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WO2024092985A1 - 用于蛋白质凝胶电泳的内源性荧光成像检测系统及方法 - Google Patents

用于蛋白质凝胶电泳的内源性荧光成像检测系统及方法 Download PDF

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WO2024092985A1
WO2024092985A1 PCT/CN2022/140350 CN2022140350W WO2024092985A1 WO 2024092985 A1 WO2024092985 A1 WO 2024092985A1 CN 2022140350 W CN2022140350 W CN 2022140350W WO 2024092985 A1 WO2024092985 A1 WO 2024092985A1
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imaging detection
electrophoresis
light source
chip
gel electrophoresis
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French (fr)
Inventor
曹成喜
薛静晶
张强
刘伟文
田佑吏
曹毅仁
刘小平
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Shanghai Jiao Tong University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44713—Particularly adapted electric power supply
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • 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
    • G01N2021/6417—Spectrofluorimetric devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6463—Optics

Definitions

  • the invention relates to the field of biological and biomedical analysis and detection technology, and in particular to an endogenous fluorescence imaging detection system for protein gel electrophoresis.
  • PAGE Polyacrylamide gel electrophoresis
  • capillary electrophoresis and microfluidic chip electrophoresis have been widely studied in the past three decades and applied to the separation and analysis of biomacromolecules such as proteins and nucleic acids.
  • These electrophoretic separations based on micro-nano-sized channels usually have the characteristics of fast speed, low sample volume, low reagent consumption, and miniaturization of equipment. They can also realize column end/point detection of the substance to be tested after separation.
  • this method also has certain limitations.
  • capillary electrophoresis or microfluidic chip electrophoresis often use laser induced fluorescence (LIF) detectors with a relatively matched size and sensitivity to detect proteins, while most protein molecules need to be derivatized to have fluorescence, which increases the experimental steps and difficulty.
  • LIF laser induced fluorescence
  • the ultraviolet absorbance of the quartz capillary column end or the entire column can be detected.
  • matrix materials such as PMMA, PC and PDMS of electrophoresis chips
  • using the 415nm wavelength absorption of colored proteins (such as myoglobin and hemoglobin) to achieve online detection a meat quality identification method based on isoelectric focusing electrophoresis technology, CN201910219220.6
  • most proteins are colorless proteins, so the application of this detection technology is very limited.
  • the online UV-VIS imaging detection device (CN202010596037.0) provides a new mode for online detection of chip gel electrophoresis, but because the chip needs to be placed between the light source and the innovative equipment, it is not compatible with the existing gel electrophoresis technology; and the deep ultraviolet CCD imager is extremely expensive.
  • the purpose of the present invention is to provide an online endogenous fluorescence imaging (IFI) detection device for a protein gel electrophoresis chip, so as to solve the problems of online imaging detection including chip electrophoresis, and portable electrophoresis separation detection.
  • IFI endogenous fluorescence imaging
  • the first aspect of the present invention provides an endogenous fluorescence imaging detection system for protein gel electrophoresis, characterized in that it comprises a portable suitcase, a chip electrophoresis device, an imaging detection device and a computer terminal, wherein specifically:
  • a chip electrophoresis device is arranged inside the portable suitcase
  • An imaging detection device is arranged inside the portable suitcase and placed on one side of the chip electrophoresis device. After the chip electrophoresis in the chip electrophoresis device is completed, the imaging detection device collects protein band images and triggers the light source to be turned on instantaneously for exposure while imaging, thereby capturing a fluorescent signal image;
  • the computer terminal is connected to the imaging detection device for communication, processes and stores the fluorescent signal image captured by the imaging detection device, and obtains the gel electrophoresis test result.
  • the imaging detection device includes a device cover, an ultraviolet-sensitive CCD camera disposed in the device cover, and a light source component disposed on one side of the ultraviolet-sensitive CCD camera.
  • the photosensitive side of the ultraviolet-sensitive CCD camera faces the chip electrophoresis device.
  • the light source assembly includes a light source cover arranged at an opening of the device outer cover and a deep ultraviolet LED array arranged on the light source cover.
  • the light source cover comprises four light source carrier plates and a light source cover outer frame;
  • the light source cover outer frame is fixed at the opening of the device cover
  • each light source carrier is connected to the frame of the ultraviolet-sensitive CCD camera, and the other side is connected to the frame of the ultraviolet-sensitive CCD camera.
  • the ultraviolet-sensitive CCD camera is provided with a bandpass filter with a cut-off wavelength of 280-488 nm, and the selection of the cut-off wavelength parameter also needs to take the LED wavelength into consideration.
  • the deep ultraviolet LED array includes a plurality of deep ultraviolet LED lamp beads with a wavelength of 240-300nm arranged in an array.
  • the portable suitcase body is provided with an openable and closable opening, through which the chip electrophoresis device and the imaging detection device can be placed in and taken out.
  • the portable suitcase body also includes a power supply unit for supplying power to the active device;
  • the portable suitcase body is made of light-proof material.
  • the second aspect of the present invention provides a protein gel electrophoresis endogenous fluorescence online imaging detection method using the above detection system, comprising the following steps:
  • IFI fluorescence imaging acquisition is started. While the UV-sensitive CCD camera is capturing, the LED array light source is triggered to automatically turn on and expose. The computer terminal analyzes the obtained fluorescence imaging results.
  • the present invention Compared with the existing traditional and chip gel electrophoresis technology equipment, the present invention has the following significant advantages:
  • the device is integrated, portable and automated.
  • the deep ultraviolet LED array light source of this system is compactly designed, and the chip electrophoresis device can be inserted into it, and the device is powered by a lithium battery, which realizes the small size (38 ⁇ 15 ⁇ 15cm) of the entire electrophoresis and detection system. It has the ability to be portable and applied on site, and the electrophoresis process and online imaging are all automated software operations. Compared with the traditional PAGE technology, the degree of automation is greatly improved.
  • FIG. 1 is a structural perspective view of an imaging detection device in the present invention.
  • FIG. 2 is a front view and a side view of the imaging detection device of the present invention.
  • 3 and 4 are schematic diagrams of the overall structure of the imaging detection device used for chip electrophoresis in the present invention.
  • FIG. 5 is a non-uniformity experiment of a single-chip deep ultraviolet LED imaging PAGE in the present invention.
  • FIG6 is a uniformity experiment of imaging a spot with the same BSA concentration using four deep ultraviolet LEDs in the present invention.
  • FIG7 shows that the imaging detection device of the present invention is used for three standard model proteins (gamma globulin ⁇ -Glb (arrow 1), bovine serum albumin BSA (arrow 2) and egg white lysozyme LZ (arrow 3)) chip gel electrophoresis-IFI imaging analysis residual linear range experiment and reproducibility experiment.
  • standard model proteins gamma globulin ⁇ -Glb (arrow 1), bovine serum albumin BSA (arrow 2) and egg white lysozyme LZ (arrow 3)
  • FIG8 is a comparison diagram of the results of two electrophoresis modes of human serum using the chip gel electrophoresis-IFI imaging detection device of the present invention and the classic flat plate electrophoresis-Coomassie brilliant blue staining method.
  • FIG9 is a comparison diagram of experiments on separation of pure and mixed samples of three model proteins by the chip gel electrophoresis-IFI imaging detection device of the present invention.
  • FIG. 10 is a comparison chart of the sensitivity of separating serum albumin by the chip gel electrophoresis-IFI imaging detection device of the present invention and traditional PAGE-Coomassie Brilliant Blue staining.
  • FIGS. 1-2 light source cover 1 , deep ultraviolet LED array 2 , ultraviolet sensitive CCD camera 3 , device cover 4 .
  • FIGS. 3 and 4 an imaging detection device 5 , a power supply unit 6 , a computer terminal 7 , a portable case 8 , and a chip electrophoresis device 9 .
  • the present invention designs an online intrinsic fluorescence imaging (IFI) detection device for a protein gel electrophoresis chip, and the design ideas are as follows.
  • a deep ultraviolet array light source is designed to excite the intrinsic fluorescence of tryptophan residues that are commonly present in protein molecules, avoiding the use of visible dyes, fluorescent dyes and chemical derivatization;
  • a UV imaging device integrated with the deep ultraviolet array light source is designed to solve the problem that traditional and chip gel electrophoresis cannot achieve universal protein label-free imaging detection;
  • the designed online imaging analysis detection device can be adapted and integrated with the protein gel electrophoresis chip, solving the problem of rapid, direct, imaging detection of the protein band to be tested after electrophoresis separation.
  • a deep ultraviolet excitation array light source including: an array surface light source composed of a number of 275nm deep ultraviolet LED lamp beads; a cover-type deep ultraviolet excitation light source composed of four array surface light sources, the light source power is 50-200W, and the uniform irradiation range of the surface light source is 4.0-15.0cm (length) ⁇ 4.0-15.0cm (width);
  • an imaging device integrated with a deep ultraviolet excitation array light source including: a UV-sensitive CCD camera; a bandpass filter with a cutoff wavelength of 365nm; and a sealed housing that integrates the cover-type light source and the imaging device;
  • a power supply and control module including: a group of lithium batteries (12V); a boost module (DC power supply 100 ⁇ 200V); control software and data system.
  • the method for detecting the protein bands separated by chip electrophoresis using the above device comprises the following steps:
  • the protein band image is collected through the control module, and the light source is triggered to turn on instantly during imaging.
  • the exposure time is 10ms to 30sec to capture the fluorescence signal image.
  • the present invention can realize direct online imaging detection of protein bands in an electrophoresis chip, and the device is simple to use, integrated and portable.
  • Figure 6 shows the uniformity of the excitation light source when using four deep UV LEDs for IFI imaging analysis.
  • the concentration of native BSA is 2.0 ⁇ g/ ⁇ L; in insets B1 and B2, the concentration of native BSA is 0.2 ⁇ g/ ⁇ L; in insets C1C2, the concentration of native BSA is 0.02 ⁇ g/ ⁇ L.
  • Illustration A is an IFI imaging analysis and linear range experimental test obtained by using the imaging detection device of the present invention for chip gel electrophoresis separation of three standard model proteins (gamma globulin ⁇ -Glb (arrow 1), bovine serum albumin BSA (arrow 2) and egg white lysozyme LZ (arrow 3).
  • Illustration B is an experimental test of the reproducibility and consistency of IFI imaging analysis obtained by using the imaging detection device of the present invention for chip gel electrophoresis separation of three standard model proteins ( ⁇ -Glb, BSA and LZ).
  • Illustration A shows the separation and analysis of 8 adult serum samples (a-h) using chip gel electrophoresis and the present fluorescence imaging detection device
  • Illustration B shows the separation and analysis of 8 adult serum samples (a-h) using classic slab gel electrophoresis and Coomassie Brilliant Blue staining.
  • Illustration A is the result of chip gel electrophoresis-IFI imaging analysis of denatured BSA, LZ and ⁇ -Glb standard samples
  • Illustration B is the result of chip gel electrophoresis-IFI imaging analysis of denatured BSA, LZ and ⁇ -Glb mixed samples.
  • Illustration A is the result of IFI imaging analysis of BSA standard sample after traditional PAGE electrophoresis
  • Illustration B is the result of ordinary optical imaging analysis of BSA standard sample after traditional PAGE electrophoresis and Coomassie brilliant blue staining.
  • Table 1 is an experimental comparison of the linear range of 2 ⁇ L protein spots of three non-denatured model proteins (BSA, LZ and ⁇ -Glb) and three denatured model proteins (BSA, LZ and ⁇ -Glb) of the chip gel electrophoresis-IFI imaging analysis detection system, fluorescence microplate reader and UV absorption microplate reader of the present invention.
  • Table 2 shows the uniformity test of 2 ⁇ l BSA spots in the chip gel electrophoresis-IFI light source of the present invention (corresponding to the experimental data of Figures 6A, 5B and 5C).
  • Table 3 shows the protein separation and imaging analysis performance of the chip gel electrophoresis-IFI of the present invention (corresponding to the data in FIG. 7 ).
  • Table 4 shows the repeatability of the chip gel electrophoresis-IFI test for detecting denatured model proteins of the present invention (corresponding to the data in FIG. 7B ).
  • the chip electrophoresis-IFI fluorescence imaging detection device system is shown in Figures 3 and 4.
  • three model proteins and three standard model proteins gamma globulin ⁇ -Glb, bovine serum albumin BSA and egg white lysozyme LZ are selected and prepared into a certain concentration gradient, and the prepared model protein samples are injected.
  • the electrophoresis chip is placed in front of the imaging detection device 1 so that it is on the same horizontal axis as the center of the surface irradiated by the LED array light source 2 and the center of the CCD camera 3 in FIG. 1 and FIG. 2 .
  • IFI fluorescence imaging acquisition is started. While the CCD camera is capturing, the LED array light source is triggered to automatically turn on. The exposure time is 320ms. The computer terminal 3 analyzes the obtained fluorescence imaging results.
  • Illustration A is the chip electrophoresis-IFI imaging analysis and linear range experiment of three standard model proteins (gamma globulin ⁇ -Glb (arrow 1), bovine serum albumin BSA (arrow 2) and egg white lysozyme LZ (arrow 3). The linear range is 0.02-2.0 ⁇ g/uL.
  • Illustration B is the reproducibility and consistency experimental test of the imaging detection device of the present invention for three standard model proteins ( ⁇ -Glb, BSA and LZ.
  • system of the present invention can not only be used for quantitative analysis of protein gel electrophoresis, but also has good linear range, stability and consistency.
  • the chip electrophoresis-fluorescence imaging detection device system is shown in Figures 3 and 4.
  • the electrophoresis chip is placed in front of the imaging detection device 1 so that it is on the same horizontal axis as the center of the surface irradiated by the LED array light source 2 and the center of the CCD camera 3 in Figures 1 and 2.
  • fluorescence imaging is collected. While the CCD camera is capturing, the LED array light source is triggered to automatically turn on. The exposure time is 320ms.
  • the computer terminal 3 analyzes the obtained fluorescence imaging results.

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Abstract

一种用于蛋白质凝胶电泳的内源性荧光成像检测系统及方法，系统包括便携手提箱体(8)、芯片电泳装置(9)、成像检测装置(5)和计算机终端(7)，其中芯片电泳装置(9)设于便携手提箱体(8)内部；成像检测装置(5)设于便携手提箱体(8)内部且置于芯片电泳装置(9)一侧，待芯片电泳装置(9)中的芯片电泳结束后，成像检测装置(5)采集蛋白条带图像，在成像的同时触发光源瞬时打开进行曝光，以此捕捉荧光信号图像；计算机终端(7)与成像检测装置(5)通信连接，对成像检测装置(5)捕捉荧光信号图像进行处理并储存，得到凝胶电泳测试结果。检测系统具有集成化、操作简便、在线成像检测、高通量以及低消耗等多种优点。

Description

用于蛋白质凝胶电泳的内源性荧光成像检测系统及方法 技术领域
本发明涉及生物、生物医学分析和检测技术领域，尤其是涉及一种用于蛋白质凝胶电泳的内源性荧光成像检测系统。
背景技术
凝胶电泳技术(poly acrylamide gel electrophoresis，PAGE)是分子生物学、检验医学、蛋白组学等领域中广泛使用的蛋白分离鉴定手段，其具有装置简单，结果直观，且使用成本低廉等优势。然而，由于大多数蛋白质大分子在可见光区是无色的，电泳后蛋白区带的检测需利用可见染料或荧光试剂标记蛋白分子，这些检测方法存在以下问题，其一，繁杂的染色、脱色、孵育等操作步骤耗时费力，效率低下；其二，由于蛋白的氨基酸组成和结构不同，可能使不同蛋白分子被标记和染色的程度不均一，导致显色分布的异质性而影响后续检测；其三，银等一些染料与蛋白共价结合后，会导致蛋白活性或结构改变，与后续的蛋白制备、质谱鉴定等过程难以兼容。另外，染色、标记、脱色所使用的染料试剂、还原剂等对人体、环境有害，成本较高。因此，传统凝胶电泳难以满足现代分析检验的高效、安全、环保的需求。
为了解决这些问题，近三十年来，毛细管电泳以及微流控芯片电泳被广泛研究，并应用于蛋白、核酸等生物大分子分离分析，这些基于微纳尺寸通道的电泳分离，通常具有速度快、所需样品量低、试剂消耗小，设备微型化等特点，并且可以实现分离后待测物质的柱端/点检测。然而此方法也存在一定的局限性，比如，毛细管电泳或微流控芯片电往往使用尺寸和灵敏度较匹配的激光诱导荧光(Laser Induce Fluorescence，LIF)检测器来检测蛋白，而大多数蛋白分子需要通过衍生化才具有荧光，增加了实验步骤和难度。
或利用蛋白等大分子的紫外吸收基团，可实现石英毛细管柱端或整柱的紫外吸光度的检测，然而因电泳芯片PMMA、PC和PDMS等基质材料UV透光性能较差，仍然缺少与芯片电泳兼容的紫外检测和成像技术装置。或利用有色蛋白(如肌红蛋白和血红蛋白)415nm波长吸收来实现在线检测(一种基于等电聚焦电泳技术的肉类品质鉴定方法，CN201910219220.6)，然而绝大多数蛋白为无色蛋白，因此这一检测技术应用面非常有限。在线UV-VIS成像检测装置(CN202010596037.0) 为芯片凝胶电泳在线检测提供了新模式，但因芯片需要放置在光源与创新设备之间无法与现有凝胶电泳技术兼容；并且深紫外CCD成像仪极其昂贵。
因此，亟待发展新创新分析技术解决包括芯片电泳在内的在线成像检测问题、以及便携式电泳分离检测问题。
发明内容
本发明的目的就是提供一种用于蛋白凝胶电泳芯片的在线内源性荧光成像(Intrinsic Fluorescence Imaging，IFI)检测装置，以此解决包括芯片电泳在内的在线成像检测问题、以及便携式电泳分离检测问题。
本发明的目的可以通过以下技术方案来实现：
本发明第一方面提供一种用于蛋白质凝胶电泳的内源性荧光成像检测系统，其特征在于，包括便携手提箱体、芯片电泳装置、成像检测装置和计算机终端，其中具体地：
芯片电泳装置，设于所述便携手提箱体内部；
成像检测装置，设于所述便携手提箱体内部且置于芯片电泳装置一侧，待所述芯片电泳装置中的芯片电泳结束后，所述成像检测装置采集蛋白条带图像，在成像的同时触发光源瞬时打开进行曝光，以此捕捉荧光信号图像；
计算机终端，与所述成像检测装置通信连接，对成像检测装置捕捉荧光信号图像进行处理并储存，得到凝胶电泳测试结果。
进一步地，所述成像检测装置包括装置外罩、设于装置外罩中的紫外敏感CCD相机、设于紫外敏感CCD相机一侧的光源组件。
进一步地，所述紫外敏感CCD相机的光敏侧朝向芯片电泳装置。
进一步地，所述光源组件包括设于装置外罩开口处的光源罩、设于光源罩上的深紫外LED阵列。
进一步地，所述光源罩包括4块光源载板和光源罩外框；
所述光源罩外框固定于所述装置外罩开口处；
各片光源载板的一侧与紫外敏感CCD相机的边框连接，另一侧与所述紫外敏感CCD相机的边框连接。
进一步地，所述紫外敏感CCD相机上设有截止波长为280-488nm的带通滤光片，截止波长参数选取还需考虑LED波长。
进一步地，所述深紫外LED阵列包括多个阵列式设置的波长为240-300nm深紫外LED灯珠。
进一步地，所述便携手提箱体的上设有可开闭开口，通过可开闭开口实现芯片电泳装置、成像检测装置的置入和拿出。
进一步地，所述便携手提箱体中还包括向有源器件供电的供电单元；
所述便携手提箱体为不透光材料。
本发明第二方面提供一种采用上述检测系统的蛋白质凝胶电泳内源性荧光在线成像检测方法，包括以下步骤：
将配置好的模式蛋白样品进样，将电泳芯片置于成像检测装置正前方，使深紫外LED阵列光源辐照面的中心、紫外敏感CCD相机的中心轴在同一水平轴上，并置于一个不透明的便携箱中；
电泳芯片上样后，打开供电单元，开始电泳；
电泳结束时，启动IFI荧光成像采集，紫外敏感CCD相机捕捉的同时，触发LED阵列光源自动开启，曝光，由计算机终端对所获荧光成像结果进行分析。
与现有传统和芯片凝胶电泳技术设备相比，本发明具有以下显著优点：
1、实现电泳后蛋白区带无标记快速成像检测。传统的PAGE电泳后需要3小时固定染色和脱色，然后再用凝胶成像仪成像分析，耗时费力、费试剂、费空间。而采用本装置，无色蛋白区带无需传统固定、染色、脱色或衍生化等耗时手工步骤，即可直接成像检测，显著提升区带凝胶电泳效率。
2、提高芯片凝胶电泳检测灵敏度。利用深紫外LED阵列光源，辐射光在一定面积内均匀性强，激发芯片凝胶中蛋白分子的色氨酸残基内源荧光，从而对蛋白条带在线成像并定量检测，其灵敏度优于传统考马斯亮蓝蛋白染色法和蛋白紫外吸光度成像检测。
3、实现蛋白芯片/传统凝胶电泳定量。传统蛋白凝胶电泳采用染色观测蛋白区带，能够实现半定量分析，但很难实现定量分析。本发明能够实现蛋白凝胶电泳的在线定量分析，并具有较好的线性范围和稳定性。
4、装置集成、便携、自动化。本系统的深紫外LED阵列光源设计紧凑，可将芯片电泳装置插入其中，并使用锂电池为装置供电，实现了整个电泳和检测系统的小尺寸(38×15×15cm)，具有便携和现场应用能力，且电泳过程和在线成像均为自动化软件操作，相比于传统PAGE技术，自动化程度大幅提升。
5、大幅度降低试剂耗材消耗。传统凝胶电泳需要蛋白核酸固定液、染色液和脱色液等。使用本技术方案中检测系统，蛋白成像无需使用染料、衍生试剂，且配合芯片凝胶电泳，极大减少化学试剂的消耗。
6、降低实验室室内污染。传统的PAGE电泳固定、染色和脱色需要大量三氯乙酸、冰醋酸、乙醇和考马斯亮蓝等有机溶剂。造成实验室室内污染严重。
7、节省大量设备和空降。传统PAGE电泳需要电泳电源、电泳槽、制胶架、摇床或自动染色仪、凝胶成像仪、以及台式计算机等。不仅各种设备多，而且占用实验面积大。
附图说明
图1为本发明中成像检测装置的结构透视图。
图2为本发明中成像检测装置的前视图和侧视图。
图3、4为本发明中成像检测装置用于芯片电泳的整体结构示意图。
图5为本发明中单片深紫外LED成像PAGE的非均匀性实验。
图6为本发明中四片深紫外LED成像同一BSA浓度斑点的均匀性实验。
图7本发明中成像检测装置用于三种标准模式蛋白(丙种球蛋白γ-Glb(箭头1)、牛血清蛋白BSA(箭头2)和蛋清溶菌酶LZ(箭头3)芯片凝胶电泳-IFI成像分析余线性范围实验、以及重现性实验。
图8为本发明中芯片凝胶电泳-IFI成像检测装置与经典平板电泳-考马斯亮蓝染色法的人血清的二种电泳模式结果对比图。
图9为本发明中芯片凝胶电泳-IFI成像检测装置分离三种模式蛋白纯品与混样的实验对比图。
图10为本发明中芯片凝胶电泳-IFI成像检测装置分离血清白蛋白与传统PAGE-考马斯亮蓝染色的灵敏度对比图。
在图1～2中：光源罩1、深紫外LED阵列2、紫外敏感CCD相机3、装置外罩4。
在图3、4中：成像检测装置5、供电单元6、计算机端7、便携手提箱体8、芯片电泳装置9。
具体实施方式
本发明的设计了一种用于蛋白凝胶电泳芯片的在线内源性荧光成像(Intrinsic Fluorescence Imaging，IFI)检测装置，设计思路如下。设计一深紫外阵列光源用于激发蛋白分子普遍存在的色氨酸残基内源荧光，避免了使用可见染料、荧光染料及化学衍生；设计一与深紫外阵列光源集成的紫外成像装置，解决传统和芯片凝胶电泳无法实现普适性的蛋白免标记成像检测的问题；设计的在线成像分析检测装置可以与蛋白凝胶电泳芯片适配和集成，解决了电泳分离后待测蛋白区带的快速、直接、成像检测问题。
本发明的目的通过以下技术方案实现：
1、设计一种深紫外激发阵列光源，包括：由若干个275nm深紫外LED灯珠组成的阵列面光源；由四块阵列面光源构成的罩式深紫外激发光源，光源功率50～200W，面光源均匀辐照范围为4.0～15.0cm(长度)×4.0～15.0cm(宽度)；
2、设计一与深紫外激发阵列光源集成为一体的成像装置，包括：一紫外敏感型CCD相机；一截止波长365nm的带通滤光片；一密闭外壳将罩式光源和成像装置集成；
3、设计一供电和控制模块，包括：一组锂电池(12V)；一个增压模块(直流供电100～200V)；控制软件和数据系统。
利用上述装置对芯片电泳分离后的蛋白区带进行检测的方法，包括如下步骤：
(1)芯片电泳结束后，通过控制模块采集蛋白条带图像，在成像的同时触发光源瞬时打开，曝光时间10ms～30sec，捕捉荧光信号图像。
(2)获得成像结果后，通过计算机软件记录和分析。计算机软件为市面上采购，在此不再赘述。
本发明可以实现电泳芯片中蛋白区带的直接在线成像检测，且装置使用简单、集成化、便携化。
下面结合附图和具体实施例对本发明进行详细说明。本技术方案中如未明确说明的部件型号、材料名称、连接结构、控制方法、算法等特征，均视为现有技术中公开的常见技术特征。
在图5中：当使用单片深紫外LED光源做IFI成像分析时表现出来的激发光源的非均匀性。
在图6中：当使用四片深紫外LED光源做IFI成像分析时表现出来的激发光源的均匀性。在插图A1和A2中，非变性BSA浓度为2.0μg/μL；在插图B1和 B2中，非变性BSA浓度为0.2μg/μL；在插图C1C2中，非变性BSA浓度为0.02μg/μL。
在图7中：插图A为本发明成像检测装置用于三种标准模式蛋白(丙种球蛋白γ-Glb(箭头1)、牛血清蛋白BSA(箭头2)和蛋清溶菌酶LZ(箭头3)芯片凝胶电泳分离获得的IFI成像分析及线性范围实验测试。插图B为本发明成像检测装置用于三种标准模式蛋白(γ-Glb、BSA和LZ)芯片凝胶电泳分离的IFI成像分析的重现性和一致性实验测试。
在图8中：插图A为使用芯片凝胶电泳及本荧光成像检测装置分离分析8个成人血清样本(a-h)；插图B为使用经典平板凝胶电泳及考马斯亮蓝染色法分离分析8个成人血清样本(a-h)。
在图9中：插图A为变性BSA、LZ和γ-Glb标样经芯片凝胶电泳-IFI成像分析结果；插图B为变性BSA、LZ和γ-Glb混样经芯片凝胶电泳-IFI成像分析结果。
在图10中：插图A为BSA标样经传统PAGE电泳后经IFI成像分析结果；插图B为BSA标样经传统PAGE电泳后考马斯亮蓝染色普通光学成像的分析结果。
表1为本发明芯片凝胶电泳-IFI成像分析检测系统、荧光微孔板读出仪及紫外吸收微孔板读出仪的三种非变性模式蛋白(BSA、LZ和γ-Glb)与三种变性模式蛋白(BSA、LZ和γ-Glb)2μL蛋白斑点线性范围的实验对比。
表2为本发明芯片凝胶电泳-IFI光源的2微升BSA斑点均匀性测试(对应图6A、5B和5C实验数据)。
表3为本发明芯片凝胶电泳-IFI的蛋白分离与成像分析性能表征(对应图7数据)。
表4为本发明芯片凝胶电泳-IFI检测变性模式蛋白的重复性实验(对应图7B数据)。
实施例1
标准模式蛋白的芯片凝胶电泳-IFI荧光成像检测
芯片电泳-IFI荧光成像检测装置系统如图3、4所示。首先挑选三种模式蛋白三种标准模式蛋白(丙种球蛋白γ-Glb、牛血清蛋白BSA和蛋清溶菌酶LZ，配制成一定浓度梯度，将配置好的模式蛋白样品进样。
之后，将电泳芯片置于成像检测装置1正前方，使其与图1和图2中LED阵列光源2所辐照的面的中心、CCD相机3的中心在同一水平轴上。
然后，如图3、4所示，使所有部件置于一个不透明的便携箱中5。电泳芯片上样后，打开供电单元6，设置100V恒压模式，时间6min，开始电泳。
电泳结束时，启动IFI荧光成像采集，CCD相机捕捉的同时，触发LED阵列光源自动开启，曝光时间320ms，由计算机终端3对所获荧光成像结果进行分析。
结果如图5所示，插图A为三种标准模式蛋白(丙种球蛋白γ-Glb(箭头1)、牛血清蛋白BSA(箭头2)和蛋清溶菌酶LZ(箭头3)芯片电泳-IFI成像分析及线性范围实验。线性范围在0.02-2.0μg/uL。插图B为本发明成像检测装置用于三种标准模式蛋白(γ-Glb、BSA和LZ的重现性和一致性实验测试。
实验证明，本发明系统不仅能够用于蛋白凝胶电泳的定量分析，而且具有较好的线性范围、稳定性和一致性。
实施例2
人血清蛋白的芯片凝胶电泳-荧光成像检测
芯片电泳-荧光成像检测装置系统如图3、4所示。首先将电泳芯片置于成像检测装置1正前方，使其与图1和图2中LED阵列光源2所辐照的面的中心、CCD相机3的中心在同一水平轴上。
然后，如图3、4所示，使所有部件置于一个不透明的便携箱中5。电泳芯片上样后，打开供电单元6，设置100V恒压模式，时间6min，开始电泳。
电泳结束后，进行荧光成像采集，CCD相机捕捉的同时，触发LED阵列光源自动开启，曝光时间320ms，由计算机终端3对所获荧光成像结果进行分析。
结果如图5所示，首先，芯片电泳-荧光成像检测(插图A)与平板电泳-考马斯亮蓝染色(插图B)分离检测人血清蛋白获得的蛋白条带位置分布具有较高的一致性。
同时，可以观察到，使用考马斯亮蓝染色法的平板凝胶中的一较弱蛋白条带(插图B中箭头1)，在芯片电泳荧光成像检测中可以清晰的观察到(插图A中箭头1)，条带信号强度明显增强，并且，插图A中箭头2所示区域比插图B中相同区域多分辨出1-3个条带。以上对比结果表明，本装置相比于传统平板电泳及考马斯亮蓝染色法具有更好的检测灵敏度，且方法简单、快速，无需染色、衍生等操作，实现了电泳后蛋白的快速在线成像检测。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发 明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。

Claims (10)

  1. 一种用于蛋白质凝胶电泳的内源性荧光成像检测系统，其特征在于，包括：
    便携手提箱体(8)；
    芯片电泳装置(9)，设于所述便携手提箱体(8)内部；
    成像检测装置(5)，设于所述便携手提箱体(8)内部且置于芯片电泳装置(9)一侧，待所述芯片电泳装置(9)中的芯片电泳结束后，所述成像检测装置(5)采集蛋白条带图像，在成像的同时触发光源瞬时打开进行曝光，以此捕捉荧光信号图像；
    计算机终端(7)，与所述成像检测装置(5)通信连接，对成像检测装置(5)捕捉荧光信号图像进行处理并储存，得到凝胶电泳测试结果。
  2. 根据权利要求1所述的一种用于蛋白质凝胶电泳的内源性荧光成像检测系统，其特征在于，所述成像检测装置(5)包括装置外罩(4)、设于装置外罩(4)中的紫外敏感CCD相机(3)、设于紫外敏感CCD相机(3)一侧的光源组件。
  3. 根据权利要求2所述的一种用于蛋白质凝胶电泳的内源性荧光成像检测系统，其特征在于，所述紫外敏感CCD相机(3)的光敏侧朝向芯片电泳装置(9)。
  4. 根据权利要求2所述的一种用于蛋白质凝胶电泳的内源性荧光成像检测系统，其特征在于，所述光源组件包括设于装置外罩(4)开口处的光源罩(1)、设于光源罩(1)上的深紫外LED阵列(2)。
  5. 根据权利要求4所述的一种用于蛋白质凝胶电泳的内源性荧光成像检测系统，其特征在于，所述光源罩(1)包括4块光源载板和光源罩外框；
    所述光源罩外框固定于所述装置外罩(4)开口处；
    各片光源载板的一侧与紫外敏感CCD相机(3)的边框连接，另一侧与所述紫外敏感CCD相机(3)的边框连接。
  6. 根据权利要求2所述的一种用于蛋白质凝胶电泳的内源性荧光成像检测系统，其特征在于，所述紫外敏感CCD相机(3)上设有截止波长为280-488nm的带通滤光片。
  7. 根据权利要求4所述的一种用于蛋白质凝胶电泳的内源性荧光成像检测系统，其特征在于，所述深紫外LED阵列(2)包括多个阵列式设置的波长为240-300nm深紫外LED灯珠。
  8. 根据权利要求1所述的一种用于蛋白质凝胶电泳的内源性荧光成像检测系统，其特征在于，所述便携手提箱体(8)的上设有可开闭开口，通过可开闭开口实现芯片电泳装置(9)、成像检测装置(5)的置入和拿出。
  9. 根据权利要求1所述的一种用于蛋白质凝胶电泳的内源性荧光成像检测系统，其特征在于，所述便携手提箱体(8)中还包括向有源器件供电的供电单元(6)；
    所述便携手提箱体(8)为不透光材料。
  10. 一种采用如权利要求1至9中任意一项检测系统的蛋白质凝胶电泳内源性荧光在线成像检测方法，其特征在于，包括以下步骤：
    将配置好的模式蛋白样品进样，将电泳芯片置于成像检测装置正前方，使深紫外LED阵列光源辐照面的中心、紫外敏感CCD相机(3)的中心轴在同一水平轴上，并置于一个不透明的便携箱中(5)；
    电泳芯片上样后，打开供电单元(6)，开始电泳；
    电泳结束时，启动IFI荧光成像采集，紫外敏感CCD相机(3)捕捉的同时，触发LED阵列光源自动开启，曝光，由计算机终端(7)对所获荧光成像结果进行分析。
PCT/CN2022/140350 2022-10-31 2022-12-20 用于蛋白质凝胶电泳的内源性荧光成像检测系统及方法 Ceased WO2024092985A1 (zh)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000146910A (ja) * 1998-09-02 2000-05-26 Sankyo Co Ltd 電気泳動システム
WO2002093144A1 (en) * 2001-05-10 2002-11-21 Regents Of The University Of Minnesota Imaging of biological samples using electronic light detector
CN103079032A (zh) * 2012-11-16 2013-05-01 上海培清科技有限公司 一种高清晰度的毛细管电泳凝胶成像系统
US20160370319A1 (en) * 2015-05-20 2016-12-22 ProteinSimple Systems and methods for electrophoretic separation and analysis of analytes
US10591488B2 (en) * 2018-01-29 2020-03-17 Intabio, Inc. Devices, methods and kits for sample characterization
CN112964881A (zh) * 2021-03-05 2021-06-15 上海交通大学 一种高通量高灵敏度的单细胞转染蛋白质分析芯片
CN113866425A (zh) * 2021-09-24 2021-12-31 上海交通大学 一种单细胞蛋白数字化成像检测方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102648309B (zh) * 2009-08-24 2015-08-12 生命技术公司 电泳凝胶盒以及电泳梳
CN101949886A (zh) * 2010-08-27 2011-01-19 亚亚科技股份有限公司 实时荧光电泳装置
CN203037567U (zh) * 2013-01-24 2013-07-03 上海领成生物科技有限公司 一种新型的凝胶成像分析仪
CN103713033A (zh) * 2013-12-31 2014-04-09 南京山诺生物科技有限公司 一种实时可视及成像共享的核酸电泳装置
CN107064497B (zh) * 2017-06-13 2019-03-01 福建省农业科学院畜牧兽医研究所 一种便携式电泳和拍照设备及其使用方法
CN119534433A (zh) * 2017-12-15 2025-02-28 生物辐射实验室股份有限公司 测定的组合成像
CN111812091B (zh) * 2020-06-28 2023-09-05 上海交通大学 芯片凝胶电泳及其在线uv-vis成像检测装置

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000146910A (ja) * 1998-09-02 2000-05-26 Sankyo Co Ltd 電気泳動システム
WO2002093144A1 (en) * 2001-05-10 2002-11-21 Regents Of The University Of Minnesota Imaging of biological samples using electronic light detector
CN103079032A (zh) * 2012-11-16 2013-05-01 上海培清科技有限公司 一种高清晰度的毛细管电泳凝胶成像系统
US20160370319A1 (en) * 2015-05-20 2016-12-22 ProteinSimple Systems and methods for electrophoretic separation and analysis of analytes
US10591488B2 (en) * 2018-01-29 2020-03-17 Intabio, Inc. Devices, methods and kits for sample characterization
CN112964881A (zh) * 2021-03-05 2021-06-15 上海交通大学 一种高通量高灵敏度的单细胞转染蛋白质分析芯片
CN113866425A (zh) * 2021-09-24 2021-12-31 上海交通大学 一种单细胞蛋白数字化成像检测方法

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