Real-time quantitative WesternBlot experiment method and system

Abstract

The invention relates to the technical field of optical detection, in particular to a real-time quantitative WesternBlot experimental method and system.The method comprises the following steps that an incubation box soaked with a detection film is put into an equipment detection table, shooting is controlled through LuQAS client software, and exposure is conducted through a preset or self-edited time gradient; the software intelligently assists in identifying the strip, automatically marks the print contour and the IOD value, and automatically detects overexposure; determining a proper exposure duration according to a time gradient exposure result, then performing continuous exposure by using the set duration, selecting a corresponding picture and using a'statistics' function to obtain a luminescence curve; the incubation box has the beneficial effect that a biological sample is quantitatively detected in real time in the incubation box. The optimized exposure duration ensures high quality and accuracy of image data, and provides powerful support for subsequent biological research and clinical application. And meanwhile, the traceability and the safety of experimental data are effectively guaranteed.

Description

Technical Field

[0001] The invention relates to the technical field of optical detection, in particular to a real-time quantitative Western Blot experimental method and system. Background Art

[0002] In biomedical research, protein detection is one of the key technologies in understanding biological processes, disease mechanisms, and drug development. Although traditional protein detection methods such as Western Blotting are highly specific and sensitive, they involve multiple tedious steps in the experimental process, and the quantitative analysis of the results often relies on the subjective judgment of the experimenter, which challenges the accuracy and repeatability of the results.

[0003] In recent years, with the development of chemiluminescence imaging technology, especially the emergence of electrochemiluminescence (ECL) technology, a new and efficient means for protein detection has been provided. ECL technology excites the luminescent substance labeled on the protein to emit visible light, which is then captured and converted into digital signals by imaging equipment to achieve quantitative analysis of proteins. However, there are still many problems in the selection of exposure time, analysis of image data, and accuracy of experimental results in the ECL imaging process.

[0004] At present, although there are some devices for ECL imaging on the market, these devices have deficiencies in exposure control, image analysis, and data security. For example, some devices lack intelligence in image analysis and require experimenters to manually identify imprints and calculate data, which not only reduces experimental efficiency but also increases the subjectivity of the results; in addition, some devices have loopholes in data security and are prone to tampering or falsification of experimental results; an important problem with traditional experimental methods and systems is that it is impossible to confirm whether the test results are obtained during the stable peak stage of luminescence, and therefore it is impossible to confirm whether the test data is correct. Summary of the invention

[0005] The purpose of the present invention is to provide a real-time quantitative Western Blot experimental method and system to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a real-time quantitative Western Blot experimental method, the method comprising the following steps:

[0007] Place the incubation box with the test membrane soaked in it on the test bench of the equipment, and control the shooting through the LuQAS client software. First, use the preset or self-edited time gradient for exposure;

[0008] The software intelligently assists in identifying bands, automatically marks the imprint outline and IOD value, and automatically detects overexposure;

[0009] Determine the appropriate exposure time based on the time gradient exposure results, then use this predetermined time for continuous exposure, select the corresponding image and use the "Statistics" function to obtain the luminescence curve;

[0010] Select the image data of the stable peak stage of luminescence from the luminescence curve as the experimental result;

[0011] The system supports encrypted signature experiment images to store experiment results, making the experiment traceable.

[0012] Preferably, the device parameters include:

[0013] The analysis modes are fully automatic and assisted blot recognition;

[0014] The number of electrons in the full well is 50ke+;

[0015] Data transmission speed is 1000Mbps;

[0016] The acquisition time is 1-30 seconds for extremely fast imaging;

[0017] The imaging device is a 9-megapixel, high-sensitivity cooled camera with a quantum efficiency of 80%+.

[0018] Preferably, the method further includes the steps of storing and analyzing the image data:

[0019] Image data is signed with an asymmetric encryption to ensure that the source of the data is traceable and cannot be tampered with;

[0020] Support saving as tiff image file, and exporting experimental report containing image, experimental background data and notes;

[0021] The experimental data is calculated based on the original RAW image of the photosensitive data to ensure the accuracy of the data.

[0022] Preferably, the experimental method further comprises: using a transparent box to incubate the ECL luminescent liquid, and adjusting the dilution factor of the ECL luminescent liquid according to the intensity of the luminescent signal.

[0023] Preferably, the method further includes the step of experiment management: supporting experiment management by user, independent experiment archiving, and providing preset and customized experiment parameters.

[0024] A real-time quantitative Western Blot experimental system is applied to the real-time quantitative Western Blot experimental method, characterized in that the system comprises:

[0025] LuQAS client software is used to control image acquisition equipment, set exposure parameters, and automatically and assisted blot recognition and analysis of Western Blot data; the software automatically recognizes blot contours, annotates IOD values, and supports one-click setting to display absolute values ​​or normalized values, while providing the functions of manually adding, deleting, and fine-tuning blots;

[0026] Image acquisition equipment, consisting of a highly sensitive refrigerated camera, lens, smart board and light box, can capture weak fluorescence, support 1-30 seconds ultra-fast imaging, and connect to the client computer system via USB;

[0027] The system supports automatic or manual photography mode, which can be used to perform continuous exposure according to the preset time gradient or user-defined time sequence to obtain the luminescence curve of each blot;

[0028] The system automatically marks overexposed images and determines the optimal exposure time based on the luminescence curve or print clarity to output stable quantitative data.

[0029] Preferably, the account management system supports multi-user use, each user can create an independent profile, and provides the function of presetting and customizing experimental parameters to ensure the independence and traceability of experimental data;

[0030] Image data security measures include encrypting image data using asymmetric encryption signatures and supporting traceability through RSA signatures to ensure that the source of the data is traceable and cannot be tampered with.

[0031] Preferably, the LuQAS client software also has the following functions:

[0032] During the exposure sequence, thumbnails are displayed in real time, allowing the user to observe and analyze the blot data on the images that have appeared, without having to wait for the entire sequence to complete;

[0033] After selecting a desired trace on a satisfactory image, it can automatically identify and display the outline and IOD, and automatically apply the currently selected trace range to all images;

[0034] Provides statistical functions that can display the luminescence curve of each blot and automatically identify stable peak phase data as quantitative data.

[0035] Preferably, the image acquisition device has the following parameters:

[0036] The number of full well electrons reaches 50ke+, the data transmission speed is 1000Mbps, and chemiluminescence and single wavelength narrow band light source are used;

[0037] The imaging device is a highly sensitive cooled camera with 9 million pixels and a quantum efficiency of over 80%, which can easily capture weak fluorescence signals.

[0038] Preferably, the system also includes an experimental method guidance function, specifically:

[0039] Provide ECL luminescent solution dilution recommendations and guidance on using transparent boxes for incubation, as well as how to monitor real-time data of luminescent blots through time gradient exposure and select the optimal exposure time;

[0040] Provide suggestions for whole membrane detection, including the order of antibody detection, cross-use of primary antibodies to reduce background interference, and suggestions for the protein molecular weight span corresponding to two antibodies detected consecutively.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The real-time quantitative Western Blot experimental method and system proposed by the present invention perform exposure through a preset time gradient or a self-edited gradient time series, and monitor the real-time data of the luminescent blot in real time. When overexposure occurs, the time before overexposure is selected as the exposure time for continuous shooting. This automatic exposure control function not only improves the experimental efficiency, but also reduces the introduction of human errors. Real-time quantitative detection of biological samples is performed in an incubation box. The optimized exposure time ensures the high quality and accuracy of the image data, providing strong support for subsequent biological research and clinical applications. At the same time, the traceability and security of the experimental data are effectively guaranteed; the problem that the traditional experimental methods and systems cannot confirm whether the detection results are obtained by the blot at the stable peak stage of luminescence, and therefore cannot confirm whether the detection data is correct, is overcome. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The figure is a flow chart of the method of the present invention. DETAILED DESCRIPTION

[0044] In order to make the purpose and technical solution of the present invention clearly and completely described, and the advantages more clearly understood, the embodiments of the present invention are further described in detail with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are part of the embodiments of the present invention, rather than all of the embodiments, and are only used to explain the embodiments of the present invention, and are not used to limit the embodiments of the present invention. All other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] Embodiment 1, refer to the attached Figure 1 As shown, the present invention provides a technical solution: a real-time quantitative Western Blot experimental method, the method comprising the following steps:

[0046] Place the incubation box with the immersed test membrane on the equipment test table, control the shooting through the LuQAS client software, and first use the preset or self-edited time gradient for exposure; the equipment parameters include: the analysis mode is fully automatic and assisted imprint recognition; the full well electron number is 50ke+; the data transmission speed is 1000Mbps; the acquisition time is 1-30 seconds for ultra-fast imaging; the imaging device is a 9-megapixel, high-sensitivity cooled camera with a quantum efficiency of 80%+.

[0047] The software intelligently assists in identifying bands, automatically marks the imprint outline and IOD value, and automatically detects overexposure;

[0048] Determine the appropriate exposure time based on the time gradient exposure results, then use this predetermined time for continuous exposure, select the corresponding image and use the "Statistics" function to obtain the luminescence curve;

[0049] Select the image data of the stable peak stage of luminescence from the luminescence curve as the experimental result;

[0050] The system supports encrypted signature experiment images to store experiment results, making the experiment traceable.

[0051] It also includes steps for saving and analyzing image data: image data is signed with asymmetric encryption to ensure that the data source is traceable and cannot be tampered with; it supports saving as tiff image files and exporting experimental reports containing images, experimental background data and notes; experimental data is calculated based on the original RAW image of photosensitive data to ensure data accuracy.

[0052] The experimental method also includes: using a transparent box to incubate the ECL luminescent liquid, and adjusting the dilution multiple of the ECL luminescent liquid according to the luminescent signal intensity; it also includes the steps of experimental management: supporting user-based experimental management, independent experimental filing, and providing preset and customized experimental parameters.

[0053] Example 2, based on Example 1, proposes a bioluminescence imaging experiment based on real-time quantitative detection technology in an incubation box.

[0054] 1. Experimental purpose: To use the bioluminescence imaging system to perform real-time quantitative detection of specific biological samples. By precisely controlling the exposure time, the image data of the luminescence peak stage of each blot in the sample is obtained to achieve high-sensitivity and high-precision quantitative analysis.

[0055] 2. Experimental materials: biological samples (such as protein, DNA, etc.), detection membrane, incubation box, bioluminescence imaging system (equipped with LuQAS client software), buffer, labeled probe.

[0056] 3. Experimental steps:

[0057] 1) Sample preparation and incubation: Spread the biological sample evenly on the test membrane. Add an appropriate amount of labeled probe and buffer to ensure that the sample and probe are fully combined. Place the test membrane in an incubation box and incubate under appropriate temperature and humidity conditions to ensure that the labeled probe fully reacts with the target molecule in the sample.

[0058] 2) Set up the imaging system: Place the incubation box soaked with the detection membrane on the detection table of the bioluminescence imaging system and close the door. Open the LuQAS client software and set the shooting parameters. Select a preset time gradient or edit the gradient time series to expose to explore the optimal exposure time.

[0059] 3) Real-time quantitative detection: Initial exposure exploration: Use a preset time gradient (such as increasing by 1 second per second) for continuous exposure, observe and record the image data at each time point.

[0060] Luminescence peak selection: During the exposure sequence, the images appear in thumbnails in the software interface one by one. By observing and analyzing the imprint data, the image data at the luminescence peak stage is selected as the experimental result.

[0061] Optimize exposure time:

[0062] If overexposure occurs during the exposure process, select the exposure time of the last picture before the overexposure mark appears as the exposure time for subsequent shooting.

[0063] If the target blot has a clear and full outline, stop shooting immediately and use the current exposure time as the duration of the continuous shooting sequence.

[0064] Continuous shooting: Starts a new shooting sequence with an optimized exposure time (e.g. 5 seconds per frame) and takes 10 images in a row.

[0065] 4) Data analysis: Select the target blot on a satisfactory image, and the software will automatically identify and display the blot outline and integrated optical density (IOD) value. Click the "Apply to All" button to automatically mark the currently selected blot range on all images. Check the target image sequence and display the luminescence curve of each blot through the software statistics function. Among them, the stable peak stage data is the quantitative data.

[0066] 5) Data export and report: Supports saving image data as tiff format files for subsequent analysis and archiving. Export the experimental report, which contains the image, background data of the experiment (such as exposure time, shooting parameters, etc.) and notes. All data are calculated based on the original RAW image of the photosensitive data to ensure the accuracy and traceability of the data.

[0067] 6) Experiment management and data security: Supports user-specific experimental management functions to ensure that each user can only access and operate their own experimental data to avoid misuse. Image data is processed using asymmetric encryption signature technology to ensure that the data source is traceable and cannot be tampered with, thereby ensuring the reliability and security of experimental results.

[0068] 2. Experimental results: Through the method of this embodiment, real-time quantitative detection of biological samples in the incubation box was successfully achieved. The optimized exposure time ensured the high quality and accuracy of the image data, providing strong support for subsequent biological research and clinical applications. At the same time, the traceability and security of the experimental data were effectively guaranteed.

[0069] Embodiment 3, based on embodiment 1, proposes a real-time quantitative Western Blot experimental system, which is applied to the real-time quantitative Western Blot experimental method, and the system comprises:

[0070] LuQAS client software is used to control image acquisition equipment, set exposure parameters, and automatically and auxiliaryly identify and analyze Western Blot data; the software automatically identifies the blot outline, marks the IOD value, and supports one-click setting to display absolute values ​​or normalized values, while providing the function of manually adding, deleting and fine-tuning blots; LuQAS client software also has the following functions: during the exposure sequence, thumbnails are displayed in real time, allowing users to observe and analyze blot data on the images that have appeared without waiting for the entire sequence to be completed; after the blot is framed on a satisfactory image, it automatically identifies and displays the outline and IOD, and automatically applies the currently selected blot range to all images; it provides statistical functions, can display the luminescence curve graph of each blot, and automatically identify the stable peak stage data as quantitative data.

[0071] The image acquisition device consists of a high-sensitivity cooled camera, lens, intelligent board and light box. It can capture weak fluorescence, support 1-30 seconds ultra-fast imaging, and connect to the client computer system via USB. The image acquisition device has the following parameters: the number of full-well electrons reaches 50ke+, the data transmission speed is 1000Mbps, and chemiluminescence and single-wavelength narrow-band light source are used. The imaging device is a high-sensitivity cooled camera with 9 million pixels and a quantum efficiency of more than 80%, which can easily capture weak fluorescence signals.

[0072] The system supports automatic or manual photography mode, which can be used to perform continuous exposure according to the preset time gradient or user-defined time sequence to obtain the luminescence curve of each blot;

[0073] The system automatically marks overexposed images and determines the optimal exposure time based on the luminescence curve or print clarity to output stable quantitative data.

[0074] It also includes: an account management system that supports multi-user use. Each user can create an independent profile and provide the function of pre-setting and customizing experimental parameters to ensure the independence and traceability of experimental data; image data security measures, including the use of asymmetric encryption signatures to encrypt image data, and support traceability through RSA signatures to ensure that the source of the data is traceable and cannot be tampered with.

[0075] The system also includes experimental method guidance functions, specifically: providing ECL luminescent solution dilution recommendations and guidance on using transparent boxes for incubation, as well as how to monitor real-time data of luminescent blots through time gradient exposure and select the optimal exposure time; providing recommendations for full-membrane detection, including antibody detection order, cross-use of primary antibodies to reduce background interference, and recommendations on the protein molecular weight span corresponding to two antibodies for consecutive adjacent detections.

[0076] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Real-time quantitative Western Blot experimental method, characterized in that: The method comprises the following steps: Place the incubation box with the test membrane soaked in it on the test bench of the equipment, and control the shooting through the LuQAS client software. First, use the preset or self-edited time gradient for exposure; The software intelligently assists in identifying bands, automatically marks the imprint outline and IOD value, and automatically detects overexposure; Determine the appropriate exposure time based on the time gradient exposure result, then use this set time for continuous exposure, select the corresponding picture and use the "Statistics" function to obtain the luminescence curve; Select the image data of the stable peak stage of luminescence from the luminescence curve as the experimental result; The system supports encrypted signature experiment images to store experiment results, making the experiment traceable.

2. The real-time quantitative Western Blot experimental method according to claim 1, characterized in that: Device parameters include: The analysis modes are fully automatic and assisted blot recognition; The number of electrons in the full well is 50ke+; Data transmission speed is 1000Mbps; The acquisition time is 1-30 seconds for extremely fast imaging; The imaging device is a 9-megapixel, high-sensitivity cooled camera with a quantum efficiency of 80%+.

3. The real-time quantitative Western Blot experimental method according to claim 1, characterized in that: It also includes steps for saving and analyzing image data: Image data is signed with an asymmetric encryption to ensure that the source of the data is traceable and cannot be tampered with; Supports saving as tiff image files and exporting experimental reports containing images, experimental background data and notes; The experimental data is calculated based on the original RAW image of the photosensitive data to ensure the accuracy of the data.

4. The real-time quantitative Western Blot experimental method according to claim 1, characterized in that: The experimental method also includes: using a transparent box to incubate the ECL luminescent liquid, and adjusting the dilution multiple of the ECL luminescent liquid according to the intensity of the luminescent signal.

5. The real-time quantitative Western Blot experimental method according to claim 1, characterized in that: It also includes the steps of experiment management: supporting experiment management by user, independent experiment filing, and providing preset and customized experiment parameters.

6. A real-time quantitative Western Blot experimental system, applied to the real-time quantitative Western Blot experimental method according to any one of claims 1 to 5, characterized in that: The system comprises: LuQAS client software is used to control image acquisition equipment, set exposure parameters, and automatically and assisted blot recognition and analysis of Western Blot data; the software automatically recognizes blot contours, annotates IOD values, and supports one-click setting to display absolute values ​​or normalized values, while providing the functions of manually adding, deleting, and fine-tuning blots; Image acquisition equipment, consisting of a highly sensitive refrigerated camera, lens, smart board and light box, can capture weak fluorescence, support 1-30 seconds ultra-fast imaging, and connect to the client computer system via USB; The system supports automatic or manual photography mode, which can be used to perform continuous exposure according to the preset time gradient or user-defined time sequence to obtain the luminescence curve of each blot; The system automatically marks overexposed images and determines the optimal exposure time based on the luminescence curve or print clarity to output stable quantitative data.

7. The real-time quantitative Western Blot experimental system according to claim 6, characterized in that: Also includes: The account management system supports multi-user use. Each user can create an independent profile and provide the function of presetting and customizing experimental parameters to ensure the independence and traceability of experimental data. Image data security measures include encrypting image data using asymmetric encryption signatures and supporting traceability through RSA signatures to ensure that the source of the data is traceable and cannot be tampered with.

8. The real-time quantitative Western Blot experimental system according to claim 6, characterized in that: LuQAS client software also has the following functions: During the exposure sequence, thumbnails are displayed in real time, allowing the user to observe and analyze the blot data on the images that have appeared, without having to wait for the entire sequence to complete; After selecting a desired trace on a satisfactory image, it can automatically identify and display the outline and IOD, and automatically apply the currently selected trace range to all images; Provides statistical functions that can display the luminescence curve of each blot and automatically identify stable peak phase data as quantitative data.

9. The real-time quantitative Western Blot experimental system according to claim 6, characterized in that: The image acquisition device has the following parameters: The number of full well electrons reaches 50ke+, the data transmission speed is 1000Mbps, and chemiluminescence and single wavelength narrow band light source are used; The imaging device is a highly sensitive cooled camera with 9 million pixels and a quantum efficiency of over 80%, which can easily capture weak fluorescence signals.

10. The real-time quantitative Western Blot experimental system according to claim 6, characterized in that: The system also includes experimental method guidance functions, specifically: Provide ECL luminescent solution dilution recommendations and guidance on using transparent boxes for incubation, as well as how to monitor real-time data of luminescent blots through time gradient exposure and select the optimal exposure time; Provide suggestions for whole membrane detection, including the order of antibody detection, cross-use of primary antibodies to reduce background interference, and suggestions for the protein molecular weight span corresponding to two antibodies detected consecutively.

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