WO2024251172A1 - 一种荧光光源装置、荧光检测光路系统及pcr检测系统 - Google Patents

一种荧光光源装置、荧光检测光路系统及pcr检测系统 Download PDF

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Publication number
WO2024251172A1
WO2024251172A1 PCT/CN2024/097617 CN2024097617W WO2024251172A1 WO 2024251172 A1 WO2024251172 A1 WO 2024251172A1 CN 2024097617 W CN2024097617 W CN 2024097617W WO 2024251172 A1 WO2024251172 A1 WO 2024251172A1
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Prior art keywords
fluorescence
optical fiber
detection
temperature
carrier
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PCT/CN2024/097617
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English (en)
French (fr)
Inventor
徐强
韦嘉
赵蒙
徐涛
蓝银涛
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Guangzhou National Laboratory
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Guangzhou National Laboratory
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Priority claimed from CN202321422229.5U external-priority patent/CN220376678U/zh
Priority claimed from CN202310661917.5A external-priority patent/CN119086434A/zh
Application filed by Guangzhou National Laboratory filed Critical Guangzhou National Laboratory
Publication of WO2024251172A1 publication Critical patent/WO2024251172A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • 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
    • C12M1/02—Apparatus for enzymology or microbiology with agitation means; with heat exchange means
    • 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
    • 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
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • 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
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • 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

Definitions

  • the present application relates to the field of in vitro diagnostic technology, and in particular to a fluorescent light source device, a fluorescent detection optical path system and a PCR detection system.
  • PCR polymerase chain reaction
  • the reaction sample Before PCR amplification, the reaction sample needs to be placed in a carrier, wherein the reaction sample is composed of samples such as collected throat swabs or nasal swabs and reagents for PCR amplification.
  • the reaction sample Before PCR amplification, the reaction sample needs to be heated by a heater and cooled by a cooling mechanism, so that the reaction sample is cycled in high temperature denaturation, low temperature annealing and suitable temperature extension stages.
  • fluorescence detection is often used for reaction samples. During the detection, excitation light is emitted to the reaction sample. After the reaction sample interacts with the excitation light, another fluorescent signal is generated. By analyzing the intensity of the fluorescent signal, the reaction sample can be detected, such as detecting whether the reaction sample contains a certain DNA fragment.
  • the fluorescence detection optical path in the prior art is mainly aimed at the carrier with a larger area for the reaction sample to receive the excitation light.
  • the interference signal received by the detector is high, but because the area receiving the excitation light is large, the fluorescence signal generated is high. Therefore, the fluorescence signal received by the detector is high, and the interference signal has little effect on the detection result, so a more accurate detection result can be obtained.
  • the fluorescence signal generated by the reaction sample is low, it is difficult for the detector to obtain an accurate detection result.
  • the carrier is a tube structure such as an ep tube structure, the diameter of the area of the carrier for holding the reaction sample is large, the heat equalization speed is slow, and the temperature rise and fall speed of the reaction sample is slow.
  • the temperature of the reaction sample needs to be detected.
  • the temperature detection method commonly used in the prior art has low accuracy, resulting in inaccurate detection results.
  • One purpose of the present application is to provide a fluorescent light source device to solve one of the above technical problems.
  • a fluorescent light source device in a first aspect, comprising:
  • At least two fluorescence emission units wherein the at least two fluorescence emission units are used to emit excitation light
  • control unit wherein the control unit controls the at least two fluorescent emission units to respectively control the at least two fluorescent emission units in different time periods. Emit excitation light.
  • the fluorescence emission unit includes a light source and an excitation optical fiber, and the excitation optical fiber is used to transmit the excitation light emitted by the light source.
  • the wavelengths of the excitation lights emitted by the at least two fluorescence emission units are different from each other.
  • the excitation light is monochromatic light or polychromatic light.
  • Another object of the present application is to provide a PCR detection system to solve one of the above-mentioned technical problems.
  • a fluorescence detection optical path system comprises the fluorescence light source device and a fluorescence detection unit.
  • the fluorescence detection unit includes at least two fluorescence transmission light paths, and the at least two fluorescence emission units correspond one-to-one to the at least two fluorescence transmission light paths.
  • each of the fluorescence transmission light paths includes a filter that allows a preset fluorescence signal to pass through.
  • the fluorescence detection unit further includes a detector, and the plurality of fluorescence transmission light paths are all connected to the detector.
  • the number of the detector is one, and the detector is electrically connected to the control unit to record the intensity of the fluorescence signal in different time periods.
  • the fluorescence detection optical path system also includes a fiber optic holder
  • the fluorescence transmission optical path includes a collecting optical fiber, an end of the excitation optical fiber of the fluorescence light source device that emits the excitation light, and an end of the collecting optical fiber that injects the fluorescence signal form a fiber group
  • the fiber group is arranged in the fiber optic holder
  • the optical fibers in the fiber optic holder are arranged along a first direction, which is the radial direction of the optical fiber, so that the emitting end of the collecting optical fiber and the injecting end of the excitation optical fiber are arranged flat in the fiber optic holder.
  • At least the two fluorescence transmission light paths and the at least two fluorescence emission units form at least two groups of optical fiber groups, and the at least two groups of optical fiber groups are arranged in sequence along the first direction.
  • a group of the optical fiber groups includes at least two collecting optical fibers, and at least one collecting optical fiber is arranged on both sides of the first direction of the excitation optical fiber in a group of the optical fiber groups.
  • the detector includes a silicon photomultiplier tube, a photon detector or a photomultiplier tube.
  • Another object of the present application is to provide a PCR detection system to solve one of the above-mentioned technical problems.
  • the third aspect of this application adopts the following technical solution:
  • a PCR detection system comprises a carrier and the fluorescence detection optical path system, wherein the fluorescence detection optical path system is used to detect a reaction sample in the carrier.
  • the carrier includes a first wall and a second wall arranged opposite to each other, and a side wall arranged between the first wall and the second wall, the first wall, the second wall and the side wall form a accommodating cavity, the accommodating cavity and/or the carrier are flat structures, at least part of the side wall is light transmissive, and the fluorescence detection optical path system detects the reaction sample through the light-transmissive side wall.
  • the fluorescent emission unit is disposed on one side or at least both sides of the carrier.
  • the light-transmissive sidewall material is polydimethylsiloxane, polypropylene or polycarbonate.
  • the carrier includes at least one built-in heater, and the first wall and/or the second wall is the built-in heater.
  • the built-in heater includes a heating element.
  • the built-in heater includes at least two independently controlled heating elements.
  • the built-in heater further includes a temperature calibration unit for reflecting the temperature of the heating element.
  • the built-in heater further includes a rapid conduction portion, and the rapid conduction portion is used to conduct the heat of the heating element to the temperature calibration portion.
  • the PCR detection system may further include a resistance detection element, and the resistance detection element is used to detect the resistance of the heating element.
  • the flat structure means that a dimension of the accommodating cavity or the carrier in a direction perpendicular to its thickness direction is larger than a dimension in its thickness direction.
  • the ratio of the dimension of the accommodating cavity or the carrier in a direction perpendicular to its thickness direction to its thickness direction is greater than 5:1.
  • the ratio of the dimensions is 50:1 to 100:1.
  • the technical solution provided by the present application is that the fluorescence emission unit is independently arranged, and the excitation light transmitted in the fluorescence emission unit will not be reflected into the optical path of the fluorescence signal, thereby greatly reducing the amount of excitation light in the optical path of the fluorescence signal, greatly reducing the background in the fluorescence signal, and improving the detection accuracy.
  • the control unit can control the opening of different fluorescence emission units in sequence in different time periods, such as the first fluorescence emission unit works within the 1st to 2s from the start of detection, and the second fluorescence emission unit works within the 2nd to 3s from the start of detection, and so on. Only one fluorescence emission unit works in a time period, and the control unit controls the conduction of the fluorescence emission units of each channel to achieve millisecond-level switching of the excitation light channel, which can achieve rapid detection of reaction samples.
  • the fluorescence detection unit is used to detect fluorescence signals.
  • the fluorescence emission unit is completely separated from the fluorescence detection unit that receives the excitation signal, which reduces the background of the fluorescence signal and improves the detection sensitivity of the fluorescence detection optical path system.
  • the fluorescence emission unit is completely separated from the fluorescence detection unit, which reduces the optical elements in the fluorescence detection unit, such as dichroic mirrors, etc., thereby reducing costs and improving the detection efficiency of fluorescence.
  • FIG. 1a is a schematic diagram of the structure of a carrier provided in an embodiment of the present application.
  • Fig. 1b is a cross-sectional view of the carrier at C-C in Fig. 1a;
  • Fig. 2 is a cross-sectional view of the carrier at D-D in Fig. 1b;
  • FIG3a is a schematic structural diagram of a fluorescence detection optical path system in the prior art
  • FIG3b is a schematic diagram of the structure of a fluorescence detection optical path system provided in an embodiment of the present application.
  • FIG3c is a schematic diagram of the structure of the optical fiber holder provided in an embodiment of the present application.
  • FIG3d is a schematic structural diagram of the optical fiber holder provided by an embodiment of the present application from another perspective;
  • FIG4 is a schematic diagram of the structure of a fluorescence detection unit in the prior art
  • FIG5 is a schematic diagram of the structure of another fluorescence detection unit provided in an embodiment of the present application.
  • FIG6a is a schematic structural diagram of an optical fiber group disposed on one side of a carrier provided in an embodiment of the present application;
  • FIG6b is an amplification curve obtained by the PCR detection system of this embodiment.
  • FIG7 is a schematic structural diagram of a carrier provided in an embodiment of the present application in which optical fiber groups are arranged on both sides;
  • FIG8 is a schematic diagram of a carrier provided in an embodiment of the present application being heated by an external heater
  • FIG9 is a schematic diagram of the internal structure of an external heater, a cooling assembly and a carrier provided in an embodiment of the present application
  • FIG10 is a schematic diagram of the internal structure of a carrier provided in an embodiment of the present application including two built-in heaters;
  • FIG11 is a graph of temperature calibration provided in an embodiment of the present application.
  • FIG. 12 is a schematic diagram of the structure of the carrier provided in an embodiment of the present application.
  • Fluorescence emission unit 11. Excitation optical fiber; 12. Light source; 2. Fluorescence detection unit; 21. Fluorescence transmission optical path; 211. Filter; 212. Collection optical fiber; 213. Collimating lens; 22. Detector; 3. Optical fiber holder; 31. Optical fiber group; 32. Holder body; 33. Flat groove; 4. Carrier; 41. Accommodating chamber; 42. Side wall; 43. First wall; 44. Second wall; 45. Built-in heater; 521, cooling assembly; 5221, flow channel; 522, external heater; 91. Heating element; 92. Upper conducting component; 921. Heat-saturating layer; 93. Temperature calibration unit; 94. Fast conducting unit; 941. SMD; 942.
  • a first feature being “above” or “below” a second feature may include the first and second features being in direct contact, or the first and second features being in contact not directly but through another feature between them.
  • “Above” and “above” include the first feature being directly above and obliquely above the second feature, or simply means that the first feature is higher in level than the second feature.
  • connection should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements.
  • connection can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements.
  • this embodiment provides a carrier 4, which includes a first wall 43 and a second wall 44 arranged opposite to each other, and a side wall 42 arranged between the first wall 43 and the second wall 44.
  • the first wall 43, the second wall 44 and the side wall 42 form a accommodating cavity 41, and the accommodating cavity 41 and/or the carrier 4 are flat structures, and at least a portion of the side wall 42 is light-transmissive.
  • the flat structure may refer to that the dimension of the accommodating cavity 41 or the carrier 4 in the thickness direction (i.e., the direction in which the first wall 43 and the second wall 44 are arranged) is smaller than the dimension in the direction perpendicular to the thickness direction.
  • the ratio of the dimension in the direction perpendicular to the thickness direction to the dimension in the thickness direction is greater than 5:1. More preferably, the dimension of the carrier 4 or the accommodating cavity 41 in the thickness direction is much smaller than the dimension in the direction perpendicular to the thickness direction, such as the dimension ratio is 50:1 to 100:1. As an example, the dimension ratio is 90:1.
  • the accommodating cavity 41 is a cuboid, and the ratio of the length and thickness of the cuboid may be greater than 5:1, such as 90:1.
  • the dimension of the accommodating cavity 41 in the thickness direction may be 0.3-1.0 mm.
  • the thickness of the accommodating cavity 41 is 0.3-0.6 mm, and the width and length of the accommodating cavity 41 are about 10 mm and 20 mm, respectively, wherein the arrangement direction of the first wall 43 and the second wall 44 in the thickness direction.
  • the accommodating cavity 41 can also be a cylindrical structure, with a diameter-to-thickness ratio greater than 5:1, such as a thickness of 0.3-1.0 mm and a diameter of 5-20 mm.
  • the cross-section of the accommodating cavity 41 can be a polygon or an ellipse, etc.
  • the cross-section of the accommodating cavity 41 can be a polygon or an ellipse, etc.
  • At least one side of the accommodating chamber 41 can be heated to heat the reaction sample in the accommodating chamber 41.
  • the first wall 43 and the second wall 44 of the carrier 4 are both provided with an external heater 522 as shown in FIG8 or as shown in FIG10, the first wall 43 and the second wall 44 are built-in heaters 45, and the external heater 522 or the built-in heater 45 heats the reaction sample, thereby achieving rapid temperature rise and fall of the reaction sample and rapid nucleic acid detection.
  • the side wall 42 of the accommodating cavity 41 is light-transmissive, and the fluorescence detection optical path system 100 detects the reaction sample through the light-transmissive side wall 42 of the carrier 4, which not only meets the need for rapid temperature rise and fall, but also can accurately complete the detection.
  • the fluorescence detection optical path in the prior art is an integral structure.
  • the excitation light path 200 and the fluorescence signal light path 201 are transmitted through an optical fiber, so that the excitation light emitted by the light source 205 enters the fluorescence signal light path 201 after being reflected by the optical fiber.
  • a dichroic mirror 203 and a filter 204 are arranged on the path of the optical fiber transmission. Both the dichroic mirror 203 and the filter 204 allow the fluorescence signal to pass through, and filter the excitation light in the fluorescence signal light path 201.
  • the dichroic mirror 203 and the filter 204 do not filter the excitation light 100%, they also cannot allow the fluorescence signal to pass 100%, that is, the fluorescence signal will be attenuated when passing through the dichroic mirror 203 and the filter 204, and part of the filtered excitation light will enter the detection circuit 206 for detecting the fluorescence signal.
  • the area of the reaction sample excited by the excitation light is large, the number of fluorescence signals generated is also large. Therefore, the excitation light passing through the filter 204 and the attenuated fluorescence signal have little effect on the detection result, and a relatively accurate detection result can be obtained.
  • the thickness of the accommodating cavity 41 in this embodiment is thin (0.3-0.6 mm), and when fluorescence detection is performed through the side wall 42 of the accommodating cavity 41, the number of reaction samples that can receive the excitation light is greatly reduced. Therefore, the number of fluorescence signals excited each time is about one order of magnitude less. If the existing fluorescence detection optical path is used for detection, the signal detected by the detection circuit is low, and the excitation light passing through the filter 211 will drown the fluorescence signal, which will lead to large errors in the detection, and even the fluorescence detection circuit cannot detect the fluorescence signal.
  • the fluorescent light source device includes a control unit and at least two fluorescent emission units 1, and at least two fluorescent emission units 1 are used to emit excitation light, and the excitation light is monochromatic light or polychromatic light, such as polychromatic light can be white light.
  • the control unit controls at least two fluorescent emission units 1 to emit excitation light in different time periods respectively, so as to allow the reaction sample to receive only one kind of excitation light at the same time.
  • the control unit is connected to the fluorescent emission unit 1 by a wire, and the wire can enable the control unit to control the fluorescent emission unit 1 without delay.
  • the control unit and the fluorescent emission unit 1 can also be connected wirelessly by Bluetooth or other wireless means.
  • control unit can be a centralized or distributed control unit.
  • control unit can be a single single-chip microcomputer, or it can be composed of multiple distributed single-chip microcomputers.
  • the control program can be run in the single-chip microcomputer to control each component to realize its function.
  • the fluorescence emission unit 1 is independently arranged, and the excitation light transmitted in the fluorescence emission unit 1 will not be reflected into the optical path of the fluorescence signal, thereby greatly reducing the amount of excitation light in the optical path of the fluorescence signal, greatly reducing the background in the fluorescence signal, and improving the detection accuracy.
  • the control unit can control the opening of different fluorescence emission units 1 in sequence in different time periods, such as the first fluorescence emission unit 1 works within the 1st-2s from the start of the detection, and the second fluorescence emission unit 1 works within the 2nd-3s from the start of the detection, and so on.
  • Only one fluorescence emission unit 1 works in a time period, and the control unit controls the conduction of the fluorescence emission units 1 of each channel to achieve millisecond-level switching of the excitation light channel, which can achieve rapid detection of the reaction sample.
  • the excitation light is monochromatic light
  • a fluorescence emission unit 1 only needs to emit one excitation light, so that the wavelength of the excitation light is single in a time period, and the type of the fluorescence signal is single.
  • other fluorescence signals will not generate noise, which can further improve the accuracy of the detection result.
  • the excitation light is monochromatic light, and the wavelengths of the excitation lights emitted by at least two fluorescence emission units 1 are different from each other, so as to improve the utilization rate of the fluorescence emission unit 1.
  • a spare fluorescence emission unit 1 can also be provided, such as at least two fluorescence emission units 1.
  • the wavelengths of the excitation lights emitted by the two fluorescence emission units 1 are the same.
  • the fluorescence emission unit 1 includes a light source 12 and an excitation optical fiber 11.
  • the light source 12 can be an LED or the like.
  • the light source 12 is used to emit excitation light, and the light source 12 is electrically connected to the control unit.
  • the excitation optical fiber 11 is used to transmit the excitation light emitted by the light source 12 to transmit the excitation light to the reaction sample.
  • the light source 12 and the reaction sample are connected by the excitation optical fiber 11.
  • the excitation optical fiber 11 is convenient for isolating the heat source, making the experimental data more stable, and the connection between the excitation optical fiber 11 and the light source 12 is simple, which is conducive to earthquake resistance.
  • one light source 12 corresponds to one excitation optical fiber 11, and each excitation channel has a separate optical fiber, which is easy to couple and has small light loss.
  • This embodiment also provides a fluorescence detection optical path system 100, which includes the above-mentioned fluorescence light source device and the fluorescence detection unit 2.
  • the fluorescence detection unit 2 is used to detect the fluorescence signal, and the fluorescence emission unit 1 is completely separated from the fluorescence detection unit 2 that receives the excitation signal, which reduces the background of the fluorescence signal and improves the detection sensitivity of the fluorescence detection optical path system 100.
  • the fluorescence emission unit 1 is completely separated from the fluorescence detection unit 2, which reduces the optical elements in the fluorescence detection unit 2, such as the dichroic mirror, etc., which reduces the cost and improves the detection efficiency of the fluorescence.
  • the fluorescence detection unit 2' includes a detector 22', a fluorescence transmission optical path 21' and a turntable 23'.
  • a plurality of filters 211' are arranged on the turntable 23'.
  • the fluorescence transmission optical path 21' includes an optical fiber 212'.
  • a plurality of fluorescence signals are transmitted to the filter 211' through the optical fiber 212'.
  • the plurality of filters 211' can pass a fluorescence signal respectively.
  • the channels are switched by rotating the turntable 23' so that different fluorescence signals can pass through and then be received by the detector 22'. However, this requires a motor or the like to drive the turntable 23' to rotate. Switching between channels takes tens of milliseconds, which reduces the detection efficiency. In addition, the rotation of the turntable 23' will generate vibrations, which will affect the detection results.
  • the fluorescence detection unit 2 includes at least two fluorescence transmission optical paths 21, and the at least two fluorescence transmission optical paths 21 correspond to at least two fluorescence emission units 1 one by one, that is, one fluorescence emission unit 1 can correspond to one fluorescence transmission optical path 21.
  • the reaction sample at the location opposite to the excitation optical fiber 11 of the fluorescence emission unit 1 can receive stronger excitation light, so the reaction sample at this location can generate a higher fluorescence signal.
  • the at least two fluorescence transmission optical paths 21 correspond to the at least two fluorescence emission units 1 one by one, so that the fluorescence transmission optical path 21 can be set close to the reaction sample excited by the corresponding fluorescence emission unit 1, thereby allowing more fluorescence signals to enter the fluorescence transmission optical path 21, further improving the accuracy of the detection result.
  • Each fluorescence transmission optical path 21 includes a filter 211 that allows a preset fluorescence signal to pass through, that is, each fluorescence transmission optical path 21 can only allow a specific fluorescence signal to pass through.
  • a filter 211 is set in each fluorescence transmission optical path 21, which can reduce the time required for mechanical switching when the fluorescence signal passes through the optical path, realize rapid detection of the fluorescence signal, and eliminate the influence of mechanical vibration caused by mechanical switching on the detection result.
  • the excitation light is polychromatic light, other fluorescent signals other than the preset fluorescent signal cannot pass through the filter 211, so the accuracy of the detection result can be guaranteed.
  • the fluorescence detection unit 2 further includes a detector 22 , and the plurality of fluorescence transmission optical paths 21 are all connected to the detector 22 , and the detector 22 is used to detect fluorescence signals.
  • the number of detectors 22 is one, and the detector 22 is electrically connected to the control unit to record the intensity of the fluorescence signal in different time periods.
  • Different fluorescence transmission optical paths 21 converge on one detector 22 after passing through different filters 211.
  • the fluorescence detector 22 can only detect the intensity of the signal fluorescence but not the type of the signal fluorescence, it can determine what kind of signal fluorescence it is according to the receiving time period, and then determine the reaction sample corresponding to the signal fluorescence. This embodiment determines what kind of signal fluorescence it is according to the time period, has low equipment cost, simple equipment structure, no mechanical switching, and fast detection speed.
  • the fluorescence detection optical path system 100 also includes a fiber holder 3, a fluorescence transmission optical path 21 including a collecting optical fiber 212, an end of the excitation optical fiber 11 emitting excitation light, and an end of the collecting optical fiber 212 injecting a fluorescence signal to form a fiber group 31, the fiber group 31 is arranged in the fiber holder 3, and the optical fibers in the fiber group 31 are arranged along a first direction, the first direction is the radial direction of the optical fiber (the direction as shown by the arrow R in FIG3b is the radial direction of the optical fiber), so that the emission end of the collecting optical fiber 212 and the injection end of the excitation optical fiber 11 are arranged flat in the fiber holder 3, that is, the optical fibers (collecting optical fiber 212 and excitation optical fiber 11) are arranged flat at one end close to the side wall 42 of the carrier 4, the first direction can be the length direction of the side wall 42, and in the thickness direction of the carrier 4, the optical
  • the diameter of the optical fiber can be smaller than the thickness of the carrier 4, so that the optical fiber in the optical fiber holder 3 does not protrude from the carrier 4 in the thickness direction of the carrier 4, and therefore, the excitation light emitted by the collection optical fiber 212 can enter the accommodating cavity 41, and the entire excitation optical fiber 11 can collect fluorescence.
  • the excitation optical fiber and the collection optical fiber are arranged at an angle, it is often hoped that the excitation light injection point of the laser optical fiber into the reaction sample coincides with the fluorescence signal injection point injected into the collection optical fiber, so that the maximum fluorescence signal enters the collection optical fiber, but in this way, more excitation light will also enter the collection optical fiber.
  • the excitation optical fiber 11 and the collection optical fiber 212 are arranged in parallel along the radial direction of the optical fiber (the direction shown by the arrow R in FIG. 3 b is the radial direction of the optical fiber), which can reduce the amount of excitation light entering the excitation optical fiber 11, and at the same time, it can ensure that as many collection optical fibers 212 as possible receive more fluorescence signals, so as to obtain the optimal sensitivity and signal-to-noise ratio.
  • the optical fiber group 31 is coupled to the carrier 4
  • the first direction of optical fiber arrangement is perpendicular to the thickness direction of the carrier 4
  • both the optical fiber group 31 and the optical fiber holder 3 are flat structures, thereby matching the carrier 4 .
  • At least two groups of optical fiber groups 31 are formed accordingly.
  • the at least two groups of optical fiber groups 31 in the optical fiber holder 3 are arranged in sequence along the first direction, that is, when multiple optical fiber groups 31 are provided, the multiple optical fiber groups 31 are also arranged in a flat manner.
  • the optical fibers in the optical fiber holder 3 are also arranged in a single layer, so as to adapt to the flat structure of the carrier 4.
  • the optical fibers in the optical fiber holder 3 do not protrude from the carrier 4 in the thickness direction of the carrier 4. Therefore, the excitation light emitted by the collection optical fiber 212 can enter the accommodating cavity 41, and the entire excitation optical fiber 11 can collect fluorescence.
  • multiple optical fiber groups 31 are arranged in sequence, and the collection optical fiber 212 and the excitation optical fiber 11 in the same optical fiber group 31 are adjacent to each other.
  • the reaction sample at the corresponding position of the excitation optical fiber 11 can generate more fluorescence signals, and the collection optical fiber 212 is close to the corresponding excitation optical fiber 11, so that more fluorescence signals can enter the fluorescence transmission optical path 21, thereby further improving the accuracy of the detection results.
  • the optical fiber seat 3 includes a seat body 32 and a flat plate 34 formed on one side of the seat body 32.
  • the flat groove 33 in which the optical fiber group 32 is arranged.
  • a group of optical fiber sets 31 includes at least two collecting optical fibers 212, and at least one collecting optical fiber 212 is arranged on both sides of the first direction of the excitation optical fiber 11 in a group of optical fiber sets 31.
  • increasing the number of collecting optical fibers 212 to at least two and performing multi-point detection on the reaction sample can effectively improve the detection efficiency of fluorescence, solve the problem of low signal, and reduce the demand for the sensitivity of the detector 22.
  • one of the collecting optical fibers 212 is affected by bubbles in the reaction sample, it can be corrected through the detection results of other collecting optical fibers 212.
  • the detector 22 includes a silicon photomultiplier tube (i.e., SiPM), a photon detector (i.e., PD) or a photomultiplier tube (i.e., PMT).
  • SiPM silicon photomultiplier tube
  • PD photon detector
  • PMT photomultiplier tube
  • the fluorescence detector 22 using a silicon photomultiplier tube, a photon detector or a photomultiplier tube has high sensitivity and can achieve ultrafast, high-sensitivity detection of fluorescence signals in milliseconds when performing fluorescence detection.
  • the heating element 91 of this embodiment is a resistor, and there is a specific relationship between the resistor and its temperature. Therefore, the real-time resistance change of the heating element 91 can be measured while heating, and the average temperature of the heating element 91 can be deduced through the resistance temperature coefficient and the nominal resistance value.
  • the temperature reflects the current temperature of the carrier 4 in real time without delay, so it can be used for rapid feedback control of the carrier 4 and the reaction sample temperature. Compared with the prior art, the sample temperature can be controlled more accurately and the overall response speed of the temperature control system can be improved.
  • the PCR detection system may further include a resistance detection element, which is used to detect the resistance of the heating element 91 so as to measure the temperature of the heating element 91 by a resistance temperature measurement method.
  • the disadvantage of the resistance temperature measurement method is that for the same type of resistors, such as copper wire resistors, there are slight differences in the nominal resistance value and the resistance temperature coefficient (the resistance value at the nominal temperature is referred to as the nominal resistance value, and the nominal resistance means that at this temperature, the declared (or marked) resistance value is true, where this temperature is the nominal temperature, and the nominal temperature can be selected arbitrarily according to needs) between the resistors, resulting in a slight difference between the actual resistance temperature coefficient of a single heating element 91 and the nominal resistance value, which may cause temperature measurement errors. Therefore, preferably, as shown in Figures 9 and 10, the built-in heater 45 provided in this embodiment also includes a temperature calibration unit 93 for reflecting the temperature of the heating element 91. The temperature calibration unit 93 can be used for the temperature detection unit 10 to detect the temperature, so that the carrier 4 can be controlled by a dual temperature measurement method of resistance temperature measurement and calibration of the temperature of the carrier 4.
  • the temperature calibration part 93 can reflect the temperature of the heating element 91 and the temperature detection unit 10 can detect the temperature of the temperature calibration part 93, since the temperature of the reaction sample changes very quickly during the amplification stage, when the temperature detection unit 10 such as the temperature sensor detects the temperature of the carrier 4, it takes a certain amount of time for the heat to be transferred from the temperature calibration part 93 to the temperature detection unit 10. Therefore, under normal circumstances, the detection result measured by the temperature detection unit 10 will have a temperature measurement delay of 1 to 2 seconds. During the rapid heating and cooling process, the temperature change of the carrier 4 can reach more than 30°C within 1-2 seconds. Therefore, during the rapid heating and cooling process, it is relatively difficult to control the carrier 4 through the temperature detection unit 10.
  • This embodiment does not completely rely on the temperature value measured by the uncalibrated resistance temperature measurement method, nor does it completely rely on
  • the temperature of the carrier 4 is controlled not by the temperature detected by the temperature detection unit 10, but by combining the two, the temperature calibration part 93 of the carrier 4 is measured and the temperature of the heating element 91 is measured by the resistance temperature measurement method, so that the temperature of the carrier 4 can be quickly and accurately controlled to achieve the purpose of accurate temperature control, thereby overcoming the problems of temperature detection delay and large temperature measurement error caused by the temperature detection method commonly used in the prior art.
  • each heating element 91 is correspondingly provided with a temperature calibration portion 93 and a resistance detection element so that the temperature of each heating element 91 can be calibrated.
  • FIG11 shows a process of calibrating the resistance temperature measurement method by the temperature detection unit 10 in an actual detection.
  • an initial RT temperature curve i.e., a preset temperature curve
  • a very small current is applied to the heating element 91 of the carrier 4, such as a current of less than 1 mA.
  • the purpose of applying a very small current is to read the resistance of the heating element 91 without causing the heating element 91 to heat up.
  • the temperature calibration value can be detected throughout the entire process of nucleic acid amplification, so the temperature can be calibrated multiple times in the subsequent process to further improve the detection accuracy.
  • the two first contacts of the temperature detection unit 10 are respectively in contact with the two temperature calibration parts 93, and the two temperature calibration parts 93 are not conductive.
  • the built-in heater 45 can also include external electrical connection contacts 97 and electrical connection leads 98.
  • the number of external electrical connection contacts 97 and electrical connection leads 98 can be two.
  • the two external electrical connection contacts 97 are respectively located on the side away from each other of the two temperature calibration parts 93.
  • One external connection contact is electrically connected to one temperature calibration part 93 through an electrical connection lead 98, and the other external connection contact is electrically connected to the other temperature calibration part 93 through another electrical connection lead 98.
  • the heat of the heat-dissipating layer 921 of the upper conducting component 92 is conducted to the temperature calibration part 93, and the temperature calibration part 93 is electrically connected to the outside at the external electrical connection contact 97 through the electrical connection lead 98, wherein the diameter of the electrical connection lead 98 is smaller than that of the temperature calibration part 93 and the external electrical connection contact 97, thereby reducing the heat loss generated by the temperature calibration part 93 through the electrical connection lead 98, so that the temperature calibration part 93 can better reflect the upper conducting component 92,
  • the temperature of the heat-dissipating layer 921 of the upper conductive component 92 is achieved by the temperature detection unit 10 and the temperature calibration part 93 through the solder joints.
  • the temperature detection unit 10 can quickly and accurately sense the temperature change.
  • the temperature change causes the resistance value of the temperature detection unit 10 to change.
  • the resistance value change of the temperature detection unit 10 is detected in real time at the external electrical connection contact 97, thereby achieving real-time temperature detection.
  • the carrier 4 may further include a fast conduction part 94, and the fast conduction part 94 is used to conduct the heat of the heating element 91 to the temperature calibration part 93.
  • the heat of the heating element 91 is indirectly conducted to the temperature calibration part 93, such as the heating element 91 heats the heat-averaging layer 921, and the heat of the heat-averaging layer 921 is conducted to the temperature calibration part 93 through the fast conduction part 94, thereby, the temperature calibration part 93 accurately reflects the temperature of the heat-averaging layer 921, and then the temperature detection unit 10 can accurately measure the temperature of the heat-averaging layer 921. Since the thickness of the reaction sample is very small, the temperature of the reaction sample is basically consistent with the temperature of the heat-averaging layer 921, so the temperature of the reaction sample can be obtained by detecting the temperature of the temperature calibration part 93.
  • one side of the fast conduction part 94 is connected to one side of the upper conduction component 92 close to the heating element 91 or to one side of the lower conduction component 95 close to the heating element 91, and the other side is connected to the temperature calibration part 93.
  • the lower surface of the upper conduction component 92 and the upper surface of the lower conduction component 95 are closest to the heating element 91, and their temperatures are the first to approach the temperature of the heating element 91. Therefore, the arrangement of the fast conduction part 94 can make the temperature of the fast conduction part 94 and the temperature of the heating element 91 reach the same level in the shortest time.
  • the fast conduction part 94 is made of a material with high thermal conductivity, such as a metal material such as copper or aluminum, or a thermally conductive ceramic.
  • the thermal conductivity of the fast conduction part 94 is particularly better than that of the lower conduction component 95, so as to quickly transfer heat to the temperature calibration part 93.
  • the fast conduction part 94 includes a patch 941 and one or more guide pillars 942.
  • the patch 941 is attached to the side of the upper conduction component 92 close to the heating element 91 or to the side of the lower conduction component 95 close to the heating element 91.
  • One end of the one or more guide pillars 942 is connected to the patch 941, and the other end is passed through the lower conduction component 95 and connected to the temperature calibration part 93.
  • the lower surface of the upper conduction component 92 and the upper surface of the lower conduction component 95 are closest to the heating element 91, and their temperatures are first close to the temperature of the heating element 91.
  • the arrangement of the patch 941 can make the temperature of the fast conduction part 94 consistent with the temperature of the heating element 91 as quickly as possible.
  • the patch 941 can increase the contact area between the fast conduction part 94 and the upper conduction component 92 or the lower conduction component 95, and improve the conduction efficiency.
  • the cross-sectional area of the guide pillar 942 can be smaller than the cross-sectional area of the patch 941, and the temperature of the patch 941 can be quickly conducted to the temperature calibration part 93, ensuring that the thermal resistance layer generates the required thermal resistance as designed.
  • patch 941 and guide pillar 942 are made of materials with high thermal conductivity such as copper. When patch 941 and guide pillar 942 are required to be insulating materials to avoid short circuit of carrier 4, patch 941 or guide pillar 942 can be made of materials such as high thermal conductivity ceramics.
  • the temperature calibration part 93 can be arranged in a one-to-one correspondence with the patch 941, and two temperature calibration parts 93 can also be connected to one patch 941.
  • One temperature calibration part 93 can be connected to one guide post 942, and in order to improve the temperature uniformity of the temperature calibration part 93, the temperature calibration part 93 can also be connected to multiple guide posts 942.
  • a plurality of second contacts 96 are provided on the outer surface of the carrier 4, and the second contacts 96 are electrically connected to the heating element 91.
  • the current and voltage of the heating element 91 can be obtained through the second contacts 96, and then the resistance value of the heating element 91 can be obtained. It is understood that when the plurality of heating elements 91 are independently controlled, each heating element 91 is correspondingly provided with a second contact 96 to calibrate the heating element 91 and provide heat to the heating element 91 , respectively.
  • the second contact 96 enables the carrier 4 to realize its own temperature measurement function. Compared with the traditional structure that can only measure the temperature through an external temperature measurement unit, this embodiment can directly measure the temperature of the carrier 4 itself, so the temperature measurement is more accurate and faster, which can improve the accuracy and control speed of the temperature control system.

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Abstract

本申请涉及一种荧光光源装置、荧光检测光路系统及PCR检测系统。荧光光源装置，包括：至少两个荧光发射单元，至少两个荧光发射单元用于发射激发光；控制单元，控制单元控制至少两个荧光发射单元分别在不同时间段发射激发光。

Description

一种荧光光源装置、荧光检测光路系统及PCR检测系统
相关申请的引用
本申请要求2023年06月05日提交的中国专利申请CN2023106619175、2023年06月05日提交的中国专利申请CN2023214222295的优先权，通过援引加入的方式将上述专利申请的内容整体并入本申请，并用于所有目的。
技术领域
本申请涉及体外诊断技术领域，尤其涉及一种荧光光源装置、荧光检测光路系统及PCR检测系统。
背景技术
现有技术中，PCR(聚合酶链式反应)是指体外酶促合成特异DNA片段的一种分子生物学实验方法，主要由高温变性、低温退火和适温延伸三个步骤反复的热循环构成。反应样品在PCR扩增前，需要放入到载体中，其中，反应样品由采集的咽拭子或鼻拭子等样本以及用于PCR扩增的试剂组成。在PCR扩增时，需要通过加热器加热反应样品，以及通过冷却机构冷却反应样品，从而使得反应样品在高温变性、低温退火和适温延伸阶段循环。
PCR扩增后，往往采用荧光检测反应样品，在检测时，向反应样品发射激发光，反应样品与激发光作用后，会产生另一种荧光信号，分析荧光信号的强度，进而可以实现对反应样品的检测，如检测反应样品中是否含有某种DNA片段。
当载体内的反应样品存在多种荧光探针/染料时，需要发射多种荧光激发反应样品，现有技术中的荧光检测光路导致进入到探测器检测内的光源种类多，检测结果不准确。以及光路结构复杂，设备成本高。
现有技术中的荧光检测光路主要针对反应样品接收激发光的区域较大的载体，探测器接收的干扰信号高，但由于接收激发光的区域较大，产生的荧光信号高，因此，探测器接收到的荧光信号高，干扰信号对检测结果的影响很小，可以得到较为准确的检测结果。但是，对于反应样品产生的荧光信号较低的情况，探测器难以得到准确的检测结果。
另外，载体为管类结构如ep管结构，载体盛放反应样品的区域直径大，均热速度慢，反应样品升降温速度慢。
此外，为了便于控制反应样品进行扩增，需要检测反应样品的温度，现有技术中常用的温度检测方法准确度低，导致检测结果不准确。
发明内容
本申请的一个目的在于提出一种荧光光源装置，以解决上述技术问题之一。
为实现上述目的，本申请第一方面提供了一种荧光光源装置，包括：
至少两个荧光发射单元，所述至少两个荧光发射单元用于发射激发光；
控制单元，所述控制单元控制所述至少两个荧光发射单元分别在不同时间段 发射激发光。
可选的，所述荧光发射单元包括光源和激发光纤，所述激发光纤用于传输所述光源的发射的所述激发光。
可选的，所述至少两个荧光发射单元发射的所述激发光的波长互不相同。
可选的，所述激发光为单色光或多色光。
本申请的另一个目的在于提供一种PCR检测系统，以解决上述技术问题之一。
为达此目的，本申请第二方面采用以下技术方案：
一种荧光检测光路系统，包括所述的荧光光源装置和荧光探测单元。
可选的，所述荧光探测单元包括至少两个荧光传输光路，至少两个所述荧光发射单元与至少两个所述荧光传输光路一一对应。
可选的，每个所述荧光传输光路均包括允许预设荧光信号通过的滤光片。
可选的，所述荧光探测单元还包括探测器，多个所述荧光传输光路均连接于所述探测器。
可选的，所述探测器的数量为一个，所述探测器与所述控制单元电连接，以分时间段记录荧光信号的强度。
可选的，所述荧光检测光路系统还包括光纤座，所述荧光传输光路包括收集光纤，所述荧光光源装置的激发光纤射出所述激发光的一端，以及所述收集光纤射入荧光信号的一端形成光纤组，所述光纤组设置在所述光纤座内，所述光纤座内的光纤沿第一方向排列，所述第一方向为光纤的径向，以使所述收集光纤的射出端和所述激发光纤的射入端在所述光纤座中呈扁平排列。
可选的，至少所述两个荧光传输光路和所述至少两个荧光发射单元形成至少两组所述光纤组，至少两组所述光纤组沿所述第一方向依次排列。
可选的，一组所述光纤组包括至少两个所述收集光纤，一组所述光纤组内的所述激发光纤的所述第一方向的两侧至少设置有一个所述收集光纤。
可选的，所述探测器包括硅光电倍增管、光子型探测器或光电倍增管。
本申请的又一个目的在于提供一种PCR检测系统，以解决上述技术问题之一。
为达此目的，本申请第三方面采用以下技术方案：
一种PCR检测系统，包括载体和所述的荧光检测光路系统，所述荧光检测光路系统用于检测所述载体内的反应样品。
可选的，所述载体包括相对设置的第一壁和第二壁，以及设置在所述第一壁和所述第二壁之间的侧壁，所述第一壁、所述第二壁和所述侧壁形成容纳腔，所述容纳腔和/或所述载体为扁平结构，至少部分所述侧壁透光，所述荧光检测光路系统通过透光的所述侧壁检测反应样品。
可选的，所述载体的一侧或至少两侧均设置有所述荧光发射单元。
可选的，所述透光的所述侧壁材料为聚二甲基硅氧烷、聚丙烯或聚碳酸酯。
可选的，所述载体包括至少一个内置加热器，所述第一壁和/或所述第二壁为所述内置加热器。
可选的，所述内置加热器包括加热件。
可选的，所述内置加热器包括至少两个独立控制的所述加热件。
可选的，所述内置加热器还包括用于体现所述加热件温度的温度校准部。
可选的，所述内置加热器还包括快速传导部，所述快速传导部用于将所述加热件的热量传导至所述温度校准部。
可选的，所述PCR检测系统还可以包括电阻检测件，所述电阻检测件用于检测所述加热件的电阻。
可选的，所述扁平结构指，所述容纳腔或所述载体垂直于其厚度方向的方向的尺寸大于其厚度方向的尺寸。
可选的，所述容纳腔或所述载体垂直于其厚度方向的方向的尺寸与其厚度方向的尺寸之比大于5：1。
可选的，所述尺寸之比为50：1～100：1。
由上可见，本申请提供的技术方案，荧光发射单元独立设置，荧光发射单元中传输的激发光不会反射到传输荧光信号的光路中，从而大大降低荧光信号的光路中的激发光的量，大大降低荧光信号中的背底，提高检测准确度。同时，载体内的反应样品具有多种荧光探针/染料时，控制单元可以控制在不同时间段依次开启不同的荧光发射单元，如第一个荧光发射单元在检测开始的第1-2s内工作，第二个荧光发射单元在检测开始的第2-3s内工作，依次类推，一个时间段内只有一个荧光发射单元工作，通过控制单元控制各通道的荧光发射单元的导通，实现毫秒级切换激发光通道，可以实现快速检测反应样品。
荧光探测单元用于探测荧光信号，荧光发射单元与接收激发信号的荧光探测单元完全分离，降低荧光信号背底，提高了荧光检测光路系统的检测灵敏度，同时，荧光发射单元与荧光探测单元完全分离，减少荧光探测单元中的光学元件，如二向色镜等，降低成本，提高了荧光的检测效率。
附图说明
图1a是本申请实施例提供的载体的结构示意图；
图1b是图1a中载体的C-C处的剖视图；
图2是图1b中载体的D-D处的剖视图；
图3a是现有技术的荧光检测光路系统的结构示意图；
图3b是本申请实施例提供的荧光检测光路系统的结构示意图；
图3c是本申请实施例提供的光纤座的结构示意图；
图3d是本申请实施例提供的光纤座另一视角的结构示意图；
图4是现有技术中荧光探测单元的结构示意图；
图5是本申请实施例提供的另一种荧光探测单元的结构示意图；
图6a是本申请实施例提供的载体的一侧设置光纤组的结构示意图；
图6b是本实施例PCR检测系统检测得到的扩增曲线；
图7是本申请实施例提供的载体的两侧均设置光纤组的结构示意图；
图8是本申请实施例提供的载体被外置加热器加热的示意图；图9是本申请实施例提供的外置加热器、冷却组件和载体的内部结构示意图；
图10是本申请实施例提供的载体包括两个内置加热器的内部结构示意图；
图11是本申请实施例提供的温度校准的曲线图；
图12是本申请实施例提供的载体的结构示意图。
图中：
1、荧光发射单元；11、激发光纤；12、光源；
2、荧光探测单元；21、荧光传输光路；211、滤光片；212、收集光纤；213、
准直透镜；22、探测器；
3、光纤座；31、光纤组；32、座本体；33、扁平槽；
4、载体；41、容纳腔；42、侧壁；43、第一壁；44、第二壁；
45、内置加热器；
521、冷却组件；5221、流道；522、外置加热器；
91、加热件；92、上传导组件；921、均热层；93、温度校准部；94、快速
传导部；941、贴片；942、导柱；95、下传导组件；951、绝缘热阻层；952、导热层；96、第二触点；97、外部电连接触点；98、电连接引线；
10、温度检测单元；100、荧光检测光路系统。
图3a中：
200、激发光光路；201、荧光信号光路；203、二向色镜；204、滤光片；205、
光源；206、探测电路；
图4中：
2'、荧光探测单元；21'、荧光传输光路；22'、探测器；23'、转盘；211'、滤
光片；212'、光纤。
具体实施方式
下面结合附图并通过具体实施方式来进一步说明本申请的技术方案。可以理解的是，此处所描述的具体实施例仅仅用于解释本申请，而非对本申请的限定。另外还需要说明的是，为了便于描述，附图中仅示出了与本申请相关的部分而非全部。
本申请中限定了一些方位词，在未作出相反说明的情况下，所使用的方位词如“上”、“下”、“左”、“右”、“内”、“外”这些方位词是为了便于理解而采用的，因而不构成对本申请保护范围的限制。
在本申请中，除非另有明确的规定和限定，第一特征在第二特征之“上”或之“下”可以包括第一和第二特征直接接触，也可以包括第一和第二特征不是直接接触而是通过它们之间的另外的特征接触。而且，第一特征在第二特征“之上”、 “上方”和“上面”包括第一特征在第二特征正上方和斜上方，或仅仅表示第一特征水平高度高于第二特征。第一特征在第二特征“之下”、“下方”和“下面”包括第一特征在第二特征正下方和斜下方，或仅仅表示第一特征水平高度小于第二特征。
在本申请的描述中，除非另有明确的规定和限定，术语“相连”、“连接”、“固定”应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或成一体；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通或两个元件的相互作用关系。对于本领域的普通技术人员而言，可以具体情况理解上述术语在本申请中的具体含义。
如图1a、图1b和图2所示，本实施例提供一种载体4，载体4包括相对设置的第一壁43和第二壁44，以及设置在第一壁43和第二壁44之间的侧壁42，第一壁43、第二壁44和侧壁42形成容纳腔41，容纳腔41和/或载体4为扁平结构，至少部分侧壁42透光，。
扁平结构可以指，容纳腔41或载体4的厚度方向(也即第一壁43和第二壁44设置的方向)的尺寸小于垂直于厚度方向的方向的尺寸，作为示例性的，垂直于厚度方向的方向的尺寸与厚度方向的尺寸之比大于5：1。更优选地，载体4或容纳腔41的厚度方向的尺寸远小于垂直于厚度方向的方向的尺寸，如尺寸之比为50：1～100：1，作为示例性的，尺寸之比为90:1。作为示例性的，容纳腔41为长方体，长方体的长度和厚度的比例可以为大于5：1，如90:1，如容纳腔41厚度方向的尺寸可以为0.3-1.0mm，在本实施例中，容纳腔41的厚度为0.3-0.6mm，容纳腔41的宽度和长度分别为10mm和20mm左右，其中，厚度方向第一壁43和第二壁44的布置方向。作为示例性的，容纳腔41还可以为圆柱结构，直径和厚度比为大于5：1，如厚度为0.3-1.0mm，直径为5-20mm。当然，容纳腔41的横截面可以为多边形或椭圆形等。当然，容纳腔41的横截面可以为多边形或椭圆形等。
现有的PCR检测系统往往会选择较大的面进行核酸检测，这样接收激发光的反应样品面积大，产生的荧光信号强，容易得到准确的检测结果。按照上述思维习惯，由于本实施例中的第一壁43或第二壁44的面积大，往往会考虑通过第一壁43或第二壁44进行荧光检测。
容纳腔41的至少一面可以被加热，以为容纳腔41内的反应样品加热。为了实现快速地扩增，实现快速核酸检测，优选地，载体4的第一壁43和第二壁44均设置如图8所示的外置加热器522或如图10所示，第一壁43和第二壁44为内置加热器45，外置加热器522或内置加热器45对反应样品进行加热，进而实现反应样品的快速升降温，实现快速核酸检测。
可选地，容纳腔41的至少部分侧壁42透光，荧光检测光路系统100通过载体4的透光的侧壁42检测反应样品，既满足了快速升降温的需要，又可以精准地完成检测。
如图3a所示，现有技术中的荧光检测光路为整体式结构，即荧光检测光路的 激发光光路200与荧光信号光路201通过一根光纤传输，导致光源205发射的激发光在光纤的反射后进入到荧光信号光路201上。光纤传输的路径上设置二向色镜203和滤光片204，二向色镜203和滤光片204均允许荧光信号通过，且会滤过荧光信号光路201中的激发光，但是，由于二向色镜203和滤光片204不会百分百过滤激发光，也不能允许荧光信号百分百的通过，即荧光信号会在通过二向色镜203和滤光片204时发生衰减，部分滤激发光会进入到探测荧光信号的探测电路206中。现有技术中，由于激发光激发的反应样品的区域较大，产生的荧光信号数量也多，因此，透过滤光片204的激发光以及衰减的荧光信号对检测结果的影响很小，可以得到较为准确的检测结果。
但是本实施例中的容纳腔41的厚度薄(0.3～0.6mm)，通过容纳腔41的侧壁42进行荧光检测时，能够接受激发光的反应样品大大减少，因此，每次激发的荧光信号数量大约要少一个数量级。若利用现有的荧光检测光路进行检测，探测电路检测到的信号低，透过滤光片211的激发光会淹没荧光信号，进而导致检测产生的误差大，甚至荧光探测电路无法检测到荧光信号。
如图3b，本实施例提供的荧光光源装置包括控制单元和至少两个荧光发射单元1，至少两个荧光发射单元1用于发射激发光，激发光为单色光或多色光，如多色光可以为白光。控制单元控制至少两个荧光发射单元1分别在不同时间段发射激发光，以允许反应样品在同一时刻只能接收一种激发光。在本实施例中，控制单元与荧光发射单元1通过导线连接，导线可以使得控制单元无延时地控制荧光发射单元1。当然，在其他可选的实施例中，控制单元与荧光发射单元1之间还可以通过蓝牙等无线方式连接。可以理解的是，控制单元可以是集中式或分布式的控制单元，比如，控制单元可以是一个单独的单片机，也可以是分布的多块单片机构成，单片机中可以运行控制程序，进而控制各部件实现其功能。
荧光发射单元1独立设置，荧光发射单元1中传输的激发光不会反射到传输荧光信号的光路中，从而大大降低荧光信号的光路中的激发光的量，大大降低荧光信号中的背底，提高检测准确度。同时，载体4内的反应样品中具有多种荧光探针/染料时，控制单元可以控制在不同时间段依次开启不同的荧光发射单元1，如第一个荧光发射单元1在检测开始的第1-2s内工作，第二个荧光发射单元1在检测开始的第2-3s内工作，依次类推，一个时间段内只有一个荧光发射单元1工作，通过控制单元控制各通道的荧光发射单元1的导通，实现毫秒级切换激发光通道，可以实现快速检测反应样品。同时，当激发光为单色光时，一荧光发射单元1只需要发射一种激发光，因此，在一个时间段内激发光的波长单一，荧光信号的种类单一，在检测荧光信号时，其他荧光信号不会产生噪音，可以进一步提高检测结果的准确性。
可选地，激发光为单色光，且至少两个荧光发射单元1发射的激发光的波长互不相同，以提高荧光发射单元1的利用率。当然，为了避免荧光发射单元1故障导致检测结果不准确，还可以设置备用的荧光发射单元1，如至少两个荧光发射 单元1中，两荧光发射单元1发射的激发光的波长相同。
如图3b所示，荧光发射单元1包括光源12和激发光纤11，光源12可以为LED等，光源12用于发射激发光，光源12与控制单元电连接。激发光纤11用于传输光源12的发射的激发光，以将激发光传输至反应样品。通过激发光纤11将光源12和反应样品连接起来，激发光纤11便于隔离热源，使得实验数据更稳定，且激发光纤11与光源12连接简单，利于抗震。同时，由于光纤本身尺寸较小，可以满足扁平结构的载体4需要。另外，一光源12对应一激发光纤11，每个激发通道单独一根光纤，容易耦合，光损小。
本实施例还提供了一种荧光检测光路系统100，其包括上述的荧光光源装置和荧光探测单元2。荧光探测单元2用于探测荧光信号，荧光发射单元1与接收激发信号的荧光探测单元2完全分离，降低荧光信号背底，提高了荧光检测光路系统100的检测灵敏度，同时，荧光发射单元1与荧光探测单元2完全分离，减少荧光探测单元2中的光学元件，如二向色镜等，降低成本，提高了荧光的检测效率。
如图4所示，现有技术中，荧光探测单元2'包括一个探测器22'、一个荧光传输光路21'和转盘23'，转盘23'上设置多个滤光片211'，荧光传输光路21'包括一根光纤212'，多种荧光信号通过一根光纤212'传输至滤光片211'，多个滤光片211'能够分别通过一种荧光信号，通过转动转盘23'切换通道，以使不同的荧光信号通过，进而被探测器22'接受。但是这样需要电机等驱动转盘23'转动，通道之间切换需要十数毫秒，降低检测效率，且转盘23'转动会产生震动，对检测结果造成影响。
为解决上述技术问题，在本实施例中，如图3b和图5所示，优选地，荧光探测单元2包括至少两个荧光传输光路21，至少两个荧光传输光路21与至少两个荧光发射单元1一一对应，即一个荧光发射单元1可以对应一个荧光传输光路21。荧光发射单元1的激发光纤11相对处的反应样品能够接受更强的激发光，因此，该处的反应样品能够产生较高的荧光信号，至少两个荧光传输光路21与至少两个荧光发射单元1一一对应，使得荧光传输光路21可以靠近与其对应的荧光发射单元1激发的反应样品设置，进而使得较多的荧光信号进入到荧光传输光路21中，进一步提高检测结果的准确度。
每个荧光传输光路21均包括允许预设荧光信号通过的滤光片211，即，每条荧光传输光路21中只能允许特定的荧光信号通过相对于上述只设置一个荧光传输光路21'，通过机械切换不同的滤光片211'而言，本实施例中，在每个荧光传输光路21均设置滤光片211，可以减少荧光信号通过光路时，机械切换所需时间，实现了荧光信号的快速检测，同时还可以消除机械切换造成的机械震动对检测结果的影响。当激发光为多色光时，预设荧光信号之外的其他荧光信号等光无法通过滤光片211，因此，可以保证检测结果的准确度。
如图3b和图5，荧光探测单元2还包括探测器22，多个荧光传输光路21均连接于探测器22，探测器22用于检测荧光信号。
优选地，探测器22的数量为一个，探测器22与控制单元电连接，以分时间段记录荧光信号的强度，不同荧光传输光路21通过不同的滤光片211后汇聚到一个探测器22上。通过荧光发射单元1分时间段开启不同的荧光发射单元1，实现激发通道的切换，从而分时间段激发出不同的信号荧光，相应的信号荧光可以穿过与其对应的滤光片211，虽然荧光探测器22只能检测信号荧光的强度，不能检测信号荧光的种类，但是，可以根据接受时间段判断为何种信号荧光，进而确定信号荧光对应的反应样品。本实施例根据时间段判断为何种信号荧光，设备成本低，设备结构简单，无机械切换，检测速度快。
如图3b所示，并结合图6a，荧光检测光路系统100还包括光纤座3荧光传输光路21包括收集光纤212，激发光纤11射出激发光的一端，以及收集光纤212射入荧光信号的一端形成光纤组31，光纤组31设置在光纤座3内，光纤组31内的光纤沿第一方向排列，第一方向为光纤的径向(如图3b中箭头R所示方向为光纤的径向)，以使收集光纤212的射出端和激发光纤11的射入端在光纤座3中呈扁平排列，即光纤(收集光纤212和激发光纤11)靠近载体4的侧壁42的一端扁平排列，第一方向可以为侧壁42的长度方向，在载体4的厚度方向上，光纤座3内的光纤呈单层排列，从而适应扁平结构的载体4。可以理解的是，光纤的直径可以小于载体4的厚度，从而使得光纤座3内的光纤在载体4的厚度方向上不突出于载体4，因此，收集光纤212射出的激发光均能够进入到容纳腔41内，以及整根激发光纤11均能够收集荧光。现有技术中，当激发光纤和收集光纤成夹角设置时，往往希望激光光纤射入到反应样品的激发光射入点与射入到收集光纤中的荧光信号射入点重合，以期最多的荧光信号进入到收集光纤，但是，这样也会有更多的激发光进入到收集光纤，本实施例中，激发光纤11和收集光纤212沿光纤的径向平行排列(如图3b中箭头R所示方向为光纤的径向)，可以降低激发光进入到激发光纤11的量，同时，可以保证尽量多的收集光纤212接受较多的荧光信号，从而可以获取最优的灵敏度与信噪比。同时，将光纤组31与载体4耦合时，光纤排列的第一方向与载体4的厚度方向垂直，光纤组31和光纤座3均为扁平结构，从而配合载体4。
当设置有至少两个荧光传输光路21和至少两个荧光发射单元1时，对应形成至少两组光纤组31，光纤座3内的至少两组光纤组31沿第一方向依次排列，即当设置多个光纤组31时，多个光纤组31也呈扁平排列，在载体4的厚度方向上，光纤座3内的光纤也呈单层排列，从而适应扁平结构的载体4，光纤座3内的光纤在载体4的厚度方向上不突出于载体4，因此，收集光纤212射出的激发光均能够进入到容纳腔41内，以及整根激发光纤11均能够收集荧光。
同时，多个光纤组31依次排列，同一组光纤组31内的收集光纤212和激发光纤11相邻，激发光纤11对应处的反应样品能够产生较多的荧光信号，收集光纤212靠近与其对应的激发光纤11，进而使得较多的荧光信号进入到荧光传输光路21中，进一步提高检测结果的准确度。
如图3c和图3d所示，光纤座3包括座本体32和开设于座本体32本体一侧的扁 平槽33，光纤组32设置于扁平槽33中。
如图6a所示，一组光纤组31包括至少两个收集光纤212，一组光纤组31内的激发光纤11的第一方向的两侧至少设置有一个收集光纤212。在一光纤组31中，将收集光纤212增加到至少两个，对反应样品进行多点探测，可有效提升荧光的检出效率，解决信号低的问题，并降低对探测器22灵敏度的需求。同时，当其中一个收集光纤212被反应样品内的气泡影响时，可以通过其他的收集光纤212的探测结果进行纠正。
探测器22包括硅光电倍增管(即SiPM)、光子型探测器(即PD)或光电倍增管(即PMT)，采用硅光电倍增管、光子型探测器或光电倍增管的荧光探测器22灵敏度高，进行荧光检测时，实现了荧光信号毫秒级的超快、高灵敏度检测。
如图6a所示，在一个具体的实施例中，设置有四个荧光发射单元1，当然，荧光发射单元1的数量不限于四个，还可以少于四个或多于四个，其中，一激发光纤11的两侧分别设置一根收集光纤212，并形成一个光纤组31，即每种荧光可以通过两根收集光纤212进行收集，当然一个光纤组31中也可以只设置一根收集光纤212或多于两根收集光纤212。
本实施例还提供了一种PCR检测系统，其包括载体4和上述的荧光检测光路系统100，荧光检测光路系统100用于检测载体4内的反应样品。
如图6a所示，可以仅在载体4的一侧设置荧光发射单元1，或，如图7所示，在载体4的两侧均设置有荧光发射单元1，一荧光发射单元1的激发光纤11对应设置有收集光纤212，在载体4的两侧的各个收集光纤212优选地连接至一个探测器22，当然每侧的收集光纤212可以分别连接到一个探测器22上。对反应样品的两侧均进行探测，可有效提升荧光的检出效率，解决信号低的问题，并降低对探测器22灵敏度的需求。并且，当其中一侧收集光纤212因被反应样品中的气泡遮挡等造成探测结果不准确时，可以通过另外一侧的探测结果进行纠正。当然，还可以在载体4的三侧或多于三侧的位置进行检测。优选地，均是通过载体4的透光的侧壁42进行荧光检测。可选地，透光的侧壁42材料为可以为聚二甲基硅氧烷(即PDMS)、聚丙烯(即PP)、有机玻璃(即PMMA)或聚碳酸酯(即PC)，PMMA、PDMS、PP和PC是具有较好生物相容性的光学透明的材料，因此可以满足荧光检测需求以及对反应样品没有影响。图6b为通过荧光检测光路系统100获得的扩增曲线，通过该荧光检测光路系统100可有效提升荧光的检出效率，解决信号低的问题。
可选地，第一壁43和/或第二壁44为导热材料制成的膜，尤其是，载体4被加热的一面为导热材料制成的膜。具体地，膜为铝膜和隔离膜组成，可选地，隔离膜为聚丙烯膜(即pp膜)。隔离膜与反应液直接接触，可防止铝膜对反应样品的影响。铝膜的厚度可以为几十μm，如30μm，60μm等，该厚度的铝膜既可以变形，也具有一定的强度，隔离膜的厚度可以为10-30μm，如20微米等，隔离膜只要能将反应样品与铝膜隔开即可。当然，膜还可以为铝膜，铝膜的厚度可以为 几十μm，如30μm，60μm等，该厚度的铝膜既可以变形，也具有一定的强度。
如图8所示，第一壁43和第二壁44分别通过外置加热器522加热，两个外置加热器522可以分别被冷却组件521冷却，以实现反应样品的快速升降温。冷却组件521用于为载体4降温。冷却组件521可以为水冷或气冷，如图9所示，如冷却组件521内部设置有使液态冷却介质流动的流道5221，冷却介质不断流动，带走载体4和外置加热器522传导至冷却组件521的热量。或冷却介质内开设有空腔，泵等动力件向空腔内喷射液态或气态冷却介质，液态或气态冷却介质带走载体4和外置加热器522传导至冷却组件521的热量。
如图10所示，当然，在其他可选的实施例中，可以不设置外置加热器522，第一壁43和/或第二壁44不是膜，而是载体4包括至少一个内置加热器45，第一壁43和/或第二壁44为内置加热器45。具体地，载体4可以包括两个内置加热器45，两个内置加热器45可以分别被冷却组件521冷却，以实现反应样品的快速升降温。
可选地，容纳腔41内的反应样品直接与内置加热器45接触，以提高内置加热器45与反应样品之间的导热效率。侧壁42位于两个内置加热器45之间，侧壁42由透明材料制成，荧光检测光路系统100通过侧壁42进行荧光检测。
如图10所示，内置加热器45包括加热件91。电源与加热件91连接，加热件91为内置加热器45内部的可控加热源，可以为电阻，如可以用铜材料制作成电阻细线结构，通过控制流经电阻的电流大小来控制发热功率，从而实现温度控制。在其他可选的实施例中，加热件91也可以采用线圈结构或通过铁磁材料等进行电磁感应加热。
如图10所示，优选地，内置加热器45包括至少两个独立控制的加热件91，加热件91可以被独立控制，以提高反应样品温度的均匀性。如，若一加热件91的温度未达到预设温度(下面详细介绍如何检测加热件91的温度)，则增加该加热件91的电流，使反应样品快速升至预设温度。在本实施例中，由于内置加热器45与反应样品直接接触，内置加热器45与反应样品之间的导热效率高，内置加热器45的加热件91的温度可以等同于反应样品的温度，因此，控制每个加热件91的温度均达到预设温度，可以使得各处的反应样品均处于预设温度，进而保证反应样品温度均匀性。
如图10所示，内置加热器45还可包括上传导组件92和下传导组件95，加热件91夹设于上传导组件92和下传导组件95之间。上传导组件92和下传导组件95具有传导热量作用和绝缘作用。
内置加热器45包括均热层921，具体地，上传导组件92还可包括均热层921。均热层921与容纳腔41内的反应样品直接接触，均热层921可以保证热量在纵、横(也即反应样品的厚度方向和与厚度方向垂直的面)两个方向的均匀传导，保证样品液体的温度均匀性。可选地，均热层921由绝缘材料制成，如采用高导热陶瓷等绝缘材料制成。
均热层921由绝缘材料制成，均热层921与加热件91相邻，此时可以减少载体 4的层数，缩短加热件91的热量传递至载体4内的反应样品的时间以及缩短载体4散热所需的时间。
如图10所示，下传导组件95还包括绝缘热阻层951。绝缘热阻层951具有一定热阻特性和绝缘特性。绝缘热阻层951除了为加热件91绝缘以外，还可以形成一个纵向热阻。热阻的大小可以通过材料选择与厚度选择进行设计。通常该层的热阻远大于结构其他层的热阻，因而缘热阻层是载体4向冷却组件521散热降温的主要热阻来源。绝缘热阻层951是载体4热学性能的主要影响因素之一。
可选地，下传导组件95还包括导热层952，导热层952位于绝缘热阻层951远离加热件91的一侧。进一步地，导热层952为下传导组件95的最外层，其直接与冷却组件521接触。导热层952由铜等金属或其他导热率高的材料制成。由于成本控制或加工技术限制等原因，下传导组件95与冷却组件521接触的表面难以避免存在点接触。当下传导组件95的最外层为导热层952时，即使导热层952与冷却组件521存在点接触，导热层952也可以因其良好的传导性，使得热量均匀分布在整个导热层952，进而使得下传导组件95的其他层的热量均匀分布。
优选地，本实施例的加热件91为电阻，电阻与其温度之间存在特定的关系，因此，可以在加热的同时测量加热件91的实时阻值变化，并通过电阻温度系数与标称电阻值，推导加热件91的平均温度。该温度无延时的实时体现了载体4的当前温度，从而可以用于快速的反馈控制载体4以及反应样品温度，相对于现有技术，可以更精准地控制样品温度，并提高温度控制系统的整体反应速度。
为了检测加热件91的电阻，可选地，PCR检测系统还可以包括电阻检测件，电阻检测件用于检测加热件91的电阻，以通过电阻测温法对加热件91测温。
但是，电阻测温法的缺点是对于同一类型的电阻，如铜线电阻，电阻之间的标称电阻值和电阻温度系数(标称温度下的电阻值简称为标称电阻值，标称电阻是指在这个温度下，所宣称的(或者标注的)的电阻值是真实的，其中，这个温度即为标称温度，标称温度可以根据需求任意选择)稍有差异，导致单一加热件91的真实的电阻温度系数与标称电阻值略有差异，这有可能会造成温度测量误差，因此优选地，如图9和图10所示，本实施例提供的内置加热器45还包括用于体现加热件91温度的温度校准部93，温度校准部93可以供温度检测单元10检测温度，从而使得可以通过电阻测温法和对载体4的温度进行标定的双测温方式控制载体4。
虽然温度校准部93能够体现加热件91温度，温度检测单元10可以检测温度校准部93的温度，但是，由于反应样品在扩增阶段温度变化很快，温度传感器等温度检测单元10检测载体4的温度时，热量从温度校准部93传递至温度检测单元10需要一定时间，因此正常情况下温度检测单元10测量的检测结果会存在1～2s的测温延时，在快速升降温过程中1-2s的时间，载体4的温度变化可以达到30℃以上，因此，在快速升降温过程中，通过温度检测单元10控制载体4相对困难。
本实施例既没有完全依赖未校准的电阻测温法测得的温度值，也没有完全依 赖温度检测单元10检测的温度控制载体4，而是通过将两者结合，采用对载体4的温度校准部93进行测温以及使用电阻测温法对加热件91测温，从而得以快速精确地控制载体4的温度，达到准确控温的目的，从而克服了现有技术中常用的温度检测方法带来的温度检测延时以及温度测量误差大的问题。
可以理解的是，当内置加热器45设置有至少两个加热件91时，每个加热件91均对应设置有温度校准部93和电阻检测件，以使得加热件91的温度均能够被校准。
为了更清楚的表述本实施例中的如何利用温度校准部93对电阻检测件检测的温度进行校准，结合图11所示，展示了一个实际检测中，通过温度检测单元10对电阻测温法进行校准的过程。在校准温度值前，预设初始的RT温度曲线，即温度预设曲线，然后向载体4的加热件91施加一个很小的电流，如电流可以小于1毫安。其中，施加很小的电流目的是为了读取到加热件91的电阻，又不会使加热件91发热。
第一次校准：温度检测单元10测得第一个温度标定值T1，电阻检测单元检测加热件91在T1温度下的第一电压U1和第一电流I1，根据R＝U/I可以得到加热件91在T1温度下的电阻R1。
第二次校准：随后温度检测单元10测得第二个温度标定值T2，电阻检测单元检测加热件91在T2温度下的第二电压U2和第二电流I2，根据R＝U/I可以得到加热件91在T2温度下的电阻R2。
最后根据两组二元一次方程：R1＝R0(1+αΔT1)和R2＝R0(1+αΔT2)(其中，ΔT1＝T1-T0，ΔT2＝T2-T0，R1是加热件91在温度T1下对应的电阻值，R2是加热件91在温度T2下对应的电阻值，α是材料的电阻温度系数，T0为标称温度，R0是标称电阻值)，得到R0和α的具体值，即得到了准确的R-T曲线，后续便可以以电阻测温法测得的加热件91温度为反馈进行准确控温。
在核酸扩增的整个过程中均可以检测温度标定值，因此在后续过程中还可以对温度进行多次校准，以进一步提高检测精度。
如图12所示，为便于温度检测单元10测温，温度检测单元10的两个第一触点分别与两个温度校准部93接触，两个温度校准部93之间不导电，此时，可选地，内置加热器45还可以包括外部电连接触点97和电连接引线98，外部电连接触点97和电连接引线98的数量可以均为两个，两个外部电连接触点97分别位于两个温度校准部93相互远离的一侧，一外部连接触点与一温度校准部93通过一电连接引线98电连接，另一外部连接触点与另一温度校准部93通过另一电连接引线98电连接。
如图10所示，上传导组件92的均热层921的传导至温度校准部93，温度校准部93通过电连接引线98在外部电连接触点97与外部实现电连接，其中，电连接引线98直径小于温度校准部93和外部电连接触点97，由此，减小温度校准部93通过电连接引线98产生的热量损失，因而温度校准部93能较好地体现上传导组件92， 如上传导组件92的均热层921的温度，温度检测单元10通过焊点与温度校准部93实现良好的电和热接触，当上传导组件92，如上传导组件92的均热层921温度发生变化时，温度检测单元10能快速且精准低感知到温度变化，温度变化导致温度检测单元10的阻值变化，在外部电连接触点97实时检测温度检测单元10的阻值变化，即可实现实时温度检测。
如图10所示，可选地，为缩短温度校准部93的温度与加热件91的温度一致的时间，可选地，载体4还可以包括快速传导部94，快速传导部94用于将加热件91的热量传导至温度校准部93。具体而言，在本实施例中，加热件91的热量间接传导至温度校准部93，如加热件91将均热层921加热，均热层921的热量通过快速传导部94传导至温度校准部93，由此，温度校准部93准确体现均热层921的温度，进而温度检测单元10可以准确测量均热层921的温度。又由于反应样品的厚度很小，故反应样品的温度基本与均热层921的温度一致，故通过检测温度校准部93的温度可以得到反应样品的温度。
优选地，快速传导部94的一侧连接上传导组件92靠近加热件91的一侧或连接于下传导组件95靠近加热件91的一侧，另一侧连接于温度校准部93。上传导组件92的下表面和下传导组件95的上表面距离加热件91最近，其温度最先接近加热件91的温度，因此，快速传导部94的设置方式可以使快速传导部94的温度与加热件91的温度在最短的时间达到一致。可选地，快速传导部94由导热率较高的材料制成，如铜或铝等金属材料，或导热陶瓷等。快速传导部94的导热率尤其优于下传导组件95的导热率，以快速将热量传递至温度校准部93。
快速传导部94包括贴片941和一个或多个导柱942，贴片941与上传导组件92靠近加热件91的一侧贴合或与下传导组件95靠近加热件91的一侧贴合，一个或多个导柱942的一端连接于贴片941，另一端穿设于下传导组件95并与温度校准部93连接。上传导组件92的下表面和下传导组件95的上表面距离加热件91最近，其温度最先接近加热件91的温度，因此，贴片941的设置方式可以最快使快速传导部94的温度与加热件91的温度一致。贴片941可以增加快速传导部94与上传导组件92或下传导组件95的接触面积，提高传导效率。导柱942的横截面面积可以小于贴片941的横截面面积，可以将贴片941的温度快速传导至温度校准部93，保证缘热阻层按照设计产生所需的热阻。可选地，贴片941和导柱942由铜等高导热率的材料制成，当需要贴片941和导柱942为绝缘材料，以避免载体4出现短路时，贴片941或导柱942可以由高导热陶瓷等材料制成。
可以理解的是，温度校准部93可以与贴片941一一对应设置，两个温度校准部93也可以连接在一个贴片941上。一个温度校准部93可以与一个导柱942连接，为提高温度校准部93的温度均匀性，温度校准部93也可以与多个导柱942连接。
如图10所示，为便于得到加热件91的电阻以及为加热件91供电，可选地，载体4的外表面设置有多个第二触点96，第二触点96与加热件91电连接。通过第二触点96可以得到加热件91的电流和电压，进而可以得到加热件91的电阻值。可以 理解的是，当多个加热件91独立被控制时，每个加热件91均对应设置有第二触点96，以分别校准加热件91和为加热件91供热。
本实施例中，第二触点96使得载体4能够实现自身的温度测量功能，相比较于传统结构只能通过外部测温单元测量温度，本实施例可以直接测量载体4本身的温度，因而测温更加准确和快速，可以提升控温系统的准确性和控制速度。
虽然，上文中已经用一般性说明、具体实施方式及试验，对本申请作了详尽的描述，但在本申请基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本申请精神的基础上所做的这些修改或改进，均属于本申请要求保护的范围。

Claims (26)

  1. 一种荧光光源装置，包括：
    至少两个荧光发射单元(1)，所述至少两个荧光发射单元(1)用于发射激发光；
    控制单元，所述控制单元控制所述至少两个荧光发射单元(1)分别在不同时间段发射激发光。
  2. 根据权利要求1所述的荧光光源装置，其特征在于，所述荧光发射单元(1)包括光源(12)和激发光纤(11)，所述激发光纤(11)用于传输所述光源(12)的发射的所述激发光。
  3. 根据权利要求1所述的荧光光源装置，其特征在于，所述至少两个荧光发射单元(1)发射的所述激发光的波长互不相同。
  4. 根据权利要求1所述的荧光光源装置，其特征在于，所述激发光为单色光或多色光。
  5. 一种荧光检测光路系统，包括权利要求1-4任意一项所述的荧光光源装置和荧光探测单元(2)。
  6. 根据权利要求5所述的荧光检测光路系统，其特征在于，所述荧光探测单元(2)包括至少两个荧光传输光路(21)，至少两个所述荧光发射单元(1)与至少两个所述荧光传输光路(21)一一对应。
  7. 根据权利要求6所述的荧光检测光路系统，其特征在于，每个所述荧光传输光路(21)均包括允许预设荧光信号通过的滤光片(211)。
  8. 根据权利要求6或7所述的荧光检测光路系统，其特征在于，所述荧光探测单元(2)还包括探测器(22)，多个所述荧光传输光路(21)均连接于所述探测器(22)。
  9. 根据权利要求8所述的荧光检测光路系统，其特征在于，所述探测器(22)的数量为一个，所述探测器(22)与所述控制单元电连接，以分时间段记录荧光信号的强度。
  10. 根据权利要求6所述的荧光检测光路系统，其特征在于，所述荧光检测光路系统还包括光纤座(3)，所述荧光传输光路(21)包括收集光纤(212)，所述荧光光源装置的激发光纤(11)射出所述激发光的射出端，以及所述收集光纤(212)射入荧光信号的射入端形成光纤组(31)，所述光纤组(31)设置在所述光纤座(3)内，所述光纤座(3)内的光纤沿第一方向排列，所述第一方向为光纤的径向，以使所述收集光纤(212)的射出端和所述激发光纤(11)的射入端在所述光纤座(3)中呈扁平排列。
  11. 根据权利要求10所述的荧光检测光路系统，其特征在于，至少所述两个荧光传输光路(21)和所述至少两个荧光发射单元(1)形成至少两组所述光纤组(31)，至少两组所述光纤组(31)沿所述第一方向依次排列。
  12. 根据权利要求10所述的荧光检测光路系统，其特征在于，一所述光纤组 (31)包括至少两个所述收集光纤(212)，一所述光纤组(31)内的所述激发光纤(11)的所述第一方向的两侧至少设置有一所述收集光纤(212)。
  13. 根据权利要求8所述的荧光检测光路系统，其特征在于，所述探测器(22)包括硅光电倍增管、光子型探测器或光电倍增管。
  14. 一种PCR检测系统，包括载体(4)和权利要求5-13任意一项所述的荧光检测光路系统(100)，所述荧光检测光路系统(100)用于检测所述载体(4)内的反应样品。
  15. 根据权利要求14所述的PCR检测系统，其特征在于，所述载体(4)包括相对设置的第一壁(43)和第二壁(44)，以及设置在所述第一壁(43)和所述第二壁(44)之间的侧壁(42)，所述第一壁(43)、所述第二壁(44)和所述侧壁(42)形成容纳腔(41)，所述容纳腔(41)和/或所述载体(4)为扁平结构，至少部分所述侧壁(42)透光，所述荧光检测光路系统(100)通过透光的所述侧壁(42)检测反应样品。
  16. 根据权利要求14所述的PCR检测系统，其特征在于，所述载体(4)的一侧或至少两侧均设置有所述荧光发射单元(1)。
  17. 根据权利要求15所述的PCR检测系统，其特征在于，所述透光的所述侧壁(42)材料为聚二甲基硅氧烷、聚丙烯或聚碳酸酯。
  18. 根据权利要求15所述的PCR检测系统，其特征在于，所述载体(4)包括至少一个内置加热器(45)，所述第一壁(43)和/或所述第二壁(44)为所述内置加热器(45)。
  19. 根据权利要求18所述的PCR检测系统，其特征在于，所述内置加热器(45)包括加热件(91)。
  20. 根据权利要求19所述的PCR检测系统，其特征在于，所述内置加热器(45)包括至少两个独立控制的所述加热件(91)。
  21. 根据权利要求19所述的PCR检测系统，其特征在于，所述内置加热器(45)还包括用于体现所述加热件(91)温度的温度校准部(93)。
  22. 根据权利要求21所述的PCR检测系统，其特征在于，所述内置加热器(45)还包括快速传导部(94)，所述快速传导部(94)用于将所述加热件(91)的热量传导至所述温度校准部(93)。
  23. 根据权利要求19所述的PCR检测系统，其特征在于，所述PCR检测系统还包括电阻检测件，所述电阻检测件用于检测所述加热件(91)的电阻。
  24. 根据权利要求15所述的PCR检测系统，其特征在于，所述扁平结构指，所述容纳腔(41)或所述载体垂直于其厚度方向的方向的尺寸大于其厚度方向的尺寸。
  25. 根据权利要求24所述的PCR检测系统，其特征在于，所述容纳腔(41)或所述载体垂直于其厚度方向的方向的尺寸与其厚度方向的尺寸之比大于5：1。
  26. 根据权利要求25所述的PCR检测系统，其特征在于，所述尺寸之比为50： 1～100：1。
PCT/CN2024/097617 2023-06-05 2024-06-05 一种荧光光源装置、荧光检测光路系统及pcr检测系统 Ceased WO2024251172A1 (zh)

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CN101705280A (zh) * 2009-11-16 2010-05-12 杭州博日科技有限公司 定量pcr的多波长荧光检测方法及其装置
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