CN114018891A - Optical detection system of droplet type digital PCR - Google Patents
Optical detection system of droplet type digital PCR Download PDFInfo
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- CN114018891A CN114018891A CN202111333612.9A CN202111333612A CN114018891A CN 114018891 A CN114018891 A CN 114018891A CN 202111333612 A CN202111333612 A CN 202111333612A CN 114018891 A CN114018891 A CN 114018891A
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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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
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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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6463—Optics
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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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6463—Optics
- G01N2021/6478—Special lenses
Abstract
The invention relates to an optical detection system of a droplet type digital PCR, belonging to the technical field of optical detection. The invention provides an optical detection system of a droplet type digital PCR (polymerase chain reaction), which comprises a light source group, a light source beam combining system, an optical fiber coupling system and a light splitting detection system, wherein the light source beam combining system is arranged on a light path of emergent light of the light source group, the optical fiber coupling system is arranged on a light path of emergent light of the light source beam combining system, and the light splitting detection system is arranged on a light path of emergent light of the optical fiber coupling system; the light source beam combining system combines the emergent light of the light source group into one beam and then outputs the beam, thereby solving the problems of complexity, low integration level and poor stability of the existing micro-drop type digital PCR light source module; the light-splitting detection system splits light to be detected according to wave bands, and solves the problems of fluorescence crosstalk and laser crosstalk existing in the existing micro-drop digital PCR detection result.
Description
Technical Field
The invention relates to an optical detection system of a droplet type digital PCR, belonging to the technical field of optical detection.
Background
Droplet Digital PCR (DDPCR) is a third generation PCR (Polymerase Chain Reaction) technique, which is an absolute method for the quantification of nucleic acid molecules. The principle of droplet-type digital PCR is to carry out micro-droplet treatment on a sample before PCR amplification, namely, a reaction system containing nucleic acid molecules is divided into thousands of nano-upgrade micro-droplets, wherein each micro-droplet contains no nucleic acid target molecules to be detected or contains one to a plurality of nucleic acid target molecules to be detected. After PCR amplification, each microdroplet is detected one by one, the microdroplet with a fluorescent signal is judged to be 1, the microdroplet without the fluorescent signal is judged to be 0, and the initial copy number or the concentration of the target molecule can be obtained according to the Poisson distribution principle and the number and the proportion of the positive microdroplets.
The sensitivity of the droplet digital PCR is very high, and only a small amount of templates is needed; the method is particularly suitable for trace samples to be detected which are difficult to obtain; the defects that a second generation PCR system is difficult to accurately determine the gene copy number, cannot qualitatively and quantitatively determine the trace mutation, has low accuracy and the like are overcome. The requirement of more clinical examinations is satisfied, and the method is more accurate and more digital. The kit is very suitable for the accurate detection of nucleic acid in some rare samples, and is used for early screening of tumors, secondary drug resistance detection of tumors, real-time monitoring of tumor loads and the like.
The droplet-type digital PCR mainly comprises a droplet generation system, an optical detection system, a liquid path system and a sample adding system, wherein the droplet generation system is mainly used for generating droplets with uniform size; the optical detection system is mainly used for exciting the micro-droplets of the micro-droplet generation system to form light to be detected and detecting the light intensity of the light to be detected; the liquid path system is mainly used for forming a micro-droplet array to be detected; the sample adding system is mainly used for automatic sample adding. However, the existing optical detection system usually uses free space light output, which may cause poor consistency of multi-channel excitation light, complex installation and adjustment difficulty and system layout deadlines, so that the existing droplet digital PCR has problems of complex light source module, low integration level and poor stability. In addition, because the fluorescence spectrum of part of reagents used by the existing droplet generation system is obviously overlapped, and a plurality of excitation wavelengths are possibly close to the fluorescence spectrum band, the existing droplet digital PCR also has the problems of fluorescence crosstalk and laser crosstalk.
Disclosure of Invention
In order to solve the above problems, the present invention provides an optical detection system of droplet digital PCR, which comprises a light source group, a light source beam combining system, an optical fiber coupling system and a light splitting detection system;
the light source beam combining system is arranged on a light path of emergent light of the light source group and is used for combining the received light;
the optical fiber coupling system is arranged on a light path of emergent light of the light source beam combining system and is used for focusing received light on a micro-droplet position of the micro-droplet flow channel to form a light spot after secondary transmission and shaping, and the light spot focused on the micro-droplet position excites fluorescent dye in the micro-droplet to form light to be detected;
the light splitting detection system is arranged on a light path of emergent light of the optical fiber coupling system and used for splitting the received light to be detected and detecting light intensity of the split light to be detected.
In one embodiment of the present invention, the light source beam combining system comprises a first collimating lens group and a dichroic lens group;
the first collimating lens group is arranged on a light path of emergent light of the light source and is used for collimating the received light;
the dichroic mirror group is arranged on a light path of emergent light of the first collimating lens group, the emergent light of the first collimating lens group is reflected at the dichroic mirror group, and the emergent light reflected by the dichroic mirror group is combined into a beam.
In one embodiment of the present invention, the fiber coupling system comprises a coupling lens group, an optical fiber and a laser focusing lens group;
the coupling lens group is arranged on a light path of emergent light of the dichroic mirror group and is used for coupling the received light;
the input end of the optical fiber is arranged on a light path of emergent light of the coupling lens group and is used for carrying out secondary transmission on the received light;
the laser focusing lens group is arranged on a light path of emergent light at the output end of the optical fiber and is used for shaping received light;
emergent light of the laser focusing lens group is focused on a droplet of the droplet channel to form a light spot, and the light spot focused on the droplet excites fluorescent dye in the droplet to form light to be detected.
In one embodiment of the present invention, the coupling lens group includes a first convex mirror, a second convex mirror, a third convex mirror, and a first concave mirror; the first concave mirror is located between the second convex mirror and the third convex mirror.
In one embodiment of the invention, the surface of the first convex mirror far away from the second convex mirror is a spherical surface with the curvature radius of-3.105 mm, and the surface close to the second convex mirror is a spherical surface with the curvature radius of-3.665 mm;
the surface of the second convex mirror close to the first convex mirror is a spherical surface with the curvature radius of-7.398 mm, and the surface close to the concave mirror is a spherical surface with the curvature radius of 1.821 mm;
the surface of the first concave mirror, which is close to the second convex mirror, is a spherical surface with the curvature radius of 1.821mm, and the surface of the first concave mirror, which is close to the third convex mirror, is a spherical surface with the curvature radius of-7.661 mm;
the surface of the third convex mirror close to the concave mirror is a spherical surface with the curvature radius of-7.661 mm, and the surface far away from the concave mirror 24 is a spherical surface with the curvature radius of 9.827 mm.
In one embodiment of the invention, the thickness of the first convex mirror is 3 mm; the thickness of the second convex mirror is 3 mm; the thickness of the first concave mirror is 0.2 mm; the thickness of the third lens is 2 mm.
In one embodiment of the invention, the optical fiber is a single mode fiber having a core diameter of 50 μm.
In one embodiment of the present invention, the spectroscopic detection system comprises a dispersive element, a fluorescence focusing lens group, a slit group and a detector group;
the dispersion element is arranged on the light path of the light to be measured and is used for dispersing the received light so as to separate the fluorescence with different wavelengths in the light to be measured;
the fluorescence focusing lens group is arranged on a light path of emergent light of the dispersion element and is used for converging received fluorescence with different wavelengths into light beams at different positions;
the position of the slit group corresponds to the emission spectrum position of the fluorescent dye in the microdroplet, and the slit group is used for selecting the emergent light beam of the fluorescent focusing lens group, so that the light to be detected by the detector group is emitted into the detector group.
In one embodiment of the invention, the dispersive element is a prism or a grating.
In one embodiment of the invention, the dispersive element is a transmission grating with 600 grating lines/mm of material BK 7.
In one embodiment of the present invention, the fluorescence focus lens group includes a fourth convex mirror, a second concave mirror, and a fifth convex mirror.
In one embodiment of the invention, the surface of the fourth convex mirror close to the second concave mirror is a spherical surface with the curvature radius of-63.582 mm, and the surface far away from the second concave mirror is a spherical surface with the curvature radius of 115.15 mm;
the surface of the second concave mirror, which is close to the fourth convex mirror, is a spherical surface with the curvature radius of 115.15mm, and the surface far away from the fourth convex mirror is a plane;
the surface of the fifth convex mirror close to the second concave mirror is a plane, and the surface far away from the second concave mirror is a spherical surface with the curvature radius of 32.115 mm.
In one embodiment of the invention, the thickness of the fourth convex mirror is 5 mm; the thickness of the second concave mirror is 3 mm; the thickness of the fifth convex mirror is 5 mm.
In an embodiment of the present invention, the light splitting detection system further includes a second collimating lens group; the second collimating lens group is arranged on a light path of the light to be measured and is used for collimating the received light.
In one embodiment of the present invention, the second collimating lens group includes a sixth convex mirror and a third concave mirror.
In one embodiment of the invention, the surface of the sixth convex mirror far away from the third concave mirror is a spherical surface with the curvature radius of-27.36 mm, and the surface close to the third concave mirror is a spherical surface with the curvature radius of 22.54 mm;
the surface of the third concave mirror close to the sixth convex mirror is a spherical surface with the curvature radius of 22.54mm, and the surface close to the sixth convex mirror is a spherical surface with the curvature radius of 91.83 mm.
In one embodiment of the invention, the thickness of the sixth convex mirror is 3.5 mm; the thickness of the third concave mirror is 1.5 mm.
The invention provides a droplet type digital PCR instrument, which comprises the optical detection system.
The invention provides a nucleic acid detection method, which uses the micro-drop digital PCR instrument to carry out quantitative detection on nucleic acid in a sample to be detected.
The invention provides the application of the optical detection system or the micro-drop digital PCR instrument or the nucleic acid detection method in nucleic acid detection.
In one embodiment of the present invention, the nucleic acid detection is a quantitative nucleic acid detection.
The technical scheme of the invention has the following advantages:
1. the invention provides an optical detection system of a micro-drop digital PCR (polymerase chain reaction), which comprises a light source group, a light source beam combining system, an optical fiber coupling system and a light splitting detection system, wherein the light source beam combining system is arranged on a light path of emergent light of the light source group and used for combining the received light, the optical fiber coupling system is arranged on the light path of the emergent light of the light source beam combining system and used for focusing the received light on a micro-drop position of a micro-drop flow passage to form a light spot after secondary transmission and shaping, the light spot focused on the micro-drop position excites fluorescent dye in the micro-drop to form light to be detected, and the light splitting detection system is arranged on the light path of the emergent light of the optical fiber coupling system and used for splitting the received light to be detected according to wave bands and detecting the light intensity of the split light to be detected; the light source beam combining system can combine the emergent light of the light source group into one beam and then output the beam, and solves the problems of complexity, low integration level and poor stability of a droplet type digital PCR light source module caused by the output of free space light by the existing optical detection system; the light splitting detection system can split light to be detected, and solves the problems of fluorescence crosstalk and laser crosstalk existing in a droplet type digital PCR detection result caused by obvious superposition of a part of fluorescence spectra of reagents used by the existing droplet generation system and the possibility of approaching of a plurality of excitation wavelengths to a fluorescence spectrum waveband.
The optical fiber coupling system comprises a coupling lens group, an optical fiber and a laser focusing lens group, wherein the coupling lens group is arranged on a light path of emergent light of the dichroic mirror group and used for coupling received light, an input end of the optical fiber is arranged on the light path of emergent light of the coupling lens group and used for carrying out secondary transmission on the received light, the laser focusing lens group is arranged on a light path of emergent light at an output end of the optical fiber and used for shaping the received light, the emergent light of the laser focusing lens group is focused at a droplet of the droplet flow channel to form a light spot, and the light spot focused at the droplet excites fluorescent dye in the droplet to form light to be detected; the arrangement of the optical fiber can break through the problem of physical space limitation of the existing optical detection system, and the flexibility is higher.
Further, the coupling lens group includes a first convex mirror, a second convex mirror, a third convex mirror and a first concave mirror, the first concave mirror is located between the second convex mirror and the third convex mirror, wherein a surface of the first convex mirror away from the second convex mirror is a spherical surface with a curvature radius of-3.105 mm, a surface close to the second convex mirror is a spherical surface with a curvature radius of-3.665 mm, a surface of the second convex mirror close to the first convex mirror is a spherical surface with a curvature radius of-7.398 mm, a surface close to the concave mirror is a spherical surface with a curvature radius of 1.821mm, a surface close to the second convex mirror is a spherical surface with a curvature radius of 1.821mm, a surface close to the third convex mirror is a spherical surface with a curvature radius of-7.661 mm, a surface close to the concave mirror of the third convex mirror is a spherical surface with a curvature radius of-7.661 mm, a surface far from the concave mirror 24 is a spherical surface with a curvature radius of 9.827mm, and a thickness of the first convex mirror is 3mm, a thickness of the second convex mirror is 3mm, the thickness of the first concave mirror is 0.2mm, and the thickness of the third lens is 2 mm; the spherical surface and the thickness of the coupling lens group can effectively achieve the effect of eliminating aberration, light spots of light with various wavelengths in the range of 400-700 nm are far smaller than 50 microns after passing through the coupling lens group, and the light spots enter the optical fiber with high coupling efficiency.
Further, the light splitting detection system comprises a dispersion element, a fluorescence focusing lens group, a slit group and a detector group, wherein the dispersion element is arranged on a light path of light to be detected and is used for dispersing received light so as to separate fluorescence with different wavelengths in the light to be detected, the fluorescence focusing lens group is arranged on the light path of emergent light of the dispersion element and is used for converging the received fluorescence with different wavelengths into light beams with different positions, and the position of the slit group corresponds to the emission spectrum position of fluorescent dye in a droplet and is used for selecting the emergent light beam of the fluorescence focusing lens group so as to enable the emergent light of the detector group to be detected to be emitted into the detector group; the slit group can screen the light to be detected after light splitting, and further avoids the fluorescent crosstalk and laser crosstalk of a detection result caused by obvious superposition of a part of fluorescence spectra of reagents used by the conventional optical detection system and the possibility that a plurality of excitation wavelengths are close to the band of the fluorescence spectra.
Furthermore, the light splitting detection system further comprises a second collimating lens group, and the second collimating lens group is arranged on a light path of the light to be detected and is used for collimating the received light; the second collimating lens group is arranged to contribute to the splitting by the dispersive element.
Further, the fluorescence focusing lens group comprises a fourth convex lens, a second concave lens and a fifth convex lens, wherein the surface of the fourth convex lens close to the second concave lens is a spherical surface with the curvature radius of-63.582 mm, the surface far away from the second concave lens is a spherical surface with the curvature radius of 115.15mm, the surface of the second concave lens close to the fourth convex lens is a spherical surface with the curvature radius of 115.15mm, the surface far away from the fourth convex lens is a plane, the surface of the fifth convex lens close to the second concave lens is a plane, the surface far away from the second concave lens is a spherical surface with the curvature radius of 32.115mm, the thickness of the fourth convex lens is 5mm, the thickness of the second concave lens is 3mm, and the thickness of the fifth convex lens is 5 mm; the second collimating lens group comprises a sixth convex lens and a third concave lens, wherein the surface of the sixth convex lens, which is far away from the third concave lens, is a spherical surface with the curvature radius of-27.36 mm, the surface close to the third concave lens is a spherical surface with the curvature radius of 22.54mm, the surface of the third concave lens, which is close to the sixth convex lens, is a spherical surface with the curvature radius of 22.54mm, the surface close to the sixth convex lens is a spherical surface with the curvature radius of 91.83mm, the thickness of the sixth convex lens is 3.5mm, and the thickness of the third concave lens is 1.5 mm; the arrangement of the spherical surfaces and the thicknesses of the fluorescence focusing lens group and the second collimating lens group can effectively collimate and eliminate aberration of fluorescence in microdroplets, so that light spots of light with wavelengths within the range of 400-700 nm pass through the fluorescence focusing lens group and are smaller than 10 mu m and uniformly distributed, the distance between two wavelengths spaced by 30nm is larger than 1mm, and the slit gating requirement is met.
Further, the dispersive element is a prism or a grating; the fluorescence with different wavelengths in the light to be measured is split by selecting a specific prism material and a prism apex angle or selecting the number of gratings of a specific grating.
2. The invention provides a droplet type digital PCR instrument, which comprises the optical detection system; the micro-drop digital PCR instrument can solve the problems of complexity, low integration level and poor stability of a micro-drop digital PCR light source module caused by the fact that an existing optical detection system uses free space light output, and can solve the problems of fluorescence crosstalk and laser crosstalk of a micro-drop digital PCR detection result caused by the fact that fluorescence spectra of part of reagents used by an existing micro-drop generation system are obviously overlapped and excitation wavelengths and fluorescence spectrum wave bands are close to each other.
3. The invention provides a nucleic acid detection method, which uses the micro-drop digital PCR instrument to carry out quantitative detection on nucleic acid in a sample to be detected; the method for quantitatively detecting the nucleic acid in the sample to be detected can solve the problems of complexity, low integration level and poor stability of a droplet type digital PCR light source module caused by the fact that an existing optical detection system uses free space light output, and can solve the problems of fluorescence crosstalk and laser crosstalk of droplet type digital PCR detection results caused by the fact that fluorescence spectra of partial reagents used by an existing droplet generation system are obviously overlapped and excitation wavelengths are probably close to fluorescence spectrum wave bands.
Drawings
FIG. 1: the overall structure of one embodiment of the optical detection system is schematically shown.
FIG. 2: the overall structure of one embodiment of the coupling lens group is schematic.
FIG. 3: the overall structure of one embodiment of the light splitting detection system is schematic.
FIG. 4: and (3) a two-dimensional light path diagram of the coupling lens group.
FIG. 5: coupled lens group optical dot alignment.
FIG. 6: and the coupling lens group has an optical path aberration diagram.
FIG. 7: three-dimensional light path diagram of the light splitting detection system.
FIG. 8: and (5) an image surface footprint of the light splitting detection system.
In FIGS. 1 to 3, 1, a light source group; 2. a first collimating lens group; 3. a dichroic mirror group; 4. a coupling lens group; 41. a first convex mirror; 42. a second convex mirror; 43. a third convex mirror; 44. a first concave mirror; 5. an optical fiber; 6. a laser focusing lens group; 7. a droplet flow channel; 8. a second collimating lens group; 81. a sixth convex mirror; 82. a third concave mirror; 9. a dispersive element; 10. a fluorescent focusing lens group; 101. a fourth convex mirror; 102. a second concave mirror; 103. a fifth convex mirror; 11. a slit group; 12. a set of detectors.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
Example 1: optical detection system
As shown in fig. 1 to 3, the present embodiment provides an optical detection system of droplet digital PCR, where the optical detection system includes a light source group 1, a light source beam combining system, an optical fiber coupling system, and a light splitting detection system;
the light source beam combining system is arranged on a light path of emergent light of the light source group 1 and is used for combining the received light;
the optical fiber coupling system is arranged on a light path of emergent light of the light source beam combining system and is used for focusing received light on a micro-droplet position of the micro-droplet flow channel 7 to form a light spot after secondary transmission and shaping, and the light spot focused on the micro-droplet position excites fluorescent dye in the micro-droplet to form light to be detected;
the light splitting detection system is arranged on a light path of emergent light of the optical fiber coupling system and used for splitting the received light to be detected and detecting light intensity of the split light to be detected. The light source beam combining system can combine the emergent light of the light source group 1 into one beam and then output the beam, thereby solving the problems of complicated micro-drop digital PCR light source module, low integration level and poor stability caused by the output of free space light by the existing optical detection system; the light splitting detection system can split light to be detected, and solves the problems of fluorescence crosstalk and laser crosstalk existing in a droplet type digital PCR detection result caused by obvious superposition of a part of fluorescence spectra of reagents used by the existing droplet generation system and the possibility of approaching of a plurality of excitation wavelengths to a fluorescence spectrum waveband.
Preferably, the light source beam combining system comprises a first collimating lens group 2 and a dichroic lens group 3;
the first collimating lens group 2 is arranged on a light path of emergent light of the light source and is used for collimating the received light;
the dichroic mirror group 3 is arranged on a light path of emergent light of the first collimating mirror group 2, the emergent light of the first collimating mirror group 2 is reflected at the dichroic mirror group 3, and the emergent light reflected by the dichroic mirror group 3 is combined into a beam.
Preferably, the optical fiber coupling system comprises a coupling lens group 4, an optical fiber 5 and a laser focusing lens group 6;
the coupling lens group 4 is arranged on a light path of emergent light of the dichroic lens group 3 and is used for coupling the received light;
the input end of the optical fiber 5 is arranged on the light path of emergent light of the coupling lens group 4 and is used for carrying out secondary transmission on the received light;
the laser focusing lens group 6 is arranged on a light path of emergent light at the output end of the optical fiber 5 and is used for shaping received light;
emergent light of the laser focusing lens group 6 is focused on a droplet of the droplet channel 7 to form a light spot (the light spot is focused to be 50-100 mu m in size), and the light spot focused on the droplet excites fluorescent dye in the droplet to form a to-be-detected light (the wavelength of fluorescence in the to-be-detected light corresponds to the type of the fluorescent dye, and the emitted fluorescence is a broad spectrum). The arrangement of the optical fiber 5 can break through the problem of physical space limitation existing in the existing optical detection system, and the flexibility is higher.
Preferably, the coupling lens group 4 comprises a first convex mirror 41, a second convex mirror 42, a third convex mirror 43 and a first concave mirror 44; the first concave mirror 44 is located between the second convex mirror 42 and the third convex mirror 43.
In one embodiment of the present invention, the surface of first convex mirror 41 away from second convex mirror 42 is a spherical surface with a radius of curvature of-3.105 mm, and the surface close to second convex mirror 42 is a spherical surface with a radius of curvature of-3.665 mm;
the surface of the second convex mirror 42 close to the first convex mirror 41 is a spherical surface with the curvature radius of-7.398 mm, and the surface close to the concave mirror is a spherical surface with the curvature radius of 1.821 mm;
the surface of the first concave mirror 44 close to the second convex mirror 42 is a spherical surface with the curvature radius of 1.821mm, and the surface close to the third convex mirror 43 is a spherical surface with the curvature radius of-7.661 mm;
the surface of the third convex mirror 43 close to the concave mirror is a spherical surface with the curvature radius of-7.661 mm, and the surface far away from the concave mirror 24 is a spherical surface with the curvature radius of 9.827 mm.
Preferably, the thickness of the first convex mirror 41 is 3 mm; the thickness of the second convex mirror 42 is 3 mm; the thickness of the first concave mirror 44 is 0.2 mm; the thickness of the third lens is 2 mm. The spherical surface and the thickness of the coupling lens group 4 can effectively achieve the effect of eliminating aberration, so that light with various wavelengths in the range of 400-700 nm passes through the coupling lens group 4, the light spot is far smaller than 50 mu m, and enters the optical fiber 5 with high coupling efficiency (see fig. 4-6 in particular).
Preferably, the specific parameters of the first convex mirror 41, the second convex mirror 42, the third convex mirror 43 and the first concave mirror 44 are as follows:
TABLE 1 concrete parameters of the first, second, third and first concave mirrors
| Lens | Radius of curvature (mm) | Thickness (mm) | Caliber (mm) | Material |
| First convex mirror | R1＝-3.105，R2＝-3.665 | 3 | 3.6 | S-FPL53 |
| Second convex mirror | R1＝-7.398，R2＝-1.821 | 3 | 3.6 | S-FPL53 |
| First concave mirror | R1＝1.821，R2＝-7.661 | 0.2 | 3.6 | N-BAK1 |
| Third lens | R1＝-7.661，R2＝9.827 | 2 | 3.6 | H-ZBAF3 |
In Table 1, S-FPL53, N-BAK1 and H-ZBAF3 are all glass-numbered, and the specific parameters are shown in the reference: GB903-87 colorless optical glass.
Preferably, the optical fiber 5 is a single mode fiber having a core diameter of 50 μm.
Preferably, the spectroscopic detection system comprises a dispersive element 9, a fluorescence focusing lens group 10, a slit group 11 and a detector group 12;
the dispersion element 9 is arranged on the light path of the light to be measured and is used for dispersing the received light so as to separate the fluorescence with different wavelengths in the light to be measured;
the fluorescence focusing lens group 10 is arranged on the light path of the emergent light of the dispersion element 9 and is used for converging the received fluorescence with different wavelengths into light beams at different positions;
the position of the slit group 11 corresponds to the emission spectrum position of the fluorescent dye in the droplet, and is used for selecting the emergent light beam of the fluorescence focusing lens group 10, so that the light to be detected by the detector group 12 is incident to the detector group 12. The slit group 11 can screen the split light to be detected, and further avoids the fluorescent crosstalk and laser crosstalk in the detection result caused by obvious superposition of a part of fluorescence spectra of reagents used by the conventional optical detection system and the possibility of proximity of a plurality of excitation wavelengths to the band of the fluorescence spectra.
Preferably, the dispersive element 9 is a prism or a grating. The fluorescence with different wavelengths in the light to be measured is split by selecting a specific prism material and a prism apex angle or selecting the number of gratings of a specific grating.
Preferably, the dispersive element 9 is a transmission grating, the grating lines being 600 lines/mm, material BK 7.
Preferably, the fluorescence focusing lens group 10 includes a fourth convex mirror 101, a second concave mirror 102, and a fifth convex mirror 103.
Preferably, the surface of the fourth convex mirror 101 close to the second concave mirror 102 is a spherical surface with a curvature radius of-63.582 mm, and the surface far away from the second concave mirror 102 is a spherical surface with a curvature radius of 115.15 mm;
the surface of the second concave mirror 102 close to the fourth convex mirror 101 is a spherical surface with the curvature radius of 115.15mm, and the surface far away from the fourth convex mirror 101 is a plane;
the surface of the fifth convex mirror 103 close to the second concave mirror 102 is a plane, and the surface far away from the second concave mirror 102 is a spherical surface with the curvature radius of 32.115 mm.
Preferably, the thickness of the fourth convex mirror 101 is 5 mm; the thickness of the second concave mirror 102 is 3 mm; the thickness of the fifth convex mirror 103 is 5 mm.
Preferably, the specific parameters of the fourth convex mirror 101, the second concave mirror 102 and the fifth convex mirror 103 are as follows:
TABLE 2 concrete parameters of the fourth, second and fifth convex mirrors
| Lens | Radius of curvature (mm) | Thickness (mm) | Caliber (mm) | Material |
| Fourth convex mirror | R1＝-63.582，R2＝115.15 | 5 | 12 | N-SK16 |
| Second concave mirror | R1＝115.15，R2＝∞ | 3 | 12 | F2 |
| Fifth convex mirror | R1＝∞，R2＝32.115 | 3 | 12 | N-SK16 |
In Table 2, BK7, N-SK16 and F2 are all glass numbers, and the specific parameters are shown in the reference: GB903-87 colorless optical glass.
Preferably, the light splitting detection system further comprises a second collimating lens group 8; the second collimating lens group 8 is disposed on a light path of the light to be detected, and is configured to collimate the received light. The second collimating lens group 8 is arranged to assist the dispersive element 9 in splitting light.
Preferably, the second collimating lens group 8 comprises a sixth convex mirror 81 and a third concave mirror 82.
Preferably, the surface of the sixth convex mirror 81 far from the third concave mirror 82 is a spherical surface with the curvature radius of-27.36 mm, and the surface close to the third concave mirror 82 is a spherical surface with the curvature radius of 22.54 mm;
the surface of the third concave mirror 82 close to the sixth convex mirror 81 is a spherical surface with the curvature radius of 22.54mm, and the surface close to the sixth convex mirror 81 is a spherical surface with the curvature radius of 91.83 mm.
Preferably, the thickness of the sixth convex mirror 81 is 3.5 mm; the thickness of the third concave mirror 82 is 1.5 mm. The arrangement of the spherical surfaces and the thickness of the fluorescence focusing lens group 10 and the second collimating lens group 8 can effectively collimate and eliminate aberration of fluorescence in the microdroplets, so that light spots of wavelengths within the range of 400-700 nm pass through the fluorescence focusing lens group 10 and are smaller than 10 micrometers and uniformly distributed, the distance between two wavelengths spaced by 30nm is larger than 1mm, and the slit gating requirement is met (see fig. 7-8 in particular).
Preferably, the specific parameters of the sixth convex mirror 81 and the third concave mirror 82 are as follows:
TABLE 3 concrete parameters of the sixth convex mirror and the third concave mirror
| Lens | Radius of curvature (mm) | Thickness (mm) | Caliber (mm) | Material |
| Sixth convex mirror | R1＝-27.36，R2＝22.54 | 3.5 | 12 | N-BK7 |
| Third concave mirror | R1＝22.54，R2＝91.83 | 1.5 | 12 | SF2 |
In Table 3, both N-BK7 and SF2 are glass numbers, and the specific parameters are shown in the references: GB903-87 colorless optical glass.
Example 2: micro-drop type digital PCR instrument
This example provides a digital PCR machine in droplet form comprising the optical detection system of example 1.
Example 3: nucleic acid detection method
The present embodiment provides a nucleic acid detection method, which uses the above-mentioned droplet-type digital PCR instrument to quantitatively detect nucleic acids in a sample to be detected.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. An optical detection system of a droplet type digital PCR is characterized by comprising a light source group, a light source beam combining system, an optical fiber coupling system and a light splitting detection system;
the light source beam combining system is arranged on a light path of emergent light of the light source group and is used for combining the received light;
the optical fiber coupling system is arranged on a light path of emergent light of the light source beam combining system and is used for focusing received light on a micro-droplet position of the micro-droplet flow channel to form a light spot after secondary transmission and shaping, and the light spot focused on the micro-droplet position excites fluorescent dye in the micro-droplet to form light to be detected;
the light splitting detection system is arranged on a light path of emergent light of the optical fiber coupling system and used for splitting the received light to be detected and detecting light intensity of the split light to be detected.
2. The optical detection system of claim 1, wherein the light source combining system comprises a first collimating lens group and a dichroic lens group;
the first collimating lens group is arranged on a light path of emergent light of the light source and is used for collimating the received light;
the dichroic mirror group is arranged on a light path of emergent light of the first collimating lens group, the emergent light of the first collimating lens group is reflected at the dichroic mirror group, and the emergent light reflected by the dichroic mirror group is combined into a beam.
3. The optical inspection system of claim 2, wherein the fiber coupling system includes a coupling lens group, an optical fiber, and a laser focusing lens group;
the coupling lens group is arranged on a light path of emergent light of the dichroic mirror group and is used for coupling the received light;
the input end of the optical fiber is arranged on a light path of emergent light of the coupling lens group and is used for carrying out secondary transmission on the received light;
the laser focusing lens group is arranged on a light path of emergent light at the output end of the optical fiber and is used for shaping received light;
emergent light of the laser focusing lens group is focused on a droplet of the droplet channel to form a light spot, and the light spot focused on the droplet excites fluorescent dye in the droplet to form light to be detected.
4. The optical detection system of claim 3, wherein the spectroscopic detection system comprises a dispersive element, a fluorescence focusing lens group, a slit group, and a detector group;
the dispersion element is arranged on the light path of the light to be measured and is used for dispersing the received light so as to separate the fluorescence with different wavelengths in the light to be measured;
the fluorescence focusing lens group is arranged on a light path of emergent light of the dispersion element and is used for converging received fluorescence with different wavelengths into light beams at different positions;
the position of the slit group corresponds to the emission spectrum position of the fluorescent dye in the microdroplet, and the slit group is used for selecting the emergent light beam of the fluorescent focusing lens group, so that the light to be detected by the detector group is emitted into the detector group.
5. The optical detection system of claim 4 wherein the spectroscopic detection system further comprises a second collimating lens group; the second collimating lens group is arranged on a light path of the light to be measured and is used for collimating the received light.
6. An optical detection system according to claim 4 or 5 wherein the dispersive element is a prism or a grating.
7. A droplet-type digital PCR apparatus, comprising the optical detection system according to any one of claims 1 to 6.
8. A method for detecting nucleic acid, which comprises quantitatively detecting nucleic acid in a sample to be detected using the digital PCR machine of claim 7.
9. Use of the optical detection system of any one of claims 1 to 6 or the digital droplet PCR machine of claim 7 or the nucleic acid detection method of claim 8 for nucleic acid detection.
10. The use of claim 9, wherein the nucleic acid assay is a quantitative nucleic acid assay.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114413944A (en) * | 2022-03-29 | 2022-04-29 | 安徽省国盛量子科技有限公司 | Distributed optical fiber sensor based on quantum dots |
| CN115901702A (en) * | 2022-11-02 | 2023-04-04 | 苏州中科医疗器械产业发展有限公司 | Digital microdroplet quantitative detection system, detection method and medium |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114413944A (en) * | 2022-03-29 | 2022-04-29 | 安徽省国盛量子科技有限公司 | Distributed optical fiber sensor based on quantum dots |
| CN114413944B (en) * | 2022-03-29 | 2022-06-21 | 安徽省国盛量子科技有限公司 | Distributed optical fiber sensor based on quantum dots |
| CN115901702A (en) * | 2022-11-02 | 2023-04-04 | 苏州中科医疗器械产业发展有限公司 | Digital microdroplet quantitative detection system, detection method and medium |
| CN115901702B (en) * | 2022-11-02 | 2024-02-09 | 苏州中科医疗器械产业发展有限公司 | Digital microdroplet quantitative detection system, detection method and medium |
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