Sequencing wafer for continuous single molecule nucleic acid sequencing

Abstract

The invention discloses a sequencing wafer for continuous single molecule nucleic acid sequencing. The sequencing wafer includes a substrate, a waveguide, at least one optical coupler, at least one sequencing site, and a beam adjustment mechanism. The waveguide is provided on the substrate and includes a core layer and an overlying layer provided on the core layer. The optical coupler extends from the overlying layer into the core layer. The at least one sequencing site is located in the overlying layer. The light beam adjusting mechanism is used for adjusting the total projection area of the light beam from the optical coupler when the light beam is transmitted towards the sequencing site.

Description

[0001] This invention is a divisional application. The application number of the parent application is 202011495199.1, the application date is December 17, 2020, and the name of the invention is “Equipment and system for single-molecule nucleic acid detection”.

[0002] The present invention claims U.S. Provisional Application No. 62 / 950,551 filed on December 19, 2019, which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates to a bioassay system; in particular, a system comprising a detection module capable of detecting single molecule nucleic acid sequencing. Background Art

[0004] The continuous single-molecule nucleic acid sequencing method is a high-speed, long-sequence sequencing method with great potential, which can be used to sequence DNA / RNA / mRNA / modified DNA / modified RNA. When executing this method, an optical system is required to continuously monitor each polymerase that replicates the template DNA / RNA molecule (molecule to be sequenced), and identify the fluorescent signal associated with the introduction event, and thus be able to read the nucleic acid base sequence of the template molecule. "Science" magazine (02Jan 2009, Vol.323, Issue5910, pp.133-138) discloses a sequencing method, which uses a dye determination method for DNA sequencing to determine the fluorescently labeled nucleotide (A, T, G, C) sequence of a given DNA fragment at a specific reaction site on a DNA nanostructure array.

[0005] The sequencing site is a location that can fix, hold or restrict the polymerase replication complex (i.e., the complex of polymerase combined with template and primer) at a fixed position. Usually, a large number of sequencing sites are arranged in an array on a substrate, which is called a sequencing chip.

[0006] The continuous single-molecule sequencing system includes an excitation light source, a sequencing chip, a fluorescent optical device, and an optical sensor. The excitation light source can supply excitation light to each sequencing site. The fluorescent optical device is used to collect and guide the fluorescent signal emitted by each sequencing site on the sequencing chip to the optical sensor. The optical sensor is formed by an array of photodetectors (also called an image sensor or an area sensor). Each photodetector on the sensor is called a pixel. Since four nucleotide bases need to be distinguished, the optical sensor needs to be able to distinguish multiple wavelength bands of labeled fluorophores.

[0007] Performance of the sequencing system. In order for the sequencing system to capture the fluorescence emitted by the introduction event without interference from all the labeled dNTPs floating around the polymerase, the replication complex (also called sequencing complex, formed by polymerase / template / primer) needs to be fixed in one position (sequencing site). And the system needs to provide a light source and a sensor, the light source only excites the fluorophore that is close enough to the polymerase (limited excitation space), and the sensor only receives signals from the vicinity of the polymerase (limited observation space). In order to improve the efficiency of the system, a system with multiple sequencing sites acting in parallel is preferred. System performance can be defined as "number of sequencing sites / system cost".

[0008] Lens-based sequencing system: In the laboratory, a high-resolution total internal reflection fluorescence (TIRF) microscope can be converted into a continuous nucleic acid DNA single-molecule sequencer. Figure 1 Schematic diagram of an inverted microscope, which is a typical TIRF microscope. The laser beam is emitted from the laser light emitter 90 through the filter 91 into the high NA (usually 1.5NA) 60X or 100X oil immersion objective 92, which forms TIRF conditions on the upper surface of the cover glass 93 for continuous nucleic acid DNA single molecule sequencing. The objective lens 92 is infinity corrected. The fluorescence image collected by the objective lens 92 and passed through the filter cube 91 removes the laser light wavelength, and only the fluorescence of interest is retained. Using some other optical elements, such as mirrors, prisms, and relay lenses (not shown), the image beam is projected onto the sensor device 94 to form an enlarged image through the projection lens 95 (also called an objective lens or tube lens). The fluorescence image collected by the sensor device 94 is then analyzed and converted into sequencing data. Since lenses are used to provide excitation light and collect fluorescence, this type of system is called a lens-based sequencing system. The number of sequencing sites in a lens-based system ranges from thousands to tens of thousands, depending on the specifications of the lens and sensor.

[0009] U.S. Patent No. 8,318,094 and U.S. Patent No. 7,170,050 disclose a sequencing system that provides more than 100,000 sequencing sites. The reaction sites on these system chips usually have microlenses or micromirrors to increase the efficiency of fluorescence collection. However, these micro-optical devices make it impossible to reduce the spacing between the reaction sites, and therefore cannot further increase the density. Micro-optical devices increase the chip manufacturing process and cost. Moreover, such systems usually use a variety of different optical elements to guide, modify and otherwise manipulate the light directed to and / or received from the reaction site. Such systems are bulky and sensitive to environmental vibrations because their array of excitation light spots needs to be precisely aligned with the array of sequencing sites. These systems that use lenses to collect fluorescence from sequencing chips can be classified as special cases of lens-based sequencing systems. The total number of sequencing sites that can be monitored by the best existing lens-based commercial sequencing systems is 150,000. In contrast, the sequencing chips in lens-based systems are simple and relatively low in cost.

[0010] Regarding chip-based sequencing systems. In another embodiment, U.S. Patent No. 7,767,441 discloses a sequencing system that integrates a sequencing site array, an excitation light distribution waveguide, and a sensor into a single chip. This system that integrates a sequencing chip and an optical sensor can be called a chip-based sequencing system. The number of sequencing sites depends on the size of the chip and can be millions or tens of millions. However, the manufacturing cost of the chip is very high and is designed for disposable use, which makes the operating cost higher than most applications can afford. Summary of the invention

[0011] In an exemplary embodiment, a device for single-molecule nucleic acid detection is disclosed. The device includes a detection module for detecting fluorescence generated by a sequencing chip. The detection module includes a sensor device, an objective lens with a first magnification, and a projection lens with a second magnification. The objective lens and the projection lens are used to transmit the fluorescence from the sequencing chip to the sensor device. The second magnification is less than 1.

[0012] In another exemplary embodiment, a device for continuous single-molecule nucleic acid detection is disclosed. The device includes a sequencing chip having a plurality of sequencing sites arranged at a pitch; and a detection module for detecting fluorescence generated by the sequencing sites. The detection module includes a sensor device having a plurality of pixels, each of which has a pixel size; and a lens group having an objective lens and a projection lens. The objective lens and the projection lens are used to transmit fluorescence from the sequencing site to the sensor device, and the lens group has an overall magnification. The product of the pitch and the overall magnification is equal to or greater than 1 times the pixel size, and equal to or less than 2 times the pixel size.

[0013] In another exemplary embodiment, an apparatus for continuous single-molecule nucleic acid sequencing is disclosed. The apparatus includes a detection module for detecting a fluorescence generated by a sequencing chip. The detection module includes a sensor device having a plurality of pixels, each of which has a pixel size; and a lens assembly having an objective lens and a projection lens, wherein the objective lens and the projection lens are used to transmit the fluorescence from the sequencing chip to the sensor device. A projected light spot size of the fluorescence on the sensor device is less than or equal to 1.5 times the pixel size.

[0014] In yet another exemplary embodiment, a sequencing chip for continuous single-molecule nucleic acid sequencing is disclosed. The sequencing chip comprises a substrate, a waveguide, an optical coupler, a sequencing site, and a beam adjustment mechanism. The waveguide is disposed on the substrate. The waveguide comprises a core layer and an upper cladding layer disposed on the core layer. The optical coupler extends from the upper cladding layer into the core layer. The sequencing site is located in the upper cladding layer. The beam adjustment mechanism is used to adjust a total projected area of ​​a light beam from the optical coupler when it is transmitted toward the sequencing site. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A cross-sectional schematic diagram of a conventional TIRF microscope is shown.

[0016] Figure 2 A schematic diagram of the design of a lens-based single-molecule sequencing system according to certain embodiments of the present disclosure is shown.

[0017] Figure 3 A schematic diagram showing a conventional microscope objective.

[0018] Figure 4 FIG. 4 is a schematic diagram showing the numerical aperture (NA) of an objective lens according to some embodiments of the present disclosure.

[0019] Figure 5 The field of view (FOV) of a lens system is shown.

[0020] Figure 6 Depicting the spatial resolution of an optical system.

[0021] Figure 7 A schematic diagram of a nanowell according to some embodiments of the present disclosure is shown.

[0022] Fig. 8A and Figure 8B The field intensity distributions of epi excitation and evanescent wave excitation are shown.

[0023] Fig.9A and Fig. 9BLens-type and prism-type TIRF are shown according to certain embodiments of the present disclosure.

[0024] Fig.10 It is shown that according to some embodiments of the present disclosure, the projected light spot size may affect the signal intensity at a pixel.

[0025] Fig.11 A schematic diagram of an apparatus for continuous single-molecule nucleic acid sequencing according to certain embodiments of the present disclosure is shown.

[0026] Fig.12 A schematic cross-sectional view of a sequencing chip according to certain embodiments of the present disclosure is shown.

[0027] Fig.13 A schematic top view of a substantially circular nanowell is shown according to certain embodiments of the present disclosure.

[0028] Fig.14 A schematic top view of a groove-shaped nanowell according to certain embodiments of the present disclosure is shown.

[0029] Fig.15 A schematic top view of a groove-shaped nanowell according to certain embodiments of the present disclosure is shown.

[0030] Fig.16 A schematic top view of a substantially circular nanowell is shown according to certain embodiments of the present disclosure.

[0031] Fig.17 A schematic top view of a groove-shaped nanowell according to certain embodiments of the present disclosure is shown.

[0032] Fig.18 A schematic top view of a groove-shaped nanowell according to certain embodiments of the present disclosure is shown.

[0033] Fig.19A A schematic top view of a waveguide having a beam adjustment mechanism according to some embodiments of the present disclosure is shown.

[0034] Fig.19B A schematic top view of a spacer according to certain embodiments of the present disclosure is shown.

[0035] Fig.19C A schematic top view of a cover according to certain embodiments of the present disclosure is shown.

[0036] Fig. 20 The pitch of the nanowells according to some embodiments of the present disclosure is shown, and this pitch is also the size of the pixels on the sensor device.

[0037] Fig.21 The spectral bands of the labeled fluorophores detected by the three channels are shown.

[0038] Fig. 22 A Philips-type prism is depicted according to certain embodiments of the present disclosure.

[0039] Fig.23 A dispersive device for spectral detection according to certain embodiments of the present disclosure is shown.

[0040] Fig.24 FIG. 1 is a SEM cross-sectional image of a sequencing wafer having nanowells according to certain embodiments of the present disclosure.

[0041] Fig.25A and Fig.25B A snapshot of an image frame showing the sequencing raw data.

[0042] Fig.26 Plotted are sequencing time traces from a nanowell of one channel.

[0043] The main component symbols are as follows:

[0044] 10: Sequencing sites

[0045] 11: Projection lens

[0046] 12: Optical sensor group

[0047] 13: Sequencing chip

[0048] 14: Objective lens

[0049] 20: Molecules being sequenced

[0050] 21: Nucleotide triphosphate

[0051] 22: Big light spot

[0052] 23: Small light spot

[0053] 24: Pixels

[0054] 31: Sequencing chip

[0055] 32: Detection module

[0056] 32': Detection module

[0057] 33: Laser light source

[0058] 34: Laser coupler

[0059] 35: Waveguide

[0060] 36: Filter

[0061] 41: Beam adjustment mechanism

[0062] 42: Holes

[0063] 43: Spacer

[0064] 44: First opening

[0065] 45: Fluid channel

[0066] 46: Cover

[0067] 47: Second opening

[0068] 50: Philips type prism

[0069] 51: Dispersion device

[0070] 90: Laser light transmitter

[0071] 91: Filter

[0072] 92: Oil immersion objective

[0073] 93: Cover glass

[0074] 94: Sensor device

[0075] 95: Projection lens

[0076] 120: Sensor

[0077] 150: Lens FOV

[0078] 151: Effective FOV

[0079] 310: Substrate

[0080] 311: Optocoupler

[0081] 312: Thin film or channel waveguide

[0082] 313: Upper layer

[0083] 314: Lower cladding

[0084] 315: Nanowell Sequencing Site Array

[0085] 315A: Top opening

[0086] 315B: Cylindrical wall

[0087] 316: Nanowell

[0088] 317: Core layer

[0089] 318: Nanogrooves

[0090] 321: Sensor device

[0091] 322: Objective lens

[0092] 323: Projection lens

[0093] S: Spacing

[0094] P: Spacing DETAILED DESCRIPTION

[0095] The following disclosure provides a variety of different embodiments or illustrations, which can be used to implement the different features of the present disclosure provided. The specific examples of the components and configurations described below are used to simplify the present disclosure. It is conceivable that these descriptions are only illustrative and are not intended to limit the present disclosure. For example, in the following detailed description, the first feature is formed above the second feature or on the second feature, which may include an embodiment in which the first and second features are formed as direct contact, and may also include an embodiment in which additional features can be formed between the first and second features so that the first and second features are not in direct contact. In addition, the present disclosure may reuse component symbols and / or labels in multiple embodiments. This repetition is based on the purpose of simplicity and clarity, and does not itself represent the relationship between the different embodiments and / or configurations discussed.

[0096] Additionally, spatially relative terms (e.g., "beneath," "below," "lower," "above," "upper," and the like) are used herein to facilitate describing the relative relationship of one element or feature illustrated in the figures to another or more elements or features. These spatially relative terms are intended to encompass other orientations of the device in use or operation in addition to the orientation depicted in the figures. The device may be positioned in other orientations (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

[0097] In the present disclosure, terms such as "first", "second", and "third" are used to describe various elements, components, regions, layers, and / or parts, but the elements, components, regions, layers, and / or parts are not limited thereto. These terms are only used to distinguish one element, component, region, layer, or part from another. Unless the context clearly indicates otherwise, the use of terms such as "first", "second", and "third" in the present disclosure does not indicate a particular sequence or order.

[0098] Figure 2A schematic diagram of the design of a lens-based single-molecule sequencing system according to certain embodiments of the present disclosure is shown, and this design mainly follows the design principle of single-molecule detection. An infinity-corrected objective lens is used to collect fluorescence from the sequencing site 10, and a projection lens 11 is used to project the signal of the sequencing site 10 to an optical sensor group 12. Generally speaking, the objective lens is used to collect fluorescence from the sequencing site, and the projection lens (also called an image field lens or a tube lens) is used to project the signal of the sequencing site to the optical sensor group. In certain embodiments, the sequencing system further includes a microfluidic structure, which is coupled to the nanowell, and the sample can be loaded onto the sequencing chip 13 through this microfluidic structure. The microfluidic structure can evenly distribute the sample on the sequencing chip 13 or can guide the sample to a designated nanowell.

[0099] Figure 3 A schematic diagram of a typical microscope objective lens 14 is shown. Generally speaking, the magnification of the objective lens 14 is marked on its body, such as "100X" as shown in the figure. In ordinary microscopes, a 100X objective lens is used in conjunction with a preset or pre-set 1X projection lens, such as Figure 1 As shown, the overall magnification of this lens group is 100X. Since the microscope is used to magnify the detailed structure of the specimen, the magnification of the projection lens is always equal to or greater than 1. Therefore, the magnification of the common projection lens is 1X, so as to achieve the magnification marked on the objective body, but 2X or greater magnifications can also be seen.

[0100] Figure 4 A schematic diagram of the numerical aperture (NA) of the objective lens 14 is shown. The NA of the objective lens 14 is a dimensionless number that describes the angle range over which the objective lens 14 can collect emitted fluorescence, and an objective lens with a higher NA can collect more light. Generally speaking, the NA of the objective lens 14 is also indicated on its body, such as "1.5" as shown in the figure. The relevant formula can be expressed as: NA = n × sinθ, where n is the refractive index of the immersion medium and θ is the collection angle of the objective lens. In order to achieve higher single-molecule sensitivity, the NA of the objective lens 14 must be higher than a certain level. In general, the minimum NA is affected by the sensor performance specifications and the arrangement of the optical lenses.

[0101] In the lens-based single molecule sequencing system, the NA of the objective lens 14 is less than 1, 0.9, 0.8 or 0.7.

[0102] The field of view (FOV) of a lens-based optical module is determined by Figure 5The diameter of the circular area imaged by the system shown. Since a sensor 120 is used to receive the image projected by the optical module, the effective FOV 151 can be determined by the overlap between the lens FOV 150 and a corresponding area of ​​the sensor 120. If the size of the sensor 120 is larger than the projection of the lens FOV 150 on it, the effective FOV 151 depends on the lens. Otherwise, the effective FOV 151 is limited by the size of the sensor 120.

[0103] The effective chip area refers to the area on the sequencing chip 13 corresponding to the effective FOV 151 of the optical module. In order to achieve higher performance, the sequencing sites 10 should be arranged as much as possible within the effective chip area to ensure that the sequencing sites 10 can be accommodated. The spacing can be defined as the distance between every two adjacent sequencing sites 10. There are many factors that prevent the sequencing sites 10 from being too close.

[0104] For example, optical spatial resolution is the minimum distance between signals from two adjacent sequencing sites 10 that the optical module can distinguish. Due to the nature of light, signals from different sequencing sites 10 usually present a Gaussian distribution. Figure 6 As shown in the figure, the fluorescence signals from single-molecule sequencing sites A and B are projected onto the sensor plane. There is no clear boundary between each signal beam point, and they always overlap and cannot be completely separated. As long as the distance between site A and site B is far enough and the overlapping part of their signal beam points does not exceed the resolvable degree, the signals from these two sites can be distinguished. Therefore, the minimum distance between the signals from site A and site B is called the spatial resolution of the optical system.

[0105] Generally speaking, when the numerical aperture (NA) of the objective lens is larger, higher spatial resolution can be provided, which is based on the relevant equation: resolution (r) = 0.61×(λ) / NA, so the sequencing sites 10 can be arranged closer or more closely with a smaller spacing to resolve smaller features, which means that more sequencing sites 10 can be arranged in the same area. Therefore, the design of the optical system is to provide a combination of a lens module and a sensor, which can be used to identify more sequencing sites and thus provide the best performance.

[0106] An example is given below to illustrate the design of a conventional lens-based single-molecule system. In this example, the sensor has a 2000 x 1500 pixel array, and each pixel is 3.45 square microns in size. Table 1 shows the spatial resolution of lenses with different magnifications and NA values.

[0107] When using a 60X oil immersion lens with NA 1.5, the spatial resolution is 0.24μm, that is, the minimum spacing of sequencing sites is 0.24μm. A 60X objective lens with a 1X projection lens can magnify the 0.24μm spacing to a 14.4μm spacing on the sensor plane. The maximum total number of sequencing sites that can be imaged is (3.45x2000) / 14.4x(3.45x 1500) / 14.4=479x 359=171,961 (sequencing sites).

[0108] Table 1

[0109] lens 100X Oil 60X Oil 40X Oil 20X Air Magnification 100 60 40 20 NA 1.5 1.5 1.3 0.7 FOV(D,μm) 220 366 550 1100 SR(μm) 0.24 0.24 0.28 0.5 Number of sites: 1 61,705 171,961 284,592 356,730 Number of sites: 2 61,705 136,354 100,178 31,416 Number of pixels per pixel 48 17 11 8.4

[0110] In Table 1, the results in the column “Number of sites: 1” only take into account the optical spatial resolution of the lens module.

[0111] Continuous single-molecule sequencing requires a certain substrate concentration value to keep the polymerase introduced at a reasonable rate. Figure 7 As shown, even if some of the labeled triphosphate nucleotides 21 are not introduced like the molecules 20 being sequenced, once excited, the fluorophores or fluorescent dyes attached to those triphosphate nucleotides may emit unwanted fluorescence with the same wavelength. That is, all fluorescent dyes entering the observation space (i.e., the sequencing nanowell) may produce fluorescence, thus becoming unwanted "fluorescence background". The details of the nanowell will be further described later. Resolvable and detected introduction events should have a high ratio of "(introduction signal + fluorescence background) / (fluorescence background)", where the fluorescence background can be estimated from the substrate concentration, the excitation field distribution, and the observation volume of the corresponding pixel.

[0112] For this case, the meaningful volume observation is the space where the fluorescence can pass through the lens to the corresponding pixel. Fig. 8A For epi excitation as shown, all lenses listed in Table 1 above cannot detect single molecule events with a mass of 1 μm, because a considerable number of background molecules will be excited and overwhelm the single molecule signal. To overcome this interference problem, single molecule sequencing systems can deploy Figure 8B The evanescent wave shown provides excitation energy to the sequencing site 10 without increasing the background fluorescence signal. The evanescent wave may decay exponentially and quickly decay beyond the range of 100 nm.

[0113] like Fig.9A Lens-based total internal reflection (TIRF) as shown or Fig. 9BThe prism-based TIRF shown can limit the effective excitation energy to within 100nm from the reflective surface. Due to the limitation of laser light power, lens-based TIRF or prism-based TIRF can only excite a smaller area, such as about 100 microns in diameter. The "Number of sites: 1" column in Table 1 only considers the effective FOV. The "Number of sites: 2" column in Table 1 is the result of assuming that the size of the excitation area is about 100μm. Table 1 shows that the available sequencing sites for a lens-based system based on a traditional design (i.e., by using a 60X oil immersion lens) are about 150,000. The effective FOV can be increased by applying a large area sensor, but the limitation of the system is the area of ​​the evanescent wave excitation field. In the present disclosure, the laser light power is guided by a 250nm waveguide core and is within the evanescent wave range of 100nm, rather than within the beam size range of 100μm, thereby generating a strong field intensity across the entire sequencing chip, thereby extending an area of ​​2mm instead of 100μm. Therefore, more sequencing sites 10 can be arranged in the excitation area. In some embodiments, more than one million sequencing sites 10 can be arranged and observed in an area of ​​2 mm x 2 mm.

[0114] Regarding the sensitivity of the sensor, due to the magnifying nature of the microscope, the sequencing site emission can be spread over several pixels 24, e.g. Fig.10 The "large light spot" 22 shown. Therefore, the signal intensity will be divided among these pixels, which makes the signal too weak to be detected by each pixel 24 of the CMOS image sensor. Although some scientific-grade CCD, ICCD, EMCCD or specially designed scientific-grade CMOS sensors can be used to detect weak signals, they are usually bulky and expensive components that may significantly increase the size, weight and overall cost of the system. Ideally, when the sequencing site emission is projected onto only one or two or at most four pixels 24, such as Fig.10 The "small light spot" 23 shown in the figure, at this time the signal is relatively concentrated and the detected signal intensity may be greatly increased. In this way, a reasonably priced modern CMOS sensor is sensitive enough to detect the signal. Today, high-quality CMOS sensors can achieve a peak quantum efficiency of 60%, which is not far from the scientific level of 90%. The present disclosure can use modern industrial or surveillance grade CMOS sensors and avoid the use of expensive scientific grade sensors by properly arranging the sequencing chip and optical elements. In fact, although the projected light spot size can be as small as or smaller than the pixel 24, the projection may overlap with the boundary of the pixel 24, resulting in two or at worst four adjacent pixels 24 sharing the same signal from one sequencing site. However, when the signal is divided into four parts, the signal intensity dispersed over each of the four pixels is still strong enough to be detected.

[0115] Design rules for lens-based continuous single-molecule sequencing devices. Based on the disclosed design rules, a lens-based continuous single-molecule sequencing system can have more than 150,000, 300,000, 500,000, or 1,000,000 sequencing sites. According to the Nyquist sampling theorem, one sequencing well can be resolved by 2 x 2 pixels of the sensor. Therefore, a 5 million pixel sensor will be able to resolve more than one million sequencing wells.

[0116] like Fig.11 As shown, in some embodiments, it includes a sequencing chip 31 and a detection module 32. In some embodiments, the detection module 32 includes a sensor device 321, an objective lens 322 with a first magnification, and a projection lens 323 with a second magnification. In some embodiments, the objective lens 322 and the projection lens 323 are used to transmit the fluorescence from the sequencing chip 31 to the sensor device 321. In some embodiments, the second magnification is less than 1. In some embodiments, a laser light source 33 can be coupled to the sequencing chip 31 through a laser coupler 34, so the waveguide 35 of the sequencing chip 31 can be used to guide the laser light to the sequencing site 10. The laser light source 33 and the sequencing chip 31 should be as close as possible to avoid unwanted scattered light from entering the closed space. The distance between the two is less than 1 mm, less than 0.5 mm or less than 0.3 mm. In some embodiments, the sequencing signal is collected by the objective lens 322 and then passes through a filter 36 to block the laser light before the projection lens 323 projects the sequencing signal onto the sensor device 321 .

[0117] In some embodiments, the first magnification of the objective lens 322 is used to collect fluorescence. In some embodiments, the second magnification of the projection lens 323 is used to project the fluorescence from the objective lens onto the sensor device. In some embodiments, the second magnification of the projection lens 323 is less than 1, less than 0.5X, or less than 0.25X.

[0118] like Fig.12As shown, in some embodiments, the sequencing chip 31 includes a substrate 310, a thin film or a channel waveguide 312 disposed on the substrate 310, and an optical coupler 311 formed at the waveguide 312. In some embodiments, the waveguide 312 includes an upper cladding layer 313, a lower cladding layer 314, and a nano-well sequencing site array 315. In some embodiments, the nano-well sequencing site array 315 includes a plurality of nano-wells 316. In some embodiments, a core layer 317 is sandwiched between the upper cladding layer 313 and the lower cladding layer 314, and the core layer 317 has a lower surface and an upper surface opposite to the lower surface. In some embodiments, the optical coupler 311 is used to couple the excitation light into the waveguide 312, and the waveguide 312 can limit and guide the excitation light to reach the nano-well sequencing site array 315 and form an evanescent wave excitation field from the bottom of each nano-well 316, as shown above. Figure 8B In other words, the light field intensity is strongest at the bottom and gradually decreases exponentially, as shown in Figure 8B As shown. In a preferred application scenario, the field intensity on the upper cladding layer 313 is weak and can be ignored or approach zero. This makes it possible that the fluorophores suspended or not introduced on the upper surface of the upper cladding layer 313 may not produce unwanted fluorescent background signals. In some embodiments, the nanowell sequencing site array 315 is manufactured to have an upper opening 315A and a cylindrical wall 315B in the upper cladding layer 313, and the bottom of the nanowell sequencing site array 315 is located at or adjacent to the upper surface of the core layer 317. In some embodiments, the cylindrical wall 315B is formed in the upper cladding layer 313 of the waveguide 312. In some embodiments, the bottom of each nanowell 316 may be coplanar with the upper surface of the core layer 317 or have a thin bottom layer (not shown) covering the upper surface of the core layer 317. The thin bottom layer of the above-mentioned nanowell 316 can be prepared using the same or different material as the upper cladding layer 313.

[0119] In some embodiments, the shape of the nanowell 316 is substantially circular, such as Fig.13 In some embodiments, the nanowell 316 can be made into other shapes, such as a square, hexagonal, etc. when viewed from above. In some embodiments, the nanowell 316 can also extend laterally to form a groove shape instead of a general well shape. For example, the sequencing site array can include the following: Fig.14 and Fig.15 A plurality of nano-trench 318 are shown arranged in parallel.

[0120] Fig.16A schematic top view of a substantially circular nanowell is shown according to some embodiments of the present disclosure. As shown, a beam adjustment mechanism 41 of the sequencing chip can be disposed between the point-type optical coupler 311 and the first side of the nanowell sequencing site array 315 to adjust a total projected area of ​​each light beam from the optical coupler 311 when passing toward the sequencing site 10. For example, the beam adjustment mechanism 41 can convert a narrower light beam (e.g., injected laser light) from the optical coupler 311 into a wider light beam entering the sequencing site therein, while the strip-type optical coupler 311 is located at a second side adjacent to the sequencing site in the array 315. In some embodiments, the second side is opposite to the first side, so that the sequencing site array 315 is substantially between different types of optical couplers 311.

[0121] Fig.17 A schematic top view of a grooved nanowell according to some embodiments of the present disclosure is shown. As shown, a beam adjustment mechanism 41 may be disposed between a point-type optical coupler 311 and a sidewall of a nano-groove 318, while a strip-type optical coupler 311 is located adjacent to a sidewall of another nano-groove 318. In some embodiments, a plurality of nano-grooves 318 are arranged in parallel and parallel to the strip-type optical coupler 311.

[0122] Fig.18 A schematic top view of a grooved nanowell according to some embodiments of the present disclosure is shown. Fig.17 Similar to the embodiment shown, a beam adjustment mechanism 41 may be disposed between a point-type optical coupler 311 and a plurality of nano-grooves 318, and the nano-grooves 318 are arranged in parallel and parallel to a strip-type optical coupler 311. The point-type optical coupler 311 and the beam adjustment mechanism 41 are disposed on a first side of the array of nano-grooves 318, and the strip-type optical coupler 311 is disposed on a second side of the array of nano-grooves 318, wherein the second side is opposite to the first side. In this embodiment, the beam adjustment mechanism 41 is adjacent to one end of each nano-grooves 318.

[0123] Reference Fig.19AIn some embodiments, more than one beam adjustment mechanism 41 may be used. For example, the beam adjustment mechanism 41 may be disposed between each optical coupler 311 and the nanowell sequencing site array 315 to adjust a total projected area of ​​each light beam from the optical coupler 311 when it is transmitted toward the sequencing site 10. For example, the beam adjustment mechanism 41 may convert a narrow light beam (e.g., injected laser light) from the optical coupler 311 into a wider light beam entering the sequencing site 10. In some embodiments, the optical coupler 311 includes a nanometer-scale grating structure. In some embodiments, the optical coupler 311 is a bar type. In some embodiments, the optical coupler 311 is a dot type. In some embodiments, the waveguide 312 is a film type. In some embodiments, the waveguide 312 is a channel type. In some embodiments, the sequencing sites 10 form the sequencing site array 315, and the beam width of the wider light beam substantially covers the sequencing site array 315. In some embodiments, the beam adjustment mechanism 41 comprises a nanostructure beam expander disposed between the optical coupler 311 and the sequencing site 10. In some embodiments, the nanostructure beam expander is at least a portion of the waveguide 312, and thus can be a thin film waveguide or a channel waveguide. In some embodiments, the sequencing site array 315 comprises 2000 x 2000 pixels, and the optical coupler 311 has a diameter of about 50 μm. In some embodiments, the sequencing chip comprises a pair of holes 42 for fluid input and output for carrying samples.

[0124] In other embodiments, the beam adjustment mechanism may include a plurality of beam splitters to multiply the number of beams from the optical coupler 311, thereby allowing the multiplied beams to be synchronously transmitted toward the sequencing site 10. Compared with a single beam, the multiplied beams can provide a larger coverage area at the sequencing site.

[0125] like Fig.19B As shown in FIG. 1 , in some embodiments, a spacer 43 may be disposed above the sequencing chip. The spacer 43 may include a plurality of first openings 44. Fig.19A The plurality of optical couplers 311 are shown aligned to allow the laser light to pass through. The spacer 43 may include a fluid channel 45 located at its center and located above the spacer 43. Fig.19A The sequencing site array 315 is shown to store fluid including samples. In some embodiments, the diameter of the opening 44 is larger than the diameter of the optical coupler 311. Fig.19C As shown, in some embodiments, a cover 46 may be disposed above the spacer 43. The cover 46 may include a plurality of second openings 47, and the second openings 47 are aligned with the first openings 44 of the spacer 43. In some embodiments, the spacer 43 is sandwiched between the cover 46 and the sequencing chip.

[0126] In some embodiments, the polymerase replication complex can be attached to the bottom of the nanowell 316 via a linker site, as described above. Figure 7 In some embodiments, the polymerase replication complex can be fixed to the linker by using a biotin-streptavidin complexing method or covalent bonding or by using charge attraction or physical restriction.

[0127] When the sequencing site array includes a plurality of parallel nanogrooves 318, the linker sites in different nanogrooves 318 may be arranged to have substantially the same intervals therebetween. In some embodiments, it may be considered whether this arrangement can prevent the fluorescence generated by each linker site from interfering with each other, thereby ensuring better optical spatial resolution.

[0128] In some embodiments, the interval between adjacent nanowells 316 is between less than 1 μm and about 5 μm. In some embodiments, the interval between linker sites is about 3 μm. Although not shown in the present disclosure, in some embodiments, no nanowells are formed on the sequencing chip 31. In such embodiments, a plurality of linker sites are arranged on the flat surface of the sequencing chip 31 at appropriate intervals.

[0129] In certain embodiments, as above Figure 7 As shown, the width of each nanowell 316 (along the Y axis) is in the range of about 50nm to about 650nm. The height of each nanowell (along the Z axis) is in the range of about 20nm to about 600nm. In some embodiments, the spacing between two adjacent nanowells (along the Y axis) is in the range of less than 1μm to about 5μm. In the case where the width of the nanowell 316 is less than about 50nm or the height of the nanowell 316 is greater than about 600nm, it may be difficult for the sample to be loaded into the nanowell 316 and reach its bottom due to capillary forces. On the other hand, in the case where the width of the nanowell 316 is greater than about 650nm, the spatial confinement of the sample by the nanowell 316 may be insufficient. In one embodiment, the spacing between two adjacent nanowells 316 is less than about 1μm. In some cases, the spacing between two adjacent nanowells is greater than about 5μm, and the density of the nanowell 316 is too small to maintain the detection flux. In certain embodiments, the nanowell 316 has a width of about 80 nm and a height of about 100 nm, and a spacing between two adjacent nanowells 316 is about 1.3 μm.

[0130] In certain embodiments, in order to improve the stability of the sequencing complex (e.g., polymerase replication complex) in the nanowell 316 of the sequencing chip 31, the lower surface or sidewall of the nanowell 316 is chemically modified to enhance the adsorption capacity of the sequencing complex at the linker site. For example, a hydrophilic treatment may be performed on the lower surface or sidewall of the nanowell 316. Optionally, a hydrophobic treatment may be performed on the upper surface of the covering layer (e.g., the upper cover layer 313) defining the nanowell 316, thereby avoiding unintroduced fluorophore labeling and thus reducing unwanted background fluorescence signals. Furthermore, the above-mentioned hydrophobic treatment may also prevent the fluid containing unintroduced fluorophores from flowing through the non-reaction area, thereby reducing unwanted fluorescence background signals.

[0131] In certain embodiments, Fig.12 The illustrated substrate 310 may be an opaque silicon wafer, transparent glass, quartz, fused silica, sapphire, or the like.

[0132] In some embodiments, the space of the nanowell 316 can be defined by patterning the overlayer 313. In other words, two adjacent nanowells on the sequencing chip 31 are separated by the patterned overlayer 313. The overlayer 313 is used to reduce background signals, such as scattered light from the non-information space (i.e., the space between adjacent linker sites). In some embodiments, the overlayer 313 can be transparent, but physically prevent the fluorescent dye-labeled substrate from reaching the excitation field other than the sequencing site 10 (i.e., the linker site). The patterned cover layer can be opaque, thereby partially or completely blocking the light from the non-information space. In some embodiments, the overlayer 313 is a metal sheet. In some embodiments, the overlayer 313 is a chemically modified surface that can reduce the chance of the fluorescent dye-labeled substrate being closer to the excitation field. In some embodiments, the overlayer 313 can be a plastic film. In some embodiments, the overlayer 313 can be SiO2 or other inorganic oxides having a low refractive index compared to the core layer 317.

[0133] Fig. 20 The spacing of the nanowells 316 and the pixel spacing on the sensor device 321 are shown. In some embodiments, the spacing (S) of the sequencing sites can be designed as:

[0134] 2P>=S×F>=1P

[0135] That is, the product of the pitch and the overall magnification is equal to or greater than one pitch and equal to or less than two pitches, where (F) is the overall magnification of the detection module 32', and (P) is the pitch of the pixels of the sensor device 321, which is equal to the pixel size. In some embodiments, the pixel size is less than or equal to about 10 μm, about 5 μm, or about 3 μm. In some embodiments, the pitch between two adjacent sequencing sites is less than or equal to about 5 μm, about 3 μm, or about 1 μm.

[0136] In some embodiments, the fluorescence signal from a sequencing site is first collected by the objective lens 322 and then projected onto the sensor device 321 at a size less than 1.5 or 1 times the pixel size P. The fluorescence signal can be appropriately concentrated to one or at most four pixels, with overlapping boundaries, thereby producing the above Fig.10 The "small light spot" 23 shown here can provide a well-detectable signal intensity.

[0137] An embodiment of an apparatus following the above design rules includes a sensor device 321 having 2000 x 1500 pixels, each pixel having a pixel size and pitch of 3.45 μm; an objective lens 322 having a NA of 0.7 and a magnification of 20X; a projection lens 323 having a magnification of 0.25X; and a sequencing chip 31 having a nanowell sequencing site pitch of 1.38 μm.

[0138] Where X1 x X2 = 5

[0139] (2 x 3.45 μm) / 5 = 1.38 μm.

[0140] That is, the spacing of the sequencing sites is 1.38 μm, which meets the above design rules. In some embodiments, the number of effective sequencing sites is 750,000.

[0141] An embodiment of an apparatus following the above design rules includes a sensor device 321 having 2000 x 1500 pixels, each pixel having a pixel size and pitch of 3.45 μm; an objective lens 322 having a NA of 0.7 and a magnification of 20X; a projection lens 323 having a magnification of 0.25X; and a sequencing chip 31 having a nanowell sequencing site pitch of 1.04 μm.

[0142] Where X1 x X2 = 5

[0143] (2 x 3.45 μm) / 5 = 1.38 μm

[0144] (1x 3.45μm) / 5=0.69μm

[0145] That is, the spacing of the sequencing sites is 1.04 μm, and 1.04 μm is greater than 0.69 μm and less than 1.38 μm, which meets the above design rules. In some embodiments, the number of effective sequencing sites is 1,333,333.

[0146] An embodiment of an apparatus following the above design rules includes a sensor device 321 having 2000 x 1500 pixels, and each pixel has a pixel size and pitch of 3.45 μm; an objective lens 322 having a NA of 0.7 and a magnification of 20X; a projection lens 323 having a magnification of 0.25X; and a sequencing chip 31 having a nanowell sequencing site pitch of 0.7 μm.

[0147] Where X1 x X2 = 5

[0148] (1x 3.45μm) / 5=0.69μm

[0149] That is, the spacing of the sequencing sites is 0.7 μm, and 0.7 μm is greater than 0.69 μm, which meets the above design rules. In some embodiments, the number of effective sequencing sites is 3,000,000.

[0150] The device and system proposed here can be used to efficiently detect single-molecule nucleic acids, that is, a larger number of sequencing sites can be achieved using a lens-based optical module. In addition, the detection cost is reduced by using a simpler and replaceable sequencing chip. The sequencing signal from a site is projected onto a size of less than 1.5 pixels or even less than 1 pixel on the sensor device to concentrate the signal intensity thereon and greatly improve the detection signal intensity. In this way, there is no need to use bulky, highly sensitive and expensive scientific research-grade CCD or CMOS sensors, thereby reducing the cost of the detection module.

[0151] Fig.21Spectral bands of labeled fluorophores are depicted. In certain embodiments, each nucleotide sequence used for sequencing the four d-NTPs of ATCG may be labeled with a different fluorophore (fluorescent dye) capable of emitting at different wavelengths. As shown in the figure, by calculating the weights of different channels, three spectral range channels can be used to resolve these different fluorophores, and they can be analyzed and converted into sequencing data. For example, when laser light is used as excitation energy and is thus coupled into a waveguide and guided to a sequencing site; dye 1 located at a specific sequencing site in the array will be evanescently excited, and channel 1 and channel 2 can collect the light emitted thereby. Based on the information of the emission spectrum, for example, only channel 1 will show a pixel signal value above a threshold, so the weight (1,0,0) represents that the nucleotide specifically introduced at the reaction site is labeled by dye 1. However, if the dye is changed to dye 2, the result will show a weight (1,1,0), which means that the pixel values ​​shown by channels 1 and 2 are above the threshold, so the weight (1,1,0) represents that the nucleotide specifically introduced is labeled by dye 2. Dye 3 is also evanescently excited by the laser light, and its results show weights (0,1,1). However, if the dye is changed to dye 4, its results show weights (0,0,1). Please note that these weights (1,0,0), (1,1,0), (0,1,1), and (0,0,1) are for illustration purposes only and are not exact measured numbers.

[0152] In one embodiment, the trichromatic prism is designed so that channel 1 can receive wavelengths from 530nm to 610nm, channel 2 can receive wavelengths from 610nm to 675nm, and channel 3 can receive wavelengths from 675nm to 800nm, for example only. The light intensity that can be collected by each pixel of the three optical sensors, and therefore the signal value thereof, can be adjusted by changing the type of dye or modifying the spectral range of each channel on the trichromatic beam splitter prism.

[0153] Fig. 22A Philips type prism is shown according to some embodiments of the present disclosure. In some embodiments, a splitter is used to direct the fluorescence having different wavelength spectral ranges to the at least two sensor devices. In some embodiments, a Philips type prism 50 is used to separate the sequencing signal into three channels. The sensor device 321 is firmly adhered (adhesive not shown in the figure) to the prism and thus maintains excellent mechanical stability in the operating environment. By adhering several components into a working piece, the device is more resistant to vibrations and thus the pixel correlation in the three sensors can also be fixed. Philips type prism 50 is commercially available and very practical, but other types of color separators are also possible, such as a cross color prism or a more commonly used cubic color splitter. Compared to the Bayer filter used in common color sensors, the Philips type prism 50 can separate colors instead of filtering light, so it can absorb unwanted colors and provide higher overall transmission efficiency. In fact, no photons are wasted using a prismatic three-color beam splitter.

[0154] Fig.23 A dispersion device for spectral detection according to some embodiments of the present disclosure is shown. In some embodiments, a multi-channel sensor device is not used, but a dispersion device 51, such as a wedge prism or a grating, is used to separate light of different spectral ranges into different pixels in an array. For example, different spectral ranges can be separated into 4, 5 or 6 pixels arranged in an array.

[0155] Fig.24 The cross-sectional SEM image of a sequencing wafer with a nano-well 316 according to some embodiments of the present disclosure is shown. The nano-well 316 is constructed in the upper cover layer 313 , and the bottom of the nano-well is adjacent to the upper surface of the core layer 317 .

[0156] Fig.25A and Fig.25B A snapshot of an image frame of sequencing raw data according to certain embodiments of the present disclosure is shown. Fig.25A A portion of a snapshot of an image frame from a dye landing event. The image shows the fluorophore signal captured by the sensor assembly. With this optical arrangement, the signal only overlaps four adjacent pixels at most, ensuring a solid signal strength. Fig.25B A typical pixel is shown. It can be seen from the figure that most of the light is concentrated in one pixel, and only a small part is scattered on adjacent pixels. Overall, the sequencing site signal is quite concentrated.

[0157] Fig.26The sequencing time trajectory of a nanowell in one channel is shown. The rise and fall of the fluorescence signal can be observed along with the dye landing event. By arranging the optical elements according to the present disclosure, the signal to noise ratio (SNR) obtained is quite ideal (e.g., signal to noise ratio = 54976 / 2441 = 22.52), and can be commercialized by using a reasonably priced industrial-grade SMOS camera.

[0158] item

[0159] 1. A sequencing chip for continuous single-molecule nucleic acid sequencing, comprising:

[0160] substrate;

[0161] A waveguide, disposed on the substrate, comprising:

[0162] Core layer; and

[0163] an upper covering layer, disposed on the core layer;

[0164] at least one optical coupler extending from the cladding layer into the core layer;

[0165] at least one sequencing site located in the overlying layer; and

[0166] The beam adjustment mechanism is used to adjust the total projection area of ​​the beam from the optical coupler when it is transmitted toward the sequencing site.

[0167] 2. A sequencing chip according to item 1, wherein the sequencing sites form a sequencing site array, and the beam width of the light beam substantially covers the sequencing site array.

[0168] 3. A sequencing chip according to item 1, wherein the beam adjustment mechanism comprises a nanostructure beam expander disposed between the optical coupler and the sequencing site.

[0169] 4. A sequencing chip according to item 1, wherein the substrate of the sequencing chip comprises silicon, transparent glass, quartz or fused quartz.

[0170] 5. A sequencing chip according to item 2, wherein the sequencing site array is between the strip-type optical coupler and the point-type optical coupler.

[0171] 6. A sequencing chip according to item 2, wherein the sequencing site array is between different types of optical couplers.

[0172] 7. A sequencing chip according to item 1, wherein the sequencing sites are respectively located in nanowells defined by the upper covering layer of the waveguide.

[0173] 8. The sequencing chip according to item 7, wherein the spacing between two adjacent nanowells is in the range of less than 1 μm to 5 μm.

[0174] 9. A sequencing chip according to item 7, wherein the width of the nanowell is in the range of 50nm to 650nm.

[0175] 10. A sequencing chip according to item 7, wherein the height of the nanowell is in the range of 20 nm to 600 nm.

[0176] 11. A sequencing chip according to item 1, wherein the sequencing sites are respectively located in nano-grooves defined by the upper cladding layer of the waveguide, and the nano-grooves are parallel to the optical coupler.

[0177] 12. A sequencing chip according to item 1, wherein the sequencing sites are respectively located in nano-grooves defined by the upper cladding layer of the waveguide, the beam adjustment mechanism is located between the nano-groove and the optical coupler, and the beam adjustment mechanism is adjacent to one end of the nano-groove.

[0178] 13. A sequencing chip according to item 1, wherein the bottom of the sequencing site comprises a modified surface for selectively immobilizing sequencing complexes.

[0179] 14. A sequencing chip according to item 1, wherein the beam adjustment mechanism comprises a plurality of beam splitters for multiplying the number of the beams from the optical coupler.

[0180] 15. A sequencing chip according to item 1, wherein a spacer is disposed above the sequencing chip, the spacer comprising a first opening, and the first opening is aligned with the optical coupler.

[0181] 16. A sequencing chip according to item 15, wherein the diameter of the first opening is larger than the diameter of the optical coupler.

[0182] 17. A sequencing chip according to item 15, wherein the spacer comprises a fluid channel, and the fluid channel is located above the sequencing site.

[0183] 18. The sequencing chip according to item 15 further comprises a cover disposed above the spacer, the cover comprising a second opening aligned with the first opening.

[0184] The above description briefly presents the features of certain embodiments of the present invention, so that those skilled in the art can more fully understand the various embodiments of the present disclosure. Those skilled in the art can easily use the disclosure as a basis to design or modify other processes and structures to achieve the same purpose and / or achieve the same advantages as the embodiments described herein. Those skilled in the art should understand that these equivalent embodiments still belong to the spirit and scope of the disclosure, and various changes, substitutions and modifications can be made without departing from the spirit and scope of the disclosure.

Claims

1. A sequencing chip for continuous single-molecule nucleic acid sequencing, comprising: substrate; A waveguide, disposed on the substrate, comprising: Core layer; and an upper covering layer, disposed on the core layer; at least one optical coupler extending from the cladding layer into the core layer; at least one sequencing site located in the overlying layer; as well as The beam adjustment mechanism is used to adjust the total projection area of ​​the beam from the optical coupler when it is transmitted toward the sequencing site.

2. The sequencing chip according to claim 1, wherein the sequencing sites form a sequencing site array, and the beam width of the light beam substantially covers the sequencing site array.

3. The sequencing chip according to claim 1, wherein the beam adjustment mechanism comprises a nanostructure beam expander, which is disposed between the optical coupler and the sequencing site.

4. The sequencing chip according to claim 1, wherein the substrate of the sequencing chip comprises silicon, transparent glass, quartz or fused silica.

5. The sequencing chip according to claim 2, wherein the sequencing site array is between the strip-type optical coupler and the dot-type optical coupler.

6. The sequencing chip according to claim 2, wherein the sequencing site array is between different types of optical couplers. 7 . The sequencing chip according to claim 1 , wherein the sequencing sites are respectively located in nanowells defined by the upper cladding layer of the waveguide. 8 . The sequencing chip according to claim 7 , wherein a distance between two adjacent nanowells is in a range of less than 1 μm to 5 μm.

9. The sequencing chip according to claim 7, wherein the width of the nanowell is in the range of 50 nm to 650 nm.

10. The sequencing chip according to claim 7, wherein the height of the nanowell is in the range of 20 nm to 600 nm. 11 . The sequencing chip according to claim 1 , wherein the sequencing sites are respectively located in nano-trench defined by the upper cladding layer of the waveguide, and the nano-trench is parallel to the optical coupler.

12. The sequencing chip according to claim 1, wherein the sequencing sites are respectively located in nano-trench defined by the upper cladding layer of the waveguide, the beam adjustment mechanism is located between the nano-trench and the optical coupler, and the beam adjustment mechanism is adjacent to one end of the nano-trench.

13. The sequencing chip according to claim 1, wherein the bottom of the sequencing site comprises a modified surface for selectively immobilizing a sequencing complex.

14. The sequencing chip according to claim 1, wherein the beam adjustment mechanism comprises a plurality of beam splitters for multiplying the quantity of the light beam from the optical coupler. 15 . The sequencing chip according to claim 1 , wherein a spacer is disposed above the sequencing chip, the spacer comprising a first opening, and the first opening is aligned with the optical coupler.

16. The sequencing chip according to claim 15, wherein a diameter of the first opening is larger than a diameter of the optical coupler.

17. The sequencing chip according to claim 15, wherein the spacer comprises a fluid channel, and the fluid channel is located above the sequencing site. 18 . The sequencing chip according to claim 15 , further comprising a cover disposed above the spacer, the cover comprising a second opening aligned with the first opening.

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