HK1225406A1

HK1225406A1 - Single molecule gene sequencer

Info

Publication number: HK1225406A1
Application number: HK16113667.7A
Authority: HK
Inventors: 颜钦; 姜泽飞; 郑焦; 吴平; 周志良; 葛良进
Original assignee: 深圳市真迈生物科技有限公司
Filing date: 2016-11-30
Publication date: 2017-09-08

Abstract

The present invention discloses a single-molecule gene sequencer,A technical solution belonging to the field of gene sequencing,It includes a machine base,The machine base is equipped with a clamping platformReagent storage deviceFluid control deviceMobile platform and total reflection microscope;The clamping platform is equipped with a gene sequencing chip;The reagent storage device is used to store gene sequencing reagents;The fluid control device is used to pump the gene sequencing reagent from the reagent storage device to the gene sequencing chip;The mobile platform is used to drive the clamping platform to move towards and away from below the total reflection microscope;The total reflection microscope is used to detect the gene sequence of samples within the gene sequencing chip;This plan does not have a database construction process,Without PCR,Simple and convenient operation,Reduce sequencing costs,It is a highly suitable diagnostic and therapeutic method for clinical applications.

Description

Single molecule gene sequencer

Technical Field

The invention relates to gene sequencing equipment, in particular to a monomolecular gene sequencer.

Background

With the continuous development of gene sequencing technology, the second generation high-throughput sequencing technology has been widely applied in various research fields, but with the popularization of application, the shortcomings of the second generation sequencing technology are increasingly highlighted. For example, library construction is required, which is complex and time-consuming; PCR amplification is needed, preference is easy to generate, and the proportion of original genes is distorted; the sequencing reading length is short, and the difficulty is brought to the subsequent bioinformatics analysis such as sequence splicing and assembling. Therefore, the third generation of single molecule sequencing technology comes, which adopts the single molecule reading technology, has higher sensitivity and faster data reading speed, does not need PCR (polymerase Chain reaction) amplification, ensures the real information of a detection sample, and further reduces the sequencing cost. Among them, PCR is called polymerase chain reaction, which is a molecular biology technique for amplifying and amplifying a specific DNA fragment, and can be regarded as special DNA replication in vitro, and the biggest characteristic of PCR is that a trace amount of DNA can be greatly increased.

Currently, single-molecule sequencing technologies include single-molecule real-time synthesis sequencing technology and nanopore sequencing technology. The single-molecule real-time synthesis sequencing technology has the advantage of long sequencing read length, but the manufacturing process of the gene sequencing chip and the sequencing technical route limit the sequencing flux to be incapable of reaching a high level. In another nanopore sequencing technology, the detected electric signal is a very weak signal from nanoampere to picoampere, and the nanopore of the gene sequencing chip is difficult to manufacture, so that the sequencing error rate is high at present, and the level of mass chip production and mass sequencing cannot be achieved.

In summary, it is important to provide an efficient and low-cost sequencing apparatus.

Disclosure of Invention

The invention aims to provide a single-molecule gene sequencer, which aims to solve the problems of low efficiency and high cost in the prior art.

In order to solve the technical problem, the invention provides a single-molecule gene sequencer, which comprises a machine base, wherein a clamping platform, a reagent storage device, a fluid control device and a moving platform are arranged on the machine base;

a gene sequencing chip is arranged on the clamping platform;

the reagent storage device is used for storing a gene sequencing reagent;

the fluid control device is used for pumping the gene sequencing reagent from the reagent storage device to the gene sequencing chip;

the machine base is also provided with a total reflection microscope, the moving platform is arranged below the total reflection microscope, the clamping platform is arranged on the moving platform, and the moving platform is used for driving the clamping platform to move towards and away from the position below the total reflection microscope;

the total reflection microscope comprises a laser emission mechanism, a microscope objective, a filter set, an automatic focusing device, a guide mechanism, a detection camera and a computer;

the laser emission mechanism is used for emitting two lasers with different wavelengths to the filter set;

the filter set comprises a first double-bandpass filter, a second double-bandpass filter and a first dichroic mirror;

the first double-bandpass filter is used for filtering the laser and emitting the filtered laser to the first dichroic mirror;

the first dichroic mirror is used for reflecting the laser to the microscope objective;

the microscope objective is used for focusing the laser on the gene sequencing chip in a state that the incidence angle is larger than the critical angle, so as to excite the sample in the gene sequencing chip to generate fluorescence;

the fluorescence sequentially passes through the microscope objective, the first dichroic mirror and the second double-bandpass filter, and the second double-bandpass filter is used for filtering the fluorescence and then transmitting the filtered fluorescence to the guide mechanism;

the guide mechanism is used for transmitting the fluorescence to the detection camera, and the detection camera is used for carrying out image information acquisition on the fluorescence and sending the image information to the computer so as to enable the computer to measure the gene sequence of the sample in the gene sequencing chip according to the image information;

the automatic focusing device is used for transmitting infrared light to the guide mechanism, the guide mechanism transmits the infrared light to the second double-bandpass optical filter, the infrared light sequentially passes through the second double-bandpass optical filter, the first dichroic mirror and the microscope, then is transmitted to the gene sequencing chip, and returns back to the automatic focusing device according to the original way, so that the automatic focusing device can continuously focus samples in the gene sequencing chip.

Preferably, the second double-bandpass filter is arranged in parallel with and opposite to the mirror surface of the microscope objective, and the first dichroic mirror is arranged between the second double-bandpass filter and the microscope objective in a manner of being inclined by 45 °; the mirror surface of the first dichroic mirror opposite to the microscope objective is also opposite to the first double-bandpass filter, and the included angle between the first dichroic mirror and the opposite surface of the first double-bandpass filter is 45 degrees.

Preferably, the laser emission mechanism includes a first laser emitter, a second dichroic mirror, and a first reflecting mirror;

the first laser emitter is used for emitting laser light with a first wavelength to the second dichroic mirror, so that the laser light with the first wavelength passes through the second dichroic mirror to be reflected to the first double-bandpass filter;

the second laser emitter is configured to emit laser light with a second wavelength to the first mirror, and the first mirror is configured to reflect the laser light with the second wavelength to the second dichroic mirror, so that the second dichroic mirror reflects the laser light with the second wavelength to the first dual-bandpass filter.

Preferably, the emitting end of the first laser emitter is arranged opposite to the first dual-band-pass filter, so that the laser light with the first wavelength can vertically enter the first dual-band-pass filter;

the second dichroic mirror is arranged between the emission end of the first laser emitter and the first double-bandpass filter in a 45-degree inclined mode, the mirror surface of the first reflecting mirror is parallel to and opposite to the mirror surface of the second dichroic mirror, and the mirror surface of the first reflecting mirror is arranged in an inclined mode of 45 degrees relative to the emission end of the second laser emitter.

Preferably, the laser emission mechanism comprises a first laser emitter, a second laser emitter and a second dichroic mirror;

the second laser emitter is used for emitting laser light with a second wavelength to the second dichroic mirror, so that the second dichroic mirror reflects the laser light with the second wavelength to the first double-bandpass filter.

the second dichroic mirror is arranged between the emission end of the first laser emitter and the first double-bandpass filter in a 45-degree inclined mode; and one surface of the second dichroic mirror, which is opposite to the first double-bandpass filter, is also opposite to the emission end of the second laser emitter in an inclined manner of 45 degrees.

Preferably, the guiding mechanism comprises a third dichroic mirror and a second reflecting mirror;

the third dichroic mirror is used for reflecting the infrared light to the second double-bandpass filter and reflecting the infrared light reflected from the gene sequencing chip to the automatic focusing device;

the fluorescent light is reflected through the third dichroic mirror to the second mirror for reflecting the fluorescent light to the detection camera.

Preferably, one surface of the third dichroic mirror is opposite to the second double-bandpass filter and the automatic focusing device, and the third dichroic mirror is arranged in an inclined manner of 45 ° with respect to the second double-bandpass filter and the transceiving end of the automatic focusing device;

the second reflecting mirror is arranged in parallel and opposite to the other surface of the third dichroic mirror, and the second reflecting mirror is obliquely arranged at 45 degrees relative to the collecting end of the detection camera.

Preferably, the guiding mechanism comprises a third dichroic mirror;

the fluorescent light is mirrored through the third dichroic mirror to the detection camera.

Preferably, one surface of the third dichroic mirror is opposite to the second double-bandpass filter and the automatic focusing device, and the other surface of the third dichroic mirror is opposite to the detection camera, and the third dichroic mirror is arranged in an inclined manner of 45 ° with respect to the second double-bandpass filter, the transceiving end of the automatic focusing device, and the acquisition end of the detection camera.

Preferably, the clamping platform comprises a platform base, a temperature control chip and a clamping frame for positioning and fixing the gene sequencing chip;

the upper surface of the platform base is provided with an installation area for installing the gene sequencing chip, the temperature control chip is installed in the installation area, and the gene sequencing chip is installed above the temperature control chip;

the clamping frame is connected with the rotating shaft of the platform base, so that the clamping frame can be turned to and away from the platform base.

Preferably, reagent diversion holes are formed in two sides of the installation area, the lower port of each reagent diversion hole is communicated with the fluid control device, and the upper port of each reagent diversion hole is communicated with the gene sequencing chip.

Preferably, the platform base is provided with a rotating shaft platform on each of two sides of the mounting area;

the clamping frame comprises two clamping edges and a positioning edge, one ends of the two clamping edges are respectively and vertically connected with two ends of the positioning edge, and the other ends of the two clamping edges are respectively connected with the rotating shaft of the rotating shaft table, so that when the clamping frame is turned to the platform base, the clamping frame can surround the periphery of the mounting area.

Preferably, a lock catch is arranged on the positioning edge, a lock platform is arranged on the platform base corresponding to the lock catch, and an unlocking button is arranged on the lock platform;

the lock catch is inserted into the lock platform, so that locking and positioning between the clamping frame and the platform base are realized;

the unlocking button is used for releasing the fixation of the locking platform on the clamping frame.

Preferably, the base bottom is equipped with a plurality of first shock attenuation callus on the sole.

Preferably, the base is further provided with a support, the total reflection microscope and the mobile platform are mounted on the support, the bottom of the support is provided with a plurality of second shock absorption foot pads, and the second shock absorption foot pads are supported on the base.

Preferably, the reagent storage device comprises a refrigeration storage chamber, a refrigeration reagent bottle and an electric lifting mechanism are arranged in the refrigeration storage chamber, the electric lifting mechanism is arranged above the refrigeration reagent bottle, a first puncture needle communicated with the fluid control device is arranged on the electric lifting mechanism, and the electric lifting mechanism is used for driving the first puncture needle to be inserted into and separated from the refrigeration reagent bottle.

Preferably, reagent strorage device still includes the normal atmospheric temperature apotheca, be equipped with normal atmospheric temperature reagent bottle and manual elevating system in the normal atmospheric temperature apotheca, manual elevating system locates the top of normal atmospheric temperature reagent bottle, be equipped with the second pjncture needle on the manual elevating system, manual elevating system is used for driving second pjncture needle inserts and leaves normal atmospheric temperature reagent bottle.

The invention has the following beneficial effects:

the invention adopts a single-molecule fluorescence sequencing technology, is based on a total internal reflection fluorescence microscopic imaging technology, adopts a sequencing principle of sequencing while synthesizing, directly sequences DNA molecular fragments, has no library building process and no PCR, is simple and convenient to operate, reduces the sequencing cost, and is a diagnosis and treatment means very suitable for clinical application.

Drawings

In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic diagram showing the structure of a single-molecule gene sequencer according to a preferred embodiment of the present invention;

FIG. 2 is a schematic structural view of a total reflection microscope of the single molecule gene sequencer according to the preferred embodiment of the present invention;

FIG. 3 is a schematic structural diagram of a second embodiment of a laser emitting mechanism of the single molecule gene sequencer according to the preferred embodiment of the present invention;

FIG. 4 is a schematic structural diagram of a second embodiment of a guide mechanism of a single-molecule gene sequencer according to a preferred embodiment of the present invention;

FIG. 5 is a schematic view of a single molecule gene sequencer using oil immersion according to a preferred embodiment of the present invention;

FIG. 6 is a schematic view showing an open state of a clamping platform of the single molecule gene sequencer according to the preferred embodiment of the present invention when a gene sequencing chip is not mounted thereon;

FIG. 7 is an enlarged schematic view of portion A of FIG. 6;

FIG. 8 is a schematic view showing a closed state of a clamping platform of the single molecule gene sequencer according to the preferred embodiment of the present invention when a gene sequencing chip is not mounted thereon;

FIG. 9 is a schematic diagram showing the opened state of a gene sequencing chip mounted on a clamping platform of the single-molecule gene sequencer according to the preferred embodiment of the present invention;

FIG. 10 is a schematic diagram showing a closed state of a clamping platform of the single molecule gene sequencer according to the preferred embodiment of the present invention after a gene sequencing chip is mounted thereon;

FIG. 11 is a schematic cross-sectional side view of a clamping platform of the single molecule gene sequencer according to the preferred embodiment of the present invention;

FIG. 12 is a schematic diagram showing the structure of a gene sequencing chip of a single-molecule gene sequencer according to a preferred embodiment of the present invention;

FIG. 13 is a schematic view showing a state where the clamping platform of the single molecule gene sequencer is removed from the microscope objective lens according to the preferred embodiment of the present invention;

FIG. 14 is a schematic view showing a state in which a clamping stage of the single molecule gene sequencer is moved toward a microscope objective lens according to the preferred embodiment of the present invention;

FIG. 15 is a schematic diagram showing the construction of a reagent storage device of a clamping platform of the single molecule gene sequencer according to the preferred embodiment of the present invention;

FIG. 16 is a schematic view of the structure of a fluid control apparatus of a single-molecule gene sequencer according to a preferred embodiment of the present invention.

The reference numbers are as follows:

1. a machine base; 11. a support;

2. clamping the platform; 21. a platform base; 211. an installation area; 212. reagent diversion holes; 213. a spindle table; 214. locking the platform; 215. an unlock button; 22. a temperature control chip; 23. clamping the frame; 231. clamping edges; 232. positioning the edge; 233. locking; 234. a boss; 24. a torsion spring;

3. a reagent storage device; 31. a refrigerated storage compartment; 32. refrigerating the reagent bottle; 33. an electric lifting mechanism; 34. a first puncture needle; 35. a normal temperature storage chamber; 36. a normal temperature reagent bottle; 37. a manual lifting mechanism; 38. a second puncture needle;

4. a fluid control device; 41. a multi-way valve; 411. a reagent extraction port; 412. a liquid outlet; 42. a first three-way valve; 421. a liquid suction port; 422. a first diversion port; 423. a second diversion port; 43. a drive assembly; 431. a first syringe pump; 432. a second syringe pump; 433. a second three-way valve; 434. a third three-way valve; 435. a first waste liquid bottle; 436. a second waste bottle;

5. a mobile platform;

6. gene sequencing chip; 61. positioning holes; 62. a first gene sequencing channel; 63. a second gene sequencing channel;

7. a total reflection microscope; 71. a laser emitting mechanism; 711. a first laser transmitter; 712. a second laser transmitter; 713. a second dichroic mirror; 714. a first reflector; 72. a microscope objective; 73. a filter set; 731. a first dual band pass filter; 732. a second dual bandpass filter; 733. a first dichroic mirror; 74. an automatic focusing device; 75. a guide mechanism; 751. a third dichroic mirror; 752. a second reflector; 76. a detection camera; 77. a computer; 78. a cylindrical mirror;

81. a first shock absorbing foot pad; 82. a second shock absorbing foot pad;

9. and (5) oil immersion.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

As can be seen from fig. 1 to 16, the single molecule gene sequencer of the present invention comprises a base 1, wherein the base 1 is provided with a clamping platform 2, a reagent storage device 3, a fluid control device 4 and a moving platform 5; a gene sequencing chip 6 is arranged on the clamping platform 2; the reagent storage device 3 is used for storing a gene sequencing reagent; the fluid control device 4 is used for pumping the gene sequencing reagent from the reagent storage device 3 to the gene sequencing chip 6; the machine base 1 is also provided with a total reflection microscope 7, the moving platform 5 is arranged below the total reflection microscope 7, the clamping platform 2 is arranged on the moving platform 5, and the moving platform 5 is used for driving the clamping platform 2 to move towards and away from the position below the total reflection microscope 7; the total reflection microscope 7 comprises a laser emission mechanism 71, a microscope objective 72, a filter set 73, an automatic focusing device 74, a guide mechanism 75, a detection camera 76 and a computer 77; the laser emitting mechanism 71 is configured to emit two kinds of laser with different wavelengths to the filter set 73; the filter set 73 includes a first dual bandpass filter 731, a second dual bandpass filter 732, and a first dichroic mirror 733; the first dual-bandpass filter 731 is configured to filter the laser light and emit the filtered laser light to the first dichroic mirror 733; the first dichroic mirror 733 is used for reflecting the laser light to the microscope objective 72; the microscope objective 72 is used for focusing the laser on the gene sequencing chip 6 in a state that the incidence angle is larger than the critical angle, so as to excite the sample in the gene sequencing chip 6 to generate fluorescence; the fluorescence sequentially passes through the microscope objective 72, the first dichroic mirror 733 and the second double-bandpass filter 732, and the second double-bandpass filter 732 is configured to filter the fluorescence and emit the filtered fluorescence to the guiding mechanism 75; the guiding mechanism 75 is used for transmitting the fluorescence to the detection camera 76, the detection camera 76 is used for collecting image information of the fluorescence and sending the image information to the computer 77, so that the computer 77 can measure the gene sequence of the sample in the gene sequencing chip 6 according to the image information; the auto-focusing device 74 is configured to emit infrared light to the guiding mechanism 75, the guiding mechanism 75 transfers the infrared light to the second dual-band-pass filter 732, and the infrared light sequentially passes through the second dual-band-pass filter 732, the first dichroic mirror 733 and the microscope objective 72, and then is emitted to the genetic sequencing chip 6, and returns back to the auto-focusing device 74 according to the original path, so that the auto-focusing device 74 can perform continuous focusing on a sample in the genetic sequencing chip 6.

Further, as shown in fig. 2, the second dual bandpass filter 732 is disposed in parallel with and opposite to the mirror surface of the microscope objective lens 72, and the first dichroic mirror 733 is disposed between the second dual bandpass filter 732 and the microscope objective lens 72 with an inclination of 45 °; the mirror surface of the first dichroic mirror 733 opposite to the microscope objective 72 is also opposite to the first dual-bandpass filter 731, and the included angle between the opposing surfaces of the first dichroic mirror 733 and the first dual-bandpass filter 731 is 45 °.

With reference to fig. 2, the microscope objective 72 is now disposed horizontally above; the first dichroic mirror 733 is disposed below the microscope objective 72, and an upper surface of the first dichroic mirror 733 is disposed at an inclination of 45 ° with respect to the microscope objective 72; the second double bandpass filter 732 is disposed below the first dichroic mirror 733, the second double bandpass filter 732 is parallel to the microscope objective lens 72, and the lower surface of the first dichroic mirror 733 is inclined at 45 ° with respect to the second double bandpass filter 732; the first dual-bandpass filter 731 is disposed on the left side of the first dichroic mirror 733, and an included angle between the first dual-bandpass filter 731 and the upper surface of the first dichroic mirror 733 is 45 °, that is, the first dual-bandpass filter 731 is perpendicular to the microscope objective 72 and the second dual-bandpass filter 732. By this arrangement, it is ensured that the laser light passing through the first dual-band pass filter 731 perpendicularly reflects onto the microscope objective 72, so as to ensure that the microscope objective 72 can focus the laser light on the gene sequencing chip 6 at an incident angle larger than the critical angle, thereby exciting the sample in the gene sequencing chip 6 to generate fluorescence.

Further, as shown in fig. 2, the laser emission mechanism 71 includes a first laser emitter 711, a second laser emitter 712, a second dichroic mirror 713, and a first reflecting mirror 714; the first laser emitter 711 is configured to emit laser light with a first wavelength to the second dichroic mirror 713, so that the laser light with the first wavelength passes through the second dichroic mirror 713 and is incident on the first dual-bandpass filter 731; the second laser emitter 712 is configured to emit laser light with a second wavelength to the first reflecting mirror 714, and the first reflecting mirror 714 is configured to reflect the laser light with the second wavelength to the second dichroic mirror 713, so that the second dichroic mirror 713 reflects the laser light with the second wavelength to the first dual-bandpass filter 731.

In actual production, the laser light emitted by the first laser emitter 711 and the second laser emitter 712 can be directly emitted to the second dichroic mirror 713, and then the two laser lights are transmitted to the first double-bandpass filter 731 by the second dichroic mirror 713; however, the first laser emitter 711 and the second laser emitter 712 are respectively arranged in the horizontal direction and the longitudinal direction by the scheme, so that the arrangement of the whole machine is not uniform, and the whole machine occupies a space; this problem can be solved by adding the first reflecting mirror 714, because even if the first laser transmitter 711 and the second laser transmitter 712 are arranged in the same manner, the two lasers can be emitted to the first dual bandpass filter 731 by the cooperation of the second dichroic mirror 713 and the first reflecting mirror 714.

Further, as shown in fig. 2, the emitting end of the first laser emitter 711 is disposed opposite to the first dual-band-pass filter 731, so that the laser light with the first wavelength can vertically enter the first dual-band-pass filter 731; the second dichroic mirror 713 is disposed between the emission end of the first laser light emitter 711 and the first double bandpass filter 731 with an inclination of 45 °, the mirror surface of the first reflecting mirror 714 is parallel-opposed to the mirror surface of the second dichroic mirror 713, and the mirror surface of the first reflecting mirror 714 is arranged with an inclination of 45 ° with respect to the emission end of the second laser light emitter 712.

Referring to FIG. 2, the first laser emitter 711 is positioned on the left side and the first dual-bandpass filter 731 is positioned on the right side of the first laser emitter 711; the second dichroic mirror 713 is provided between the first laser emitter 711 and the first dual band pass filter 731, and the upper surface of the second dichroic mirror 713 is arranged to be inclined at 45 ° with respect to the emission end of the first laser emitter 711 and the lower surface is arranged to be inclined at 45 ° with respect to the first dual band pass filter 731; the first reflecting mirror 714 is arranged below the second dichroic mirror 713, and the first reflecting mirror 714 is parallel to and opposite to the lower surface of the second dichroic mirror 713; the second laser transmitter 712 is disposed below the first laser transmitter 711, i.e. disposed on the left side of the first reflecting mirror 714, and the mirror surface of the first reflecting mirror 714 is arranged at an inclination of 45 ° with respect to the transmitting end of the second laser transmitter 712. With this arrangement, the first laser transmitter 711 can emit laser light of a first wavelength that passes through the first dual-bandpass filter 731 vertically, and the second laser transmitter 712 can emit laser light of a second medium wavelength that passes through the first dual-bandpass filter 731 vertically after two reflections.

Further, in the second embodiment of the laser emission mechanism shown in fig. 3, the laser emission mechanism 71 includes a first laser emitter 711, a second laser emitter 712, and a second dichroic mirror 713; the first laser emitter 711 is configured to emit laser light with a first wavelength to the second dichroic mirror 713, so that the laser light with the first wavelength passes through the second dichroic mirror 713 and is incident on the first dual-bandpass filter 731; the second laser emitter 712 is configured to emit laser light with a second wavelength to the second dichroic mirror 713, so that the second dichroic mirror 713 reflects the laser light with the second wavelength to the first dual-bandpass filter 731.

As mentioned above, this implementation would increase the footprint of the device, but would save costs by eliminating the first mirror 714, and is an alternative to actual production.

Further, as shown in fig. 3, the emitting end of the first laser emitter 711 is disposed opposite to the first dual-band-pass filter 731, so that the laser light with the first wavelength can vertically enter the first dual-band-pass filter 731; the second dichroic mirror 713 is disposed between the emission end of the first laser light emitter 711 and the first double bandpass filter 731 with an inclination of 45 °; the surface of the second dichroic mirror 713 opposite to the first dual bandpass filter 731 is also inclined at 45 ° to the emission end of the second laser emitter 712.

Referring to FIG. 3, the first laser emitter 711 is positioned on the left side and the first dual-bandpass filter 731 is positioned on the right side of the first laser emitter 711; the second dichroic mirror 713 is provided between the first laser emitter 711 and the first dual band pass filter 731, and the upper surface of the second dichroic mirror 713 is arranged to be inclined at 45 ° with respect to the emission end of the first laser emitter 711 and the lower surface is arranged to be inclined at 45 ° with respect to the first dual band pass filter 731; the second laser light emitter 712 is disposed below the second dichroic mirror 713, an emitting end of the second laser light emitter 712 is opposed to a lower surface of the second dichroic mirror 713, and the lower surface of the second dichroic mirror 713 is arranged at an inclination of 45 ° with respect to the emitting end of the second laser light emitter 712. With this arrangement, the first laser transmitter 711 can emit laser light of a first wavelength that passes through the first dual-bandpass filter 731 vertically, and the second laser transmitter 712 can emit laser light of a second intermediate wavelength that passes through the first dual-bandpass filter 731 vertically after a single reflection.

In both schemes, the laser with the first wavelength is preferably 527-. Of course, the laser with the first wavelength may be 635-645nm, and the laser with the second wavelength may be 527-537nm, in which case, it is only necessary to select a suitable second dichroic mirror 713, so that the second dichroic mirror 713 still maintains the characteristics that the laser with the first wavelength can penetrate through and the laser with the second wavelength can only reflect.

Further, in the first embodiment of the guide mechanism shown in fig. 2, the guide mechanism 75 includes a third dichroic mirror 751 and a second reflecting mirror 752; the third dichroic mirror 751 is used for reflecting the infrared light to the second double-bandpass filter 732 and reflecting the infrared light reflected from the gene sequencing chip 6 to the automatic focusing device 74; the fluorescence passes through the third dichroic mirror 751 to the second mirror 752, and the second mirror 752 is used to reflect the fluorescence to the detection camera 76.

In actual production, the fluorescence may be received directly with the detection camera 76; however, the solution makes the automatic focusing device 74 and the detection camera 76 respectively arranged in the horizontal and vertical directions, so that the arrangement of the whole machine is not uniform and the whole machine occupies space; the addition of the second mirror 752 can solve this problem because the infrared light can be emitted to the autofocus device 74 and the fluorescence can be emitted to the detection camera 76 by the cooperation of the third dichroic mirror 751 and the second mirror 752 even if the autofocus device 74 and the detection camera 76 are arranged in the same manner.

Further, as shown in fig. 2, one surface of the third dichroic mirror 751 is opposite to the second dual band pass filter 732 and the autofocus device 74, and the third dichroic mirror 751 is arranged to be inclined at 45 ° with respect to the transceiving ends of the second dual band pass filter 732 and the autofocus device 74; the second reflecting mirror 752 is disposed in parallel to and opposed to the other surface of the third dichroic mirror 751, and the second reflecting mirror 752 is disposed obliquely at 45 ° with respect to the collecting end of the detection camera 76.

With reference to fig. 2, in this case, the third dichroic mirror 751 is disposed below the second dual-band pass filter 732, the upper surface of the third dichroic mirror 751 is simultaneously opposite to the second dual-band pass filter 732 and the transceiving end of the autofocus device 74, and the upper surface of the third dichroic mirror 751 is disposed at an inclination of 45 ° with respect to both the second dual-band pass filter 732 and the transceiving end of the autofocus device 74; the second reflecting mirror 752 is arranged below the third dichroic mirror 751, and the upper surface of the second reflecting mirror 752 is parallel to and opposite to the lower surface of the third dichroic mirror 751; the detection camera 76 is arranged at the right side of the second reflector 752, and the upper surface of the second reflector 752 is arranged at an inclination of 45 ° with respect to the collecting end of the detection camera 76. As can be seen from fig. 2, a tube mirror 78 may be additionally disposed between the second reflecting mirror 752 and the detection camera 76, and the tube mirror 78 is used for converging light rays so as to image the DNA image in the gene sequencing chip 6 onto the detection camera 76.

Further, in the second embodiment of the guiding mechanism as shown in fig. 4, the guiding mechanism 75 includes a third dichroic mirror 751; the third dichroic mirror 751 is used for reflecting the infrared light to the second double-bandpass filter 732 and reflecting the infrared light reflected from the gene sequencing chip 6 to the automatic focusing device 74; the fluorescence passes through the third dichroic mirror 751 to the detection camera 76.

As mentioned above, this implementation would increase the space occupied by the device, but it would also be an alternative to actual production, since the second mirror 752 is omitted, thereby saving costs.

Further, as shown in fig. 4, one surface of the third dichroic mirror 751 is opposite to the second dual band pass filter 732 and the auto focusing device 74, and the other surface is opposite to the detection camera 76, and the third dichroic mirror 751 is arranged to be inclined at 45 ° with respect to the second dual band pass filter 732, the transmitting/receiving end of the auto focusing device 74, and the collecting end of the detection camera 76.

With reference to fig. 4, in this case, the third dichroic mirror 751 is disposed below the second dual-band pass filter 732, the upper surface of the third dichroic mirror 751 is simultaneously opposite to the second dual-band pass filter 732 and the transceiving end of the autofocus device 74, and the upper surface of the third dichroic mirror 751 is disposed at an inclination of 45 ° with respect to both the second dual-band pass filter 732 and the transceiving end of the autofocus device 74; the detection camera 76 is provided below the third dichroic mirror 751, the collection end of the detection camera 76 is opposed to the lower surface of the third dichroic mirror 751, and the lower surface of the third dichroic mirror 751 is arranged at an inclination of 45 ° with respect to the collection end of the detection camera 76. As can be seen from fig. 4, a tube mirror 78 may be additionally disposed between the third dichroic mirror 751 and the detection camera 76, and the tube mirror 78 is used for light convergence, so that the DNA image in the gene sequencing chip 6 is imaged on the detection camera 76.

It should be noted that a dichroic mirror, also called a dichroic mirror, is commonly used in laser technology, and is characterized in that light with a certain wavelength is almost completely transmitted, light with other wavelengths is almost completely reflected, and what light can be reflected and what light can pass through can be made and selected according to needs. For example, the first dichroic mirror 733 transmits fluorescence and infrared light, but reflects laser light completely. In addition, the double-band-pass filter is one of the optical filters, and can separate monochromatic light of certain two wave bands; as described above, the first dual band pass filter 731 can filter only two types of laser light emitted from the laser emitting mechanism 71.

As shown in FIG. 5, oil immersion 9 can be added between the gene sequencing chip 6 and the microscope objective lens 72 when sampling, because the surface where total internal reflection occurs is located at the interface between the gene sequencing chip 6 and the DNA water environment. Two conditions must be met for total reflection to occur: (1) from optically dense media to optically sparse media; (2) the angle of incidence a is greater than the critical angle. The numerical aperture NA (NA-n sin theta) of the microscope objective 72 can be effectively increased by adding the immersion oil 9, and then the excitation light emitted from the off-axis can be emitted at a larger angle, so that the requirement of being larger than the critical angle is met at the interface between the gene sequencing chip 6 and the DNA water environment.

In addition, by definition of optical reflectivityWherein n1 and n2 are optical refractive indexes of media on two sides of the interface. It will be appreciated that there will also be some optical reflections at the interfaces where there is a refractive index difference, which will attenuate the energy of the incident light, and the light reflected back is detected by the detection camera 76 to form a background noise signal. The addition of the immersion oil 9 between the microscope objective 72 and the gene sequencing chip 6 reduces the primary optical reflection, thereby reducing the background noise to some extent.

Further, as shown in fig. 6 to 11, the clamping platform 2 includes a platform base 21, a temperature control chip 22 and a clamping frame 23 for positioning and fixing the gene sequencing chip 6; the upper surface of the platform base 21 is provided with an installation region 211 for installing the gene sequencing chip 6, the temperature control chip 22 is installed in the installation region 211, and the gene sequencing chip 6 is installed above the temperature control chip 22; the clamping frame 23 is rotatably coupled to the platform base 21 so that the clamping frame 23 can be turned toward and away from the platform base 21.

When the clamping frame 23 is turned away from the platform base 21, the gene sequencing chip 6 can be installed in the installation region 211, or the gene sequencing chip 6 can be removed from the installation region 211; after the gene sequencing chip 6 is installed in the installation region 211, the clamping frame 23 can be turned over to the platform base 21, so that the gene sequencing chip 6 can be clamped and fixed firmly; in addition, the temperature control chip 22 is used for controlling the reaction temperature in the gene sequencing chip 6, the temperature control is accurate, and the regulation is convenient, which can not be realized by adopting a heating wire to heat in the prior art.

Furthermore, as shown in fig. 6, reagent guiding holes 212 are formed on both sides of the mounting region 211, and a lower port of each reagent guiding hole 212 is connected to the fluid control device 4, and an upper port thereof is connected to the gene sequencing chip 6.

Further, as shown in fig. 6, the platform base 21 is provided with a pivot platform 213 on each side of the mounting region 211; the clamping frame 23 comprises two clamping edges 231 and a positioning edge 232, one ends of the two clamping edges 231 are respectively and vertically connected with two ends of the positioning edge 232, and the other ends of the two clamping edges 231 are respectively connected with the rotating shaft of the rotating shaft table 213, so that the clamping frame 23 can surround the periphery of the mounting area 211 when the clamping frame 23 is turned over to the platform base 21.

Furthermore, as shown in fig. 6, a lock 233 is disposed on the positioning edge 232, a lock platform 214 is disposed on the platform base 21 at a position corresponding to the lock 233, and an unlocking button 215 is disposed on the lock platform 214; the lock 233 is used for being inserted into the lock platform 214, so as to realize the locking and positioning between the clamping frame 23 and the platform base 21; the unlock button 215 is used to release the lock stage 214 from fixing the clip frame 23.

Further, as shown in fig. 6 and 9, the clamping edge 231 can be provided with a boss 234 facing the surface of the platform base 21, the two sides of the gene sequencing chip 6 are provided with positioning holes 61, and the boss 234 is used for positioning the gene sequencing chip 6 by being embedded into the positioning holes 61.

Further, as shown in fig. 6 and 7, a torsion spring 24 is disposed at a joint of the clamping edge 231 and the pivot base 213, and one end of the torsion spring 24 abuts against the clamping edge 231 and the other end abuts against the pivot base 213, so that the clamping frame 23 is maintained in the flipped open state.

As shown in fig. 6, in the normal state, since the torsion spring 24 continuously applies force to the clamping edge 231, the clamping frame 23 will be kept in the opened state, and at this time, if the clamping frame 23 is turned to the platform base 21, the latch 233 will be inserted into the latch 214, so that the clamping frame 23 can surround the periphery of the mounting area 211 and be kept fixed in this state, as shown in fig. 8 in particular; when the unlock button 215 is pressed, the lock platform 214 unlocks the lock 233, so that the torsion spring 24 pushes the clamping frame 23 away from the platform base 21 again, and the gene sequencing chip 6 can be installed in the installation region 211, wherein the installed state is as shown in fig. 9; finally, the clamping frame 23 is turned over to the platform base 21, so that the latch 233 is embedded into the locking platform 214 to fix the gene sequencing chip 6, wherein the boss 234 is embedded into the positioning hole 61, thereby further enhancing the fixation of the gene sequencing chip, as shown in fig. 10 and 11, at this time, the gene sequencing chip 6, the temperature control chip 22, the platform base 21 and the mobile platform 5 are sequentially arranged from top to bottom.

In addition, as shown in fig. 12, a first gene sequencing channel 62 and a second gene sequencing channel 63 are arranged on the gene sequencing chip 6, and when the gene sequencing chip 6 is installed in the installation region 211, the first gene sequencing channel 62 and the second gene sequencing channel 63 are communicated with the reagent flow-through hole.

Further, as shown in fig. 13 and 14, the moving platform 5 can drive the clamping platform 2 to move towards and away from the microscope objective 72, and in particular, after the clamping platform 2 moves towards the microscope objective 72, the microscope objective 72 is always kept above the gene sequencing chip 6 due to the blocking of the clamping frame 23, thereby ensuring that the microscope objective 72 is always aligned with the gene sequencing chip.

Further, as shown in fig. 1, a plurality of first shock absorbing foot pads 81 are disposed at the bottom of the machine base 1.

Furthermore, as shown in fig. 1, a support 11 is further disposed on the base 1, the total reflection microscope 7 and the mobile platform 5 are mounted on the support 11, and a plurality of second shock-absorbing foot pads 82 are disposed at the bottom of the support 11 and supported on the base.

The single-molecule fluorescence detection system is very sensitive to external vibration, and a two-stage damping structure is designed for the instrument in order to avoid the optical image shake caused by the external vibration. That is, the first shock-absorbing foot pad 81 is isolated from the outside and the whole shock of the instrument, and the second shock-absorbing foot pad 82 is isolated from the shock between the internal shock of the instrument and the total reflection microscope, and simultaneously eliminates the residual shock of the first-level shock absorption.

Furthermore, as shown in fig. 15, the reagent storage device 3 includes a refrigerated storage chamber 31, a refrigerated reagent bottle 32 and an electric lifting mechanism 33 are disposed in the refrigerated storage chamber 31, the electric lifting mechanism 33 is disposed above the refrigerated reagent bottle 32, a first puncture needle 34 communicated with the fluid control device 4 is disposed on the electric lifting mechanism 33, and the electric lifting mechanism 33 is configured to drive the first puncture needle 34 to be inserted into and separated from the refrigerated reagent bottle 32.

Furthermore, as shown in fig. 15, the reagent storage device 3 further includes a normal temperature storage chamber 35, a normal temperature reagent bottle 36 and a manual lifting mechanism 37 are arranged in the normal temperature storage chamber 35, the manual lifting mechanism 37 is arranged above the normal temperature reagent bottle 36, a second puncture needle 38 is arranged on the manual lifting mechanism 37, and the manual lifting mechanism 37 is used for driving the second puncture needle 38 to be inserted into and separated from the normal temperature reagent bottle 36.

Obviously, for reagents requiring refrigeration, the fluid control device 4 can directly extract the reagents through the first puncture needle 34, while for reagents stored at normal temperature, the user can take the reagents through the manual lifting mechanism 37 and the second puncture needle 38, thereby improving the flexibility of the device.

Further, as shown in fig. 12 and 16, the fluid control device 4 includes a multi-way valve 41, a first three-way valve 42 and a driving assembly 43, the multi-way valve 41 includes a plurality of reagent extraction ports 411 and a liquid outlet 412, the reagent extraction port 411 is connected to the first puncture needle 34, the liquid outlet 412 can be selectively connected to any one of the reagent extraction ports 411, the first three-way valve 42 includes a liquid suction port 421, a first shunt port 422 and a second shunt port 423, the liquid suction port 421 is connected to the first shunt port 422 or the second shunt port 423, the liquid suction port 421 is connected to the liquid outlet 412 through a pipe, the gene sequencing chip 6 includes a first gene sequencing channel 62 and a second gene sequencing channel 63, the first gene sequencing channel 62 and the second gene sequencing channel 63 are respectively connected to the first shunt port 422 and the second shunt port 423 through a pipe, the drive assembly 43 includes a first syringe pump 431 and a second syringe pump 432, the first syringe pump 431 and the second syringe pump 432 being respectively piped to the first gene sequencing channel 62 and the second gene sequencing channel 63.

Wherein, when the first syringe pump 431 provides negative pressure to the first gene sequencing channel 62 to make the first gene sequencing channel 62 obtain the gene sequencing reagent for gene sequencing reaction, the second syringe pump 432 stops providing negative pressure to the second gene sequencing channel 63 to make the second gene sequencing channel 63 perform fluorescence image acquisition.

Further, as shown in fig. 12 and 16, the driving assembly 43 further includes a second three-way valve 433, a third three-way valve 434, a first waste liquid bottle 435 and a second waste liquid bottle 436, the second three-way valve 433 is connected to the first syringe pump 431 and the first gene sequencing channel 62 through a pipe, and the first waste liquid bottle 435 is also connected to the first three-way valve 434, the third three-way valve 434 is connected to the second syringe pump 432 and the second gene sequencing channel 63 through a pipe, and the second waste liquid bottle 436 is also connected to the second three-way valve 434 through a pipe.

According to the scheme, the first gene sequencing channel 62 and the second gene sequencing channel 63 are arranged in the gene sequencing chip 6, so that the gene sequencing reagent can automatically flow into the first gene sequencing channel 62 and the second gene sequencing channel 63 to perform reaction and fluorescent image acquisition, and the second gene sequencing channel 63 can perform fluorescent image acquisition when the first gene sequencing channel 62 performs fluorescent sequencing reaction, so that the gene sequencing time is effectively reduced, namely, the gene sequencing efficiency is effectively improved, the gene sequencing fluid control device 4 reduces the gene sequencing cost and improves the gene sequencing efficiency.

The sequencing reaction and image acquisition of the present invention are both performed in a 37 degree environment, i.e., both require the same temperature. The sequencing reaction and the image acquisition of the second-generation sequencing technology are required to be carried out at different temperatures, namely the sequencing reaction and the image acquisition cannot be carried out simultaneously, so that the working efficiency is greatly reduced. In the scheme, sequencing reaction and image acquisition among different units can be simultaneously carried out on the same gene sequencing chip 6 on the same temperature control platform. The parallel processing of a single camera, a single sequencing chip and multiple channels is realized, and the efficiency can be doubled.

The working process of the invention is roughly as follows:

1. placing the sample in a gene sequencing chip 6 to prepare a gene sequencing chip 6;

2. installing the gene sequencing chip 6 on the clamping platform 2, and communicating the gene sequencing chip 6 with the fluid control device 4;

3. the fluid control device sends the gene sequencing reagent to the first gene sequencing channel 62, and the temperature control chip 22 controls the sample to react at the specified temperature;

4. after the reaction in the first gene sequencing channel 62 is finished, the mobile platform 5 moves the gene sequencing chip 6 to the position under the total reflection microscope 7 for image information acquisition; at the same time, the fluid control device 4 sends the gene sequencing reagent to the second gene sequencing channel 63, and the temperature control chip 22 controls the sample to react at the specified temperature.

The gene sequencing efficiency can be improved as much as possible by the mode, and great help is provided for clinical diagnosis application.

The foregoing is directed to the preferred embodiment of the present invention, and it is understood that various changes and modifications may be made by one skilled in the art without departing from the spirit of the invention, and it is intended that such changes and modifications be considered as within the scope of the invention.

Claims

1. A single molecule gene sequencer comprises a machine base, wherein a clamping platform, a reagent storage device, a fluid control device and a mobile platform are arranged on the machine base;

a gene sequencing chip is arranged on the clamping platform;

the reagent storage device is used for storing a gene sequencing reagent;

the device is characterized in that a total reflection microscope is further arranged on the machine base, the moving platform is arranged below the total reflection microscope, the clamping platform is arranged on the moving platform, and the moving platform is used for driving the gene sequencing chip to move towards and away from the position below the total reflection microscope;

2. The single-molecule gene sequencer according to claim 1, wherein the second double-bandpass filter is disposed opposite to the mirror surface of the microscope objective lens in parallel, and the first dichroic mirror is disposed between the second double-bandpass filter and the microscope objective lens with an inclination of 45 °; the mirror surface of the first dichroic mirror opposite to the microscope objective is also opposite to the first double-bandpass filter, and the included angle between the first dichroic mirror and the opposite surface of the first double-bandpass filter is 45 degrees.

3. The single molecule gene sequencer according to claim 2, wherein the laser emitting mechanism comprises a first laser emitter, a second dichroic mirror, and a first reflecting mirror;

4. The single molecule gene sequencer according to claim 3,

the emission end of the first laser emitter is arranged opposite to the first double-bandpass filter, so that the laser with the first wavelength can vertically enter the first double-bandpass filter;

5. The single molecule gene sequencer according to claim 2,

the laser emission mechanism comprises a first laser emitter, a second laser emitter and a second dichroic mirror;

6. The single molecule gene sequencer according to claim 5,

7. The single molecule gene sequencer according to claim 2,

the guiding mechanism comprises a third dichroic mirror and a second reflecting mirror;

8. The single molecule gene sequencer according to claim 7,

one surface of the third dichroic mirror is opposite to the second double-bandpass filter and the automatic focusing device, and the third dichroic mirror is obliquely arranged at an angle of 45 degrees relative to the second double-bandpass filter and the transceiving end of the automatic focusing device;

9. The single molecule gene sequencer according to claim 2,

the guiding mechanism comprises a third dichroic mirror;

10. The single molecule gene sequencer according to claim 9,

one surface of the third dichroic mirror is opposite to the second double-bandpass filter and the automatic focusing device, and the other surface of the third dichroic mirror is opposite to the detection camera, and the third dichroic mirror is obliquely arranged at an angle of 45 degrees relative to the second double-bandpass filter, the transceiving end of the automatic focusing device and the acquisition end of the detection camera.

11. The single molecule gene sequencer according to claim 1,

the clamping platform comprises a platform base, a temperature control chip and a clamping frame for positioning and fixing the gene sequencing chip;

12. The single-molecule gene sequencer according to claim 11, wherein reagent guiding holes are formed in both sides of the mounting region, and a lower port of each reagent guiding hole is connected to the fluid control device and an upper port of each reagent guiding hole is connected to the gene sequencing chip.

13. The single molecule gene sequencer according to claim 11,

the platform base is provided with a rotating shaft platform on each of two sides of the mounting area;

14. The single molecule gene sequencer according to claim 13,

a lock catch is arranged on the positioning edge, a lock platform is arranged on the platform base at a position corresponding to the lock catch, and an unlocking button is arranged on the lock platform;

15. The single molecule gene sequencer of claim 1, wherein a plurality of first shock absorbing foot pads are disposed on a bottom of the base.

16. The single-molecule gene sequencer according to claim 15, wherein a support is further disposed on the base, the total reflection microscope and the movable platform are mounted on the support, and a plurality of second shock-absorbing foot pads are disposed at the bottom of the support and supported on the base.

17. The single-molecule gene sequencer according to claim 1, wherein the reagent storage device comprises a refrigerated storage chamber, a refrigerated reagent bottle and an electric lifting mechanism are arranged in the refrigerated storage chamber, the electric lifting mechanism is arranged above the refrigerated reagent bottle, a first puncture needle communicated with the fluid control device is arranged on the electric lifting mechanism, and the electric lifting mechanism is used for driving the first puncture needle to be inserted into and separated from the refrigerated reagent bottle.

18. The single molecule gene sequencer according to claim 17, wherein the reagent storage device further comprises a room temperature storage chamber, wherein a room temperature reagent bottle and a manual lifting mechanism are arranged in the room temperature storage chamber, the manual lifting mechanism is arranged above the room temperature reagent bottle, a second puncture needle is arranged on the manual lifting mechanism, and the manual lifting mechanism is used for driving the second puncture needle to be inserted into and separated from the room temperature reagent bottle.