3
- 239000000835 fiber Substances 0.000 description 18
- 238000000034 method Methods 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 6
- 230000005540 biological transmission Effects 0.000 description 4
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002086 nanomaterial Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000005498 polishing Methods 0.000 description 2
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- 230000009471 action Effects 0.000 description 1
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- 238000007405 data analysis Methods 0.000 description 1
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- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 239000012458 free base Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000010884 ion-beam technique Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
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- 238000006467 substitution reaction Methods 0.000 description 1
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- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
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- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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Abstract
The invention belongs to the technical field of biological detection, and provides a gene sequencing chip and a gene sequencing system. The gene sequencing chip comprises an optical fiber for transmitting exciting light, wherein at least one part of a cladding of the optical fiber is removed to form a D-shaped section, and a nanopore is modified at the removal position and is positioned in an evanescent field of the optical fiber. The nanopore structure realizes the physical local area of a nucleic acid fragment to be sequenced and sequencing enzyme, a laser enters a single optical fiber of the chip through the coupling of a light splitting element, an evanescent field generated on the upper surface of the optical fiber excites free bases carrying fluorescent dye and positioned in the nanopore, and a generated fluorescent sequence signal is detected in a microscopic imaging mode.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a gene sequencing chip and a gene sequencing system for monomolecular fluorescence sequencing.
Background
Sequencing technology based on a fluorescence labeling method is an important means for realizing high-throughput single molecule sequencing. The key to achieving high signal-to-noise ratio collection of single molecule fluorescence is to reduce background noise. Two effective ways for realizing single-molecule fluorescence excitation currently exist, (1) the number of molecules in an illumination area is reduced through nanopore constraint, and the volume of excitation light is constrained through a formed zero-mode waveguide effect, so that single-molecule fluorescence excitation is realized, (2) evanescent fields generated based on total internal reflection excite single-molecule fluorescence, and both the two technologies are applied to commercial sequencing instruments at present. However, the processing procedure of the sequencing chip based on the zero-mode waveguide is expensive due to the complicated processing technology, and the excitation method based on the total internal reflection technology generally requires an objective lens with a high numerical aperture to generate a beam satisfying the total reflection condition, which greatly increases the cost for generating the excitation beam and limits the excitation area. Therefore, designing a simple-structure and low-cost single-molecule fluorescence excitation and detection system is a technical problem to be solved by the technical personnel in the field, and has practical significance in the fields of single-molecule fluorescence sequencing and single-molecule fluorescence detection.
Disclosure of Invention
The invention provides a gene sequencing waveguide chip for monomolecular fluorescence sequencing, which aims to solve the problems of difficult preparation and high cost of the waveguide chip for gene sequencing.
In order to achieve the purpose, the invention adopts the following specific technical scheme:
the invention provides a gene sequencing chip, which comprises an optical fiber for transmitting exciting light, wherein at least one part of a cladding of the optical fiber is removed to form a D-shaped section, a nanopore is modified at the removal position, and the nanopore is positioned in an evanescent field of the optical fiber.
The gene sequencing chip provided by the invention comprises an optical fiber array formed by a plurality of optical fibers; and modifying a plurality of nano holes on each optical fiber to form a gene sequencing array.
In the gene sequencing chip provided by the invention, a plurality of optical fibers are arranged in parallel, and the distance between every two adjacent optical fibers is greater than 100 nm.
In the gene sequencing chip provided by the invention, the nanopore is positioned at a position where the evanescent field energy is stronger.
In the gene sequencing chip provided by the invention, the nanopore is cylindrical or cubic, and the axial direction of the nanopore is vertical to the center of the section of the optical fiber.
In the gene sequencing chip provided by the invention, the optical fiber comprises a core layer and a cladding layer, and the bottom of the nanopore is positioned in the cladding layer.
In the gene sequencing chip provided by the invention, the nanopore is a metal nanopore, is a cylindrical pore with the diameter of 80 nm-200 nm, and defines a physical local area for accommodating a long-chain nucleic acid sequence, sequencing enzyme and dNTPs solution.
On the other hand, the invention also provides a gene sequencing system, which comprises an excitation light source, a light splitting element, a detection unit and the gene sequencing chip;
the input end of the light splitting element is connected with the excitation light source, and the output ends of the light splitting element are connected with the optical fiber arrays in a one-to-one correspondence manner; the detection unit is aligned with a nanopore, and a fluorescence signal generated by polymerase chain reaction in the nanopore is imaged in the detection unit.
In the gene sequencing system provided by the invention, the excitation light source is matched with the excitation wavelength of fluorescent dye molecules marked on dNTPs; the spatial resolution of the detection unit is better than the minimum spatial distance of the nanopore.
In the gene sequencing system provided by the invention, a fluorescence signal generated by polymerase chain reaction is emitted from the lower part of the nanopore and enters the detection unit.
The implementation of the invention can achieve the following technical effects:
(1) the sequencing chip based on the D-type optical fiber array can realize low-cost processing of the high-throughput sequencing chip by utilizing the characteristics of independent light transmission in optical fibers, evanescent field transmission characteristics of the surface of the D-type optical fibers, arraying and assembling of the optical fibers, compatibility of an optical fiber silicon dioxide material with a micro-nano processing technology and high biocompatibility. The flexibility of the excitation of the fiber evanescent field and the high consistency of the fiber can effectively improve the consistency and efficiency of the fluorescence excitation.
(2) The time characteristic of the optical field generated based on the excitation of the optical fiber evanescent field corresponds to the response of the excitation laser one by one, namely, the continuous laser generates the evanescent field of continuous excitation, and the pulse laser generates the pulse evanescent field with time intervals. Such excitation and detection systems may be adapted for sequencing technologies based on different sequencing principles. Based on the wide-range wavelength adaptability of the optical fiber evanescent field excitation, the optical fiber structure dispersion and the modal dispersion can be regulated and controlled by regulating the proportion and the refractive index of the optical fiber core layer and the cladding layer, and the mode uniformity and the modal dispersion characteristic in a multi-wavelength range can be regulated and controlled, so that the method is also suitable for a sequencing method which needs to adopt fluorescent labels with various different wavelengths.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a schematic diagram showing the structure of a gene sequencing system according to an embodiment of the present invention;
FIG. 2 is a side view of the optical fiber array arrangement; FIG. 2a is a side view of an optical fiber array arrangement, and FIG. 2b is a side view of another optical fiber array arrangement;
FIG. 3 is a front view of the optical fiber array arrangement;
FIG. 4 is a schematic diagram of a D-type fiber nanostructure and evanescent field distribution structure.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not to be construed as limiting the invention.
The invention provides a gene sequencing system, as shown in figure 1, the gene sequencing system of the invention comprises an excitation light source 1, a light splitting element 2, a gene sequencing chip 3 and a detection unit 4; the input end of the light splitting element 2 is connected with the excitation light source 1, the output end of the light splitting element is connected with the optical fiber array 301 of the gene sequencing chip 3 in a one-to-one correspondence manner, and the excitation light is transmitted to the polymerase chain reaction site; the detection unit 4 is aligned with the nanopore 303 on the gene sequencing chip 3, and a fluorescence signal generated by polymerase chain reaction in the nanopore 303 is imaged in the detection unit 4.
In one embodiment of the present invention, the excitation light source 1 is a laser, and the light splitting element 2 is a fiber beam splitter. Emergent light of the laser enters the input end of the optical fiber beam splitter in a coupling mode through optical fiber connection, the output end of the beam splitter is connected with the array optical fibers in a one-to-one correspondence mode to input laser into the optical fiber array 301, free base dNTPs which are located inside the nanopore and carry fluorescent dye are excited, and a fluorescent signal generated by polymerase chain reaction is emitted from the lower portion of the nanopore 303 and enters the detection unit 4.
In one embodiment of the invention, in order to improve the flux of a fluorescence excitation system, improve the fluorescence excitation efficiency and reduce the cost of a sequencing fluorescence excitation system, the invention selects a mode based on fiber waveguide evanescent field excitation. The invention comprises a method for forming a plurality of optical fibers into an array, a method for polishing the surfaces of the plurality of optical fibers to form a D-shaped optical fiber array 301, and a method for forming nano structures on the surfaces of the optical fiber array 301.
In another aspect, the present invention further provides a gene sequencing chip, comprising an optical fiber for transmitting excitation light, wherein at least a portion of the cladding 304 of the optical fiber is removed to form a D-shaped cross section, and the nanopore 303 is modified at the removed position, and the nanopore 303 is in an evanescent field of the optical fiber.
In one embodiment of the present invention, a plurality of optical fibers form an optical fiber array 301; a plurality of nanopores 303 are decorated on each optical fiber to form a gene sequencing array.
In one embodiment of the present invention, the upper surfaces of the optical fibers arranged on the optical fiber array 301 are polished and filled to form a plane, and the structure of the nanopore 303 is processed or surface-treated at a position corresponding to the optical axis of each optical fiber to immobilize enzymes necessary for a sequencing reaction and nucleic acid fragments to be sequenced. When laser is transmitted in the optical fiber, an evanescent field is formed on the surface of the optical fiber array 301, and when the loaded free nucleotide labeled with fluorescent dye molecules and the nucleic acid sequence to be sequenced are subjected to polymerization reaction under the action of enzyme, fluorescence is generated by excitation of the evanescent field. The fluorescence is observed by the detection unit 4 located below the fiber array 301 and the obtained picture is transmitted to the data processing unit, completing one sequencing reading.
In one embodiment of the present invention, the emergent light of the excitation light source 1 enters the inlet of the optical fiber beam splitter through optical fiber coupling, and each output port of the optical fiber beam splitter is connected with a single optical fiber on the gene sequencing chip 3 through an optical fiber flange. The incident laser has a strong evanescent field on the top surface of the D-fiber, which excites the bases carrying fluorescent molecules dissociated on the top surface of the gene sequencing chip 3. The generated fluorescence signal is collected by a detection unit 4 arranged below the gene sequencing chip 3 in an imaging mode, and image data representing the energy distribution of the fluorescence signal is transmitted to a data processing system.
In one embodiment of the present invention, the excitation light source 1 is a high power laser and is matched with the excitation wavelength of the fluorescent dye molecules labeled on the dNTPs; the optical fiber beam splitter has low loss and uniform energy distribution proportion; the optical fiber array 301 is formed by arranging D-shaped optical fibers, the horizontal plane of the D-shaped optical fibers forms the upper surface of the optical fiber array 301 chip, the upper surface of the optical fiber array 301 is decorated with a diameter nanometer hole type metal structure, the nanometer holes 303 are cylindrical or cubic, and the axial direction of the nanometer holes is perpendicular to the center of the section of the optical fibers. These hole patterns are located directly above the D-fiber axis and the lower surface of the holes remain in the core 305 of the fiber. The spatial resolution of the micro-imaging units 1-4 should be better than the minimum spatial distance of the holes on the fiber flat plate and have sufficiently high sensitivity, and sCMOS or EMCCD can be used as an image receiver for data analysis.
An alternative form of the optical fibre array 301 is shown in figure 2, and in an alternative form in the side view of figure 2a, the process comprises:
(1) arranging cylindrical optical fibers in a V-shaped groove 302 and fixing the optical fibers by using an adhesive, wherein the center of the section of each optical fiber is positioned below the horizontal surface of the V-shaped groove, and the outer surface of each cylindrical optical fiber is ensured to be positioned above the surface of the V-shaped groove;
(2) horizontally throwing off part of the optical fiber cladding 304 on the upper part of the V-shaped groove to ensure that a single optical fiber is distributed in a D shape and each optical fiber forms a plane structure;
(3) the nanopore 303 is processed at a position right above the fiber core layer 305 by an ion beam etching method, the diameter of the nanopore 303 is in a range of tens to hundreds of nanometers, and the bottom of the nanopore 303 is ensured to be above the fiber core layer 305, i.e., the bottom of the nanopore 303 is still located in the cladding 304, so that a fiber flat plate with a nanopore 303 structure on the surface as shown in fig. 3 is formed.
In an alternative form, the side view of fig. 2b, based on the previous embodiment, and taking into account the effect of the lower V-groove and the semi-circular curvature of the fiber on the imaging, the polished side of the fiber array can be fixed to the glass plane after the D-fiber has been machined horizontally, then the fiber array can be removed from the V-groove and the other side, i.e., the side of the convex circle of the D-fiber, polished so that both sides are flat.
In an embodiment of the present invention, the optical fiber array 301 has a D-type structure, the conventional cylindrical optical fiber is composed of the core 305 and the cladding 304, a place with strong evanescent field energy generated by total reflection is concentrated in the cladding 304 near the boundary between the core 305 and the cladding 304, the influence of polishing to reduce the thickness of the cladding 304 on the energy distribution of the evanescent field is small, and the nanopore 303 can be located at a position with strong evanescent field energy to improve the fluorescence excitation efficiency.
In an embodiment of the invention, the nanopore 303 structure is a circular hole structure with a diameter of tens to hundreds of nanometers, so that physical local areas of a long-chain nucleic acid sequence, sequencing enzyme and dNTPs solution can be realized, and an incident optical fiber evanescent field can only excite fluorescent molecules in the nanopore 303. The generated fluorescence is emitted from below the nanopore 303 and is observed by the microscopic imaging unit. Preferably, the metal nanopores 303 are cylindrical pores having a diameter of 80nm to 200 nm.
In one embodiment of the present invention, the spacing of the nanopores 303 comprises two parts: the first is the distance along the axial direction of the optical fiber, and the distance is limited by the spatial resolution of the microscopic imaging unit and can be as low as hundreds of nanometers or even tens of nanometers; the second is the distance perpendicular to the axial direction of the optical fibers, the distance is determined by the distance of the V-shaped grooves, and the distance between the adjacent optical fibers is more than 1000nm and can be several microns to dozens of microns.
The gene sequencing chip scheme of the invention has the advantages that:
(1) by changing the proportion of the outer diameter and the inner diameter of the optical fiber, the integration of the nano holes with higher density can be realized.
(2) The transmission loss in the optical fiber is low, and a long-distance large-area chip is easy to realize.
(3) The optical fiber stretching process has high maturity, can easily realize mode field control and realize uniform illumination.
(4) By utilizing independent transmission of light rays in the optical fiber, energy in a waveguide core is not easy to leak to a substrate layer, so that the intensity of background light is reduced, and the signal-to-noise ratio is improved.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and should not be taken as limiting the invention. Variations, modifications, substitutions and alterations of the above-described embodiments may be made by those of ordinary skill in the art without departing from the scope of the present invention.
The above embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A gene sequencing chip is characterized by comprising an optical fiber for transmitting exciting light, wherein at least one part of a cladding of the optical fiber is removed to form a D-shaped section, and a nanopore is modified at the removal position and is positioned in an evanescent field of the optical fiber.
2. The gene sequencing chip of claim 1, comprising an optical fiber array formed by a plurality of the optical fibers; and modifying a plurality of nano holes on each optical fiber to form a gene sequencing array.
3. The gene sequencing chip of claim 2, wherein the plurality of optical fibers are arranged in parallel, and the distance between adjacent optical fibers is greater than 100 nm.
4. The gene sequencing chip of claim 1, wherein the nanopore is in a position where the evanescent field energy is stronger.
5. The gene sequencing chip of claim 1, wherein the nanopore has a cylindrical or cubic shape with an axis perpendicular to a cross-sectional center of the optical fiber.
6. The gene sequencing chip of claim 5, wherein the optical fiber comprises a core layer and a cladding layer, and wherein the bottom of the nanopore is located in the cladding layer.
7. The gene sequencing chip of claim 1, wherein the nanopore is a metal nanopore, is a cylindrical pore with a diameter of 80nm to 200nm, and defines a physical local area for holding a long-chain nucleic acid sequence, a sequencing enzyme, and a dNTPs solution.
8. A gene sequencing system, which is characterized by comprising an excitation light source, a light splitting element, a detection unit and the gene sequencing chip of any one of claims 1 to 7;
the input end of the light splitting element is connected with the excitation light source, and the output ends of the light splitting element are connected with the optical fiber arrays in a one-to-one correspondence manner; the detection unit is aligned with a nanopore, and a fluorescence signal generated by polymerase chain reaction in the nanopore is imaged in the detection unit.
9. The gene sequencing system of claim 8, wherein the excitation light source is matched to the excitation wavelength of fluorescent dye molecules labeled on dNTPs; the spatial resolution of the detection unit is better than the minimum spatial distance of the nanopore.
10. The gene sequencing system of claim 8, wherein a fluorescence signal generated by a polymerase chain reaction exits from below the nanopore into the detection unit.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007093298A (en) * | 2005-09-27 | 2007-04-12 | Univ Nagoya | Spectroscopy and probe |
| CN102279438A (en) * | 2011-07-25 | 2011-12-14 | 中国科学院光电技术研究所 | Optical-fiber evanescent field sensing optical fiber with novel micro-nano structure |
| CN103468787A (en) * | 2012-06-07 | 2013-12-25 | 许亮 | Fiber optic sensor for quantitative analysis of nucleic acid |
| CN111123428A (en) * | 2019-12-24 | 2020-05-08 | 中国科学院苏州生物医学工程技术研究所 | Modification method of zero-mode waveguide hole wall and zero-mode waveguide hole structure |
| CN111235004A (en) * | 2020-01-17 | 2020-06-05 | 中国科学院苏州生物医学工程技术研究所 | Preparation method of gene sequencing chip |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007093298A (en) * | 2005-09-27 | 2007-04-12 | Univ Nagoya | Spectroscopy and probe |
| CN102279438A (en) * | 2011-07-25 | 2011-12-14 | 中国科学院光电技术研究所 | Optical-fiber evanescent field sensing optical fiber with novel micro-nano structure |
| CN103468787A (en) * | 2012-06-07 | 2013-12-25 | 许亮 | Fiber optic sensor for quantitative analysis of nucleic acid |
| CN111123428A (en) * | 2019-12-24 | 2020-05-08 | 中国科学院苏州生物医学工程技术研究所 | Modification method of zero-mode waveguide hole wall and zero-mode waveguide hole structure |
| CN111235004A (en) * | 2020-01-17 | 2020-06-05 | 中国科学院苏州生物医学工程技术研究所 | Preparation method of gene sequencing chip |
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| Title |
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| 魏清泉等: "零模波导原理、制备及其在单分子荧光检测中的应用", 《生物技术进展》 * |
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Application publication date: 20210511 |