Device for protein separation-online imaging analysis by acrylamide gel electrophoresis, and method using device

Abstract

The present invention relates to a device for protein separation-online imaging analysis by acrylamide gel electrophoresis, and a method using the device. The device is used for protein separation and online detection by electrophoresis. The device comprises an open electrophoresis tank, which comprises an upper buffer solution chamber arranged at the upper portion of the open electrophoresis tank, a lower buffer solution chamber arranged at the lower portion of the open electrophoresis tank, and an ultraviolet light source configured to excite protein bands in gel to generate a fluorescence signal; and a CCD imaging detection mechanism, which comprises an optical filter arranged at the front end of the CCD imaging detection mechanism, an ultraviolet lens arranged between the optical filter and a CCD photosensitive chip, the CCD photosensitive chip connected to a host, and the host connected to the CCD photosensitive chip for online imaging. Compared with the prior art, the device of the present invention has various advantages of monitoring a gel-electrophoresis-based protein migration process in real time, directly acquiring a gel electrophoretogram without the need for post-treatment, automatically achieving quantitative target protein detection, involving few reagents and simple and convenient experimental processes, etc.

Description

[Corrected 07.06.2024 according to Rule 26] Protein acrylamide gel electrophoresis separation-online imaging analysis device and method Technical Field

[0001] The present invention relates to the technical field of protein acrylamide gel electrophoresis, in particular to a protein acrylamide gel electrophoresis separation-online imaging analysis device and a method thereof. Background Art

[0002] Currently, the most commonly used technology for protein separation is polyacrylamide gel electrophoresis (PAGE). However, traditional PAGE electrophoresis analysis devices and methods have many problems that greatly limit their application scenarios: First, the operation is cumbersome, reagent consumption is large, and the gel post-processing staining process is time-consuming. After electrophoresis, the PAGE gel needs to be peeled off and then placed in a protein fixative for 4-6 hours. The gel is then stained with a stain for 2-4 hours. Finally, the stained gel needs to be placed in a destaining solution for destaining. During this period, the destaining solution needs to be changed frequently, which takes 2-4 hours (Chang Shenghe, Shu Haiyan, Qin Guangyong, et al. al. Research progress of protein staining methods for gel electrophoresis [J]. Henan Agricultural Science, 2006, (5): 8-12.); Second, it is impossible to perform online imaging analysis. The traditional electrophoresis tank is a fully enclosed structure, and the front glass of the gel is ordinary glass. Ultraviolet light cannot penetrate both. The electrophoresis tank shell and the front ordinary glass greatly affect the optical signal acquisition. Therefore, for protein detection in the gel, it must be taken out after the electrophoresis is completed and stained. The stained protein bands must be imaged with the help of a professional imaging scanner, and then image processing software must be used to analyze the grayscale value of the protein bands; Third, the resolution is low. The protein in the gel is in disordered Brownian motion all the time. After the electrophoresis stops, the protein in the gel diffuses immediately. In the process of taking out the gel and fixing it, the bands diffuse, resulting in a decrease in resolution; Fourth, the data cannot be traced. The traditional electrophoresis platform is operated and organized by individuals. The source of the data is uncontrollable, and there is a possibility of data falsification and misappropriation. 5. Unable to perform quantitative detection. The current staining technology cannot perform quantitative detection of protein quality for the analysis of protein bands. The degree of staining can only reflect the amount of protein to a certain extent and cannot accurately reflect the quality of the protein in the band (Lin Fangyang, Yang Yaodong, Wan Jie, et al. Comparison of several SDS-PAGE gel electrophoresis staining methods [J]. Anhui Agricultural Sciences, 2014, 42(08): 2295-6.).

[0003] In view of the shortcomings of traditional gel electrophoresis, it is particularly important to study and develop an electrophoresis separation-online detection device. Researchers have found that most proteins contain fluorescent groups, which will emit fluorescence under ultraviolet excitation, and the fluorescence intensity emitted by proteins under ultraviolet excitation is positively correlated with the quality of the protein (Li, Q., Seeger, S. Autofluorescence Detection in Analytical Chemistry and Biochemistry [J]. Applied Spectroscopy Reviews, 2010, 45 (1): 12-43.). Therefore, the detection of protein excitation fluorescence intensity is a very promising protein label-free quantitative detection technology. However, the fluorescence intensity of ultraviolet excited proteins is weak and easily interfered by the environment. In particular, the materials used in the current traditional flat-plate PAGE electrophoresis system will also produce fluorescence interference detection under ultraviolet excitation. Traditional gel electrophoresis is incompatible with the detection method of ultraviolet excited protein fluorescence intensity. Therefore, there is a lack of key technologies and core components for combining ultraviolet excited protein fluorescence imaging with PAGE electrophoresis, which hinders its practical application and development.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide a protein acrylamide gel electrophoresis separation-online imaging analysis device and method to solve the above problems. An open electrophoresis tank is designed to monitor the migration process of gel electrophoresis proteins in real time, directly obtain gel electrophoresis patterns without post-processing, automatically achieve quantitative detection of target proteins, require less reagents, and simplify the experimental process, among other advantages.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a protein acrylamide gel electrophoresis separation-online imaging analysis device, which is used for protein electrophoresis separation and online detection;

[0008] The device includes: an open electrophoresis tank, used as a support for gel electrophoresis and protein band ultraviolet detection, including: an upper buffer chamber, arranged at the upper part of the open electrophoresis tank; a lower buffer chamber, arranged at the lower part of the open electrophoresis tank; an ultraviolet light source, used to excite protein bands in the gel to generate fluorescence signals; a CCD imaging detection mechanism, used to capture the protein band excitation fluorescence signals during the electrophoresis process for online imaging, including: a filter, arranged at the front end of the CCD imaging detection mechanism; a ultraviolet lens, arranged between the filter and the CCD photosensitive chip; a CCD photosensitive chip, connected to an online analysis host; a host, connected to the CCD photosensitive chip for online imaging; the gel is a flat gel.

[0009] Furthermore, the shell of the open electrophoresis tank is made of black light-absorbing material.

[0010] Furthermore, the shell of the open electrophoresis tank is made of materials including but not limited to black PMMA, black PC, black PVC, black PP and black PTFE.

[0011] Furthermore, the upper buffer chamber includes positive and negative electrodes symmetrically placed left and right and a flat gel slot symmetrically placed front and back.

[0012] Furthermore, the flat gel slot includes a fixed wedge and a flat structure; the flat structure includes a quartz front glass, a flat gel and a black background plate arranged in sequence.

[0013] Furthermore, the material of the black background plate includes but is not limited to polyethylene, polystyrene, polytetrafluoroethylene, polycarbonate and the like.

[0014] Furthermore, the ultraviolet light source is an ultraviolet light source with a central wavelength of 275nm-360nm, including but not limited to LED, tritium lamp, mercury lamp, laser light source with a central wavelength of 275nm-360nm, including but not limited to point light source, line light source and surface light source.

[0015] Furthermore, the ultraviolet light sources are symmetrically distributed on both sides of the detected surface in the open electrophoresis tank; and the CCD imaging detection mechanism is arranged on one side of the open electrophoresis tank.

[0016] Furthermore, the filter has a central wavelength of 250-360 nanometers, a central wavelength transmittance greater than 80%, and a half-width of 10 nm to 50 nm.

[0017] Further preferably, the half-width of the filter is 10 nm-40 nm.

[0018] Furthermore, the focal length of the ultraviolet lens is 35mm-39mm, the aperture is F#5.6, the working distance is 150mm-10m, and the working wavelength is 250-380nm.

[0019] Furthermore, the quantum conversion efficiency of the CCD photosensitive chip at the central wavelength is greater than 40%.

[0020] A second object of the present invention is to provide a method for protein electrophoresis separation and online detection using the above-mentioned protein acrylamide gel electrophoresis separation-online imaging analysis device, the method comprising the following steps:

[0021] Step 1) Install the prepared slab gel in the slab gel slot of the upper buffer chamber, then vertically install the upper buffer chamber on the lower buffer chamber. Add appropriate amounts of electrode buffer to the upper and lower buffer chambers, respectively. Add the sample to be tested to the loading channel of the slab gel, with a sample volume of 1-20 μL.

[0022] Step 2) Connect the positive and negative electrodes of the power supply to the corresponding electrodes in the upper buffer chamber, turn on the power supply, set the appropriate voltage, and start electrophoresis. The voltage is controlled between 50 V / m and 3000 V / m, and the electrophoresis time is 20-40 minutes.

[0023] Step 3) During and after electrophoresis, turn on the UV light source and use the CCD imaging detection mechanism to image the electrophoresis process online. The host computer can observe the protein bands during the electrophoresis process to obtain protein composition information and concentration information in the sample.

[0024] The present invention can not only realize online detection and imaging of PAGE protein electrophoresis, but also realize quantitative detection of protein bands without the need for subsequent complicated sample processing.

[0025] Compared with the prior art, the beneficial effects of the present invention are embodied in the following aspects:

[0026] 1) The present invention proposes a protein acrylamide gel electrophoresis separation-online imaging analysis device and method, which can achieve real-time online detection of the protein electrophoresis process. The open electrophoresis tank facilitates optical signal acquisition, and protein bands can be observed at any time during the electrophoresis process, eliminating the need for subsequent complex and time-consuming gel fixation, staining, decolorization, and scanning imaging.

[0027] 2) The present invention proposes a protein acrylamide gel electrophoresis separation-online imaging analysis device and method, which has high resolution. Online detection avoids the diffusion of protein bands in the gel, and the resolution of separated proteins is higher than that of offline staining methods.

[0028] 3) The protein acrylamide gel electrophoresis separation-online imaging analysis device and method proposed in the present invention can realize label-free detection of proteins without the need for staining reagents, saving detection time and cost.

[0029] 4) The present invention proposes a protein acrylamide gel electrophoresis separation-online imaging analysis device and method. The device is highly integrated and automated, and protein separation and detection can be automatically performed by the device. Only protein sample loading is required, and subsequent protein electrophoresis separation and imaging detection can be completed on a computer. The operation is simple, and protein separation and detection can be completed within 1 hour.

[0030] 5) The present invention proposes a protein acrylamide gel electrophoresis separation-online imaging analysis device and method. The experimental data is traceable. After the experiment is completed, the results are automatically uploaded to the database, and key information such as the experimenter's name and experimental time are recorded at the same time. Unauthorized personnel cannot change any information related to the experimental results, and the source of the experimental data is reliable.

[0031] 6) The present invention proposes a protein acrylamide gel electrophoresis separation-online imaging analysis device and method, which can achieve instantaneous quantitative detection of proteins. After electrophoretic separation, the fluorescence spectrum of protein bands is obtained by ultraviolet excitation using a highly sensitive CCD photosensitive chip. Then, image processing technology is used to reduce background noise and construct a standard curve between protein band fluorescence intensity and protein amount, thus achieving quantitative protein detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 is a schematic structural diagram of a protein acrylamide gel electrophoresis separation-online imaging analysis device in an embodiment of the present invention.

[0033] FIG2 is a schematic structural diagram of the upper buffer chamber in an embodiment of the present invention.

[0034] FIG3 is a schematic diagram of the structure of a flat gel slot in an embodiment of the present invention.

[0035] FIG4 is a schematic diagram of an optical detection light path in an embodiment of the present invention.

[0036] FIG5 is a comparison of the imaging results of a protein standard marker using ordinary front glass and quartz front glass in an embodiment of the present invention.

[0037] FIG6 is a comparison result of the fluorescence spectra of the protein standard marker and mixed protein sample bands of different masses and the protein bands stained with Coomassie brilliant blue according to an embodiment of the present invention, wherein the online imaging of the present invention and Coomassie brilliant blue staining are used.

[0038] FIG7 is a comparison result of the separation degree of proteins of different molecular weights in the protein standard marker of an embodiment of the present invention and the separation degree of Coomassie Brilliant Blue staining, wherein the online imaging of the present invention and Coomassie Brilliant Blue staining are used.

[0039] FIG8 is a comparison result of the separation degree of mixed protein samples of different masses and the separation degree of Coomassie Brilliant Blue staining in an embodiment of the present invention, wherein the online imaging of the present invention and Coomassie Brilliant Blue staining are used.

[0040] FIG9 is a standard curve diagram of proteins of different masses and fluorescence intensity according to an embodiment of the present invention.

[0041] In the figure: 1. Main unit; 2. CCD photosensitive chip; 3. UV lens; 4. Filter; 5. UV light source; 6. Upper buffer chamber; 7. Lower buffer chamber; 8. Power supply; 6.1. Fixed wedge; 6.2. Flat plate structure; 6.2.1. Quartz front glass; 6.2.2. Flat plate gel; 6.2.3. Black background plate; 6.3. Positive and negative electrodes. DETAILED DESCRIPTION

[0042] The present invention is described in detail below with reference to specific embodiments, but is by no means intended to limit the present invention. Any features, such as preparation methods, materials, structures, or composition ratios, that are not explicitly described in this technical solution are considered to be common technical features disclosed in the prior art.

[0043] Example

[0044] As shown in Figures 1 to 4, a device and method for protein acrylamide gel electrophoresis separation and online imaging analysis includes a main unit 1, a CCD photosensitive chip 2, a UV lens 3, a filter 4, a UV light source 5, an upper buffer chamber 6, a lower buffer chamber 7, a power supply 8, a fixed wedge 6.1, a quartz front glass 6.2.1, a flat gel 6.2.2, a black background plate 6.2.3, and positive and negative electrodes 6.3. The specific connections are shown in the figure. The host 1 is connected to the CCD photosensitive chip 2. The CCD photosensitive chip 2, the UV lens 3, and the filter 4 are assembled together to form a fluorescent photosensitive component. The UV light source 5 irradiates the surface of the quartz front glass 6.2.1 of the upper buffer chamber 6 at a certain angle. The UV light passes through the quartz front glass 6.2.1 and acts on the PAGE gel 6.2.2. The upper buffer chamber 6 is placed on the lower buffer chamber 7. The PAGE gel 6.2.2 is prepared between the quartz front glass 6.2.1 and the black background plate 6.2.3, and is fixed in the flat gel slot of the upper buffer chamber 6 by a fixed wedge 6.1. The positive and negative electrodes 6.3 on the upper buffer chamber 6 are connected to the power supply 8.

[0045] In this embodiment, the ultraviolet light source 5 is ultraviolet light with a central wavelength of 275 nm.

[0046] Load 10 μL of the prepared protein sample into the loading channel of the slab gel 6.2.2. Secure the PAGE gel 6.2.2 in the slab gel slot. Place the upper buffer chamber 6 vertically on the lower buffer chamber. Turn on the power supply 8 via a wire and adjust the voltage to 80 V. Perform electrophoresis for 15 minutes. Then adjust the voltage to 120 V. After 20 minutes of electrophoresis, turn on the UV light source 5 and illuminate the PAGE gel 6.2.2 through the quartz front glass 6.2.1. Connect the positive and negative electrodes 6.3 on the upper buffer chamber 6 to the power supply 8. Turn on the power supply 8 and adjust the voltage to an appropriate level for protein electrophoretic separation. During electrophoresis, the UV light source 5 can be turned on for online imaging of the protein bands.

[0047] The protein electrophoresis separation online imaging method using the above device specifically comprises the following steps:

[0048] After loading the sample into the loading channel, install the open electrophoresis tank and adjust the fluorescent photosensitive component so that the UV lens 3 faces the imaging surface of the open electrophoresis tank;

[0049] Turn on the power supply 8, set the voltage required for electrophoresis separation, turn on the host and CCD photosensitive chip 2, adjust the appropriate exposure time, and collect data in real time;

[0050] During the electrophoresis process, the UV light source 5 is turned on to perform UV excitation on the protein bands in the gel. The protein emits fluorescence under UV excitation, and the fluorescence signal is collected by the CCD photosensitive chip 2. The corresponding protein fluorescence band pattern is displayed in the host 1, realizing real-time monitoring of the protein electrophoresis separation process.

[0051] After electrophoresis, the final protein fluorescence band pattern is obtained, and image processing technology is used to reduce background noise, obtain the grayscale value of the protein fluorescence band, and realize quantitative detection of the protein.

[0052] As shown in FIG5 , ordinary front glass blocks ultraviolet light transmission, the intrinsic fluorescence of the protein in the gel cannot be excited, and the imaging system cannot observe the fluorescence spectrum ( FIG5 a ). However, the quartz front glass in this embodiment has excellent light transmittance and high ultraviolet transmittance, so the ultraviolet excitation light can smoothly reach the gel to stimulate the intrinsic fluorescence of the protein bands, and the imaging system observes the protein fluorescence bands ( FIG5 b ).

[0053] As shown in Figure 6 , for the protein samples within the red box, the sensitivity of the fluorescence spectrum is higher than that of CBB staining.

[0054] As shown in Figures 7 and 8, the resolution of this device is significantly better than that of CBB staining.

[0055] As shown in Figure 9, the device can perform quantitative analysis of protein bands with a wide dynamic range and low detection limit.

[0056] The resolution comparison of proteins with different molecular weights in the protein standard marker and the resolution comparison of mixed protein samples are shown in Tables 1 and 2, respectively. For the protein standard marker, the molecular weights of the different protein bands vary greatly, and both successfully separate them, but the resolution of the online imaging device is generally inferior to that of CBB staining. For the mixed protein samples, when the molecular weights of the proteins vary little, the online imaging resolution reaches 1.6, while CBB staining fails to separate them, with a resolution of only 0.8. The online imaging resolution is significantly better than that of CBB staining.

[0057] Table 1 Comparison of resolution of proteins with different molecular weights in protein standard markers.

[0058] Table 2 Comparison of resolution of mixed protein samples.

[0059] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A protein acrylamide gel electrophoresis separation-online imaging analysis device, characterized in that: The device is used for protein electrophoresis separation and online detection; The device comprises: an open electrophoresis tank, used as a gel electrophoresis and protein band UV detection support, comprising: An upper buffer chamber (6), arranged at the upper part of the open electrophoresis tank; A lower buffer chamber (7), arranged at the lower part of the open electrophoresis tank; An ultraviolet light source (5) is used to excite the protein bands in the gel to generate fluorescent signals; CCD imaging detection mechanism, used to capture protein band excitation fluorescence signals for online imaging during electrophoresis, including: The filter (4) is arranged at the front end of the CCD imaging detection mechanism; An ultraviolet lens (3) is arranged between the filter (4) and the CCD photosensitive chip (2); A CCD photosensitive chip (2) is connected to the host (1); A host (1) is connected to a CCD photosensitive chip (2) for online imaging; The gel is a slab gel (6.2.2).

2. The protein acrylamide gel electrophoresis separation-online imaging analysis device according to claim 1, characterized in that: The shell of the open electrophoresis tank is made of black light-absorbing material.

3. The protein acrylamide gel electrophoresis separation-online imaging analysis device according to claim 1, characterized in that: The upper buffer chamber (6) comprises positive and negative electrodes (6.3) symmetrically arranged left and right and a flat gel slot symmetrically arranged front and back.

4. The protein acrylamide gel electrophoresis separation-online imaging analysis device according to claim 3, characterized in that: The flat plate gel slot comprises a fixed wedge (6.1) and a flat plate structure (6.2); The flat plate structure (6.2) comprises a quartz front glass (6.2.1), a flat plate gel (6.2.2) and a black background plate (6.2.3) which are arranged in sequence.

5. The protein acrylamide gel electrophoresis separation-online imaging analysis device according to claim 1, characterized in that: The ultraviolet light source (5) is an ultraviolet light source with a central wavelength of 275nm-360nm.

6. The protein acrylamide gel electrophoresis separation-online imaging analysis device according to claim 1, characterized in that: The ultraviolet light sources (5) are symmetrically distributed on both sides of the detection surface in the open electrophoresis tank; The CCD imaging detection mechanism is arranged on one side of the open electrophoresis tank.

7. The protein acrylamide gel electrophoresis separation-online imaging analysis device according to claim 1, characterized in that: The optical filter (4) is an optical filter with a central wavelength of 250-360 nanometers, a central wavelength transmittance greater than 80%, and a half-width of 10 nm to 50 nm.

8. The protein acrylamide gel electrophoresis separation-online imaging analysis device according to claim 1, characterized in that: The focal length of the ultraviolet lens (3) is 35mm-39mm, the working distance is 150mm-10m, and the working wavelength is 250-380nm.

9. The protein acrylamide gel electrophoresis separation-online imaging analysis device according to claim 1, characterized in that: The quantum conversion efficiency of the CCD photosensitive chip (2) at the central wavelength is greater than 40%.

10. A method for protein electrophoresis separation and online detection using the protein acrylamide gel electrophoresis separation-online imaging analysis device according to any one of claims 1 to 9, characterized in that: The method comprises the following steps: Step 1) Install the prepared slab gel (6.2.2) in the upper buffer chamber (6), and then vertically install the upper buffer chamber (6) on the lower buffer chamber (7), add appropriate amounts of electrode buffer to the upper buffer chamber (6) and the lower buffer chamber (7), respectively, and add the sample to be tested to the loading channel of the slab gel (6.2.2), with a sample volume of 1-20 μL; Step 2) Connect the upper buffer chamber (6) to the positive and negative electrodes of the power supply (8), turn on the power supply (8), set a suitable voltage, start electrophoresis, control the voltage at 50V / m-3000V / m, and the electrophoresis time is 20-40min; Step 3) During and after the electrophoresis, the ultraviolet light source (5) is turned on, and the electrophoresis process can be imaged online using a CCD imaging detection mechanism. The protein bands during the electrophoresis process are observed through the host (1) to obtain protein component information and concentration information in the sample.

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