WO2016124083A1 - 一种超小型化多通道实时荧光光谱检测装置 - Google Patents

一种超小型化多通道实时荧光光谱检测装置 Download PDF

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WO2016124083A1
WO2016124083A1 PCT/CN2016/072014 CN2016072014W WO2016124083A1 WO 2016124083 A1 WO2016124083 A1 WO 2016124083A1 CN 2016072014 W CN2016072014 W CN 2016072014W WO 2016124083 A1 WO2016124083 A1 WO 2016124083A1
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fluorescent
channel
optical fiber
ultra
band pass
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PCT/CN2016/072014
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English (en)
French (fr)
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余家昌
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余家昌
埃捷思生物科技有限公司
奥然生物科技（上海）有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

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  • the invention relates to a device for laboratory or medical detection, in particular to an ultra-compact multi-channel real-time fluorescence spectrum detecting device.
  • real-time quantitative PCR is a method in which a fluorescent group is added to a PCR reaction system, and the entire PCR process is monitored in real time by using fluorescence signal accumulation, and finally the unknown template is quantitatively analyzed by a standard curve.
  • the real-time PCR instrument is a fluorescent detection and analysis system based on the common PCR instrument. As shown in FIG.
  • the excitation light source 101 of the instrument emits excitation light through the filter 102, the beam splitter 103, the refractor 104, and the lens 105, and finally projects to the amplification tube 106.
  • the fluorescent substance in the reaction liquid is excited by the excitation light to generate a specific emitted light, which passes through the lens 105, the refractor 104, the beam splitter 103, the filter 107 and the multi-lens 108, and finally in the photoreceiver.
  • a fluorescent signal was obtained on 109.
  • the image is transmitted to the computer software system and analyzed to show a fluorescence intensity growth curve. The intensity of the fluorescence is proportional to the amount of DNA in the specimen.
  • unused probe primers often correspond to excitation light and emission light of different wavelengths. Therefore, in practical applications, a multi-channel fluorescence detection system, that is, a plurality of excitation light sources and a plurality of emission light channels are often required. Fluorescence detection and fluorescence spectroscopic systems traditionally used in PCR instruments utilize a combination of separate beamsplitters, bandpass filters, lens groups and photoreceivers.
  • the LS32 manufactured by Idaho Corporation of the United States is an excitation light source and a fluorescence detection system for three emission channels.
  • the lightCycler 480 manufactured by Roche is a fluorescence detection system in which a plurality of excitation light sources correspond to a plurality of emission channels.
  • the technical problem solved by the present invention is to provide an ultra-compact multi-channel real-time fluorescence spectrum detecting device, which uses a multi-spectral sensor instead of a conventional filter spectroscopic system, which greatly simplifies the device structure.
  • the complexity and the size of the detection system are very important.
  • the technical problem to be solved by the present invention is to provide an ultra-compact multi-channel real-time fluorescence spectrum detecting device, which uses a fluorescent transmission channel composed of an optical fiber or a fiber bundle to reduce the energy loss of the emitted fluorescent light in the optical path propagation, and the fluorescent signal
  • the transmission mode is cleaner than the traditional open transmission structure, and is not easily contaminated by dust, which improves the accuracy and sensitivity of the experiment.
  • an ultra-small multi-channel real-time fluorescence spectrum detecting apparatus including:
  • a fluorescent transmission channel for collecting a fluorescent signal formed by the emitted light, the fluorescent transmission channel comprising an optical fiber or a fiber bundle composed of the optical fiber, and the fluorescent signal is detected by an effective output of the optical fiber or the optical fiber bundle;
  • a multi-spectral sensor comprising one or more band pass filters, and a photodiode corresponding to the number of band pass filters; the multispectral sensor receiving and detecting transmission from the fluorescent transmission channel
  • the intensity of the fluorescent signal, the bandpass filter achieves spectral selection of fluorescence at different wavelengths, and the photodiode induces the fluorescence intensity of the wavelength corresponding to the matched bandpass filter.
  • an ultra-compact multi-channel real-time fluorescence spectrum detecting apparatus provided by an embodiment of the present invention further includes part or all of the following technical features:
  • the fluorescent transmission channel comprises a mirror, a converging lens and an optical fiber or a fiber bundle composed of an optical fiber; the fluorescent substance is directly emitted by the excitation light source or reflected by the mirror and then concentrated by the converging lens to the optical fiber or the optical fiber beam. Entrance end.
  • the fluorescence transmission channel further includes a mirror array and a condensing lens lens array disposed on both sides of the sample cell, and the mirror array includes a plurality of mirrors disposed in parallel, and the reflective surface of the mirror is disposed such that a light-collecting region having a matching concave shape, the converging lens array comprising a converging lens corresponding to the number of mirrors, each of the mirrors and the matching converging lens forming a fluorescence collecting unit through which the fluorescent signal passes After collection, the fiber bundle consisting of an optical fiber or an optical fiber is transmitted to the multi-spectral sensor.
  • the fluorescent transmission channel is provided with a fiber bundle, wherein the fiber bundle includes a plurality of optical fibers whose optical inlet ends are arranged in one body, and the optical entrance end of each optical fiber corresponds to a fluorescent collecting unit in the optical fiber bundle.
  • Each fiber forms an independent wavelength-selective fluorescent channel.
  • the fluorescent transmission channel is provided with a plurality of single fibers, and the light inlet ends of each of the individual fibers correspond to one fluorescent collecting unit and form an independent wavelength selective fluorescent channel.
  • the multi-spectral sensor comprises a plurality of band pass filters integrated on a surface thereof, and a photodiode corresponding to the number of the band pass filters, the band pass filter being attached to the photodiode a surface of each of the band pass filters and the matching photodiode to form a fluorescence intensity detecting unit for detecting the fluorescence intensity of the wavelength corresponding to the band pass filter disposed on the fluorescence intensity detecting unit;
  • the light exit end of the fiber corresponds to a fluorescence intensity detecting unit.
  • the excitation light source is provided with one or two or more, and two or more excitation light sources generate excitation light of different frequencies. More preferably, the excitation light source is a laser diode or an LED light source.
  • an embodiment of the present invention provides a detection device for laboratory or medical use, comprising the ultra-miniature multi-channel real-time fluorescence spectrum detecting device described in the above scheme.
  • the embodiment of the present invention further provides a nucleic acid amplification detector, comprising the ultra-miniature multi-channel real-time fluorescence spectrum detecting device described in the above scheme; the sample pool is further provided with a temperature control for realizing nucleic acid amplification reaction Temperature controlled probe.
  • the miniaturized multi-channel real-time fluorescence spectrum detecting device replaces the traditional filter beam splitting system by using a multi-spectral sensor, which greatly simplifies the complexity of the device structure and reduces the volume of the detecting system.
  • the multi-spectral sensor comprises a band pass filter and a photodiode, and the band pass filter and the matched photodiode constitute a fluorescence intensity detecting unit, and the band pass integrated on the surface of the multispectral sensor is utilized.
  • the filter is used to realize the spectral selection of different wavelengths, and the photodiode is used to sense the fluorescence intensity of the wavelength corresponding to the matched band-pass filter, thereby realizing the replacement of the traditional spectroscopic technique by a small multi-spectral sensor with a small structure.
  • a number of discretely disposed optical components accurately deliver more information and reduce the size of the optical device; and, because the surface of the multispectral sensor integrates multiple bandpass filters, the bandpass filter is selected When the wavelengths of the light waves are different, it is possible to simultaneously detect the intensity of the fluorescent signals of a plurality of wavelengths selected via the band pass filter.
  • the miniaturized multi-channel real-time fluorescence spectrum detecting device of the present invention adopts a fluorescent transmission channel composed of an optical fiber or a fiber bundle, and each of the optical fibers or the optical fiber bundle forms an independent emission fluorescent channel, which reduces the emission of the fluorescent light in the optical path.
  • the energy loss in the light, and the transmission mode of the fluorescent signal is cleaner than the traditional open transmission structure, is not easy to be contaminated by dust, and improves the accuracy and sensitivity of the experiment.
  • FIG. 1 is a schematic diagram showing the principle of detection of a single channel real-time PCR instrument in the prior art.
  • FIG. 2 is a schematic structural view of an ultra-miniature multi-channel real-time fluorescence spectrum detecting apparatus according to a preferred embodiment 1 of the present invention.
  • FIG. 3 is a schematic structural view 1 of a multi-spectral sensor in an ultra-miniature multi-channel real-time fluorescence spectrum detecting apparatus according to a preferred embodiment 1 of the present invention.
  • FIG. 4 is a second structural diagram of a multi-spectral sensor in an ultra-miniature multi-channel real-time fluorescence spectrum detecting apparatus according to a preferred embodiment 1 of the present invention.
  • FIG. 5 is a schematic structural view of an ultra-miniature multi-channel real-time fluorescence spectrum detecting apparatus according to a preferred embodiment 2 of the present invention.
  • FIG. 6 is a schematic structural view of an ultra-miniature multi-channel real-time fluorescence spectrum detecting apparatus according to a preferred embodiment 3 of the present invention.
  • the ultra-miniature multi-channel real-time fluorescence spectrum detecting device is applied to a laboratory or medical detection experiment, and can be used for quantitative detection of, for example, a nucleic acid amplification reaction, but is not limited thereto.
  • the The ultra-compact multi-channel real-time fluorescence spectrometer can also be applied to other real-time quantitative or qualitative detection experiments based on fluorescence signal detection.
  • the ultra-miniature multi-channel real-time fluorescence spectrum detecting apparatus includes a sample cell 10, an excitation light source 20, a fluorescence transmission channel 30, and a multi-spectral sensor 40.
  • the sample cell 10 houses a sample to be tested and a fluorescent substance related to the concentration of the sample to be tested.
  • the excitation light source 20 is used to excite the fluorescent substance in the sample cell, and the fluorescent substance is excited by the excitation light generated by the excitation light source to generate emitted light (fluorescence).
  • the fluorescent transmission channel 30 is for collecting a fluorescent signal formed by the emitted light, and the fluorescent transmission channel 30 includes a bundle 31 of optical fibers 32, and the fluorescent signal is detected by the effective output of the bundle 31.
  • the multispectral sensor 40 includes a plurality of band pass filters 41 and photodiodes 42 corresponding to the number of band pass filters.
  • the multispectral sensor 40 receives and detects the intensity of the fluorescent signal transmitted from the fiber bundle 31. Since the surface of the multispectral sensor 40 integrates a plurality of band pass filters, the wavelength of the corresponding light wave selected by the different band pass filters is selected. When set to different, the spectral selection of fluorescence at different wavelengths can be simultaneously achieved, and the photodiode induces the fluorescence intensity of the wavelength corresponding to the matched bandpass filter.
  • the sample cell 10 contains a sample to be tested and a fluorescent substance related to the concentration of the sample to be tested.
  • the sample to be tested is the target nucleic acid fragment generated in the nucleic acid amplification reaction, and the fluorescence related to the target nucleic acid concentration is The substance is usually integrated into the specific primer of the nucleic acid amplification reaction, and the fluorescent substance connected to the primer is not excited by the excitation source when the amplification reaction is not performed.
  • Fluorescence usually associated with a fluorescent substance or a fluorescent group, is also attached to the nucleic acid amplification reaction primer by a fluorescent quenching group, thereby enabling the amount of the fluorescent substance or the fluorescent group to be excited by the excitation light in an effective amplification reaction.
  • the concentration of the target nucleic acid is proportional to the concentration, so that the ultra-miniature multi-channel real-time fluorescence spectrum detecting device of the present invention can determine the concentration of the sample to be tested by detecting the intensity of the excited fluorescence.
  • it is also necessary to supplement the standard in the nucleic acid amplification reaction, and determine the concentration of the sample to be tested by analytical processing.
  • the above description is merely an example of performing quantitative detection of a nucleic acid amplification reaction, and the sample cell of the present invention and its corresponding reaction principle are described, but not limited thereto. In practical applications, the ultra-small size of the present invention.
  • the multi-channel real-time fluorescence spectrometer can also be applied to other real-time quantitative or qualitative detection experiments based on fluorescence signal detection.
  • the fluorescent transmission channel includes a mirror array 33 and a converging lens lens array 34 disposed on both sides of the sample cell 10, and the mirror array 33 includes a plurality of reflective reflectors arranged in parallel.
  • the mirror 331 has a converging lens array 341 corresponding to the number of mirrors, and each of the mirrors 331 and the matching converging lens 341 constitute a fluorescence collecting unit.
  • the fluorescent signal is collected by the fluorescence collecting unit and then transmitted to the multispectral sensor 40 via the fiber bundle 31.
  • the emitted light (fluorescence) generated by the excitation light source is generated in a divergent manner, partially transmitted directly to the condenser lens 341, and the other portion is reflected by the mirror 331 and then concentrated by the converging lens 341 to the light entrance end of the fiber bundle.
  • the reflecting surface of the mirror 331 employed in the present embodiment is disposed in a concave shape that matches the range of its light collecting region.
  • the optical fiber bundle 31 disposed in the fluorescent transmission channel includes a plurality of optical fibers 32 whose optical inlet ends are integrally arranged, and the optical entrance end of each optical fiber 32 corresponds to a fluorescent collecting unit, and the optical fiber bundle
  • Each of the fibers in the fiber forms a separate wavelength selective fluorescent channel.
  • the multispectral sensor 40 includes a plurality of band pass filters 41 integrated on the surface thereof, and photodiodes 42 corresponding to the number of band pass filters 41, and the band pass filters 41 are attached.
  • each of the band pass filters 41 and the matched photodiode 42 constitute a fluorescence intensity detecting unit for detecting a wavelength corresponding to the band pass filter disposed on the fluorescence intensity detecting unit Fluorescence intensity, per The light exit end of one fiber corresponds to a fluorescence intensity detecting unit.
  • each optical fiber 32 corresponds to a fluorescent collecting unit, and each of the optical fibers forms an independent wavelength selective fluorescent channel, and each optical fiber is
  • the light exit end corresponds to a fluorescence intensity detecting unit, and therefore includes a fluorescent collecting unit composed of a mirror 331 and a matching converging lens 341, a fluorescent channel corresponding to the optical fiber 32, and a band pass filter 41 and matched thereto.
  • the photodiodes 42 constitute a one-to-one correspondence of the fluorescence intensity detecting units, thereby achieving independent transmission of the sub-channels of the fluorescent signals and independent detection of the fluorescence intensities of different wavelengths selected according to the band pass filters.
  • the multi-spectral sensor 40 includes eight band pass filters 41 integrated on the surface thereof, corresponding thereto. Further, eight photodiodes 42 are provided, and the band pass filter 41 is attached to the surface of the photodiode 42, and each of the photodiodes 42 and the band pass filter 41 attached thereto constitute a fluorescence intensity detecting unit. Each of the fluorescence intensity detecting units can select an independent emission fluorescence wavelength, and detect the fluorescence intensity of the wavelength corresponding to the band pass filter disposed on the fluorescence intensity detecting unit.
  • each optical fiber 32 corresponds to a fluorescent collecting unit, and the optical outlet end of each optical fiber ends.
  • one-to-one corresponding number is assigned to the light entrance end, the light exit end of the optical fiber 32, and the band pass filter 41 in the multi-spectral sensor 40, and each number corresponds to an independent fluorescence collection and transmission.
  • the detection channel, and each channel is connected by a corresponding fluorescence collection unit (including the mirror 331 and the matching converging lens 341), a fluorescent channel (one fiber 32 for transmitting fluorescence in the fiber bundle 31)
  • a fluorescence intensity detecting unit (composed of a photodiode 42 and a bandpass filter 41 attached thereto), when the sample cell contains a plurality of fluorescent substances, and is excited by a plurality of fluorescent substances to generate a plurality of different wavelengths
  • fluorescence when the selected wavelength of the band pass filter 41 used in each channel is set to coincide with the wavelength of one of a plurality of different wavelengths of fluorescence, the ultra-small multi-channel real-time fluorescence of the present invention can be utilized.
  • the spectrum detection device realizes scientific and accurate analysis of the experimental data of each channel, that is, simultaneous detection of multiple reactions in the sample pool.
  • the number of channels, the number of fibers in the fiber bundle, the number of band pass filters on the surface of the multi-spectral sensor, the number of converging lenses, and the number of mirrors are not limited to the technical solutions in other embodiments of the present invention.
  • the number of corresponding structures used, when specifically implemented, the number of corresponding structures and the manner of arrangement as needed should also be understood as common technical means in the art, and are within the scope of the claims of the present invention.
  • an excitation light source 20 is disposed in Embodiment 1 of the present invention.
  • the position of the light source 20 and the illumination angle of the light source are excited to enable the sample to be tested to be more Adjusted for effective reception of illumination.
  • the frequency of the excitation source is typically greater than the emitted light (fluorescence).
  • the excitation light source selects the laser diode, and the laser can enable the excitation light emitted by the excitation light source to be controlled in a single or small frequency range, thereby ensuring the excitation light having the excitation capability for the fluorescent substance.
  • the intensity and avoiding stray light interference thus improving the accuracy and sensitivity of the experiment.
  • other light sources such as ordinary LEDs, are selected as the excitation light source according to needs and specific experimental conditions, but the excitation light source must contain light waves that can effectively excite the fluorescent material to generate emitted light.
  • an excitation light source is provided in Embodiment 1 of the present invention, but in other embodiments of the present invention, the number and position of the excitation light sources are also adjusted as needed.
  • two excitation light sources 20 are disposed on both sides of the sample cell, and the excitation light beams generated by the two excitation light sources 20 have different wavelengths in the sample cell. Containing a variety of fluorescent substances, different excitation light can excite different fluorescent signals, and the selection and setting of the excitation light source 20 is more targeted, and the frequency range and power of the excitation light are controlled according to the controllability.
  • the excitation light source may be set to multiple as needed, for different purposes, and selecting the excitation light source of different frequencies can make the test reaction according to the pertinence and accuracy, and can also make the sample. Pool The sample to be tested and the fluorescent substance are subjected to more uniform excitation irradiation.
  • the ultra-miniature multi-channel real-time fluorescence spectrum detecting device provided in Embodiment 3 of the present invention has a plurality of single optical fibers 32 disposed in a fluorescent transmission channel, and a light entrance end and a single optical fiber 32 of each single optical fiber 32.
  • the fluorescence collection unit corresponds to and forms a separate wavelength selective fluorescent channel.
  • the fluorescent substance contained in each sample cell 10 is also set to one, and the excitation light source 20 also has a one-to-one correspondence with the sample cell 10, and each sample cell 10 is correspondingly provided with a fluorescent collecting unit.
  • a mirror (not shown) and a matching condenser lens 341 are included.
  • each photodiode 42 and the band pass filter 41 attached thereto constitute a fluorescence intensity detecting unit.
  • Each of the fluorescence intensity detecting units can select an independent emission fluorescence wavelength, and detect the fluorescence intensity of the wavelength corresponding to the band pass filter disposed on the fluorescence intensity detecting unit.
  • the eight individual fibers 32 form eight independent wavelength selective fluorescent channels, and the light entrance end of each single fiber corresponds to a fluorescent collecting unit, a sample cell and an excitation light source, and each single optical fiber 32 The light exit end corresponds to a fluorescence intensity detecting unit.
  • the sample cell 10 the light entrance end of the optical fiber 32 in the fiber bundle 32, the light exit end, and the band pass filter 41 in the multispectral sensor 40, each number corresponds to an independent fluorescence collection and transmission.
  • each channel is connected by a corresponding fluorescence collection unit (including the mirror 331 and the matching converging lens 341), a fluorescent channel (single fiber 32) and a fluorescence intensity detecting unit ( It is composed of a photodiode 42 and a band pass filter 41 attached thereto, so that the experimental data of each channel can be scientifically and accurately realized by the ultra-miniature multi-channel real-time fluorescence spectrum detecting device of the present invention. analysis.
  • each sample cell 10 contains a fluorescent substance, and an excitation light source corresponding to the fluorescent substance is disposed, thereby simultaneously detecting a single reaction in a plurality of different sample cells.
  • the number of channels, the number of single fibers, the number of band pass filters on the surface of the multi-spectral sensor, the number of converging lenses, and the number of mirrors are not limited to those used in the technical solutions of other embodiments of the present invention.
  • the number of corresponding structures, when specifically implemented, the number of corresponding structures and the manner of arrangement as needed should also be understood as common technical means in the art, and fall within the scope of the claims of the present invention.
  • the embodiment of the present invention further provides a detection device for laboratory or medical treatment, which comprises the ultra-miniature multi-channel real-time fluorescence spectrum detecting device disclosed in the above embodiment.
  • the ultra-miniature multi-channel real-time fluorescence spectrum detecting apparatus disclosed in the above embodiments is particularly suitable for nucleic acid amplification detecting experiments. Therefore, embodiments of the present invention further provide a nucleic acid amplification detecting apparatus, including the above embodiment.
  • the ultra-miniature multi-channel real-time fluorescence spectrum detecting device disclosed in the present invention is particularly suitable for nucleic acid amplification detecting experiments. Therefore, embodiments of the present invention further provide a nucleic acid amplification detecting apparatus, including the above embodiment.
  • temperature control is an important technical index in nucleic acid amplification reactions, such as ordinary PCR temperature cyclic amplification, and temperature control problems of constant temperature amplification reactions, it is very important to implement nucleic acid amplification in the sample cell. Reaction temperature controlled temperature controlled probe.
  • the ultra-miniature multi-channel real-time fluorescence spectrum detecting device disclosed in the above embodiment uses a multi-spectral sensor instead of the conventional filter spectroscopic system, which greatly simplifies the structure complexity and reduces the complexity. Detect the volume of the system.
  • the multi-spectral sensor comprises a band pass filter and a photodiode, and the band pass filter and the matched photodiode constitute a fluorescence intensity detecting unit, and the band pass integrated on the surface of the multispectral sensor is utilized.
  • the filter is used to achieve spectral selection at different wavelengths, and the photodiode is used to sense the fluorescence intensity of the wavelength corresponding to the matched bandpass filter.
  • the miniaturized multi-channel real-time fluorescence spectrum detecting device of the present invention adopts a fluorescent transmission channel composed of an optical fiber or a fiber bundle, and each of the optical fibers or the optical fiber bundle forms an independent emission fluorescent channel, which reduces the emission of the fluorescent light in the optical path.
  • the energy loss in the light, and the transmission mode of the fluorescent signal is cleaner than the traditional open transmission structure, is not easy to be contaminated by dust, and improves the accuracy and sensitivity of the experiment.

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Abstract

一种超小型化多通道实时荧光光谱检测装置，包括样品池（10）、激发光源（20）、荧光传输通道（30）和多光谱传感器（40），所述荧光传输通道（30）用于收集发射光形成的荧光信号，荧光信号通过光纤（32）或光纤束（31）有效输出进行检测；所述多光谱传感器（40）包括带通滤光片（41）和光电二极管（42），所述带通滤光片（41）实现不同波长荧光的光谱选择，光电二极管（42）感应出跟其相匹配的带通滤光片（41）所对应的波长的荧光强度。该检测装置简化了设备结构并减小了体积；采用由光纤（32）或光纤束（31）组成的荧光传输通道（30），降低了发射荧光在光路传播中的能量损失，且相比于传统的开放式传输结构更洁净，不易于被灰尘污染，提高了实验的准确性及灵敏度。

Description

一种超小型化多通道实时荧光光谱检测装置 技术领域
本发明涉及用于实验室或者医疗检测的设备，尤其涉及一种超小型化多通道实时荧光光谱检测装置。
背景技术
目前，在实验室或者医疗检测技术领域中，通过在反应体系中加入荧光基团，利用荧光信号积累实时监测整个反应的进程，通过荧光信号的强度确定反应体系的定性特征，或者根据标准曲线对未知待测样品进行定量分析的方法已经广为运用，其中最为典型的代表是实时荧光定量PCR。具体而言，实时荧光定量PCR是在PCR反应体系中加入荧光基团，利用荧光信号积累实时监测整个PCR进程，最后通过标准曲线对未知模板进行定量分析的方法。荧光定量PCR仪是在普通PCR仪基础上增加了荧光检测和分析系统。如图1所示，在PCR的每一循环结束时，仪器的激发光源101发射出激发光经过滤光器一102、分光镜103、折射镜104和透镜105，最后投射到扩增管106的反应液中，反应液中的荧光物质受到激发光的激发后，产生特定的发射光，经透镜105、折射镜104、分光镜103、滤光器一107和多透镜108，最后在光电接收器109上获得荧光信号。图像传输给计算机软件系统，经分析后显示出荧光强度增长曲线，荧光的强弱与标本中的DNA量成正比。
根据荧光激发原理，不用的探针引物往往对应不同波长的激发光及发射光。因此在实际应用中，往往需要多通道荧光检测系统，即多个激发光源以及多个发射光通道。传统应用于PCR仪器中的荧光检测和荧光分光系统是利用一系列独立的分光镜，带通滤光器，透镜组以及光电接收器的组合系统。例如，由美国Idaho公司生产的LS32为一个激发光源和3个发射通道的荧光检测系统。由Roche公司生产的lightCycler 480为多个激发光源对应多个发射通道的荧光检测系统。从以上几个传统荧光分光检测系统不难看出，传统的实时荧光定量PCR 仪普遍存在体积大，结构复杂，易污染的问题，而且伴随着体积大，结构复杂缺陷的同时还当然地存在设备成本过高，投入巨大的问题，普通医学或者研究实验室难以负担该技术投入等不便于技术推广应用的问题，而易污染以及由于结构复杂而带来的准确性后灵敏度的问题也因此在所难免，同时所有这些缺陷也在客观上决定了传统的实时荧光定量PCR仪存在不利于仪器小型化和一体机化设计的技术难题。
发明内容
针对现有技术中的不足，本发明解决的技术问题在于提供一种超小型化多通道实时荧光光谱检测装置，使用多光谱传感器代替了传统的滤光片分光系统，极大的简化了设备结构的复杂程度并减小了检测系统的体积。
本发明解决的技术问题还在于提供一种超小型化多通道实时荧光光谱检测装置，采用由光纤或光纤束组成的荧光传输通道，降低了发射荧光在光路传播中的能量损失，而且该荧光信号的传输方式比传统的开放式的传输结构更洁净，不易于被灰尘污染，提高了实验的准确性及灵敏度。
为了解决上述技术问题，一方面，本发明实施例提供了一种超小型化多通道实时荧光光谱检测装置包括：
样品池，该样品池内容纳有待测样品以及与待测样品浓度相关的荧光物质；
激发光源，用于激发样品池中的荧光物质，荧光物质受激发光源产生的激发光激发后产生发射光；
荧光传输通道，用于收集发射光形成的荧光信号，该荧光传输通道包括光纤或者由光纤组成的光纤束，荧光信号通过光纤或光纤束有效输出进行检测；
多光谱传感器，所述多光谱传感器包括一个或两个以上的带通滤光片，以及与所述带通滤光片数量对应的光电二极管；该多光谱传感器接收并检测从荧光传输通道传输来的荧光信号的强度，所述带通滤光片实现不同波长荧光的光谱选择，光电二极管感应出跟其相匹配的带通滤光片所对应的波长的荧光强度。
作为本发明的优选方案，本发明的实施例提供的一种超小型化多通道实时荧光光谱检测装置进一步包括以下技术特征的部分或全部：
优选地，所述荧光传输通道包括反光镜、会聚透镜和光纤或者由光纤组成的光纤束；荧光物质受激发光源产生的发射光直射或者由反光镜反射后通过会聚透镜会聚至光纤或者光纤束光入口端。
优选地，所述荧光传输通道还包括设置于所述样品池两侧的反光镜阵列和会聚透镜透镜阵列，反光镜阵列中包含多个平行设置的反光镜，该反光镜的反光面设置成与其光收集区域的范围相匹配的凹形，所述会聚透镜阵列包含与反光镜数量对应的会聚透镜，每一个反光镜和与之匹配的会聚透镜组成一个荧光收集单元，荧光信号通过该荧光收集单元收集后再经由光纤或者由光纤组成的光纤束传送至所述多光谱传感器。
优选地，所述荧光传输通道中设置有光纤束，所述光纤束中包含有多条光入口端排列为一体的光纤，每一条光纤的光入口端与一个荧光收集单元相对应，光纤束中的每根光纤均形成一个独立波长选择的荧光通道。
优选地，所述荧光传输通道中设置有多条单根光纤，每一条单根光纤的光入口端与一个荧光收集单元相对应并形成一个独立波长选择的荧光通道。
优选地，所述多光谱传感器包括集成于其表面的多个带通滤光片，以及与所述带通滤光片数量对应的光电二极管，所述带通滤光片贴合所述光电二极管的表面；每一个带通滤光片和与之匹配的光电二极管组成一荧光强度检测单元，用于检测该荧光强度检测单元上设置的带通滤光片所对应的波长的荧光强度；每一条光纤的光出口端与一个荧光强度检测单元相对应。
优选地，所述激发光源设置有一个或者两个以上，两个以上的激发光源产生不同频率的激发光。更优地，所述激发光源为是激光二极管或LED光源。
另一方面，本发明实施例还提供了一种用于实验室或者医疗的检测装置，其包含上述方案中所述的超小型化多通道实时荧光光谱检测装置。
再有，本发明实施例还提供了一种核酸扩增检测仪，包含上述方案中所述的超小型化多通道实时荧光光谱检测装置；所述样品池还设置有实现核酸扩增反应温度控制的温控探针。
相比于现有技术，本发明的技术方案至少具有如下有益效果：
本发明实施例提供的小型化多通道实时荧光光谱检测装置，使用多光谱传感器代替了传统的滤光片分光系统，极大的简化了设备结构的复杂程度并减小了检测系统的体积。在本发明的技术方案中多光谱传感器包括带通滤光片和光电二极管，且带通滤光片和与之匹配的光电二极管组成一荧光强度检测单元，利用集成在多光谱传感器表面的带通滤光片来实现不同波长的光谱选择，利用光电二极管感应跟其相匹配的带通滤光片所对应的波长的荧光强度，从而实现了由一个结构细小的多光谱传感器取代了传统分光技术中若干分散设置的光学元器件，准确地传递了更多的信息且缩小了光学设备的体积；并且，由于多光谱传感器的表面集成多个带通滤光片，而带通滤光片所选择的对应光波波长不同时，就能够同时实现对经由带通滤光片选择的多种波长的荧光信号的强度进行检测。另外，本发明的小型化多通道实时荧光光谱检测装置采用由光纤或光纤束组成的荧光传输通道，光纤或者光纤束中的每根光纤形成一个独立的发射荧光通道，降低了发射荧光在光路传播中的能量损失，而且该荧光信号的传输方式比传统的开放式的传输结构更洁净，不易于被灰尘污染，提高了实验的准确性及灵敏度。
附图说明
图1是现有技术中单通道荧光定量PCR仪的检测原理示意图。
图2是本发明优选实施例1提供的超小型化多通道实时荧光光谱检测装置的结构示意图。
图3是本发明优选实施例1提供的超小型化多通道实时荧光光谱检测装置中的多光谱传感器的结构示意图一。
图4是本发明优选实施例1提供的超小型化多通道实时荧光光谱检测装置中的多光谱传感器的结构示意图二。
图5是本发明优选实施例2提供的超小型化多通道实时荧光光谱检测装置的结构示意图。
图6是本发明优选实施例3提供的超小型化多通道实时荧光光谱检测装置的结构示意图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
图2、5、6为本发明三个优选实施例提供的超小型化多通道实时荧光光谱检测装置的结构示意图。该超小型化多通道实时荧光光谱检测装置应用于实验室或者医疗的检测实验，能够用于对例如核酸扩增反应的实施定量检测，但并不以此为限，在实际应用中，所述超小型化多通道实时荧光光谱检测装置也可应用于其他基于荧光信号探测实现的实时定量或定性检测实验。
如图2所示，本发明优选实施例提供的超小型化多通道实时荧光光谱检测装置包括样品池10、激发光源20、荧光传输通道30和多光谱传感器40。其中，样品池10内容纳有待测样品以及与待测样品浓度相关的荧光物质。激发光源20用于激发样品池中的荧光物质，荧光物质受激发光源产生的激发光激发后产生发射光(荧光)。荧光传输通道30用于收集发射光形成的荧光信号，该荧光传输通道30包括由光纤32组成的光纤束31，荧光信号通过光纤束31有效输出进行检测。多光谱传感器40包括多个带通滤光片41，以及与带通滤光片数量对应的光电二极管42。多光谱传感器40接收并检测从光纤束31传输来的荧光信号的强度，由于多光谱传感器40的表面集成多个带通滤光片，而将不同的带通滤光片所选择的对应光波波长设置为不同时，就能够同时实现对不同波长荧光的光谱选择，光电二极管感应出跟其相匹配的带通滤光片所对应的波长的荧光强度。
具体实现时，样品池10内容纳有待测样品以及与待测样品浓度相关的荧光物质。当本发明的超小型化多通道实时荧光光谱检测装置应用于核酸扩增反应的实施定量检测，待测样品即为核酸扩增反应中产生的目的核酸片度，而与目的核酸浓度相关的荧光物质通常整合于核酸扩增反应的特异性引物中，而且为了是连接于引物的荧光物质不至于在未进行扩增反应是即被激发光源激发产生 荧光，通常与荧光物质或者荧光基团共同连接于核酸扩增反应引物的还有荧光猝灭基团，从而使能够被激发光激发荧光物质或者荧光基团的数量在有效地扩增反应中与目的核酸片度的浓度成正比，从而使本发明的超小型化多通道实时荧光光谱检测装置能够通过检测被激发荧光的强度确定待测样品浓度。当然，为了确定待测样品浓度还需要辅之与核酸扩增反应中的标准品，通过分析处理确定确定待测样品浓度。在缺少标准品是也可以通过对荧光强度信号的测试对待测样品的定性特征以及核酸扩增反应的有效性进行有效地分析判断。当然，上述说明仅仅是以核酸扩增反应的实施定量检测为例，对本发明的样品池及其相应的反应原理进行说明，但并不以此为限，在实际应用中，本发明的超小型化多通道实时荧光光谱检测装置也可应用于其他基于荧光信号探测实现的实时定量或定性检测实验。
如图2所示，本发明实施例1中，荧光传输通道包括设置于所述样品池10两侧的反光镜阵列33和会聚透镜透镜阵列34，反光镜阵列33中包含多个平行设置的反光镜331，会聚透镜阵列34包含与反光镜数量对应的会聚透镜341，每一个反光镜331和与之匹配的会聚透镜341组成一个荧光收集单元。荧光信号通过该荧光收集单元收集后再经由光纤束31传送至多光谱传感器40。从光路上分析，荧光物质受激发光源产生的发射光(荧光)以发散方式产生，部分直射到会聚透镜341，另一部分由反光镜331反射后再经由会聚透镜341会聚至光纤束的光入口端。为了使荧光有效会聚，本实施中所采用的反光镜331的反光面设置成与其光收集区域的范围相匹配的凹形。
在本发明实施例1中，荧光传输通道中设置的光纤束31中包含有多条光入口端排列为一体的光纤32，每一条光纤32的光入口端与一个荧光收集单元相对应，光纤束中的每根光纤均形成一个独立波长选择的荧光通道。如图3和4所示，多光谱传感器40包括集成于其表面的多个带通滤光片41，以及与带通滤光片41数量对应的光电二极管42，带通滤光片41贴合光电二极管42的表面；每一个带通滤光片41和与之匹配的光电二极管42组成一荧光强度检测单元，用于检测该荧光强度检测单元上设置的带通滤光片所对应的波长的荧光强度，每 一条光纤的光出口端与一个荧光强度检测单元相对应。结合本实施例中对于荧光传输通道的描述，每一条光纤32的光入口端与一个荧光收集单元相对应，光纤束中的每根光纤均形成一个独立波长选择的荧光通道，同时每一条光纤的光出口端与一个荧光强度检测单元相对应，因此，包含反光镜331和与之匹配的会聚透镜341组成的荧光收集单元、光纤32对应的荧光通道以及由带通滤光片41和与之匹配的光电二极管42组成荧光强度检测单元便一一对应，从而实现荧光信号的分通道独立传输以及根据带通滤光片所选择的不同波长荧光强度的独立检测。
如图2-4所示，在本发明实施例1提供的超小型化多通道实时荧光光谱检测装置，其多光谱传感器40包括8个集成于其表面的带通滤光片41，与之对应地，光电二极管42也设置有8个，带通滤光片41贴合光电二极管42的表面，每一个光电二极管42和贴合于其上方带通滤光片41组成一个荧光强度检测单元。每个荧光强度检测单元均可选择独立的发射荧光波长，并检测该荧光强度检测单元上设置的带通滤光片所对应的波长的荧光强度。而且需要说明的是，本发明的方案中，能够有效地实现将8个甚至更多的带通滤光片(以及与之对应的光电二极管)集成于面积小于1cm2的多光谱传感器，准确地传递了更多的信息且并极大地缩小了光学设备的体积。相应地，光纤束中31中设置有8条独立光纤32，从而形成8个独立波长选择的荧光通道，每一条光纤32的光入口端与一个荧光收集单元相对应，每一条光纤的光出口端与一个荧光强度检测单元相对应。具体实现时，通过对光纤束中光纤32的光入口端、光出口端以及多光谱传感器40中的带通滤光片41进行一一对应的编号，每个编号对应一条独立的荧光收集、传送与检测通道，而每个通道均是由对应衔接的一个由荧光收集单元(包含反光镜331和与之匹配的会聚透镜341)、一个荧光通道(光纤束31中一条传输荧光的光纤32)和一个荧光强度检测单元(由光电二极管42和贴合于其上方带通滤光片41组成)组成，当样品池中含有多种荧光物质，并在多种荧光物质受激发而产生多种不同波长的荧光时，将每个通道中所用带通滤光片41的选择波长设置为与多种不同波长荧光中的一种荧光的波长一致时，就能够通过 本发明的超小型化多通道实时荧光光谱检测装置实现对每个通道的实验数据进行科学、准确的分析，即对样品池中多个反应的情况同时进行检测。当然，本发明实施例中对于通道数量、光纤束中的光纤数量、多光谱传感器表面的带通滤光片数量、会聚透镜数量和反光镜的数量并不限定本发明其他实施例中技术方案中所用的相应结构的数量，在具体实现时，根据需要调整相应结构的数量以及排布方式也应当理解为本领域的常用技术手段，属于本发明权利要求所涵盖的范围。
另外，对于激发光源20的设置，本发明实施例1中设置有一个激发光源20，为了使样品池中的荧光物质有效激发，激发光源20的位置以及光源的照射角度以能够使待测样品更为有效地接收照射为依据进行调整。另外，结合物理光学的基本原理，激发光源的频率通常大于发射光(荧光)。当样品池中含有多种荧光物质，且其受激发所产生的荧光频率不同时，该激发光源所产生的激发光需要保证能够使各种荧光物质均能够有效激发产生对应的发射光。而从光源的选择来看，本发明实施例中激发光源选择激光二极管，由于激光能够使激发光源发出的激发光控制在单一或者很小的频率范围内，保证对荧光物质具备激发能力的激发光的强度并避免杂光干扰，从而提高实验的准确性和灵敏度。当然在本发明的其他实施例中也会根据需要和具体实验条件，选择普通LED在内的其他光源作为激发光源，但是该激发光源中必须含有能使荧光物质有效激发产生发射光的光波。
关于激发光源的数量，本发明实施例1中设置有一个激发光源，但在本发明的其他实施例中，也会根据需要对激发光源的个数以及位置进行调整。如图5所示，在本发明实施例2中，激发光源20设置有两个，分别位于样品池的两侧两侧，而且两个激发光源20所产生的激发光波长不同，当样品池中含有多种荧光物质，不同的激发光能够激发产生不同的荧光信号，激发光源20选择和设置就更有针对性，激发光的频率范围和功率根据可控性。当然，在本发明的其他实施例中，也可以根据需要将激发光源设置为多个，处于不同的目的，选择不同频率的激发光源能够使测试反应根据针对性和准确性，同时也能够使样品池 待测样品及荧光物质受到更为均匀的激发照射。
如图6所示，本发明实施例3提供的超小型化多通道实时荧光光谱检测装置，其荧光传输通道中设置为多条单根光纤32，每一条单根光纤32的光入口端与一个荧光收集单元相对应并形成一个独立波长选择的荧光通道。同时，本发明实施例3中，每个样品池10中所容纳的荧光物质也设置为一种，激发光源20与样品池10也一一对应，每个样品池10对应设置有一个荧光收集单元，包括反光镜(图中未示出)和与之匹配的会聚透镜341。具体实现时，荧光传输通道中设置有8条与不同样品池10相衔接的单根光纤32，与之对应的，光电二极管42也设置有8个，带通滤光片41贴合光电二极管42的表面，每一个光电二极管42和贴合于其上方带通滤光片41组成一个荧光强度检测单元。每个荧光强度检测单元均可选择独立的发射荧光波长，并检测该荧光强度检测单元上设置的带通滤光片所对应的波长的荧光强度。相应地，8条单根光纤32形成8个独立波长选择的荧光通道，每一条单根光纤的光入口端与一个荧光收集单元、一个样品池和一个激发光源相对应，每一条单根光纤32的光出口端与一个荧光强度检测单元相对应。通过对样品池10、光纤束中光纤32的光入口端、光出口端以及多光谱传感器40中的带通滤光片41进行一一对应的编号，每个编号对应一条独立的荧光收集、传送与检测通道，而每个通道均是由对应衔接的一个由荧光收集单元(包含反光镜331和与之匹配的会聚透镜341)、一个荧光通道(单根光纤32)和一个荧光强度检测单元(由光电二极管42和贴合于其上方带通滤光片41组成)组成，这样就可以通过本发明的超小型化多通道实时荧光光谱检测装置实现对每个通道的实验数据进行科学、准确的分析。同时，每个样品池10中含有一种荧光物质，并设置与该荧光物质相对应的激发光源，从而实现对多个不同样品池中单一反应同时进行检测。当然，本发明实施例中对于通道数量、单根光纤数量、多光谱传感器表面的带通滤光片数量、会聚透镜数量和反光镜的数量并不限定本发明其他实施例中技术方案中所用的相应结构的数量，在具体实现时，根据需要调整相应结构的数量以及排布方式也应当理解为本领域的常用技术手段，属于本发明权利要求所涵盖的范围。
需要说明的是，本发明实施例还提供了一种用于实验室或者医疗的检测装置，其包含上述实施例中揭示的超小型化多通道实时荧光光谱检测装置。另外，本上述实施例中揭示的超小型化多通道实时荧光光谱检测装置尤其适用于核酸扩增检测实验，因此，本发明的实施例还提供了一种核酸扩增检测仪，包含上述实施例中揭示的超小型化多通道实时荧光光谱检测装置。而且由于温度控制是核酸扩增反应中的重要技术指标，例如普通PCR温度循环式扩增，以及恒温扩增反应的温度控制问题都非常重要，故在所述样品池还设置有实现核酸扩增反应温度控制的温控探针。
相较于现有技术，上述实施例揭示的超小型化多通道实时荧光光谱检测装置，使用多光谱传感器代替了传统的滤光片分光系统，极大的简化了结构的复杂程度并减小了检测系统的体积。在本发明的技术方案中多光谱传感器包括带通滤光片和光电二极管，且带通滤光片和与之匹配的光电二极管组成一荧光强度检测单元，利用集成在多光谱传感器表面的带通滤光片来实现不同波长的光谱选择，利用光电二极管感应跟其相匹配的带通滤光片所对应的波长的荧光强度。从而实现了由一个结构细小的多光谱传感器取代了传统分光技术中若干分散设置的光学元器件，传递了更多的信息且缩小了光学设备的体积；并且，由于多光谱传感器的表面集成多个带通滤光片，而带通滤光片所选择的对应光波波长不同时，就能够同时实现对经由带通滤光片选择的多种波长的荧光信号的强度进行检测。另外，本发明的小型化多通道实时荧光光谱检测装置采用由光纤或光纤束组成的荧光传输通道，光纤或者光纤束中的每根光纤形成一个独立的发射荧光通道，降低了发射荧光在光路传播中的能量损失，而且该荧光信号的传输方式比传统的开放式的传输结构更洁净，不易于被灰尘污染，提高了实验的准确性及灵敏度。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。

Claims (10)

  1. 一种超小型化多通道实时荧光光谱检测装置，其特征在于，包括：
    样品池，该样品池内容纳有待测样品以及与待测样品浓度相关的荧光物质；
    激发光源，用于激发样品池中的荧光物质，荧光物质受激发光源产生的激发光激发后产生发射光；
    荧光传输通道，用于收集发射光形成的荧光信号，该荧光传输通道包括光纤或者由光纤组成的光纤束，荧光信号通过光纤或光纤束有效输出进行检测；
    多光谱传感器，所述多光谱传感器包括一个或两个以上的带通滤光片，以及与所述带通滤光片数量对应的光电二极管；该多光谱传感器接收并检测从荧光传输通道传输来的荧光信号的强度，所述带通滤光片实现不同波长荧光的光谱选择，光电二极管感应出跟其相匹配的带通滤光片所对应的波长的荧光强度。
  2. 如权利要求1所述的超小型化多通道实时荧光光谱检测装置，其特征在于：所述荧光传输通道包括反光镜、会聚透镜和光纤或者由光纤组成的光纤束；荧光物质受激发光源产生的发射光直射或者由反光镜反射后通过会聚透镜会聚至光纤或者光纤束光入口端。
  3. 如权利要求1所述的超小型化多通道实时荧光光谱检测装置，其特征在于：所述荧光传输通道还包括设置于所述样品池两侧的反光镜阵列和会聚透镜透镜阵列，反光镜阵列中包含多个平行设置的反光镜，该反光镜的反光面设置成与其光收集区域的范围相匹配的凹形，所述会聚透镜阵列包含与反光镜数量对应的会聚透镜，每一个反光镜和与之匹配的会聚透镜组成一个荧光收集单元，荧光信号通过该荧光收集单元收集后再经由光纤或者由光纤组成的光纤束传送至所述多光谱传感器。
  4. 如权利要求3所述的超小型化多通道实时荧光光谱检测装置，其特征在于：所述荧光传输通道中设置有光纤束，所述光纤束中包含有多条光入口端排列为一体的光纤，每一条光纤的光入口端与一个荧光收集单元相对应，光纤束 中的每根光纤均形成一个独立波长选择的荧光通道。
  5. 如权利要求3所述的超小型化多通道实时荧光光谱检测装置，其特征在于：所述荧光传输通道中设置有多条单根光纤，每一条单根光纤的光入口端与一个荧光收集单元相对应并形成一个独立波长选择的荧光通道。
  6. 如权利要求4或5所述的超小型化多通道实时荧光光谱检测装置，其特征在于：所述多光谱传感器包括集成于其表面的多个带通滤光片，以及与所述带通滤光片数量对应的光电二极管，所述带通滤光片贴合所述光电二极管的表面；每一个带通滤光片和与之匹配的光电二极管组成一荧光强度检测单元，用于检测该荧光强度检测单元上设置的带通滤光片所对应的波长的荧光强度；每一条光纤的光出口端与一个荧光强度检测单元相对应。
  7. 如权利要求1所述的超小型化多通道实时荧光光谱检测装置，其特征在于：所述激发光源设置有一个或者两个以上，两个以上的激发光源产生不同频率的激发光。
  8. 如权利要求1所述的超小型化多通道实时荧光光谱检测装置，其特征在于：所述激发光源为是激光二极管或LED光源。
  9. 一种用于实验室或者医疗的检测装置，其特征在于：包含权利要求1所述的超小型化多通道实时荧光光谱检测装置。
  10. 一种核酸扩增检测仪，其特征在于：包含权利要求1所述的超小型化多通道实时荧光光谱检测装置；所述样品池还设置有实现核酸扩增反应温度控制的温控探针。
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