CN112147115B - Fluorescence collection device and nucleic acid detection device - Google Patents

Fluorescence collection device and nucleic acid detection device Download PDF

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Publication number
CN112147115B
CN112147115B CN202010899542.2A CN202010899542A CN112147115B CN 112147115 B CN112147115 B CN 112147115B CN 202010899542 A CN202010899542 A CN 202010899542A CN 112147115 B CN112147115 B CN 112147115B
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light
reflecting element
area
light source
reflecting
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CN112147115A (en
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周连群
张芷齐
李树力
姚佳
郭振
李超
张威
李传宇
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Suzhou Institute of Biomedical Engineering and Technology of CAS
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Suzhou Institute of Biomedical Engineering and Technology of CAS
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    • 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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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • 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
    • 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
    • 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
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • 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
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • 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
    • G01N2021/0106—General arrangement of respective parts
    • G01N2021/0112—Apparatus in one mechanical, optical or electronic block
    • 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
    • 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
    • G01N2021/6471—Special filters, filter wheel
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    • 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

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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

The invention provides a fluorescence acquisition device and a nucleic acid detection device, which comprise a light source component, a first reflecting element, a second reflecting element and a third reflecting element, wherein the second reflecting element and the third reflecting element are distributed on a first side and a second side of the light source component and at least respectively receive boundary light of the light source component at the first side and the second side; the first reflecting surface is used for respectively receiving the second light source reflected by the second reflecting element and the third light source reflected by the third reflecting element, correspondingly, the first reflecting surface respectively reflects the second reflected light and the third reflected light, and respectively irradiates the second area and the third area where the area on the pore plate is located, and the second area and the third area are respectively overlapped on the edge areas on two sides of the first area. By increasing the luminous flux of the edge area of the pore plate, the illumination intensity difference between the central area and the edge area of the pore plate of the point light source emitted by the light source component is greatly reduced, the uniform illumination of the pore plate with a large area is improved, and the detection result is more accurate.

Description

Fluorescence collection device and nucleic acid detection device
Technical Field
The invention relates to the technical field of medical detection, in particular to a fluorescence acquisition device and a nucleic acid detection device.
Background
The current real-time fluorescent quantitative PCR technology has great significance in the novel coronavirus detection, and the development of a high-flux, high-sensitivity and high-accuracy real-time nucleic acid molecule fluorescent quantitative detection system has great significance for large-scale epidemic screening. The fluorescent signal real-time optical detection system with high signal-to-noise ratio and high flux is a necessary means for realizing the quantification of nucleic acid molecules.
In the current quantitative detection of nucleic acid molecules, fluorescent dyes can be specifically combined with small grooves of double strands of DNA, and after the fluorescent dyes are combined with double strand DNA, fluorescent signals can be increased hundreds of times. As the PCR product increases, the combination amount of the PCR product and the dye also increases, and the fluorescence signal intensity of the PCR product represents the number of double-stranded DNA molecules, so that in the existing quantitative detection of nucleic acid molecules, the fluorescence signal in the PCR product is collected by a fluorescence collection device, and the PCR yield is reflected by a curve formed by the intensity of the fluorescence signal.
The existing fluorescence acquisition device comprises a camera and an excitation light source component, wherein the light source component is distributed above the central area of the pore plate, a plurality of micropores are formed in the pore plate, and PCR is continuously generated in the micropores. The light source part emits a point light source, the point light source irradiates downwards on the pore plate, fluorescence in the micropores is excited, and the excited fluorescence is imaged and is photographed and collected by the camera.
Because the light source part emits the point light source, the center energy of the point light source is high and the edge energy is low, when the point light source irradiates on the pore plate, the illumination intensity of the micropores irradiated on the center area of the pore plate is larger than the illumination intensity of the micropores irradiated on the edge area of the pore plate, so that the fluorescence excited by the center area and the edge area of the pore plate are different, the fluorescence intensity collected by the camera is large in difference, the yield of PCR (polymerase chain reaction) at different positions on the pore plate cannot be reflected more accurately, and the detection result of the nucleic acid detection device is inaccurate.
Disclosure of Invention
Therefore, the invention aims to solve the technical problem that the light intensity difference of the light source of the existing fluorescence acquisition device in the central area and the edge area of the pore plate is large, so that the detection result is inaccurate, and further provides the nucleic acid detection device.
The invention provides a fluorescence excitation mechanism, which comprises:
a light source unit for emitting a point light source;
a first reflecting element having a first reflecting surface; the first reflecting surface is used for receiving the point light source, and the first area of the first reflecting surface, where the first reflected light reflected by the first reflecting surface irradiates the pore plate, is larger than or equal to the area of the pore plate;
a second reflecting element and a third reflecting element provided between the light source part and the first reflecting element; the second and third reflective elements are distributed at the first and second sides of the light source part and receive at least boundary light of the point light source at the first and second sides, respectively;
the first reflecting surface is used for respectively receiving the second light source reflected by the second reflecting element and the third light source reflected by the third reflecting element, and correspondingly, the first reflecting surface respectively reflects second reflected light and third reflected light;
the second reflected light and the third reflected light respectively irradiate a second area and a third area where the area on the pore plate is located, and are respectively overlapped with edge areas on two sides of the first area.
Further, the light-transmitting plate is arranged on the reflecting path of the first reflecting element, and the area of the light-transmitting plate is larger than or equal to that of the pore plate.
Further, an end of the second reflecting element near the first end of the first reflecting element is aligned with a third end of the light-transmitting plate at a first projection end of the light-transmitting plate;
a second projection end of the end part, close to the first end, of the first reflecting element, of the third reflecting element on the light-transmitting plate extends out of the first projection end and is overlapped with part of the light-transmitting plate;
the protruding portion of the second projection end protruding out of the first projection end is used for receiving the point light source and the second light source to reflect to the edge area where the third end of the light-transmitting plate is located.
Further, the light-transmitting plate further comprises a fourth reflecting element, wherein two ends of the fourth reflecting element are respectively connected to the second end of the first reflecting element and the third end of the light-transmitting plate;
the fourth reflecting element is used for directly receiving the point light source and reflecting fourth reflected light, and the fourth reflected light irradiates an edge area where the third end part of the light-transmitting plate is positioned;
the first reflecting element is obliquely arranged between the third reflecting element and the fourth reflecting element.
Further, the light source component emits light between the boundaries of the first side and the second side at a divergence angle θ, wherein θ includes θ1, θ2, and θ3 distributed adjacently in order from the first side toward the second side;
the second reflecting element is used for receiving part of point light sources near the first side in theta 1, and the first reflecting element is used for receiving all point light sources in theta 2; the third reflecting element is used for receiving all point light sources in theta 3;
the fourth reflecting element is used for receiving the rest part of the point light sources close to the second side in the theta 1.
Further, the first reflecting surface of the first reflecting element is a plane, and the reflecting surfaces of the second reflecting element, the third reflecting element and the fourth reflecting element are respectively a first curved surface, a second curved surface and a third curved surface;
the first curved surface reflects part of the point light sources close to the first side in the theta 1 to the first reflecting surface, then to one side edge area in the pore plate area, and reflects the point light source with the largest part of the divergence angle to the edge end point of the pore plate area, and the point light source with the smallest part of the divergence angle to the inside of the pore plate area;
the second curved surface reflects all the point light sources in the theta 3 to the first reflecting surface and then to the edge area of the other side in the pore plate area, and reflects the point light source with the largest part of divergence angle to the edge end point of the pore plate area, and the point light source with the smallest part of divergence angle to the inside of the pore plate area;
and the third curved surface reflects the rest point light sources in the theta 1, which are close to the second side, to the first reflecting surface, and then to the edge area of one side in the pore plate area, and reflects the point light source with the smallest part of divergence angle to the edge endpoint of the pore plate area, and the point light source with the largest part of divergence angle to the inside of the pore plate area.
Further, the second reflective element and the third reflective element enclose a closed ring-shaped structure;
the light shield is arranged outside the peripheries of the first reflecting element and the light-transmitting plate in a covering mode, an avoidance hole is formed in the light shield, the avoidance hole is clung to one end, close to the first reflecting element, of the second reflecting element and one end, close to the first reflecting element, of the third reflecting element, and the avoidance hole is arranged on the first reflecting element and the light-transmitting plate in a covering mode.
The invention also provides a fluorescence acquisition device, which comprises the fluorescence excitation mechanism; the light-transmitting element is arranged on one side of the first reflecting element, which is opposite to the light-transmitting plate, and is used for allowing fluorescence to transmit only; the image collector and the first reflecting element are distributed on two sides of the light-transmitting element.
Further, a plurality of convex lenses distributed in an array are arranged on the light-transmitting plate, and the convex lenses protrude towards the direction of the first reflecting element and correspond to the micropores on the pore plate one by one.
Further, the light-transmitting element and the first reflecting element are combined into a dichroic mirror.
The invention also provides a nucleic acid detection device, which comprises the fluorescence acquisition device;
the pore plate is arranged opposite to the light-transmitting plate of the collecting device, and the light-transmitting plate is distributed between the pore plate and the first reflecting element.
The technical scheme of the invention has the following advantages:
1. the fluorescence excitation mechanism provided by the invention comprises: a light source unit for emitting a point light source;
a first reflecting element having a first reflecting surface; the first reflecting surface is used for receiving the point light source, and the first area of the first reflecting surface, where the first reflected light reflected by the first reflecting surface irradiates the pore plate, is larger than or equal to the area of the pore plate;
a second reflecting element and a third reflecting element provided between the light source part and the first reflecting element; the second and third reflective elements are distributed at the first and second sides of the light source part and receive at least boundary light of the point light source at the first and second sides, respectively;
the first reflecting surface is used for respectively receiving the second light source reflected by the second reflecting element and the third light source reflected by the third reflecting element, and correspondingly, the first reflecting surface respectively reflects second reflected light and third reflected light;
the second reflected light and the third reflected light respectively irradiate a second area and a third area where the area on the pore plate is located, and are respectively overlapped with edge areas on two sides of the first area. Through the arrangement of the technical scheme, the luminous flux of the edge area of the pore plate is increased, so that the illumination intensity difference between the central area and the edge area of the pore plate of the point light source emitted by the light source component is greatly reduced, the uniform illumination of the pore plate with a large area is improved, and the detection result is more accurate.
2. The fluorescence excitation mechanism provided by the invention further comprises a light-transmitting plate arranged on the reflection path of the first reflection element, wherein the area of the light-transmitting plate is larger than or equal to that of the pore plate. Through the arrangement of the technical scheme, the light-transmitting plate can reflect more light to the edge area of the pore plate as much as possible, and the difference of illumination intensity between the central area and the edge area of the pore plate is improved.
3. According to the fluorescence excitation mechanism provided by the invention, the end part of the second reflecting element, which is close to the first end of the first reflecting element, is aligned with the third end part of the light-transmitting plate at the first projection end of the light-transmitting plate;
a second projection end of the end part, close to the first end, of the first reflecting element, of the third reflecting element on the light-transmitting plate extends out of the first projection end and is overlapped with part of the light-transmitting plate;
the protruding part of the second projection end protruding out of the first projection end is used for receiving the point light source and the second light source so as to reflect to the edge area where the third end part of the light-transmitting plate is located. Through the arrangement of the technical scheme, the third end part of the light-transmitting plate, namely the edge of the light-transmitting plate, which is close to the second reflecting element, can receive enough illumination, and the illumination intensity difference between the central area and the edge area of the pore plate is improved.
4. The fluorescence excitation mechanism provided by the invention further comprises a fourth reflection element, wherein two ends of the fourth reflection element are respectively connected to the second end of the first reflection element and the third end of the light-transmitting plate;
the fourth reflecting element is used for directly receiving the point light source and reflecting fourth reflected light, and the fourth reflected light irradiates an edge area where the third end part of the light-transmitting plate is positioned;
the first reflecting element is obliquely arranged between the third reflecting element and the fourth reflecting element. Through the arrangement of the technical scheme, the third end part of the light-transmitting plate, namely the edge of the light-transmitting plate, which is close to the second reflecting element, can receive enough illumination, and the illumination intensity difference between the central area and the edge area of the pore plate is improved.
5. The invention provides a fluorescence excitation mechanism, wherein the divergence angle of light emitted by a light source component between the boundaries of a first side and a second side is theta, and the theta comprises theta 1, theta 2 and theta 3 which are distributed adjacently in sequence from the first side to the second side;
the second reflecting element is used for receiving part of point light sources near the first side in theta 1, and the first reflecting element is used for receiving all point light sources in theta 2; the third reflecting element is used for receiving all point light sources in theta 3;
the fourth reflecting element is used for receiving the rest part of the point light sources close to the second side in the theta 1. Through the arrangement of the technical scheme, the reflected light in the divergence angle theta 1 is intensively irradiated to the edge of the light-transmitting plate, which is close to the second reflecting element, so that the reflected light in the divergence angle theta 1 is irradiated to the whole receiving surface of the light-transmitting plate, the reflected light in the divergence angle theta 3 is intensively irradiated to the edge of the light-transmitting plate, which is away from the second reflecting element, so that the illumination intensity difference between the central area and the edge area of the pore plate of the point light source emitted by the light source component is greatly reduced, the improvement of uniform illumination of the pore plate with a large area is realized, and the detection result is more accurate.
6. The first reflecting surface of the first reflecting element is a plane, and the reflecting surfaces of the second reflecting element, the third reflecting element and the fourth reflecting element are respectively a first curved surface, a second curved surface and a third curved surface; the first curved surface reflects part of the point light sources close to the first side in the theta 1 to the first reflecting surface, then to one side edge area in the pore plate area, and reflects the point light source with the largest part of the divergence angle to the edge end point of the pore plate area, and the point light source with the smallest part of the divergence angle to the inside of the pore plate area;
the second curved surface reflects all the point light sources in the theta 3 to the first reflecting surface and then to the edge area of the other side in the pore plate area, and reflects the point light source with the largest part of divergence angle to the edge end point of the pore plate area, and the point light source with the smallest part of divergence angle to the inside of the pore plate area;
and the third curved surface reflects the rest point light sources in the theta 1, which are close to the second side, to the first reflecting surface, and then to the edge area of one side in the pore plate area, and reflects the point light source with the smallest part of divergence angle to the edge endpoint of the pore plate area, and the point light source with the largest part of divergence angle to the inside of the pore plate area. Through the arrangement of the technical scheme, the curved surface is arranged to ensure that the reflecting surface is wider and more uniform, and the improvement of uniform illumination of the pore plate with a large area can be realized.
7. The second reflecting element and the third reflecting element enclose a closed annular structure;
the light shield is arranged outside the peripheries of the first reflecting element and the light-transmitting plate in a covering mode, an avoidance hole is formed in the light shield, the avoidance hole is clung to one end, close to the first reflecting element, of the second reflecting element and one end, close to the first reflecting element, of the third reflecting element, and the avoidance hole is arranged on the first reflecting element and the light-transmitting plate in a covering mode. Through the arrangement of the technical scheme, the interference influence of ambient stray light can be reduced on one hand, so that the accuracy of reflecting the quantity of PCR reaction products by a fluorescence intensity signal is improved, the signal-to-noise ratio is improved, the detection accuracy is improved, and the loss of light energy can be greatly reduced on the other hand.
8. The fluorescence acquisition device provided by the invention comprises the fluorescence excitation mechanism; the light-transmitting element is arranged on one side of the first reflecting element, which is opposite to the light-transmitting plate, and is used for allowing fluorescence to transmit only; the image collector and the first reflecting element are distributed on two sides of the light-transmitting element. Through the arrangement of the technical scheme, large-area biomolecule fluorescence signal collection and detection can be realized.
9. According to the fluorescence acquisition device provided by the invention, the plurality of convex lenses distributed in an array are arranged on the light-transmitting plate, and the convex lenses protrude towards the direction of the first reflecting element and correspond to micropores on the pore plate one by one. Through the setting of above-mentioned technical scheme for the fluorescent light that diverges can be converged by the convex lens on the light-transmitting plate, is convenient for collect by image acquisition ware, realizes that large tracts of land biomolecule fluorescence signal collects the detection, thereby improves the collection efficiency of marginal micropore fluorescence, makes the testing result more accurate.
10. According to the fluorescence acquisition device provided by the invention, the light-transmitting element and the first reflecting element are combined into a whole to form a dichroic mirror. By the arrangement of the technical scheme, the light rays emitted by the point light source of the light source component can be totally reflected, and the excited fluorescence can be transmitted so as to be collected by the image collector.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the light path of a fluorescence excitation apparatus according to the present invention;
FIG. 2 is a schematic view of an optical path shown by a divergence angle θ1 from the light source component of FIG. 1;
FIG. 3 is a schematic view of an optical path shown by a divergence angle θ3 from the light source assembly of FIG. 1;
FIG. 4 is a schematic view of an optical path shown by a divergence angle θ2 from the light source assembly of FIG. 1;
FIG. 5 is a schematic view of the light path of the fluorescence collection device of FIG. 1;
FIG. 6 is a whole frame diagram of nucleic acid detection fluorescence imaging in the present invention;
FIG. 7 is a schematic view of a light-transmitting panel;
reference numerals:
in the figure: 100-nucleic acid detecting device, 200-orifice plate, 1-light source part, 2-light-transmitting plate, 21-convex lens, 3-image collector, 4-dichroic mirror, 5-light shield, 51-avoidance hole, 6-second reflecting element, 61-first curved surface, 7-third reflecting element, 71-second curved surface, 8-fourth reflecting element, 81-third curved surface, 9-optical filter.
Detailed Description
The following description of the embodiments of the present invention will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the description of the present invention, it should be noted that the directions or positional relationships indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it should be noted that, unless explicitly specified and limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be either fixedly connected, detachably connected, or integrally connected, for example; can be mechanically or electrically connected; the two components can be directly connected or indirectly connected through an intermediate medium, or can be communicated inside the two components, or can be connected wirelessly or in a wired way. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
In addition, the technical features of the different embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
Examples
Referring to fig. 1 to 7, the present invention provides a nucleic acid detecting apparatus 100 including a fluorescence collection apparatus including a fluorescence excitation mechanism.
The fluorescence excitation mechanism comprises: a light source unit 1 for emitting a point light source;
a first reflecting element having a first reflecting surface; the first reflecting surface is used for receiving the point light source, and the first area of the area where the first reflected light reflected by the first reflecting surface irradiates the aperture plate 200 is larger than or equal to the area of the aperture plate 200;
a second reflecting element 6 and a third reflecting element 7 provided between the light source unit 1 and the first reflecting element; the second and third reflective elements 6 and 7 are distributed at the first and second sides of the light source part 1 and receive at least boundary light of the point light source at the first and second sides, respectively;
the first reflecting surface is used for respectively receiving the second light source reflected by the second reflecting element 6 and the third light source reflected by the third reflecting element 7, and correspondingly the first reflecting surface respectively reflects second reflected light and third reflected light;
the second and third reflected lights respectively irradiate the second and third areas where the areas on the orifice plate 200 are located, and respectively overlap the edge areas on both sides of the first area. Through the arrangement of the technical scheme, the luminous flux of the edge area of the pore plate 200 is increased, so that the illumination intensity difference between the central area and the edge area of the pore plate 200 of the point light source emitted by the light source component 1 is greatly reduced, the uniform illumination of the pore plate 200 with a large area is improved, and the detection result is more accurate.
In this embodiment, the fluorescence excitation mechanism further includes a light-transmitting plate 2 disposed on the reflection path of the first reflection element, where the area of the light-transmitting plate 2 is greater than or equal to the area of the aperture plate 200. Through the arrangement of the above technical scheme, the light-transmitting plate 2 can reflect more light to the edge area of the aperture plate 200 as much as possible, and the difference of illumination intensity between the central area and the edge area of the aperture plate 200 is improved.
Further, referring to fig. 2 and 3, an end portion of the second reflecting element 6 near the first end of the first reflecting element is aligned with the third end portion of the light-transmitting plate 2 at the first projection end of the light-transmitting plate 2;
a second projection end of the end part, close to the first end, of the first reflecting element 7, on the light-transmitting plate 2 extends out of the first projection end and is overlapped with part of the light-transmitting plate 2;
the protruding portion of the second projection end protruding from the first projection end is configured to receive the point light source and the second light source, so as to reflect to an edge area where the third end of the light-transmitting plate 2 is located. Through the above technical scheme, the third end of the light-transmitting plate 2, that is, the edge of the light-transmitting plate 2 near the second reflecting element 6 can receive enough illumination, so that the difference of illumination intensity between the central area and the edge area of the aperture plate 200 is improved.
The fluorescence excitation mechanism further comprises a fourth reflection element 8 with two ends respectively connected to the second end of the first reflection element and the third end of the light-transmitting plate 2;
the fourth reflecting element 8 is configured to directly receive the point light source and reflect fourth reflected light, where the fourth reflected light irradiates an edge area where the third end of the light-transmitting plate 2 is located;
the first reflecting element is obliquely arranged between the third reflecting element 7 and the fourth reflecting element 8. Through the arrangement of the above technical solution, the third end portion of the light-transmitting plate 2, that is, the edge of the light-transmitting plate 2 near the second reflecting element 6, can receive enough illumination, so as to improve the difference of illumination intensity between the central area and the edge area of the aperture plate 200.
As shown in fig. 1, the light source part 1 emits light at a divergence angle θ between boundaries of the first side and the second side, the θ including θ1, θ2, and θ3 distributed adjacently in order from the first side toward the second side;
the second reflecting element 6 is used for receiving part of point light sources near the first side in theta 1, and the first reflecting element is used for receiving all point light sources in theta 2; the third reflecting element 7 is used for receiving all point light sources in theta 3;
the fourth reflecting element 8 is configured to receive a remaining portion of the point light sources within θ1 near the second side. The reflected light in the divergence angle theta 1 is intensively irradiated to the edge of the light-transmitting plate 2 close to the second reflecting element 6, the reflected light in the divergence angle theta 1 is irradiated to the whole receiving surface of the light-transmitting plate 2, the reflected light in the divergence angle theta 3 is intensively irradiated to the edge of the light-transmitting plate 2 away from the second reflecting element 6, the illumination intensity difference between the central area and the edge area of the aperture plate 200 of the point light source emitted by the light source component 1 is greatly reduced, the uniform illumination of the aperture plate 200 with a large area is improved, and the detection result is more accurate.
The first reflecting surface of the first reflecting element is a plane, and the reflecting surfaces of the second reflecting element 6, the third reflecting element 7 and the fourth reflecting element 8 are respectively a first curved surface 61, a second curved surface 71 and a third curved surface 81; the first curved surface 61 reflects a part of the point light sources near the first side in the θ1 to the first reflecting surface, and then to an edge region of one side in the aperture plate region, and reflects the point light source with the largest part of the divergence angle to an edge endpoint of the aperture plate region, and reflects the point light source with the smallest part of the divergence angle to the inside of the aperture plate region;
the second curved surface 71 reflects all the point light sources in the theta 3 to the first reflecting surface, and then to the other side edge area in the aperture plate area, and reflects the point light source with the largest part of the divergence angle to the edge end point of the aperture plate area, and the point light source with the smallest part of the divergence angle to the inside of the aperture plate area;
the third curved surface 81 reflects the rest of the point light sources near the second side in the θ1 to the first reflecting surface, and then to an edge region of one side in the aperture plate region, and reflects the point light source with the smallest part of the divergence angle to an edge endpoint of the aperture plate region, and reflects the point light source with the largest part of the divergence angle to the inside of the aperture plate region. Through the arrangement of the technical scheme, the curved surface enables the reflecting surface to be wider and more uniform, and the uniform illumination of the large-area pore plate 200 can be improved.
The second reflecting element 6 and the third reflecting element 7 enclose a closed ring-shaped structure; the light shield 5 is covered outside the peripheries of the first reflecting element and the light-transmitting plate 2, the light shield 5 is provided with an avoidance hole 51, the avoidance hole 51 is clung to one ends of the second reflecting element 6 and the third reflecting element 7, which are close to the first reflecting element, and the light shield is covered on the first reflecting element and the light-transmitting plate 2. Through the arrangement of the technical scheme, the interference influence of ambient stray light can be reduced on one hand, so that the accuracy of reflecting the quantity of PCR reaction products by a fluorescence intensity signal is improved, the signal-to-noise ratio is improved, the detection accuracy is improved, and the loss of light energy can be greatly reduced on the other hand.
In this embodiment, the fluorescence excitation mechanism further has a filter 9 located between the second reflection element 6 and the third reflection element 7 and close to the light source component 1, so that light in an excitation band of the excitation light source light, which is not the fluorescent dye, can be filtered, and background interference of a fluorescence signal is reduced, thereby improving accuracy of reflecting the PCR reaction product amount by the fluorescence intensity signal, improving signal-to-noise ratio, and improving detection accuracy.
The fluorescence acquisition device comprises the fluorescence excitation mechanism;
a light-transmitting element, which is arranged on one side of the first reflecting element facing away from the light-transmitting plate 2, and is used for allowing only fluorescence to transmit;
the image collector 3 and the first reflecting element are distributed on two sides of the light transmitting element. Through the arrangement of the technical scheme, large-area biomolecule fluorescence signal collection and detection can be realized.
Specifically, referring to fig. 7, a plurality of convex lenses 21 are disposed on the light-transmitting plate 2 in an array, and the convex lenses 21 protrude toward the first reflective element and are in one-to-one correspondence with the micro holes on the aperture plate 200. Through the setting of above-mentioned technical scheme for the fluorescent light that diverges can be converged by convex lens 21 on the light-transmitting plate 2, is convenient for collect by image collector 3, realizes that large tracts of land biomolecule fluorescence signal collects the detection, thereby improves the collection efficiency of marginal micropore fluorescence, makes the testing result more accurate.
In this embodiment, the light-transmitting element and the first reflecting element are combined into a dichroic mirror 4. So that the light emitted from the point light source of the light source unit 1 can be totally reflected and the excited fluorescent light can be transmitted to be collected by the image collector 3.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims (9)

1. A fluorescence excitation mechanism, comprising:
a light source unit for emitting a point light source;
a first reflecting element having a first reflecting surface; the first reflecting surface is used for receiving the point light source, and the first area of the first reflecting surface, where the first reflected light reflected by the first reflecting surface irradiates the pore plate, is larger than or equal to the area of the pore plate;
a second reflecting element and a third reflecting element provided between the light source part and the first reflecting element; the second and third reflective elements are distributed at the first and second sides of the light source part and receive at least boundary light of the point light source at the first and second sides, respectively;
the first reflecting surface is used for respectively receiving the second light source reflected by the second reflecting element and the third light source reflected by the third reflecting element, and correspondingly, the first reflecting surface respectively reflects second reflected light and third reflected light;
the second reflected light and the third reflected light respectively irradiate a second area and a third area where the area on the pore plate is located, and are respectively overlapped with edge areas on two sides of the first area;
the light-transmitting plate is arranged on the reflection path of the first reflection element, and the area of the light-transmitting plate is more than or equal to that of the pore plate;
the light-transmitting plate further comprises a second reflecting element, wherein two ends of the second reflecting element are respectively connected to the second end of the first reflecting element and the third end of the light-transmitting plate;
the fourth reflecting element is used for directly receiving the point light source and reflecting fourth reflected light, and the fourth reflected light irradiates an edge area where the third end part of the light-transmitting plate is positioned; the third end part of the light-transmitting plate is close to the edge of the second reflecting element;
the first reflecting element is obliquely arranged between the third reflecting element and the fourth reflecting element.
2. The excitation mechanism of claim 1, wherein an end of the second reflective element proximate the first end of the first reflective element is aligned with a third end of the light transmissive plate at a first projection end of the light transmissive plate;
a second projection end of the end part, close to the first end, of the first reflecting element, of the third reflecting element on the light-transmitting plate extends out of the first projection end and is overlapped with part of the light-transmitting plate;
the protruding portion of the second projection end protruding out of the first projection end is used for receiving the point light source and the second light source to reflect to the edge area where the third end of the light-transmitting plate is located.
3. The excitation mechanism of claim 2, wherein the light source component emits light at a divergence angle θ between boundaries of the first side and the second side, the θ including θ1, θ2, θ3 distributed adjacently in order from the first side toward the second side;
the second reflecting element is used for receiving part of point light sources near the first side in theta 1, and the first reflecting element is used for receiving all point light sources in theta 2; the third reflecting element is used for receiving all point light sources in theta 3;
the fourth reflecting element is used for receiving the rest part of the point light sources close to the second side in the theta 1.
4. The fluorescence excitation mechanism of claim 3, wherein the first reflective surface of the first reflective element is planar, and the reflective surfaces of the second, third, and fourth reflective elements are respectively first, second, and third curved surfaces;
the first curved surface reflects part of the point light sources close to the first side in the theta 1 to the first reflecting surface, then to one side edge area in the pore plate area, and reflects the point light source with the largest part of the divergence angle to the edge end point of the pore plate area, and the point light source with the smallest part of the divergence angle to the inside of the pore plate area;
the second curved surface reflects all the point light sources in the theta 3 to the first reflecting surface and then to the edge area of the other side in the pore plate area, and reflects the point light source with the largest part of divergence angle to the edge end point of the pore plate area, and the point light source with the smallest part of divergence angle to the inside of the pore plate area;
and the third curved surface reflects the rest point light sources in the theta 1, which are close to the second side, to the first reflecting surface, and then to the edge area of one side in the pore plate area, and reflects the point light source with the smallest part of divergence angle to the edge endpoint of the pore plate area, and the point light source with the largest part of divergence angle to the inside of the pore plate area.
5. The excitation mechanism of fluorescence according to any one of claims 1-4, wherein the second reflective element and the third reflective element enclose a closed ring structure;
the light shield is arranged outside the peripheries of the first reflecting element and the light-transmitting plate in a covering mode, an avoidance hole is formed in the light shield, the avoidance hole is clung to one end, close to the first reflecting element, of the second reflecting element and one end, close to the first reflecting element, of the third reflecting element, and the avoidance hole is arranged on the first reflecting element and the light-transmitting plate in a covering mode.
6. A fluorescence acquisition device, comprising:
an excitation mechanism for fluorescence according to any one of claims 1 to 5;
the light-transmitting element is arranged on one side of the first reflecting element, which is opposite to the light-transmitting plate, and is used for allowing fluorescence to transmit only;
the image collector and the first reflecting element are distributed on two sides of the light-transmitting element.
7. The fluorescence collection device according to claim 6, wherein a plurality of convex lenses are arranged on the light-transmitting plate in an array, and the convex lenses protrude toward the first reflecting element and correspond to the micro holes on the aperture plate one by one.
8. The fluorescence collection device of claim 6 or 7, wherein the light transmissive element and the first reflective element are combined into a dichroic mirror.
9. A nucleic acid detecting apparatus, comprising:
a fluorescence acquisition device according to any one of claims 6 to 8;
the pore plate is arranged opposite to the light-transmitting plate of the collecting device, and the light-transmitting plate is distributed between the pore plate and the first reflecting element.
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