CN115058494A - Total internal reflection single-molecule gene sequencing system - Google Patents
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Abstract
The invention provides a total internal reflection single-molecule gene sequencing system, which relates to the technical field of gene sequencing and comprises the following components: the device comprises a total internal reflection sequencing chip, an imaging temperature measuring device, an illuminating device and a precise displacement platform; the total internal reflection sequencing chip comprises a prism substrate, a cover plate, a fixing frame and a plurality of runner plates; the upper surface and the lower surface of the prism substrate are respectively attached to a runner plate, the runner plate is attached to a cover plate, and a runner penetrating through the upper surface and the lower surface is processed in the middle of the runner plate; the imaging temperature measuring device comprises a microscope objective, an imaging module and a focusing temperature measuring module; the illumination device comprises an illumination lens, an illumination lens shifter and an illumination light deflection mirror. The method can perform total internal reflection single-molecule gene sequencing of single-molecule imaging without depending on the mirror oil, and has high reliability and low maintenance cost.
Description
Technical Field
The invention relates to the technical field of gene sequencing, in particular to a total internal reflection single-molecule gene sequencing system.
Background
The gene sequencing technology is an important research means in modern biomedicine, an important diagnosis method in modern medicine and a key technology in the future gene storage technology. At present, gene sequencing technologies are mainly divided into two categories, namely fluorescence sequencing and nanopore sequencing, and high-throughput sequencing mainly depends on the fluorescence sequencing. The gene sequencer is an important instrument for running the whole gene sequencing process and generally comprises three major parts, namely a detection reagent, a sequencing chip and a sequencer. In a fluorescence high-throughput sequencer, a sequencing chip is used for bearing sequencing samples and reagents, and a sequencer is responsible for conveying the reagents to the sequencing chip and carrying out fluorescence imaging. The current fluorescence high-flux sequencers are divided into a second generation and a third generation, and the third generation fluorescence high-flux sequencers are mainly characterized in single molecule sequencing, namely, each sequencing point only contains one DNA molecule, and the imaging device of the sequencer can realize single molecule fluorescence imaging.
Single molecule imaging requires a very high signal-to-noise ratio, i.e., the ratio of the single molecule fluorescence signal to the sample background light signal. At present, the realization of high signal-to-noise ratio imaging mainly depends on a total internal reflection illumination (TIRF) imaging mode, namely, illumination light is emitted from an optically dense medium to an optically sparse medium at a certain angle and is totally reflected at an interface of the optically dense medium and the optically sparse medium, and the generated evanescent light wave can excite fluorescence photons to enable the fluorescence photons to emit fluorescence. Because the intensity of the evanescent light is sharply attenuated along with the propagation distance, the effective action range is only hundreds of nanometers, which is greatly smaller than the distance between the sample and the objective lens, and almost no background stray light enters the objective lens, the signal-to-noise ratio of the imaging mode is greatly higher than that of the common epi-illumination fluorescence imaging mode.
Total internal reflection illumination imaging is mainly achieved by two ways: one is an objective lens type, namely, an illumination light beam is emitted through an objective lens, a sample is positioned on the inner surface of a glass dish, a solution is filled in the glass dish, the bottom of the glass dish is tightly attached to the objective lens, the illumination light beam generates total internal reflection at the interface of the glass dish and the solution and generates an evanescent light to excite the sample, and the generated fluorescence is received by the objective lens so as to be imaged; the other type is prism type, the storage mode of the sample is the same as that of the former type, however, the objective lens is positioned above the glass dish, the bottom of the glass dish is tightly attached to the trapezoid prism, the illuminating light enters the interface between the glass dish and the solution from the inclined plane at one side of the trapezoid prism and is totally internally reflected to generate the evanescent light and excite the sample, the reflected light exits from the inclined plane at the other side of the prism, and the fluorescence generated by the sample is received and imaged by the objective lens positioned above the glass dish.
In fact, no matter the objective lens type or prism type total internal reflection illumination imaging, since a tiny gap exists between the glass dish for holding the sample and the prism or the objective lens, the gap must be filled with mirror oil with the same refractive index as that of glass, otherwise the illumination light beam is subjected to total internal reflection at the interface of the gap and the prism or the objective lens and cannot reach the interface between the solution and the glass dish. In the sequencer, the sequencing chip has replaced ordinary imaging glass ware, if the total internal reflection illumination imaging mode of prism type or objective type is used, must also fill mirror oil between sequencing chip and prism or objective, however sequencing chip belongs to the consumptive material, the sequencer seals the instrument again, along with the frequent change of sequencing chip, mirror oil also will produce the loss, this just needs to be equipped with automatic oil supplementing device for the sequencer, however the essential element of mirror oil is cedar oil, thereby will solidify if expose in the space for a long time and forever pollute objective or prism, this requires again to be equipped with automatic cleaning device in the system, this will improve the instrument complexity greatly, reduce the reliability of instrument. Therefore, it is an urgent need for those skilled in the art to design a sequencer capable of total internal reflection illumination single molecule imaging without relying on mirror oil.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a total internal reflection single-molecule gene sequencing system.
According to the invention, the scheme of the total internal reflection single-molecule gene sequencing system is as follows:
a total internal reflection single molecule gene sequencing system, the system comprising: the device comprises a total internal reflection sequencing chip, an imaging temperature measuring device, an illuminating device and a precise displacement platform; the total internal reflection sequencing chip is arranged on the precise displacement table, the imaging device is positioned above the total internal reflection sequencing chip, and the illuminating device is positioned on the side edge of the total internal reflection sequencing chip;
the total internal reflection sequencing chip comprises a prism substrate, a cover plate, a runner plate and a fixing frame; the prism substrate, the cover plate and the runner plate are bonded into a whole by an adhesive and are arranged on the fixed frame; the upper surface and the lower surface of the prism substrate are respectively attached to a runner plate, the runner plate is attached to a cover plate, and a runner penetrating through the upper surface and the lower surface is processed in the middle of the runner plate;
the imaging temperature measuring device comprises a microscope objective, an infrared dichroscope, a focusing shifter, an imaging module and a focusing temperature measuring module, wherein the optical axes of the microscope objective and the imaging module are coaxial with the transmission optical axis of the infrared dichroscope and are positioned at two ends of the infrared dichroscope, the optical axis of the focusing temperature measuring module is coaxial with the reflection optical axis of the infrared dichroscope and is perpendicular to the optical axes of the microscope objective and the imaging module, and the microscope objective, the infrared dichroscope and the focusing temperature measuring module are jointly installed on the focusing shifter
The illuminating device comprises an illuminating light source, an illuminating lens shifter and an illuminating light deflection mirror, wherein the illuminating lens is arranged on the illuminating lens shifter and collimates light emitted by the illuminating light source, and the illuminating light deflection mirror is arranged on the precise displacement table and is positioned on a path of emergent rays of the illuminating lens.
Preferably, the total internal reflection sequencing chip and the illumination light deflection mirror are both fixed on the precision displacement table and are driven by the precision displacement table to synchronously move along the horizontal direction;
the illumination lens is installed on the illumination lens shifter, the illumination lens shifter drives the illumination lens to move up and down, the illumination lens emits illumination light beams to the illumination light deflection mirror, and the illumination light deflection mirror deflects the illumination light and emits the illumination light into the total internal reflection sequencing chip.
Preferably, the upper surface and the lower surface of the prism substrate comprise one or more sequencing areas and coating areas;
the surface of the sequencing region is modified with a primer for connecting DNA molecules, the coating region is plated with a high-reflectivity metal film, and the sequencing region is overlapped with the region covered by the flow channel; when the illumination light is incident at an angle vertical to the left or right of the prism substrate, total internal reflection can simultaneously occur at the inner sides of the two sequencing areas corresponding to the upper surface and the lower surface, so that the evanescent light wave is emitted to the outer side of the area.
Preferably, electrodes are installed at two ends of the fixing frame, one end of the fixing frame is a positive electrode, the other end of the fixing frame is a negative electrode, and the electrodes are communicated with the film coating area of the prism substrate; when the electrodes are electrified, the film coating area of the prism substrate can generate heat so as to heat the flow channel of the total internal reflection sequencing chip.
Preferably, the focusing temperature measuring module comprises a two-quadrant photoelectric detector, an infrared light emitting diode and a signal processing module;
the working mode of the focusing temperature measurement module is divided into a focusing mode and a temperature measurement mode; the two-quadrant photoelectric detector is divided into an A quadrant and a B quadrant; when the focusing temperature measuring module works in a focusing mode, the infrared diode emits infrared beams to the rear part of the microscope objective at a specific angle, and the infrared beams reach the surface of a sample to be measured after passing through the microscope objective, are reflected and re-enter the microscope objective along a direction symmetrical to the direction of incident light;
and the two-quadrant photoelectric detector receives the light beam emitted by the infrared diode and reflected by the surface of the sample, and the signal processing module outputs a difference amplified signal of the A quadrant and the B quadrant.
Preferably, the two-quadrant detector receives a spontaneous infrared signal of the sample at the imaging focal plane, which is received by the microscope objective, and the signal processing module outputs a sum amplified signal of the quadrant A and the quadrant B.
Preferably, the light beam emitted by the illumination lens is a parallel collimated light beam emitted to the light beam deflection mirror, and when the precision displacement table drives the illumination light deflection mirror and the total internal reflection sequencing chip to move along the direction of the illumination light beam, the relative position of the total internal reflection light spot on the upper surface and the lower surface of the total internal reflection sequencing chip relative to the total internal reflection sequencing chip does not change.
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 be switched among different flow channels.
Compared with the prior art, the invention has the following beneficial effects:
1. the substrate of the gene total internal reflection sequencing chip is actually a prism with a parallelogram cross section, and an illumination light beam is injected into the substrate in a direction vertical to the side surface to enable the light beam to generate total internal reflection at the bottom of a flow channel so as to generate an evanescent wave to excite fluorescent molecules on DNA molecules, thereby forming a typical total internal reflection illumination mode;
2. conventional prism-type tir illumination microscopes include a prism that is dedicated to tir illumination and is coated with a transparent lens of refractive index equal to that of the glass between the prism and the sample to allow the illumination light to reach the surface to be imaged. However, if this imaging method is directly applied to a gene sequencer, an automatic oiling device and an automatic cleaning device are inevitably adopted due to the introduction of the mirror oil. In the invention, the prism substrate is a prism, so that a special illumination prism is not needed to be arranged;
3. because the flow channel of the total internal reflection sequencing chip is vertical to the direction of the illumination light beam, the movement of the total internal reflection sequencing chip along the flow channel direction does not change the position of the illumination light spot relative to the objective lens, and because the illumination light deflection mirror and the total internal reflection sequencing chip are relatively fixed, and the direction of the light beam entering the illumination light deflection mirror is horizontal and vertical to the flow channel, the relative position between the illumination light spot and the objective lens cannot be changed even if the total internal reflection sequencing chip moves left and right, so that the scanning imaging of the DNA sample to be tested in the flow channel can be realized by driving the total internal reflection sequencing chip to move along the front, back, left and right directions through the precision displacement platform;
4. because the illumination lens can move up and down, the light beam in the prism substrate moves left and right, so that the total internal reflection illumination light spot can also move left and right. Because 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 have a difference value, when the total internal reflection light spots irradiate one group of flow channels, the other group of flow channels cannot be irradiated, and therefore the fluorescent nucleotide in the other group of flow channels is prevented from being quenched when not being imaged;
5. for the total internal reflection sequencing chip, when the flow channel is filled with the sequencing reagent, the refractive indexes of the reagent in the area covered by the flow channel and the total internal reflection sequencing chip substrate are different, so that the illumination light can emit total internal reflection at the interface of the reagent and the total internal reflection sequencing chip substrate, and the illumination light beam can still generate total internal reflection in the area contacted with the substrate through the flow channel plate because a layer of metal thin film with high reflectivity is covered. The illumination light beams can be reflected back and forth at any position of the upper surface and the lower surface of the prism substrate, so that a plurality of total internal reflection light spots can be formed on the upper surface and the lower surface of the prism substrate, and the total internal reflection sequencing chip can comprise a plurality of flow channels, so that the sequencing flux is increased;
6. as the high-reflectivity metal film on the surface of the substrate of the total internal reflection sequencing chip has conductivity, the electrode at two ends of the fixing frame of the total internal reflection sequencing chip can generate heat by applying specific voltage to the electrode, so that the whole total internal reflection sequencing chip is heated, and proper temperature is provided for chemical reaction in a sequencing process. The structure of the instrument is simplified because an additional heating device is not needed;
7. for the focusing temperature measuring module, whether the sample is out of focus is judged by detecting a reflection signal of an infrared beam emitted by the focusing temperature measuring module on the surface of the sample, and a feedback signal can be given so that the system can adjust the distance between the microscope objective and the sample in real time, thereby realizing real-time automatic focusing;
8. the focusing temperature measurement module can directly measure the temperature of a sample through the focusing light 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 structure of the instrument and improving the operation efficiency of the instrument.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a schematic diagram of total internal reflection illumination imaging according to the present invention;
FIG. 2 is a schematic view of scanning and imaging of the total internal reflection gene sequencing chip according to the present invention;
FIG. 3 is a schematic diagram of the surface structure of a total internal reflection sequencing chip substrate according to the present invention;
FIG. 4 is a perspective view of the complete structure of the TIR sequencing chip of the present invention;
FIG. 5 is a schematic diagram of imaging a second set of flow channels of the TIR sequencing chip of the present invention;
FIG. 6 is a schematic diagram of a focusing temperature measuring module according to the present invention in a focusing mode;
FIG. 7 is a schematic diagram of a focusing temperature measurement module according to the present invention in a temperature measurement mode.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the concept of the invention. All falling within the scope of the present invention.
The embodiment of the invention provides a total internal reflection single-molecule gene sequencing system, which is shown in a reference figure 1 and a reference figure 2 and specifically comprises the following steps: the device comprises a total internal reflection sequencing chip, an imaging temperature measuring device, an illuminating device and a precise displacement platform; the total internal reflection sequencing chip is arranged on the precise displacement table, the imaging device is positioned above the total internal reflection sequencing chip, and the illuminating device is positioned on the side edge of the total internal reflection sequencing chip.
The total internal reflection sequencing chip comprises a prism substrate, a cover plate, a runner plate and a fixing frame; the prism substrate, the cover plate and the runner plate are bonded into a whole by an adhesive and are arranged on the fixed frame; the cross section of the prism substrate is a parallelogram, the upper surface and the lower surface of the prism substrate are respectively attached to a runner plate, the runner plate is attached to a cover plate, and a runner penetrating through the upper surface and the lower surface is processed in the middle of the runner plate.
The imaging temperature measuring device comprises a microscope objective, an infrared dichroscope, a focusing shifter, an imaging module and a focusing temperature measuring module, wherein the optical axes of the microscope objective and the imaging module are coaxial with the transmission optical axis of the infrared dichroscope and are positioned at two ends of the infrared dichroscope, the optical axis of the focusing temperature measuring module is coaxial with the reflection optical axis of the infrared dichroscope and is perpendicular to the optical axes of the microscope objective and the imaging module, and the microscope objective, the infrared dichroscope and the focusing temperature measuring module are jointly installed on the focusing shifter;
the illuminating device comprises an illuminating lens, an illuminating lens shifter and an illuminating light deflection mirror, wherein the illuminating lens is arranged on the illuminating lens shifter and collimates light emitted by the illuminating light source, and the illuminating light deflection mirror is arranged on the precise displacement table and is positioned on a path of emergent rays of the illuminating lens;
specifically, the total internal reflection sequencing chip and the illumination light deflection mirror are both fixed on the precise displacement table and are driven by the precise displacement table to synchronously move along the horizontal direction. The illumination lens is arranged on the illumination lens shifter, the illumination lens shifter drives the illumination lens to move up and down, the illumination lens emits illumination light beams to the illumination light deflection mirror, and the illumination light deflection mirror deflects the illumination light and emits the illumination light into the total internal reflection sequencing chip at an optimal angle.
The upper surface and the lower surface of the prism substrate comprise one or more sequencing areas and coating areas; the surface of the sequencing region is modified with a primer for connecting DNA molecules, the coating region is plated with a high-reflectivity metal film, and the sequencing region is overlapped with the region covered by the flow channel; when the illumination light is incident at an angle vertical to the left or right of the prism substrate, total internal reflection can simultaneously occur at the inner sides of the two sequencing areas corresponding to the upper surface and the lower surface, so that the evanescent light wave is emitted to the outer side of the sequencing areas.
The two ends of the fixing frame are provided with electrodes, one end of the fixing frame is a positive electrode, the other end of the fixing frame is a negative electrode, and the electrodes are communicated with the film coating area of the prism substrate; when the electrode is electrified, the coating area of the prism substrate can generate heat so as to heat the flow channel of the total internal reflection sequencing chip.
The focusing temperature measuring module comprises a two-quadrant photoelectric detector, an infrared light emitting diode and a signal processing module; the working mode of the focusing and temperature measuring module is divided into a focusing mode and a temperature measuring mode; the two-quadrant photoelectric detector is divided into an A quadrant and a B quadrant; when the focusing temperature measuring module works in a focusing mode, the infrared diode emits infrared beams to the rear of the microscope objective at a specific angle, and the infrared beams are reflected after passing through the microscope objective and reaching the surface of a sample to be measured and then re-enter the microscope objective along a direction symmetrical to the direction of incident light.
The two-quadrant photoelectric detector receives the light beam which is emitted by the infrared diode and reflected by the surface of the sample, and the signal processing module outputs a difference value amplification signal of the A quadrant and the B quadrant. And the two-quadrant detector receives a spontaneous infrared signal of the sample at the imaging focal plane, which is received by the microscope objective, and the signal processing module outputs a sum value amplification signal of the quadrant A and the quadrant B. The light beam emitted by the illumination lens is parallel collimated light beam emitted to the light beam deflection mirror, and when the precision displacement table drives the illumination light deflection mirror and the total internal reflection sequencing chip to move along the direction of the illumination light beam, the relative position of total internal reflection light spots on the upper surface and the lower surface of the total internal reflection sequencing chip relative to the total internal reflection sequencing chip is not changed.
And the illuminating lens shifter drives the illuminating lens to move up and down, so that the total internal reflection light spots in the total internal reflection sequencing chip can be switched among different flow channels.
Next, the present invention will be described in more detail.
The invention provides a total internal reflection single-molecule gene sequencing system for single-molecule imaging independent of mirror oil, aiming at solving the problems of poor reliability, high maintenance cost and the like caused by the fact that a total internal reflection illumination imaging system in a common single-molecule sequencer is complex in structure and depends on mirror oil, and the total internal reflection single-molecule gene sequencing system specifically comprises the following components: the device comprises a total internal reflection sequencing chip 11, a fixing frame 12, an imaging temperature measuring device 48, an illuminating device 123 and a precision displacement table 20. The imaging temperature measuring device 48 comprises a microscope objective 9, a focusing temperature measuring module 4, a focusing tube mirror 5, an infrared dichroscope 8, a single photon detector 6, an imaging tube mirror 7 and a focusing shifter 10. The illumination device 123 includes an illumination lens 1, an illumination light deflecting mirror 3, and an illumination lens shifter 21. The present embodiment includes two sets of imaging thermometers 48 that can simultaneously image the top and bottom of the TIR chip 11.
As shown in fig. 2, the tir sequencing chip and the illumination light deflecting mirror are fixed on the precision displacement stage 20, and can be driven by the precision displacement stage 20 to synchronously move along the horizontal direction. The illumination lens shifter 21 includes a fixing jig 206, a slider 211, and a fixing plate 212, the illumination lens 1 is clamped by the fixing jig 206 and mounted on the slider 211, and the slider 211 is driven by a motor mounted on the fixing plate 212 to move up and down along the Z axis.
The tir sequencing chip 11 includes a prism substrate 111, a flow channel plate 112, and a cover plate 113, wherein the prism substrate is made of colorless glass or quartz, and has a parallelogram cross section.
The illumination light deflecting mirror 3 is a colorless transparent prism having a trapezoidal cross section.
The upper surface and the lower surface of the prism substrate are respectively attached to a flow channel plate 111, the flow channel plate 111 is further attached to a cover plate 113, two flow channel grooves penetrating the upper and lower surfaces are formed in the middle of the flow channel plate, and the flow channel grooves are closed by the prism substrate, the prism substrate 111 and the cover plate 113 to form flow channels 101, 102, 103 and 104 through which a sequencing reagent can flow.
As shown in fig. 3, the upper surface and the lower surface of the prism substrate include a sequencing region 301 and a coating region 302, the surface of the sequencing region 301 is modified with primers for connecting DNA molecules, the coating region is coated with a high-reflectivity metal film, and the sequencing region 301 coincides with the regions covered by the flow channels 101, 102, 103, and 104.
As shown in FIG. 4, two ends of the flow channel of the sequencing chip by total internal reflection are respectively provided with a through hole penetrating through the cover plate 113, one of which is a liquid inlet 401, and the other is a liquid outlet 402, and a sequencing reagent flows in from the liquid inlet 401 and flows out from the liquid outlet 402 in the sequencing process.
In the sequencing process, fluorescent nucleotide is firstly fixed to a base to be detected under the action of DNA synthetase, and then scanning imaging is started to obtain images. When the system performs scanning imaging, the illumination lens 1 emits an illumination laser beam 2 to the right in the horizontal direction, the beam is refracted by the illumination light deflecting mirror 3 and emitted to the upper right, 9 and enters the prism substrate 111 in the direction perpendicular to the left of the prism substrate 111, and then 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. The light beam 2 forms an outward evanescent optical field in the total internal reflection area and excites fluorescent molecules of fluorescent nucleotides in the flow channel area of the prism substrate, so that the fluorescent photons are emitted. By adopting the illumination mode, the interference of background light caused by illumination light in the imaging process can be greatly reduced, so that the signal to noise ratio is improved, and the monomolecular fluorescence imaging is directly realized by adopting the air medium objective lens.
Because 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 direction does not change the position of the illumination light spot relative to the objective lens, and because the illumination light deflection mirror 3 and the total internal reflection sequencing chip 11 are relatively fixed and enter the light beam direction of the illumination light deflection mirror 3 to be horizontal and perpendicular to the flow channel, the relative position between the illumination light spot and the microscope objective lens 9 cannot be changed even if the total internal reflection sequencing chip moves left and right, so that the scanning imaging of the DNA sample to be detected in the flow channel can be realized by driving the total internal reflection sequencing chip 11 to move along the front, back, left and right directions through the precision displacement platform.
When the flow channel is filled with a sequencing reagent, the refractive index of the reagent in the area covered by the flow channel is different from that of the total internal reflection sequencing chip substrate, so that the illumination light can emit total internal reflection at the interface of the reagent and the total internal reflection sequencing chip substrate, and the illumination light beam can still generate total internal reflection in the area where the flow channel plate 112 is in contact with the prism substrate 111 due to the fact that a layer of high-reflectivity metal film is covered. The illumination beam may be reflected back and forth at any position on the upper and lower surfaces of the prism substrate.
The distance between the flow channels on the same surface of the tir sequencing chip 11 is different from the distance between the tir spots on the same surface, so that when the tir spots are irradiated to the flow channels 101 and 102, the flow channels 103 and 104 will not be irradiated, and will be reflected twice to be emitted from the right side of the prism substrate 111 along the direction perpendicular to the right side surface, thereby avoiding the fluorescent nucleotides in the flow channels 103 and 104 from being quenched when not being imaged, and generating no stray light.
As shown in fig. 5, when the system needs to image the flow channel 103 and the flow channel 104, the precision displacement stage 20 drives the tir sequencing chip 11 to move left along the X axis, so that the upper imaging device and the lower imaging device are respectively aligned with the flow channel 103 and the flow channel 104, at this time, the illumination lens shifter 21 drives the illumination lens 1 to move downward along the Z axis by a certain distance, so that the first two tir positions of the illumination beam 2 in the prism substrate 111 are deviated from the flow channel 101 and the flow channel 102 to the right, and the last two tir positions are exactly located at the central axes of the flow channel 103 and the flow channel 104.
As shown in fig. 6, the focusing temperature measuring module 4 works in a focusing mode, and the focusing temperature measuring module 4 includes an infrared light emitting diode 501, a triangular reflector 502, a focusing tube mirror 503, a two-quadrant detector 505, a signal processing module 506, and a triangular reflector displacement module 507. The photoelectric conversion chip is divided into an A quadrant and a B quadrant. The infrared diode 501 emits an infrared light beam 511 to the triangular reflector 502, the light beam is reflected by the triangular reflector 502 and then reaches the infrared dichromatic mirror 8 along a direction parallel to the main optical axis, and then is reflected downward to enter the rear of the microscope objective 9 along the direction parallel to the main optical axis, and is emitted from the front of the microscope objective 9 along a direction inclined to the main optical axis, and then reaches the sample surface 51, wherein 513 is an optimal imaging focal plane, 512 is a positive focal plane, and 514 is a negative focal plane. The light beam 511 re-enters the microscope objective lens 9 after being reflected on the surface of the sample, reaches the focusing tube lens 503 along a direction symmetrical to the previous emission path, and is converged to the two-quadrant detector 505 through the focusing tube lens 503, the position of the focusing light spot is located at the middle of the two-quadrant detector 505, the a quadrant and the B quadrant respectively account for half, and the signal intensity difference a-B between the a quadrant and the B quadrant should be 0.
When the sample surface is at the positive focal position 512, the reflection position of the light beam 511 on the sample surface is closer to the left than when the sample is at the optimal position 513, and the light spot of the final reflected light focused by the focusing tube mirror 503 will be mainly in the a quadrant of the two-quadrant detector 505, and the signal intensity difference a-B between the a quadrant and the B quadrant should be a positive value.
When the sample surface is at the negative focus position 514, the reflection position of the light beam 511 on the sample surface is closer to the right than when the sample is at the optimal position 513, and the light spot of the final reflected light focused by the focusing tube mirror 503 will be mainly in the B quadrant of the two-quadrant detector 505, and the signal intensity difference a-B between the a quadrant and the B quadrant should be a negative value.
The focus shifter 10 can drive the microscope objective 9, the focus temperature measurement module 4 and the infrared dichroic mirror 504 to move up and down along the Z axis to adjust the distance from the sample surface to the microscope objective 9.
The focusing temperature measurement module actually detects an interface between a reagent in a flow channel of the total internal reflection sequencing chip 11 and the prism substrate in a focusing mode, and the relative position of the interface and a focal plane of the microscope objective 9 has two conditions:
(1) when the interface is far from the focal plane of the microscope objective 9, since the reflected signal of the light beam 511 is hardly received by the two-quadrant detector 505, the sum a + B of the signal intensities of the a-quadrant and the B-quadrant is necessarily smaller than a certain value x.
(2) When the interface is near the focal plane of the microscope objective 9, the sum of the signal intensities of the a-and B-quadrants, a + B > x, is due to the fact that the two-quadrant detector 505 can receive a partially reflected signal of the beam 511.
Therefore, the position of the microscope objective 9 relative to the surface of the sample can be judged according to the values of the parameters A-B and A + B through a certain algorithm, and a feedback signal is sent to the control system through the signal processing module 506 so that the focusing shifter 10 can adjust the position of the microscope objective 9, thereby achieving the purpose of real-time automatic focusing.
As shown in fig. 7, when the focusing temperature measurement module works in the temperature measurement mode, the system needs to measure the temperature of the reagent in the flow channel of the tir sequencing chip 11, and the focal plane of the microscope objective 9 is located at a position close to the surface of the prism substrate in the flow channel, and since the reagent liquid always radiates infrared rays spontaneously and outwards, the microscope objective 9 can collect the 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 ray emitted from the rear of the microscope objective 9 can be completely converged to the two-quadrant detector 505 by the focusing cylindrical mirror 503. The infrared ray intensities radiated by the reagent at different temperatures are different, so that the sum of the signal intensities of the quadrant a and the quadrant B of the two-quadrant detector 505 corresponds to different values at different temperatures, and the signal processing module 506 can judge the temperature of the reagent and output a feedback signal to enable the system to adjust the heating power.
Specifically, the working principle of the invention is as follows:
step 1: a user puts the sequencing chip 11 hybridized with the DNA template to be detected on a precise displacement table 20 of a sequencer;
step 2: the precision displacement platform 20 drives the sequencing chip to make one end of the flow channels 101 and 102 of the sequencing chip 11 aligned with the microscope objective lenses 9 of the upper and lower imaging temperature measuring devices 48;
and step 3: the focusing temperature measuring modules of the upper and lower imaging temperature measuring devices 48 start a focusing mode to enable the focusing shifter 10 to be respectively brought to the upper and lower microscope objectives 9 to reach a positive focus position;
and 4, step 4: the sequencing system starts to execute the cleaning process of the flow channels 101, 102, 103 and 104, i.e. injecting a cleaning buffer solution into the flow channels at a constant flow rate and discharging, and repeating the process for x times;
and 5: the sequencing system performs a nucleotide loading procedure on the flow channels 101, 102, 103, 104. Firstly, the system injects fluorescent nucleotide solution into the flow channels 101, 102, 103, 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 channel to reach the temperature required by base pairing, and when the temperature reaches the temperature, the heating is stopped and the reaction temperature is maintained for a certain time period t 1;
and 6: the sequencing system starts to execute the washing process of the flow channels 101, 102, 103, 104 (step 4);
and 7: the sequencing system starts to execute a scanning imaging process on the flow channels 101 and 102, firstly, the system injects an imaging buffer solution into the flow channels 101 and 102, then an illumination light source is turned on, an illumination lens shifter 21 drives an illumination lens 1 to enable an illumination light beam 2 to generate total internal reflection on a solid-liquid interface between the flow channels 101 and 102 and a prism substrate 111 and enable fluorescent nucleotides in the area to be excited to generate fluorescence, at the moment, a single-photon detector starts to image and take images, the imaging time is t2, then a precision displacement platform drives a sequencing chip 11 to move step by step from an initial position to the other ends of the flow channels 101 and 102, the moving step length is L, and the time after each step of movement is kept at t2 for the single-photon detector to image.
And step 8: after the imaging of the flow channels 101 and 102 is completed, the system performs a scanning imaging process on the flow channels 103 and 104 (same as step 7); meanwhile, the system starts a nucleotide fragmentation process for the flow channels 101 and 102, injects a nucleotide fragmentation reagent into the flow channels 101 and 102 and keeps for a time t3 to fragment the fluorophore of the nucleotide currently linked to the DNA;
and step 9: the sequencing system washes the flow channels 101, 102 (same as step 4) and performs nucleotide fragmentation on the flow channels 103, 104 (same as step 8);
step 10: the sequencing system carries out nucleotide loading on the flow channels 101 and 102 (same as step 5), and cleans the flow channels 103 and 104 (same as step 4);
step 11: the sequencing system scans the flow channels 101, 102 and nucleotide loads the flow channels 103, 104.
And then the sequencing system repeats the steps 8 to 11 until the sequencing of all the basic groups of the DNA to be tested on the sequencing chip is completed.
The embodiment of the invention provides a total internal reflection single-molecule gene sequencing system, wherein the cross section of a prism substrate is a parallelogram, and illumination light can directly form total internal reflection illumination in the prism substrate, so that the imaging signal-to-noise ratio is improved, the system is free from dependence on mirror oil, an automatic oiling device and an automatic cleaning device, the instrument structure is simplified, and the sequencing efficiency is improved. The total internal reflection sequencing chip comprises a plurality of flow channels above and below, so that the sequencing flux can be improved. The total internal reflection sequencing chip comprises a heating function, and can provide proper temperature for sequencing reaction without an additional heating device. And the focusing temperature measurement module realizes automatic focusing and temperature control of the total internal reflection sequencing chip by an infrared imaging principle.
In the description of the present application, it is to be understood that the terms "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience in describing the present application and simplifying the description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present application.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.
Claims (8)
1. A total internal reflection single molecule gene sequencing system, comprising: the device comprises a total internal reflection sequencing chip, an imaging temperature measuring device, an illuminating device and a precise displacement platform; the total internal reflection sequencing chip is arranged on the precise displacement table, the imaging device is positioned above the total internal reflection sequencing chip, and the illuminating device is positioned on the side edge of the total internal reflection sequencing chip;
the total internal reflection sequencing chip comprises a prism substrate, a cover plate, a runner plate and a fixing frame; the prism substrate, the cover plate and the runner plate are bonded into a whole by an adhesive and are arranged on the fixed frame; the upper surface and the lower surface of the prism substrate are respectively attached to a runner plate, the runner plate is attached to a cover plate, and a runner penetrating through the upper surface and the lower surface is processed in the middle of the runner plate;
the imaging temperature measuring device comprises a microscope objective, an infrared dichroscope, a focusing shifter, an imaging module and a focusing temperature measuring module, wherein the optical axes of the microscope objective and the imaging module are coaxial with the transmission optical axis of the infrared dichroscope and are positioned at two ends of the infrared dichroscope, the optical axis of the focusing temperature measuring module is coaxial with the reflection optical axis of the infrared dichroscope and is perpendicular to the optical axes of the microscope objective and the imaging module, and the microscope objective, the infrared dichroscope and the focusing temperature measuring module are jointly installed on the focusing shifter;
the lighting device comprises a lighting source, a lighting lens shifter and a lighting light deflection mirror, wherein the lighting lens is arranged on the lighting lens shifter and collimates light emitted by the lighting source, and the lighting light deflection mirror is arranged on the precision displacement table and is positioned on a path of emergent light rays of the lighting lens.
2. The system for sequencing a single molecule gene by total internal reflection according to claim 1, wherein the chip for sequencing by total internal reflection and the illumination light deflecting mirror are both fixed on the precise displacement stage and are driven by the precise displacement stage to synchronously move along a horizontal direction;
the illumination lens is installed on the illumination lens shifter, the illumination lens shifter drives the illumination lens to move up and down, the illumination lens emits illumination light beams to the illumination light deflection mirror, and the illumination light deflection mirror deflects the illumination light and emits the illumination light into the total internal reflection sequencing chip.
3. The system according to claim 1, wherein the prism substrate comprises one or more sequencing and coating regions on the upper and lower surfaces;
the surface of the sequencing region is modified with a primer for connecting DNA molecules, the coating region is plated with a high-reflectivity metal film, and the sequencing region is overlapped with the region covered by the flow channel; when the illumination light is incident at an angle vertical to the left or right of the prism substrate, total internal reflection can simultaneously occur at the inner sides of the two sequencing areas corresponding to the upper surface and the lower surface, so that the evanescent light wave is emitted to the outer side of the area.
4. The system according to claim 1, wherein the two ends of the fixing frame are provided with electrodes, one end of the fixing frame is a positive electrode, the other end of the fixing frame is a negative electrode, and the electrodes are communicated with the coating area of the prism substrate; when the electrodes are electrified, the film coating area of the prism substrate can generate heat so as to heat the flow channel of the total internal reflection sequencing chip.
5. The system according to claim 1, wherein the focusing and temperature measuring module comprises a two-quadrant photodetector, an infrared light emitting diode and a signal processing module;
the working mode of the focusing and temperature measuring module is divided into a focusing mode and a temperature measuring mode; the two-quadrant photoelectric detector is divided into an A quadrant and a B quadrant; when the focusing temperature measuring module works in a focusing mode, the infrared diode emits infrared beams to the rear part of the microscope objective at a specific angle, and the infrared beams reach the surface of a sample to be measured after passing through the microscope objective, are reflected and re-enter the microscope objective along a direction symmetrical to the direction of incident light;
and the two-quadrant photoelectric detector receives the light beam emitted by the infrared diode and reflected by the surface of the sample, and the signal processing module outputs a difference amplified signal of the A quadrant and the B quadrant.
6. The totally internally reflected single-molecule gene sequencing system according to claim 5, wherein the two-quadrant detector receives spontaneous infrared signals of the sample at an imaging focal plane, which are received by the microscope objective lens, and the signal processing module outputs sum amplified signals of A-quadrant and B-quadrant.
7. The system according to claim 1, wherein the light beam emitted from the illumination lens is a parallel collimated light beam emitted to the beam deflection mirror, and when the precision displacement stage drives the illumination beam deflection mirror and the tir sequencing chip to move along the illumination beam direction, the relative position of the tir spots on the upper and lower surfaces of the tir sequencing chip with respect to the tir sequencing chip is not changed.
8. The system according to claim 1, wherein the illumination lens shifter drives the illumination lens to move up and down, so that the tir spots in the tir sequencing chip can be switched between different flow channels.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116499975A (en) * | 2023-06-29 | 2023-07-28 | 之江实验室 | Prism device for optical surface wave sensor and design and installation method thereof |
| CN116698810A (en) * | 2023-07-28 | 2023-09-05 | 深圳赛陆医疗科技有限公司 | Optical system, gene sequencing device and imaging method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116499975A (en) * | 2023-06-29 | 2023-07-28 | 之江实验室 | Prism device for optical surface wave sensor and design and installation method thereof |
| CN116499975B (en) * | 2023-06-29 | 2023-09-22 | 之江实验室 | Prism device for optical surface wave sensor and design and installation method thereof |
| CN116698810A (en) * | 2023-07-28 | 2023-09-05 | 深圳赛陆医疗科技有限公司 | Optical system, gene sequencing device and imaging method |
| CN116698810B (en) * | 2023-07-28 | 2023-11-07 | 深圳赛陆医疗科技有限公司 | Optical system, gene sequencing device and imaging method |
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