WO2024251172A1 - Dispositif source de lumière fluorescente, système de trajet de lumière de détection de fluorescence et système de détection de pcr - Google Patents

Dispositif source de lumière fluorescente, système de trajet de lumière de détection de fluorescence et système de détection de pcr 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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WIPO (PCT)
Prior art keywords
fluorescence
optical fiber
detection
temperature
carrier
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Ceased
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PCT/CN2024/097617
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English (en)
Chinese (zh)
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 CN202310661917.5A external-priority patent/CN119086434A/zh
Priority claimed from CN202321422229.5U external-priority patent/CN220376678U/zh
Application filed by Guangzhou National Laboratory filed Critical Guangzhou National Laboratory
Publication of WO2024251172A1 publication Critical patent/WO2024251172A1/fr
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

La présente invention concerne un dispositif source de lumière fluorescente, un système de trajet de lumière de détection de fluorescence et un système de détection de PCR. Le dispositif source de lumière fluorescente comprend : au moins deux unités d'émission de fluorescence, lesdites au moins deux unités d'émission de fluorescence étant utilisées pour émettre une lumière d'excitation ; et une unité de commande, l'unité de commande commandant lesdites au moins deux unités d'émission de fluorescence pour émettre la lumière d'excitation à différentes périodes de temps, respectivement.
PCT/CN2024/097617 2023-06-05 2024-06-05 Dispositif source de lumière fluorescente, système de trajet de lumière de détection de fluorescence et système de détection de pcr Ceased WO2024251172A1 (fr)

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CN202321422229.5 2023-06-05
CN202310661917.5 2023-06-05
CN202310661917.5A CN119086434A (zh) 2023-06-05 2023-06-05 一种荧光光源装置、荧光检测光路系统及pcr检测系统
CN202321422229.5U CN220376678U (zh) 2023-06-05 2023-06-05 一种荧光光源装置、荧光检测光路系统及pcr检测系统

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6597450B1 (en) * 1997-09-15 2003-07-22 Becton, Dickinson And Company Automated Optical Reader for Nucleic Acid Assays
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
US20150165438A1 (en) * 2013-12-13 2015-06-18 Canon Kabushiki Kaisha Microfluidic device and temperature control method for microfluidic device
CN106680250A (zh) * 2015-11-10 2017-05-17 北京万泰生物药业股份有限公司 用于聚合酶链式反应的检测机构及聚合酶链式反应装置
CN112432934A (zh) * 2020-11-05 2021-03-02 北京中科生仪科技有限公司 发射光检测方法
CN218893678U (zh) * 2022-11-01 2023-04-21 埃妥生物科技(杭州)有限公司 荧光采集装置以及荧光检测仪
CN220376678U (zh) * 2023-06-05 2024-01-23 广州国家实验室 一种荧光光源装置、荧光检测光路系统及pcr检测系统

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6597450B1 (en) * 1997-09-15 2003-07-22 Becton, Dickinson And Company Automated Optical Reader for Nucleic Acid Assays
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
US20150165438A1 (en) * 2013-12-13 2015-06-18 Canon Kabushiki Kaisha Microfluidic device and temperature control method for microfluidic device
CN106680250A (zh) * 2015-11-10 2017-05-17 北京万泰生物药业股份有限公司 用于聚合酶链式反应的检测机构及聚合酶链式反应装置
CN112432934A (zh) * 2020-11-05 2021-03-02 北京中科生仪科技有限公司 发射光检测方法
CN218893678U (zh) * 2022-11-01 2023-04-21 埃妥生物科技(杭州)有限公司 荧光采集装置以及荧光检测仪
CN220376678U (zh) * 2023-06-05 2024-01-23 广州国家实验室 一种荧光光源装置、荧光检测光路系统及pcr检测系统

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