CN112630148A - Integrated liquid drop digital PCR detection platform - Google Patents
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- CN112630148A CN112630148A CN202011483275.7A CN202011483275A CN112630148A CN 112630148 A CN112630148 A CN 112630148A CN 202011483275 A CN202011483275 A CN 202011483275A CN 112630148 A CN112630148 A CN 112630148A
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- 239000007788 liquid Substances 0.000 title claims abstract description 77
- 238000001514 detection method Methods 0.000 title claims abstract description 47
- 238000007847 digital PCR Methods 0.000 title abstract description 14
- 238000010438 heat treatment Methods 0.000 claims abstract description 20
- 230000003287 optical effect Effects 0.000 claims abstract description 14
- 238000000799 fluorescence microscopy Methods 0.000 claims abstract description 13
- 239000002699 waste material Substances 0.000 claims description 23
- 230000005540 biological transmission Effects 0.000 claims description 18
- 238000011304 droplet digital PCR Methods 0.000 claims description 10
- 230000005284 excitation Effects 0.000 claims description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 238000003384 imaging method Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 abstract description 13
- 108020004707 nucleic acids Proteins 0.000 abstract description 9
- 150000007523 nucleic acids Chemical class 0.000 abstract description 9
- 102000039446 nucleic acids Human genes 0.000 abstract description 9
- 230000003139 buffering effect Effects 0.000 abstract description 3
- 230000003321 amplification Effects 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 230000000149 penetrating effect Effects 0.000 description 6
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000003793 prenatal diagnosis Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention relates to the technical field of medical instruments, in particular to an integrated liquid drop digital PCR detection platform, which comprises a rack, a liquid drop generation part, a liquid drop collection part, a chip base, a driving device, a heating device and a fluorescence imaging optical reading device, wherein the liquid drop generation part is arranged on the rack; in the invention, by arranging a liquid drop generating part and generating micro liquid drops by adopting a flow focusing method, uniform and stable liquid drops are finally obtained; according to the invention, the liquid drops are collected by setting a multi-channel collection method, so that the liquid drops have speed buffering when entering the collection cavity, and bubbles at the corner of the inlet of the collection cavity are effectively prevented; meanwhile, a plurality of supporting columns are arranged in the collecting cavity, so that the collecting cavity is prevented from collapsing, and the liquid drops can be arranged more closely in the collecting cavity. Therefore, the invention provides an integrated liquid drop digital PCR detection platform, which realizes high-integration automation of nucleic acid detection, effectively improves detection efficiency, and also improves detection precision and quality.
Description
Technical Field
The invention relates to the technical field of medical instruments, in particular to an integrated liquid drop digital PCR detection platform.
Background
The digital PCR technology is used as a new generation of nucleic acid molecule absolute quantification technology, and takes Polymerase Chain Reaction (PCR) as a biological basis, nucleic acid templates are diluted and distributed into a large number of independent Reaction units, so that only a single template molecule exists in each Reaction unit, and then PCR amplification Reaction is carried out; and after the amplification is finished, the fluorescence signal of each reaction unit is subjected to statistical analysis, and the DNA amplification amount is quantitatively analyzed. With the technology becoming mature, the digital PCR technology plays an increasingly important role in the medical and biotechnological fields of cancer detection, transgene analysis, organ transplantation, prenatal diagnosis and the like. However, the problems of low integrated automation degree, low detection precision, long detection time and the like of the digital PCR detection equipment in China are urgently solved, so that the invention provides an integrated liquid drop digital PCR detection platform.
Disclosure of Invention
The invention aims to provide an integrated liquid drop digital PCR detection platform to solve the problems in the prior art.
In order to solve the technical problems, the technical scheme provided by the invention is as follows: an integrated liquid drop digital PCR detection platform is provided, which comprises a frame, a liquid drop generating part, a liquid drop collecting part, a chip base, a driving device, a heating device and a fluorescence imaging optical reading device; the liquid drop generating part and the liquid drop collecting part form a chip structure, the chip structure is fixedly arranged on a chip base, the chip base is arranged on a rack and is in sliding connection with the rack through a driving device, the heating device is fixedly arranged on the rack and is arranged at the bottom end of the chip base, and the fluorescence imaging optical reading device is fixedly arranged on the rack and is arranged at the top end of the liquid drop collecting part.
On the basis of the technical scheme, the liquid drop generating part comprises an oil hole, a reagent hole, a continuous phase channel, a disperse phase channel and a liquid drop outlet pipeline, the oil hole is communicated with the continuous phase channel, the reagent hole is communicated with the disperse phase channel, the continuous phase channel is communicated with the disperse phase channel and forms a cross structure, and the liquid drop outlet pipeline is communicated with the cross structure.
On the basis of the technical scheme, the liquid drop collecting part comprises a collecting pipeline, a collecting cavity and a supporting column, one end of the collecting pipeline is in through connection with the liquid drop outlet pipeline, the other end of the collecting pipeline is in through connection with the collecting cavity, the supporting column is provided with a plurality of liquid drops which are fixedly arranged in the collecting cavity, and an outlet of the collecting cavity is formed in one side of the collecting cavity.
On the basis of the technical scheme, the driving device comprises a pulley shaft, a crank, a connecting rod and a transmission mechanism, the pulley shaft is driven by the transmission mechanism to rotate, one end of the crank is fixedly connected with the pulley shaft, the other end of the crank is rotatably connected with the connecting rod, and the connecting rod is hinged to one side of the chip base.
On the basis of the technical scheme, the transmission mechanism comprises a stepping motor, a small belt wheel, a large belt wheel and a belt, the stepping motor is fixedly arranged on the rack, the small belt wheel is fixedly connected with an output shaft of the stepping motor, the large belt wheel is in transmission connection with the small belt wheel through the belt, and the large belt wheel is fixedly connected with a belt wheel shaft and is coaxially arranged.
On the basis of the technical scheme, the heating device comprises a heat source, a heater and a heat transfer plate, the heat source is fixedly arranged on the rack, the heater is fixedly arranged at the top end of the heat source and is electrically connected with the heat source, one side of the heat transfer plate is arranged at the top end of the heater, and the other side of the heat transfer plate is arranged at the bottom end of the collecting cavity.
On the basis of the technical scheme, the fluorescence imaging optical reading device comprises a light source, an excitation filter, a dichroic mirror, an emission filter, an imaging objective lens, a steering reflector and a CCD camera; the excitation filter is arranged at the rear end of the light source, a shading shell is fixedly arranged on one side of the excitation filter, a dichroic mirror, an emission filter, an imaging objective and a steering reflector are sequentially arranged in the shading shell along the direction of fluorescence transmission, and the CCD camera is arranged on the same horizontal plane on one side of the steering reflector and at the center of the steering reflector.
On the basis of the technical scheme, the waste liquid collecting device is fixedly arranged on the frame and comprises a waste liquid accommodating cavity and a waste liquid outlet, the waste liquid accommodating cavity is connected with the outlet of the collecting cavity, and the waste liquid outlet is formed in the side wall of the waste liquid accommodating cavity.
On the basis of the technical scheme, the top end of the rack is fixedly provided with a touch sensor, and the touch sensor is electrically connected with the chip base and the stepping motor.
The technical scheme provided by the invention has the beneficial effects that:
1. in the invention, a droplet generating part is arranged, and micro droplets are generated by adopting a flow focusing method; in the process of generating the liquid drops, the uniformity of the liquid drops is realized by controlling the flow velocity of the continuous phase and the flow velocity of the disperse phase, the generated diameter of the liquid drops is directly determined by adjusting the flow velocity of the continuous phase and the width of each channel, and the uniform and stable liquid drops are finally obtained.
2. According to the invention, the liquid drops are collected by setting a multi-channel collection method, so that the liquid drops have speed buffering when entering the collection cavity, and bubbles at the corner of the inlet of the collection cavity are effectively prevented; meanwhile, a plurality of supporting columns are arranged in the collecting cavity, so that the problem of collapse of the collecting cavity can be avoided, liquid drops can be arranged in the collecting cavity more closely, and adverse effects caused by bubbles and the like during subsequent heating amplification are reduced.
3. According to the invention, the driving device is arranged, so that the chip base can be driven to slide on the rack, and the movement position of the chip base is sensed by matching with the touch sensor, so that the automatic operation of the PCR detection platform in the nucleic acid detection process can be realized, the detection precision is effectively improved, and the detection efficiency is improved.
4. In the invention, the chip structure is heated and insulated by arranging the heating device, and more preferably, a double-spiral annular heater is selected from the heating device; the temperature requirement of the chip structure can be met, and the temperature difference of the chip during temperature rise can be ensured to be small, so that effective amplification of nucleic acid molecules in the whole cyclic heating process is guaranteed, and the detection precision is improved.
Therefore, the invention provides an integrated liquid drop digital PCR detection platform, which realizes high-integration automation of nucleic acid detection, effectively improves detection efficiency, and also improves detection precision and quality.
Drawings
FIG. 1 is a schematic view of the overall structure of the present invention;
FIG. 2 is a schematic structural view of a chip base and a droplet generating section according to the present invention;
FIG. 3 is a schematic view of a structure of a liquid droplet generating section in the present invention;
FIG. 4 is a schematic view of the structure of a droplet collecting section in the present invention;
FIG. 5 is a schematic view of the structure of the driving device of the present invention;
FIG. 6 is a schematic view of the mechanism of the belt drive mechanism of the present invention;
FIG. 7 is a schematic view of the structure of a heating apparatus according to the present invention;
FIG. 8 is a schematic view showing the structure of a double spiral heater according to the present invention;
FIG. 9 is a schematic diagram of the structure of the fluorescence imaging optical reading device of the present invention;
Detailed Description
The invention is further illustrated by the following examples in conjunction with the accompanying drawings:
in the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In the description of the present invention, it is to be understood that the terms "left", "right", "front", "back", "top", "bottom", etc. indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention.
As shown in fig. 1 to 9, an excitation filter for an integrated droplet digital PCR detection platform comprises a frame 1, a droplet generation part 2, a droplet collection part 3, a chip base 4, a driving device 5, a heating device 6 and a fluorescence imaging optical reading device 7; the liquid drop generating part 2 and the liquid drop collecting part 3 form a chip structure, the chip structure is fixedly arranged on a chip base 4, the chip base 4 is arranged on the frame 1 and is in sliding connection with the frame 1 through a driving device 5, the heating device 6 is fixedly arranged on the frame 1 and is arranged at the bottom end of the chip base 4, and the fluorescence imaging optical reading device 7 is fixedly arranged on the frame 1 and is arranged at the top end of the liquid drop collecting part 3.
On the basis of the above technical solution, as shown in fig. 3, the droplet generation part 2 includes an oil hole 21, a reagent hole 22, a continuous phase channel 23, a dispersed phase channel 24, and a droplet outlet pipeline 25, the oil hole 21 is connected to the continuous phase channel 23 in a penetrating manner, the reagent hole 22 is connected to the dispersed phase channel 24 in a penetrating manner, the continuous phase channel 23 is connected to the dispersed phase channel 24 in a penetrating manner and forms a cross structure 26, and the droplet outlet pipeline 25 is connected to the cross structure 26 in a penetrating manner. In the invention, a droplet generating part 2 is arranged, and micro droplets are generated by adopting a flow focusing method; in the process of generating the liquid drops, the uniformity of the liquid drops is realized by controlling the flow velocity of the continuous phase and the flow velocity of the disperse phase, and the generated diameter of the liquid drops is directly determined by adjusting the flow velocity of the continuous phase and the width of each channel, so that uniform and stable liquid drops are finally obtained.
On the basis of the above technical scheme, as shown in fig. 4, the droplet collecting part 3 includes a collecting pipe 31, a collecting cavity 32 and a supporting column 33, one end of the collecting pipe 31 is connected with the droplet outlet pipe 25 in a penetrating manner, the other end of the collecting pipe is connected with the collecting cavity 32 in a penetrating manner, the supporting column 33 is provided with a plurality of and fixedly arranged in the collecting cavity 32, and a collecting cavity outlet 34 is opened at one side of the collecting cavity 32. More preferably, the collecting duct 31 is provided with a plurality of branch ducts, as shown in fig. 4; therefore, the generated micro-droplets can be effectively dispersed, and the micro-droplets can be more closely arranged in the collection cavity. On the basis of the scheme, the height of the collecting cavity 32 is between 1 and 2 droplet diameters, so that the micro droplets are always distributed in a single layer in the collecting cavity 32, the detection result is not influenced by the stacking phenomenon, and the detection precision is improved. In the invention, the liquid drop collecting part 3 collects liquid drops by a multi-channel collecting method, so that the liquid drops have speed buffering when entering the collecting cavity 32, and bubbles at the corner of the inlet of the collecting cavity are effectively prevented; meanwhile, a plurality of supporting columns 33 are arranged in the collecting cavity 32, so that the problem of collapse of the collecting cavity can be avoided, liquid drops can be arranged in the collecting cavity more closely, and adverse effects caused by bubbles and the like during subsequent heating amplification are reduced.
On the basis of the above technical solution, as shown in fig. 5, the driving device 5 includes a pulley shaft 51, a crank 52, a connecting rod 53 and a transmission mechanism 54, the pulley shaft 51 is driven by the transmission mechanism 54 to rotate, one end of the crank 52 is fixedly connected with the pulley shaft 51, the other end is rotatably connected with the connecting rod 53, and the connecting rod 53 is hinged with one side of the chip base 4. On the basis of the above technical solution, as shown in fig. 6, the transmission mechanism 54 includes a stepping motor 541, a small pulley 542, a large pulley 543, and a belt 544, the stepping motor 541 is fixedly disposed on the frame 1, the small pulley 542 is fixedly connected to an output shaft of the stepping motor 541, the large pulley 543 is in transmission connection with the small pulley 542 through the belt 544, and the large pulley 543 is fixedly connected to the pulley shaft 51 and coaxially disposed. On the basis of the technical scheme, a touch sensor is fixedly arranged at the top end of the rack 1 and is electrically connected with the chip base 4 and the stepping motor 541. According to the invention, the driving device 5 is arranged, so that the chip base 4 can be driven to slide on the rack 1, and the movement position of the chip base 4 is sensed by matching with the touch sensor, so that the automatic operation of the PCR detection platform in the nucleic acid detection process can be realized, the detection precision is effectively improved, and the detection efficiency is improved.
On the basis of the above technical solution, as shown in fig. 7, the heating device 6 includes a heat source 61, a heater 62, and a heat transfer plate 63, wherein the heat source 61 is fixedly disposed on the frame 1, the heater 62 is fixedly disposed at the top end of the heat source 61 and electrically connected to the heat source 61, one side of the heat transfer plate 63 is disposed at the top end of the heater 62, and the other side is disposed at the bottom end of the collection chamber 32. In the invention, a heating device 6 is arranged to heat and preserve heat of a chip structure; more preferably, the heater 62 is a double helix ring heater, as shown in fig. 8; the temperature requirement of the chip structure can be met, and the temperature difference of the chip structure during temperature rise can be ensured to be small, so that effective amplification of nucleic acid molecules in the whole cyclic heating process is guaranteed, and the detection precision is improved.
On the basis of the above technical solution, as shown in fig. 9, the fluorescence imaging optical reading device 7 includes a light source 71, an excitation filter 72, a dichroic mirror 73, an emission filter 74, an imaging objective lens 75, a steering mirror 76, and a CCD camera 77; the excitation filter 72 is arranged at the rear end of the light source 71, a light shielding shell 8 is fixedly arranged on one side of the excitation filter 72, a dichroic mirror 73, an emission filter 74, an imaging objective 75 and a steering reflector 76 are sequentially arranged in the light shielding shell 8 along the direction of fluorescence transmission, and the CCD camera 77 is arranged on one side of the steering reflector 76 and is arranged on the same horizontal plane with the center of the steering reflector 76. Note that the rear end mentioned here is defined according to the path of the light source irradiation.
On the basis of the technical scheme, the waste liquid collecting device 9 is fixedly arranged on the rack 1, the waste liquid collecting device 9 comprises a waste liquid accommodating cavity 91 and a waste liquid outlet 92, the waste liquid accommodating cavity 91 is connected with the collecting cavity outlet 34, and the waste liquid outlet 92 is arranged on the side wall of the waste liquid accommodating cavity 91.
When the invention works, firstly, the reagent and the oil phase are respectively injected into the oil hole 21 and the reagent hole 22, and the two respectively enter the cross structure 27 through the continuous phase channel 23 and the disperse phase channel 24; forming shearing force to generate micro liquid drops under the action of the oil phase and the surface tension of the liquid drops, and finishing the generation process of the liquid drops; the generated micro-droplets are then gathered into the collecting pipe 31 through the droplet outlet pipe 25, the micro-droplets flow through the plurality of groups of collecting pipes 31 and are closely arranged inside the collecting cavity 32 under the effective buffer of the supporting pillars 33 in the collecting cavity 32, and the collecting process of the droplets is completed.
After the micro-droplet generation and collection operation is completed, the heating device 6 starts to operate, the heat source 61 provides heat, the double-helix annular heater 62 effectively isolates the boundary and the heating region, the temperature is uniformly transmitted to the chip base 4 through the heat transmission plate 63, and the temperature condition required by PCR amplification is provided.
After the amplification and quantification of the DNA are completed, the stepping motor 541 receives a signal and starts to rotate at a constant speed, the transmission mechanism 54 transmits power to the pulley shaft 51, specifically, the stepping motor 541 receives a signal and rotates at a constant speed, an output shaft of the stepping motor 541 transmits power to the small pulley 542, the large pulley 543 and the small pulley 542 rotate by being transmitted by the belt 544, and the power is output by the pulley shaft 51; then the pulley shaft 51 drives the crank 52 and the connecting rod 53 to move together, and the chip base 4 starts to move linearly under the combined action of the crank 52 and the connecting rod 53; when the chip base 4 moves to a designated position, the touch sensor is triggered, the stepping motor 541 stops working after receiving a signal, and at the moment, the fluorescent imaging optical reading device 7 detects and photographs liquid drops.
When the fluorescence imaging optical reading device 7 shoots, the chip structure moves to a position right below the fluorescence imaging optical reading device 7, and at the moment, the collection cavity 32 is aligned to an imaging area; exciting light is generated by a light source 71, exciting light suitable for a fluorescence exciting waveband is obtained through an exciting filter 72, the exciting light excites fluorescent dye on a gene to be detected in a chip structure through a dichroic mirror 73 to generate fluorescence, the fluorescence is transmitted to a CCD camera 77 through an emission filter 74, an imaging objective 75 and a steering reflector 76, real-time shooting is carried out by the CCD camera, and the fluorescence is transmitted to a computer in real time to carry out result analysis; after the shooting is finished, the stepping motor 541 receives a signal and starts to rotate in the reverse direction, and finally the chip base moves to the initial position through the transmission of the transmission mechanism 54, the pulley shaft 51, the crank 52 and the connecting rod 53.
After the detection is finished, the liquid in the collection cavity 32 is pressed into the waste liquid accommodating cavity 91 by an external vacuum pump, and is finally discharged by a waste liquid outlet 92 arranged on the side wall of the waste liquid accommodating cavity 91, so that the complete process of one-time digital PCR detection is finished.
The invention provides an integrated liquid drop digital PCR detection platform, which realizes high-integration automation of nucleic acid detection, not only effectively improves the detection efficiency, but also improves the detection precision and quality.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (9)
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113670877A (en) * | 2021-08-25 | 2021-11-19 | 华中科技大学 | Inclined top-mounted Gaussian light sheet imaging system for high-throughput digital PCR detection |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113670877A (en) * | 2021-08-25 | 2021-11-19 | 华中科技大学 | Inclined top-mounted Gaussian light sheet imaging system for high-throughput digital PCR detection |
| CN113670877B (en) * | 2021-08-25 | 2022-05-10 | 华中科技大学 | Oblique top-mounted Gaussian light sheet imaging system for high-throughput digital PCR detection |
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