WO2024092985A1 - Intrinsic fluorescence imaging detection system for protein gel electrophoresis and method - Google Patents

Abstract

An intrinsic fluorescence imaging detection system for protein gel electrophoresis and a method. The system comprises a portable hand-held box body (8), a chip electrophoresis device (9), an imaging detection device (5), and a computer terminal (7). The chip electrophoresis device (9) is arranged inside the portable hand-held box body (8); the imaging detection device (5) is arranged inside the portable hand-held box body (8) and is arranged on one side of the chip electrophoresis device (9); after chip electrophoresis in the chip electrophoresis device (9) is finished, the imaging detection device (5) collects a protein band image, and while imaging, a light source is triggered to be instantaneously turned on for exposure, so as to capture a fluorescence signal image; the computer terminal (7) is communicationally connected to the imaging detection device (5), and the fluorescence signal image captured by the imaging detection device (5) is processed and stored to obtain a gel electrophoresis test result. The detection system has multiple advantages such as integration, simple and convenient operation, online imaging detection, high throughput, and low consumption.

Description

Endogenous fluorescence imaging detection system and method for protein gel electrophoresis Technical Field

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.

Background technique

Polyacrylamide gel electrophoresis (PAGE) is a widely used protein separation and identification method in the fields of molecular biology, laboratory medicine, proteomics, etc. It has the advantages of simple equipment, intuitive results, and low cost. However, since most protein macromolecules are colorless in the visible light region, the detection of protein bands after electrophoresis requires the use of visible dyes or fluorescent reagents to label protein molecules. These detection methods have the following problems: first, the complicated operation steps such as staining, decolorization, and incubation are time-consuming and labor-intensive, and inefficient; second, due to the different amino acid composition and structure of proteins, different protein molecules may be labeled and stained to uneven degrees, resulting in heterogeneity of color distribution and affecting subsequent detection; third, some dyes such as silver will cause protein activity or structural changes after covalent binding to proteins, which is difficult to be compatible with subsequent protein preparation, mass spectrometry identification and other processes. In addition, the dye reagents, reducing agents, etc. used for staining, labeling, and decolorization are harmful to the human body and the environment, and have high costs. Therefore, traditional gel electrophoresis is difficult to meet the needs of modern analytical testing for efficiency, safety, and environmental protection.

In order to solve these problems, 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. However, this method also has certain limitations. For example, 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.

Or by using the ultraviolet absorption groups of macromolecules such as proteins, the ultraviolet absorbance of the quartz capillary column end or the entire column can be detected. However, due to the poor UV transmittance of matrix materials such as PMMA, PC and PDMS of electrophoresis chips, there is still a lack of ultraviolet detection and imaging technology devices compatible with chip electrophoresis. Or by 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), however, 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.

Therefore, it is urgent to develop new innovative analytical technologies to solve problems of online imaging detection, including chip electrophoresis, and portable electrophoresis separation detection.

Summary of the invention

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.

The purpose of the present invention can be achieved by the following technical solutions:

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.

Furthermore, 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.

Furthermore, the photosensitive side of the ultraviolet-sensitive CCD camera faces the chip electrophoresis device.

Furthermore, 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.

Furthermore, 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;

One side of 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.

Furthermore, 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.

Furthermore, the deep ultraviolet LED array includes a plurality of deep ultraviolet LED lamp beads with a wavelength of 240-300nm arranged in an array.

Furthermore, 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.

Furthermore, 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:

Inject the configured model protein sample, place the electrophoresis chip in front of the imaging detection device, make the center of the deep ultraviolet LED array light source irradiation surface and the central axis of the ultraviolet sensitive CCD camera on the same horizontal axis, and place it in an opaque portable box;

After loading the sample onto the electrophoresis chip, turn on the power supply unit and start electrophoresis;

At the end of electrophoresis, 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.

Compared with the existing traditional and chip gel electrophoresis technology equipment, the present invention has the following significant advantages:

1. Realize label-free rapid imaging detection of protein bands after electrophoresis. Traditional PAGE electrophoresis requires 3 hours of fixation, staining and decolorization, and then imaging and analysis with a gel imager, which is time-consuming, labor-intensive, reagent-intensive and space-consuming. With this device, colorless protein bands can be directly imaged and detected without traditional time-consuming manual steps such as fixation, staining, decolorization or derivatization, significantly improving the efficiency of zone gel electrophoresis.

2. Improve the sensitivity of chip gel electrophoresis detection. Using deep ultraviolet LED array light source, the radiation light is highly uniform within a certain area, which stimulates the endogenous fluorescence of tryptophan residues in protein molecules in the chip gel, thereby imaging and quantitatively detecting protein bands online, and its sensitivity is better than traditional Coomassie brilliant blue protein staining and protein ultraviolet absorbance imaging detection.

3. Realize protein chip/traditional gel electrophoresis quantification. Traditional protein gel electrophoresis uses staining to observe protein bands, which can achieve semi-quantitative analysis, but it is difficult to achieve quantitative analysis. The present invention can achieve online quantitative analysis of protein gel electrophoresis, and has a good linear range and stability.

4. 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.

5. Significantly reduce the consumption of reagents and consumables. Traditional gel electrophoresis requires protein and nucleic acid fixative, staining solution, decolorizing solution, etc. Using the detection system in this technical solution, protein imaging does not require the use of dyes and derivatization reagents, and combined with chip gel electrophoresis, it greatly reduces the consumption of chemical reagents.

6. Reduce indoor pollution in the laboratory. Traditional PAGE electrophoresis fixation, staining and decolorization require a large amount of organic solvents such as trichloroacetic acid, glacial acetic acid, ethanol and Coomassie brilliant blue, which causes serious indoor pollution in the laboratory.

7. Save a lot of equipment and space. Traditional PAGE electrophoresis requires electrophoresis power supply, electrophoresis tank, gel rack, shaker or automatic staining instrument, gel imager, and desktop computer, etc. Not only are there many kinds of equipment, but also a large experimental area is occupied.

BRIEF DESCRIPTION OF THE DRAWINGS

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 γ-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.

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.

In FIGS. 1-2 : light source cover 1 , deep ultraviolet LED array 2 , ultraviolet sensitive CCD camera 3 , device cover 4 .

In 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 .

Detailed ways

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.

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

1. Design 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);

2. Design 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;

3. Design 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:

(1) After the chip electrophoresis is completed, 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.

(2) After the imaging results are obtained, they are recorded and analyzed by computer software. The computer software is purchased on the market and will not be described in detail here.

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.

The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. Component models, material names, connection structures, control methods, algorithms and other features not clearly described in this technical solution are all considered to be common technical features disclosed in the prior art.

In Figure 5: Non-uniformity of the excitation light source when using a single-chip deep ultraviolet LED light source for IFI imaging analysis.

Figure 6 shows the uniformity of the excitation light source when using four deep UV LEDs for IFI imaging analysis. In insets A1 and A2, the concentration of native BSA is 2.0 μg/μL; in insets B1 and B2, the concentration of native BSA is 0.2 μg/μL; in insets C1C2, the concentration of native BSA is 0.02 μg/μL.

In Figure 7: 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 γ-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 (γ-Glb, BSA and LZ).

In Figure 8: 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.

In Figure 9: Illustration A is the result of chip gel electrophoresis-IFI imaging analysis of denatured BSA, LZ and γ-Glb standard samples; Illustration B is the result of chip gel electrophoresis-IFI imaging analysis of denatured BSA, LZ and γ-Glb mixed samples.

In FIG. 10 : 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 μL protein spots of three non-denatured model proteins (BSA, LZ and γ-Glb) and three denatured model proteins (BSA, LZ and γ-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 μl 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 ).

Example 1

Chip gel electrophoresis-IFI fluorescence imaging detection of standard model proteins

The chip electrophoresis-IFI fluorescence imaging detection device system is shown in Figures 3 and 4. First, three model proteins and three standard model proteins (gamma globulin γ-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.

Afterwards, 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 .

Then, as shown in Figures 3 and 4, all components are placed in an opaque portable box 5. After the electrophoresis chip is loaded with samples, the power supply unit is turned on 6, and a constant voltage mode of 100 V is set for 6 minutes to start electrophoresis.

When the electrophoresis is finished, 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.

The results are shown in Figure 5. Illustration A is the chip electrophoresis-IFI imaging analysis and linear range experiment of three standard model proteins (gamma globulin γ-Glb (arrow 1), bovine serum albumin BSA (arrow 2) and egg white lysozyme LZ (arrow 3). The linear range is 0.02-2.0 μg/uL. Illustration B is the reproducibility and consistency experimental test of the imaging detection device of the present invention for three standard model proteins (γ-Glb, BSA and LZ.

Experiments have shown that the 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.

Example 2

Chip gel electrophoresis-fluorescence imaging detection of human serum proteins

The chip electrophoresis-fluorescence imaging detection device system is shown in Figures 3 and 4. First, 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.

Then, as shown in Figures 3 and 4, all components are placed in an opaque portable box 5. After the electrophoresis chip is loaded with samples, the power supply unit is turned on 6, and a constant voltage mode of 100 V is set for 6 minutes to start electrophoresis.

After the electrophoresis is completed, 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.

The results are shown in Figure 5. First, the protein band position distribution obtained by chip electrophoresis-fluorescence imaging detection (inset A) and flat plate electrophoresis-Coomassie Brilliant Blue staining (inset B) for separation and detection of human serum proteins has a high consistency.

At the same time, it can be observed that a weak protein band in the flat gel using the Coomassie brilliant blue staining method (arrow 1 in inset B) can be clearly observed in the chip electrophoresis fluorescence imaging detection (arrow 1 in inset A), the band signal intensity is significantly enhanced, and the area indicated by arrow 2 in inset A can distinguish 1-3 more bands than the same area in inset B. The above comparison results show that the device has better detection sensitivity than traditional flat electrophoresis and Coomassie brilliant blue staining, and the method is simple and rapid, without the need for staining, derivatization and other operations, and realizes rapid online imaging detection of proteins after electrophoresis.

The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and 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 (10)

An endogenous fluorescence imaging detection system for protein gel electrophoresis, characterized by comprising: Portable suitcase body (8); A chip electrophoresis device (9) is arranged inside the portable suitcase body (8); An imaging detection device (5) is arranged inside the portable suitcase (8) and placed on one side of the chip electrophoresis device (9). After the chip electrophoresis in the chip electrophoresis device (9) is completed, the imaging detection device (5) collects a protein band image and triggers the light source to be turned on instantaneously for exposure while imaging, thereby capturing a fluorescent signal image; The computer terminal (7) is connected to the imaging detection device (5) for communication, and processes and stores the fluorescent signal image captured by the imaging detection device (5) to obtain the gel electrophoresis test result. According to claim 1, an endogenous fluorescence imaging detection system for protein gel electrophoresis is characterized in that the imaging detection device (5) comprises a device cover (4), a UV-sensitive CCD camera (3) arranged in the device cover (4), and a light source assembly arranged on one side of the UV-sensitive CCD camera (3). The endogenous fluorescence imaging detection system for protein gel electrophoresis according to claim 2, characterized in that the photosensitive side of the ultraviolet-sensitive CCD camera (3) faces the chip electrophoresis device (9). An endogenous fluorescence imaging detection system for protein gel electrophoresis according to claim 2, characterized in that the light source assembly comprises a light source cover (1) arranged at the opening of the device cover (4) and a deep ultraviolet LED array (2) arranged on the light source cover (1). The endogenous fluorescence imaging detection system for protein gel electrophoresis according to claim 4, characterized in that the light source cover (1) comprises four light source carrier plates and a light source cover outer frame; The light source cover outer frame is fixed to the opening of the device cover (4); One side of each light source carrier is connected to the frame of the ultraviolet-sensitive CCD camera (3), and the other side is connected to the frame of the ultraviolet-sensitive CCD camera (3). An endogenous fluorescence imaging detection system for protein gel electrophoresis according to claim 2, characterized in that the ultraviolet-sensitive CCD camera (3) is provided with a bandpass filter with a cut-off wavelength of 280-488 nm. The endogenous fluorescence imaging detection system for protein gel electrophoresis according to claim 4 is characterized in that the deep ultraviolet LED array (2) comprises a plurality of deep ultraviolet LED lamp beads with a wavelength of 240-300 nm arranged in an array. According to claim 1, an endogenous fluorescence imaging detection system for protein gel electrophoresis is characterized in that the portable suitcase (8) is provided with an openable and closable opening, through which the chip electrophoresis device (9) and the imaging detection device (5) can be placed in and taken out. The endogenous fluorescence imaging detection system for protein gel electrophoresis according to claim 1, characterized in that the portable suitcase (8) further comprises a power supply unit (6) for supplying power to the active device; The portable suitcase body (8) is made of light-proof material. A protein gel electrophoresis endogenous fluorescence online imaging detection method using the detection system according to any one of claims 1 to 9, characterized in that it comprises the following steps: Inject the configured model protein sample, place the electrophoresis chip in front of the imaging detection device, make the center of the irradiation surface of the deep ultraviolet LED array light source and the central axis of the ultraviolet sensitive CCD camera (3) on the same horizontal axis, and place it in an opaque portable box (5); After the sample is loaded onto the electrophoresis chip, the power supply unit (6) is turned on to start electrophoresis; When the electrophoresis is finished, IFI fluorescence imaging acquisition is started, and while the ultraviolet-sensitive CCD camera (3) is capturing, the LED array light source is triggered to automatically turn on and expose, and the obtained fluorescence imaging results are analyzed by the computer terminal (7).

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