Total internal reflection single-molecule gene sequencing system

Abstract

The present invention provides a total internal reflection single-molecule gene sequencing system, which relates to the field of gene sequencing technology and includes: a total internal reflection sequencing chip, an imaging temperature measurement device, an illumination device, and a precision displacement stage; the total internal reflection sequencing chip includes a prism substrate, a cover plate, a fixing frame, and multiple flow channel plates; wherein the upper and lower surfaces of the prism substrate are respectively bonded to a flow channel plate, which is in turn bonded to a cover plate, and the flow channel plate is machined with a flow channel running through the upper and lower surfaces; the imaging temperature measurement device includes a microscope objective lens, an imaging module, and a focus temperature measurement module; and the illumination device includes an illumination lens, an illumination lens shifter, and an illumination light deflector. The present invention can perform total internal reflection single-molecule gene sequencing without relying on immersion oil, and has high reliability and low maintenance costs.

Description

Technical Field

[0001] The present invention relates to the field of gene sequencing technology, and in particular to a total internal reflection single-molecule gene sequencing system. Background Art

[0002] Gene sequencing technology is not only a crucial research tool and diagnostic method in modern biomedicine, but also a key technology in future gene storage technology. Currently, gene sequencing technologies are primarily categorized into two main types: fluorescence sequencing and nanopore sequencing, with high-throughput sequencing primarily relying on fluorescence sequencing. A gene sequencer is a crucial instrument for the entire gene sequencing process and generally consists of three main components: detection reagents, sequencing chips, and a sequencer. In a fluorescence high-throughput sequencer, the sequencing chip carries the sequencing samples and reagents, while the sequencer delivers the reagents to the chip and performs fluorescence imaging. Currently, fluorescence high-throughput sequencers are divided into second- and third-generation fluorescence high-throughput sequencers. The key feature of third-generation fluorescence high-throughput sequencers is single-molecule sequencing, meaning each sequencing site contains only a single DNA molecule. The imaging device in the sequencer enables single-molecule fluorescence imaging.

[0003] Single-molecule imaging requires a very high signal-to-noise ratio (SNR), defined as the ratio of the single-molecule fluorescence signal to the sample's background light signal. Currently, achieving this high SNR relies primarily on total internal reflection illumination (TIRF) imaging. This involves illuminating light directed at a specific angle from a denser medium to an optically less dense medium, where it undergoes total internal reflection at the interface between the denser and less denser media. The resulting evanescent light waves excite the fluorophores, causing them to emit fluorescence. Because the intensity of evanescent light decays rapidly with propagation distance, its effective range is limited to a few hundred nanometers, significantly smaller than the distance between the sample and the objective lens. This allows for virtually no background light to enter the objective lens, resulting in a significantly higher SNR than conventional epi-illumination fluorescence imaging.

[0004] Total internal reflection illumination imaging is mainly achieved in two ways: one is the objective lens type, that is, the illumination light beam is emitted through the objective lens, the sample is located on the inner surface of the glass dish, the glass dish is filled with solution, and the bottom of the glass dish is close to the objective lens. The illumination light beam undergoes total internal reflection at the interface between the glass dish and the solution and produces evanescent light to excite the sample. The generated fluorescence is then received by the objective lens to form an image; the other is the prism type, where the sample is stored in the same way as the former, but the objective lens is located above the glass dish, and the bottom of the glass dish is close to a trapezoidal prism. The illumination light is emitted from one oblique surface of the trapezoidal prism and reaches the interface between the glass dish and the solution. After total internal reflection, it produces evanescent light and excites the sample. The reflected light is emitted from the other oblique surface of the prism. The fluorescence generated by the sample is received by the objective lens located above the glass dish and formed into an image.

[0005] In fact, whether it is an objective lens type or a prism type total internal reflection illumination imaging, since there is a tiny gap between the glass dish holding the sample and the prism or objective lens, the gap must be filled with immersion oil with the same refractive index as the glass. Otherwise, the illumination beam will undergo total internal reflection at the interface between the gap and the prism or objective lens, and cannot reach the interface between the solution and the glass dish. In sequencers, sequencing chips replace ordinary imaging glass dishes. If prism type or objective lens type total internal reflection illumination imaging is used, immersion oil must also be filled between the sequencing chip and the prism or objective lens. However, sequencing chips are consumables, and sequencers are closed instruments. With the frequent replacement of sequencing chips, immersion oil will also be lost. This requires that the sequencer be equipped with an automatic oil replenishment device. However, the main component of immersion oil is cedar oil. If exposed to space for a long time, it will solidify and permanently contaminate the immersion lens or prism. This requires that the system be equipped with an automatic cleaning device, which will greatly increase the complexity of the instrument and reduce the reliability of the instrument. Therefore, designing a sequencer that can perform total internal reflection illumination single molecule imaging without relying on immersion oil is a problem that those skilled in the art urgently need to solve. Summary of the Invention

[0006] In response to the deficiencies in the prior art, the present invention provides a total internal reflection single-molecule gene sequencing system.

[0007] According to a total internal reflection single-molecule gene sequencing system provided by the present invention, the scheme is as follows:

[0008] A total internal reflection single-molecule gene sequencing system, comprising: a total internal reflection sequencing chip, an imaging temperature measurement device, an illumination device, and a precision translation stage; the total internal reflection sequencing chip is mounted on the precision translation stage, the imaging device is located above the total internal reflection sequencing chip, and the illumination device is located to the side of the total internal reflection sequencing chip;

[0009] The total internal reflection sequencing chip comprises a prism substrate, a cover plate, a flow channel plate, and a fixing frame; the prism substrate, cover plate, and flow channel plate are bonded together as a whole with an adhesive and mounted on the fixing frame; wherein the upper and lower surfaces of the prism substrate are respectively bonded to a flow channel plate, which is in turn bonded to a cover plate; and a flow channel is machined in the middle of the flow channel plate, which runs through the upper and lower surfaces;

[0010] The imaging temperature measurement device includes a microscope objective lens, an infrared dichroic mirror, a focus shifter, an imaging module, and a focus temperature measurement module. The optical axes of the microscope objective lens and the imaging module are coaxial with the transmission optical axis of the infrared dichroic mirror and are located at both ends of the infrared dichroic mirror. The optical axis of the focus temperature measurement module is coaxial with the reflection optical axis of the infrared dichroic mirror and is perpendicular to the optical axes of the microscope objective lens and the imaging module. The microscope objective lens, the infrared dichroic mirror, and the focus temperature measurement module are jointly mounted on the focus shifter.

[0011] The lighting device includes an illumination light source, an illumination lens, an illumination lens shifter, and an illumination light deflecting mirror. The illumination lens is mounted on the illumination lens shifter and collimates the light emitted by the illumination light source. The illumination light deflecting mirror is mounted on the precision displacement stage and is located in the path of the light emitted by the illumination lens.

[0012] Preferably, the total internal reflection sequencing chip and the illumination light deflecting mirror are both fixed on the precision translation stage, and are driven by the precision translation stage to move synchronously in the horizontal direction;

[0013] The illumination lens is mounted on the illumination lens displacer, and the illumination lens displacer drives the illumination lens to move up and down. The illumination lens emits an illumination beam to the illumination light deflecting mirror, and the illumination light deflecting mirror deflects the illumination light and injects it into the total internal reflection sequencing chip.

[0014] Preferably, the upper and lower surfaces of the prism substrate include one or more sequencing areas and coating areas;

[0015] The surface of the sequencing area is modified with primers for connecting DNA molecules, and the coating area is coated with a high-reflectivity metal film. The sequencing area coincides with the area covered by the flow channel. When the illumination light is incident at an angle perpendicular to the left or right side of the prism substrate, total internal reflection can occur simultaneously on the inner sides of the two sequencing areas corresponding to the upper and lower surfaces, thereby emitting evanescent light waves to the outside of the area.

[0016] Preferably, electrodes are installed at both ends of the fixing frame, one end is a positive electrode and the other end is a negative electrode, and the electrodes are connected to the coating area of ​​the prism substrate; when the electrodes are energized, the coating area of ​​the prism substrate will generate heat, thereby heating the flow channel of the total internal reflection sequencing chip.

[0017] Preferably, the focusing and temperature measurement module comprises a two-quadrant photodetector, an infrared light emitting diode and a signal processing module;

[0018] The focusing and temperature measurement module has two operating modes: focusing and temperature measurement. The two-quadrant photodetector is divided into two areas: quadrant A and quadrant B. When the focusing and temperature measurement module operates in the focusing mode, the infrared diode emits an infrared beam at a specific angle toward the rear of the microscope objective lens. After passing through the microscope objective lens and reaching the surface of the sample to be measured, the infrared beam is reflected and re-enters the microscope objective lens in a direction symmetrical to the direction of the incident light.

[0019] The two-quadrant photoelectric detector receives the light beam emitted by the infrared diode and then reflected by the sample surface, and the signal processing module outputs the difference amplified signal between quadrant A and quadrant B.

[0020] Preferably, the two-quadrant detector receives the spontaneous infrared signal of the sample at the imaging focal plane received by the microscope objective lens, and the signal processing module outputs the sum amplified signal of the A quadrant and the B quadrant.

[0021] Preferably, the light beam emitted by the illumination lens is a parallel collimated light beam directed toward the illumination light deflecting mirror. When the precision translation stage drives the illumination light deflecting mirror and the total internal reflection sequencing chip to move along the direction of the illumination beam, the relative positions of the total internal reflection light spots on the upper and lower surfaces of the total internal reflection sequencing chip relative to the total internal reflection sequencing chip do not change.

[0022] Preferably, the illumination lens shifter drives the illumination lens to move up and down, so that the total internal reflection light spot in the total internal reflection sequencing chip can switch between different flow channels.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The substrate of the gene total internal reflection sequencing chip of the present invention is actually a prism with a parallelogram cross-section. By injecting an illumination beam into the substrate perpendicular to the side, the beam undergoes total internal reflection at the bottom of the flow channel, generating an evanescent wave that excites the fluorescent molecules on the DNA molecules, thus forming a typical total internal reflection illumination method. This illumination method can greatly reduce the background light interference caused by the illumination light during the imaging process, thereby improving the signal-to-noise ratio and enabling direct single-molecule fluorescence imaging using an air dielectric objective lens.

[0025] 2. Conventional prism-based total internal reflection illumination microscopes all contain a prism specifically designed for total internal reflection illumination. A layer of transparent immersion oil with a refractive index equal to that of glass is applied between the prism and the sample, allowing the illumination light to reach the surface to be imaged. However, if this imaging method is directly applied to a gene sequencer, the introduction of immersion oil will inevitably require automatic oiling and cleaning devices. In the present invention, since the prism substrate itself is a prism, there is no need for a dedicated illumination prism.

[0026] 3. Because the flow channel of the total internal reflection sequencing chip is perpendicular to the direction of the illumination beam, movement of the total internal reflection sequencing chip along the flow channel will not change the position of the illumination spot relative to the objective lens. Furthermore, because the illumination light deflection mirror is relatively fixed to the total internal reflection sequencing chip, and the direction of the light beam entering the illumination light deflection mirror is horizontal and perpendicular to the flow channel, left and right movement of the total internal reflection sequencing chip will not change the relative position between the illumination spot and the objective lens. Therefore, by using the precision translation stage to drive the total internal reflection sequencing chip to move in the front, back, left, and right directions, scanning imaging of the DNA sample to be tested in the flow channel can be achieved.

[0027] 4. Because the illumination lens can move up and down, the light beam in the prism substrate moves left and right, causing the total internal reflection illumination spot to also move left and right. Since there is a difference between the distance between the flow channels on the same surface of the total internal reflection sequencing chip and the distance between the total internal reflection light spots on the same plane, when the total internal reflection light spot illuminates one set of flow channels, the other set of flow channels will not be illuminated, thus preventing the fluorescent nucleotides in the other set of flow channels from being quenched when not imaging;

[0028] 5. For the total internal reflection sequencing chip of the present invention, when the flow channel is filled with sequencing reagents, the refractive index of the reagents and the total internal reflection sequencing chip substrate is different in the area covered by the flow channel. Therefore, the illumination light can emit total internal reflection at the interface between the reagents and the total internal reflection sequencing chip substrate. In the area where the flow channel plate contacts the substrate, the illumination beam can still undergo total internal reflection due to the high-reflectivity metal film covering it. The illumination beam can be reflected back and forth at any position on the upper and lower surfaces of the prism substrate, thus forming multiple total internal reflection light spots on the upper and lower surfaces of the prism substrate. This allows the total internal reflection sequencing chip to include multiple flow channels, thereby increasing sequencing throughput.

[0029] 6. The highly reflective metal film on the TIR sequencing chip substrate of the present invention is also conductive. Therefore, applying a specific voltage to it through the electrodes at both ends of the TIR sequencing chip holder can generate heat, thereby heating the entire TIR sequencing chip and providing the appropriate temperature for the chemical reactions in the sequencing process. This eliminates the need for an additional heating device, simplifying the instrument structure.

[0030] 7. The focusing and temperature measurement module of the present invention determines whether the sample is out of focus by detecting the reflection signal of the infrared beam emitted by itself on the sample surface and can provide a feedback signal so that the system can adjust the distance between the microscope objective lens and the sample in real time, thereby achieving real-time automatic focusing;

[0031] 8. For the focusing temperature measurement module in the present invention, the temperature of the sample can be directly measured through the focusing optical path and the detector, so that the system can accurately adjust the heating power of the total internal reflection sequencing chip in real time, thereby simplifying the instrument structure and improving the instrument operation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0033] Figure 1 This is a schematic diagram of the total internal reflection illumination imaging principle in the present invention;

[0034] Figure 2 This is a schematic diagram of scanning imaging of the total internal reflection gene sequencing chip of the present invention;

[0035] Figure 3 This is a schematic diagram of the surface structure of the total internal reflection sequencing chip substrate of the present invention;

[0036] Figure 4 This is a perspective view of the complete structure of the total internal reflection sequencing chip of the present invention;

[0037] Figure 5 This is a schematic diagram of the second set of flow channel imaging of the total internal reflection sequencing chip of the present invention;

[0038] Figure 6 This is a schematic diagram of the focus and temperature measurement module of the present invention in the focus mode;

[0039] Figure 7 This is a schematic diagram of the focus temperature measurement module of the present invention in the temperature measurement mode. DETAILED DESCRIPTION

[0040] The present invention will be described in detail below with reference to specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several changes and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0041] The embodiment of the present invention provides a total internal reflection single molecule gene sequencing system, referring to Figure 1 and Figure 2 As shown, it specifically includes: a total internal reflection sequencing chip, an imaging temperature measurement device, an illumination device and a precision translation stage; the total internal reflection sequencing chip is installed on the precision translation stage, the imaging device is located above the total internal reflection sequencing chip, and the illumination device is located on the side of the total internal reflection sequencing chip.

[0042] Among them, the total internal reflection sequencing chip includes a prism substrate, a cover plate, a flow channel plate and a fixing frame; the prism substrate, the cover plate and the flow channel plate are bonded into a whole by an adhesive and installed on the fixing frame; wherein, the cross-section of the prism substrate is a parallelogram, the upper surface and the lower surface of the prism substrate are respectively bonded to a flow channel plate, and the flow channel plate is bonded to a cover plate, and a flow channel is processed in the middle of the flow channel plate that runs through the upper and lower surfaces.

[0043] An imaging temperature measurement device includes a microscope objective lens, an infrared dichroic mirror, a focus shifter, an imaging module, and a focus temperature measurement module. The optical axes of the microscope objective lens and the imaging module are coaxial with the transmission optical axis of the infrared dichroic mirror and are located at both ends of the infrared dichroic mirror. The optical axis of the focus temperature measurement module is coaxial with the reflection optical axis of the infrared dichroic mirror and is perpendicular to the optical axes of the microscope objective lens and the imaging module. The microscope objective lens, the infrared dichroic mirror, and the focus temperature measurement module are jointly mounted on the focus shifter.

[0044] An illumination device comprising an illumination lens, an illumination lens shifter, and an illumination light deflecting mirror, wherein the illumination lens is mounted on the illumination lens shifter and collimates the light emitted by the illumination light source, and the illumination light deflecting mirror is mounted on the precision displacement stage and is located in the path of the light emitted by the illumination lens;

[0045] Specifically, the total internal reflection sequencing chip and the illumination light deflecting mirror are both fixed to a precision translation stage and driven by the stage to move synchronously in the horizontal direction. The illumination lens is mounted on the illumination lens shifter, which drives the illumination lens up and down. The illumination lens transmits an illumination beam toward the illumination light deflecting mirror, which deflects the illumination light and directs it at the optimal angle to the total internal reflection sequencing chip.

[0046] The upper and lower surfaces of the prism substrate contain one or more sequencing areas and coating areas; the surface of the sequencing area is modified with primers for connecting DNA molecules, and the coating area is coated with a high-reflectivity metal film. The sequencing area coincides with the area covered by the flow channel; when the illumination light is incident at an angle perpendicular to the left or right side of the prism substrate, total internal reflection can occur simultaneously inside the two sequencing areas corresponding to the upper and lower surfaces, thereby emitting evanescent light waves outside the area.

[0047] Electrodes are installed at both ends of the fixing frame, one end is positive and the other end is negative, and the electrodes are connected to the coating area of ​​the prism substrate; when the electrodes are energized, the coating area of ​​the prism substrate will heat up, thereby heating the flow channel of the total internal reflection sequencing chip.

[0048] The focusing and temperature measurement module includes a two-quadrant photodetector, an infrared light-emitting diode, and a signal processing module. The focusing and temperature measurement module has two operating modes: focusing and temperature measurement. The two-quadrant photodetector is divided into two areas, quadrant A and quadrant B. When the focusing and temperature measurement module works in focusing mode, the infrared diode emits an infrared beam at a specific angle toward the rear of the microscope objective lens. After passing through the microscope objective lens and reaching the surface of the sample to be measured, the infrared beam will be reflected and re-enter the microscope objective lens in a direction symmetrical to the direction of the incident light.

[0049] A two-quadrant photodetector receives the light beam emitted by the infrared diode and reflected from the sample surface, and the signal processing module outputs an amplified signal representing the difference between quadrants A and B. A two-quadrant detector receives the spontaneous infrared signal of the sample at the imaging focal plane, received by the microscope objective lens, and the signal processing module outputs an amplified signal representing the sum of quadrants A and B. The light beam emitted by the illumination lens is a parallel, collimated beam directed toward the illumination light deflection mirror. When the precision translation stage drives the illumination light deflection mirror and the total internal reflection sequencing chip to move along the illumination beam, the relative positions of the total internal reflection light spots on the upper and lower surfaces of the total internal reflection sequencing chip relative to the chip remain unchanged.

[0050] The illumination lens shifter drives the illumination lens to move up and down, so that the total internal reflection light spot in the total internal reflection sequencing chip can switch between different flow channels.

[0051] Next, the present invention will be described in more detail.

[0052] The present invention aims to solve the problems of the total internal reflection illumination imaging system in common single-molecule sequencers, which is complex in structure and depends on immersion oil, resulting in poor reliability and high maintenance costs. A total internal reflection single-molecule gene sequencing system that does not rely on immersion oil for single-molecule imaging is provided. Figure 1 and Figure 2 As shown, the total internal reflection single-molecule gene sequencing system specifically includes: a total internal reflection sequencing chip 11, a mounting bracket 12, an imaging temperature measurement device 48, an illumination device 123, and a precision translation stage 20. The imaging temperature measurement device 48 includes a microscope objective 9, a focus temperature measurement module 4, a focus tube lens 5, an infrared dichroic mirror 8, a single-photon detector 6, an imaging tube lens 7, and a focus shifter 10. The illumination device 123 includes an illumination lens 1, an illumination light deflection mirror 3, and an illumination lens shifter 21. This embodiment includes two sets of imaging temperature measurement devices 48, which can simultaneously image the top and bottom of the total internal reflection sequencing chip 11.

[0053] like Figure 2 As shown, the total internal reflection sequencing chip and the illumination light deflecting mirror are both fixed to the precision translation stage 20 and can be driven by the precision translation stage 20 to move synchronously in the horizontal direction. The illumination lens shifter 21 includes a fixing fixture 213, a slider 212, and a fixed plate 211. The illumination lens 1 is clamped by the fixing fixture 213 and mounted on the slider 212. The slider 212 is driven by a motor mounted on the fixed plate 211 to move up and down along the Z axis.

[0054] The total internal reflection sequencing chip 11 includes a prism substrate 111 , a flow channel plate 112 , and a cover plate 113 . The prism substrate is made of colorless glass or quartz, and its cross section is a parallelogram.

[0055] The illumination light deflecting mirror 3 is a colorless transparent prism with a trapezoidal cross section.

[0056] The upper and lower surfaces of the prism substrate are respectively bonded to a flow channel plate 112, which is in turn bonded to a cover plate 113. Two flow channel grooves are machined in the middle of the flow channel plate, running through the upper and lower surfaces. The flow channel grooves are sealed by the prism substrate 111 and the cover plate 113 to form flow channels 101, 102, 103, and 104 through which sequencing reagents can flow.

[0057] like Figure 3 As shown, the upper and lower surfaces of the prism substrate include a sequencing area 301 and a coating area 302. The surface of the sequencing area 301 is modified with primers for connecting DNA molecules, and the coating area is coated with a high-reflectivity metal film. The sequencing area 301 overlaps with the area covered by the flow channels 101, 102, 103, and 104.

[0058] like Figure 4 As shown, a through hole penetrating the cover plate 113 is opened at each end of the gene total internal reflection sequencing chip flow channel, one of which is a liquid inlet hole 401 and the other is a liquid outlet hole 402. During the sequencing process, the sequencing reagent flows in from the liquid inlet hole 401 and flows out from the liquid outlet hole 402.

[0059] During the sequencing process, fluorescent nucleotides are first fixed to the base to be tested under the action of DNA synthesizers, and then scanning and imaging begins. When the system is scanning and imaging, the illumination lens 1 emits an illumination laser beam 2 horizontally to the right. After passing through the illumination light deflection mirror 3, the beam is refracted and emitted to the upper right, 9 and enters the prism substrate 111 in a direction perpendicular to the left side of the prism substrate 111. After that, the beam 2 reaches the lower surface of the flow channel 101 and undergoes total internal reflection and reaches the upper surface of the flow channel 102. In the area of ​​total internal reflection, the beam 2 forms an outward evanescent light field and excites the fluorescent molecules of the fluorescent nucleotides in the flow channel area of ​​the prism substrate, causing them to emit fluorescent photons. The use of this illumination method can greatly reduce the background light interference caused by the illumination light during the imaging process, thereby improving the signal-to-noise ratio and enabling the direct realization of single-molecule fluorescence imaging using an air dielectric objective lens.

[0060] Since the flow channel of the total internal reflection sequencing chip 11 is perpendicular to the direction of the illumination light beam 2, the movement of the total internal reflection sequencing chip along the flow channel will not change the position of the illumination spot relative to the objective lens. Moreover, since the illumination light deflection mirror 3 is relatively fixed to the total internal reflection sequencing chip 11, and the direction of the light beam entering the illumination light deflection mirror 3 is horizontal and perpendicular to the flow channel, the left and right movement of the total internal reflection sequencing chip will not change the relative position between the illumination spot and the microscope objective lens 9. Therefore, by driving the total internal reflection sequencing chip 11 to move in the front, back, left, and right directions through the precision translation stage, scanning imaging of the DNA sample to be tested in the flow channel can be achieved.

[0061] When the flow channel is filled with sequencing reagents, the refractive index of the reagents differs from that of the total internal reflection sequencing chip substrate in the area covered by the flow channel. Therefore, the illumination light can be totally internally reflected at the interface between the reagents and the total internal reflection sequencing chip substrate. However, in the area where the flow channel plate 112 contacts the prism substrate 111, the illumination beam can still be totally internally reflected due to the highly reflective metal film covering it. The illumination beam can be reflected back and forth at any location on the upper and lower surfaces of the prism substrate.

[0062] There is a difference between the distance between the flow channels on the same surface of the total internal reflection sequencing chip 11 and the distance between the total internal reflection light spots on the same plane. Therefore, when the total internal reflection light spot illuminates flow channels 101 and 102, flow channels 103 and 104 will not be illuminated and will be emitted from the right side of the prism substrate 111 in a direction perpendicular to the right surface after two more reflections. This prevents the fluorescent nucleotides in flow channels 103 and 104 from being quenched when not imaging and prevents the generation of stray light.

[0063] like Figure 5 As shown, when the system needs to image flow channels 103 and 104, the precision translation stage 20 will drive the total internal reflection sequencing chip 11 to move leftward along the X-axis, so that the upper imaging device and the lower imaging device are respectively aligned with flow channels 103 and 104. At this time, the illumination lens shifter 21 will drive the illumination lens 1 to move downward along the Z-axis for a certain distance, so that the first two total internal reflection positions of the illumination light beam 2 in the prism substrate 111 deviate to the right from flow channels 101 and 102, while the positions of the next two total internal reflections are exactly located at the center axis positions of flow channels 103 and 104.

[0064] Electrode sheets 13 and 14 are installed at both ends of the front and back sides of the fixing frame 12, one end of which is the positive electrode sheet 13 and the other end is the negative electrode sheet 14. The positive and negative electrodes are installed in the same way. Taking the positive electrode sheet 13 as an example, the two positive electrode sheets 13 on the front and back sides are fixed to the fixing frame 12 by screws and can be in close contact with the coating area 302 of the prism substrate 111, so that the electrode sheet 13 is conductive with the coating area 302. Similarly, the negative electrode sheet 14 is also conductive with the coating area 302 in the same way. When a current of appropriate size flows from the positive electrode sheet 13 through the coating area 302 and flows out from the negative electrode sheet 14, the coating area 302 will heat up, thereby heating the flow channel of the total internal reflection sequencing chip. At the same time, the upper and lower positive electrode sheets 13 can form a clamping force to clamp one end of the corresponding sequencing chip 11. Similarly, the negative electrode sheet can also clamp the other end of the sequencing chip 11. The combined clamping action of the positive electrode sheet 13 and the negative electrode sheet 14 can firmly fix the sequencing chip 11 in the center of the fixing frame.

[0065] like Figure 6As shown, the focus temperature measurement module 4 operates in the focus mode and includes an infrared light-emitting diode 501, a triangular reflector 502, a focus tube lens 5, a two-quadrant detector 505, a signal processing module 506, and a triangular reflector displacement module 507. The photoelectric conversion chip is divided into two regions, quadrant A and quadrant B. The infrared diode 501 emits an infrared light beam 511 toward the triangular reflector 502. After being reflected by the triangular reflector 502, the infrared light beam reaches the infrared dichroic mirror 8 in a direction parallel to the main optical axis. After being reflected downward in a direction parallel to the main optical axis, it enters the rear of the microscope objective lens 9 and is emitted from the front of the microscope objective lens 9 in a direction inclined to the main optical axis, and then reaches the sample surface 51. Among them, 513 is the optimal imaging focal plane, 512 is the positive focal plane, and 514 is the negative focal plane. After being reflected on the sample surface, the light beam 511 re-enters the microscope objective lens 9 and reaches the focusing tube lens 5 along a direction symmetrical to the previous emission path, and is converged to the two-quadrant detector 505 through the focusing tube lens 5. The position of the focused light spot is in the middle of the two-quadrant detector 505, with quadrants A and B each occupying half, and the signal intensity difference AB between quadrants A and B should be 0.

[0066] When the sample surface is at the positive focus position 512, the reflection position of the light beam 511 on the sample surface is to the left of when the sample is at the optimal position 513. Then, the light spot where the reflected light is finally focused by the focusing tube lens 5 will mainly be in the A quadrant of the two-quadrant detector 505, and the signal intensity difference AB between quadrants A and B should be a positive value.

[0067] When the sample surface is in the negative focus position 514, the reflection position of the light beam 511 on the sample surface is to the right of when the sample is in the optimal position 513. Then, the light spot where the reflected light is finally focused by the focusing tube lens 5 will mainly be in the B quadrant of the two-quadrant detector 505, and the signal intensity difference AB between quadrants A and B should be a negative value.

[0068] The focus shifter 10 can drive the microscope objective lens 9 , the focus temperature measurement module 4 and the infrared dichroic mirror 8 to move up and down along the Z axis to adjust the distance from the sample surface to the microscope objective lens 9 .

[0069] In the focus mode, the focus temperature measurement module actually detects the interface between the reagent in the flow channel of the total internal reflection sequencing chip 11 and the prism substrate. The relative position of this interface and the focal plane of the microscope objective lens 9 can be in two situations:

[0070] (1) When the interface is far away from the focal plane of the microscope objective lens 9, since the two-quadrant detector 505 hardly receives the reflected signal of the light beam 511, the sum of the signal intensities A+B of quadrant A and quadrant B must be less than a certain value x.

[0071] (2) When the interface is near the focal plane of the microscope objective lens 9, since the two-quadrant detector 505 can receive the partial reflection signal of the light beam 511, the sum of the signal intensities of quadrants A and B is A+B> x .

[0072] Therefore, the position of the microscope objective lens 9 relative to the sample surface can be determined by a certain algorithm based on the values ​​of parameters AB and A+B, and a feedback signal is sent to the control system through the signal processing module 506 to enable the focus shifter 10 to adjust the position of the microscope objective lens 9, thereby achieving the purpose of real-time automatic focusing.

[0073] like Figure 7 As shown, the focusing temperature measurement module operates in temperature measurement mode. The system needs to measure the temperature of the reagent in the flow channel of the total internal reflection sequencing chip 11. At this time, the focal plane of the microscope objective lens 9 is located in the flow channel near the surface of the prism substrate. Since the reagent liquid is constantly radiating infrared rays, the microscope objective lens 9 can collect these infrared rays. The triangular reflector displacement module 507 moves the triangular reflector 502 a certain distance in the w direction to deviate from the optical axis, so that the infrared rays emitted from the rear of the microscope objective lens 9 can be completely converged by the focusing tube lens 5 to the two-quadrant detector 505. The infrared rays radiated by the reagent have different intensities at different temperatures. Therefore, the sum of the signal intensities A+B in the A and B quadrants of the two-quadrant detector 505 corresponds to different values ​​at different temperatures. Therefore, the signal processing module 506 can determine the temperature of the reagent and output a feedback signal to enable the system to adjust the heating power.

[0074] Specifically, the working principle of the present invention is as follows:

[0075] Step 1: The user places the sequencing chip 11 that has been hybridized with the DNA template to be tested on the precision translation stage 20 of the sequencer;

[0076] Step 2: The precision translation stage 20 drives the sequencing chip so that one end of the flow channels 101 and 102 of the sequencing chip 11 is aligned with the microscope objective lenses 9 of the upper and lower imaging temperature measurement devices 48;

[0077] Step 3: The focus temperature measurement modules of the upper and lower imaging temperature measurement devices 48 are turned on in focus mode to move the focus shifter 10 to move the upper and lower microscope objective lenses 9 to the focus position respectively;

[0078] Step 4: The sequencing system starts the cleaning process of flow channels 101, 102, 103, and 104, that is, the cleaning buffer is injected into the flow channels at a constant flow rate and then discharged, and this cycle is repeated x times;

[0079] Step 5: The sequencing system executes a nucleotide loading procedure on flow channels 101, 102, 103, and 104. First, the system injects a fluorescent nucleotide solution into flow channels 101, 102, 103, and 104 of the sequencing chip 11. Then, the sequencing system energizes the coating area 302 of the sequencing chip 11 to heat the liquid in the flow channels to the temperature required for base pairing. Once the temperature is reached, heating is stopped and maintained at the reaction temperature for a predetermined time, t1.

[0080] Step 6: The sequencing system starts the cleaning process of flow channels 101, 102, 103, and 104 (same as step 4);

[0081] Step 7: The sequencing system begins scanning and imaging channels 101 and 102. First, the system injects imaging buffer into channels 101 and 102. Then, the illumination light source is turned on. The illumination lens shifter 21 drives the illumination lens 1 to cause the illumination beam 2 to undergo total internal reflection at the solid-liquid interface between channels 101 and 102 and the prism substrate 111, exciting the fluorescent nucleotides in this area to produce fluorescence. At this time, the single-photon detector begins imaging and taking pictures. The imaging time is t2. Then, the precision translation stage drives the sequencing chip 11 to move stepwise from the initial position to the other end of channels 101 and 102. The movement step length is L. After each step, it stays for t2 for imaging with the single-photon detector.

[0082] Step 8: After imaging channels 101 and 102, the system scans and images channels 103 and 104 (same as step 7). Simultaneously, the system initiates the nucleotide fragmentation process for channels 101 and 102. The system injects a nucleotide fragmentation reagent into channels 101 and 102 for a retention time of t3 to fragment the fluorescent groups of the nucleotides currently attached to the DNA.

[0083] Step 9: The sequencing system cleans flow channels 101 and 102 (same as step 4) and performs nucleotide fragmentation on flow channels 103 and 104 (same as step 8);

[0084] Step 10: The sequencing system loads nucleotides into flow channels 101 and 102 (same as step 5), and cleans flow channels 103 and 104 (same as step 4);

[0085] Step 11: The sequencing system scans and images the flow channels 101 and 102 and loads nucleotides into the flow channels 103 and 104.

[0086] The sequencing system then repeats steps 8 to 11 until all bases of the DNA to be tested on the sequencing chip are sequenced.

[0087] An embodiment of the present invention provides a total internal reflection single-molecule gene sequencing system, in which the cross-section of the prism substrate is a parallelogram, and the illumination light can directly form total internal reflection illumination within the prism substrate, thereby improving the imaging signal-to-noise ratio and freeing the system from dependence on immersion oil, automatic oiling devices, and automatic cleaning devices, simplifying the instrument structure and improving sequencing efficiency. The total internal reflection sequencing chip contains multiple flow channels above and below, which can increase sequencing throughput. The total internal reflection sequencing chip includes a heating function, which can provide a suitable temperature for the sequencing reaction, without the need for an additional heating device. The focusing and temperature measurement module uses the principle of infrared imaging to achieve automatic focusing and temperature control of the total internal reflection sequencing chip.

[0088] In the description of this application, it should be understood that the terms "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on this application.

[0089] The above describes specific embodiments of the present invention. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. The embodiments of this application and the features in the embodiments may be combined with each other in any manner unless there is a conflict.

Claims

1. A total internal reflection single-molecule gene sequencing system, characterized in that: include: A total internal reflection sequencing chip, an imaging temperature measurement device, an illumination device, and a precision translation stage; the total internal reflection sequencing chip is mounted on the precision translation stage, the imaging temperature measurement device is located above the total internal reflection sequencing chip, and the illumination device is located on the side of the total internal reflection sequencing chip; The total internal reflection sequencing chip comprises a prism substrate, a cover plate, a flow channel plate, and a fixing frame; the prism substrate, cover plate, and flow channel plate are bonded together as a whole with an adhesive and mounted on the fixing frame; wherein the upper and lower surfaces of the prism substrate are respectively bonded to a flow channel plate, which is in turn bonded to a cover plate; and a flow channel is machined in the middle of the flow channel plate, which runs through the upper and lower surfaces; The imaging temperature measurement device comprises a microscope objective lens, an infrared dichroic mirror, a focus shifter, an imaging module, and a focus temperature measurement module. The optical axes of the microscope objective lens and the imaging module are coaxial with the transmission optical axis of the infrared dichroic mirror and are located at both ends of the infrared dichroic mirror. The optical axis of the focus temperature measurement module is coaxial with the reflection optical axis of the infrared dichroic mirror and is perpendicular to the optical axes of the microscope objective lens and the imaging module. The microscope objective lens, the infrared dichroic mirror, and the focus temperature measurement module are jointly mounted on the focus shifter. The lighting device comprises an illumination light source, an illumination lens, an illumination lens shifter, and an illumination light deflecting mirror. The illumination lens is mounted on the illumination lens shifter and collimates the light emitted by the illumination light source. The illumination light deflecting mirror is mounted on the precision displacement stage and is located in the path of the light emitted by the illumination lens. The total internal reflection sequencing chip and the illumination light deflection mirror are both fixed on the precision translation stage and driven by the precision translation stage to move synchronously in the horizontal direction; The illumination lens is mounted on the illumination lens displacer, which drives the illumination lens to move up and down. The illumination lens emits an illumination beam toward the illumination light deflecting mirror. The direction of the illumination beam is perpendicular to the flow channel in the total internal reflection sequencing chip. The illumination light deflecting mirror deflects the illumination light and injects it into the total internal reflection sequencing chip at an angle perpendicular to the side of the prism substrate. The cross-sectional shape of the prism substrate is a parallelogram, and the upper and lower surfaces of the prism substrate include one or more sequencing areas and coating areas; The surface of the sequencing area is modified with primers for connecting to DNA molecules, and the coating area is coated with a high-reflectivity metal film. The sequencing area coincides with the area covered by the flow channel. When the illumination light is incident at an angle perpendicular to the left or right side of the prism substrate, total internal reflection can occur simultaneously inside the two sequencing areas corresponding to the upper and lower surfaces, thereby emitting evanescent light waves outside the area. The distance between the light spots generated by total internal reflection on the same surface of the prism substrate is not equal to the distance between the two flow channels on the same surface. When one flow channel is illuminated by the light spot, the other adjacent flow channel will not be illuminated.

2. The total internal reflection single-molecule gene sequencing system according to claim 1, characterized in that: Electrodes are installed at both ends of the fixing frame, one end is a positive electrode and the other end is a negative electrode, and the electrodes are connected to the coating area of ​​the prism substrate; when the electrodes are energized, the coating area of ​​the prism substrate will generate heat, thereby heating the flow channel of the total internal reflection sequencing chip.

3. The total internal reflection single-molecule gene sequencing system according to claim 1, characterized in that: The focusing and temperature measurement module includes a two-quadrant photoelectric detector, an infrared light emitting diode and a signal processing module; The focusing and temperature measurement module has two operating modes: focusing and temperature measurement. The two-quadrant photodetector is divided into two areas: quadrant A and quadrant B. When the focusing and temperature measurement module operates in the focusing mode, the infrared light emitting diode emits an infrared beam at a specific angle toward the rear of the microscope objective lens. After passing through the microscope objective lens and reaching the surface of the sample to be measured, the infrared beam is reflected and re-enters the microscope objective lens in a direction symmetrical to the direction of the incident light. The two-quadrant photoelectric detector receives the light beam emitted by the infrared light emitting diode and then reflected by the sample surface, and the signal processing module outputs the difference amplified signal between quadrant A and quadrant B.

4. The total internal reflection single-molecule gene sequencing system according to claim 3, characterized in that: The two-quadrant photoelectric detector receives the spontaneous infrared signal of the sample at the imaging focal plane received by the microscope objective lens, and the signal processing module outputs the sum amplified signal of the A quadrant and the B quadrant.

5. The total internal reflection single-molecule gene sequencing system according to claim 1, characterized in that: The light beam emitted by the illumination lens is a parallel collimated light beam directed toward the illumination light deflecting mirror. When the precision translation stage drives the illumination light deflecting mirror and the total internal reflection sequencing chip to move along the direction of the illumination beam, the relative positions of the total internal reflection light spots on the upper and lower surfaces of the total internal reflection sequencing chip relative to the total internal reflection sequencing chip do not change.

6. The total internal reflection single-molecule gene sequencing system according to claim 1, characterized in that: The illumination lens shifter drives the illumination lens to move up and down, so that the total internal reflection light spot in the total internal reflection sequencing chip can switch between different flow channels.

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