Western blot full-automatic multi-round cyclic detection equipment
By integrating antibody denaturing solution and chemiluminescence detection into a fully automated multi-round cyclic detection device, the problems of cumbersome operation, limited throughput, and low sensitivity of traditional Western blot technology have been solved, achieving high-throughput, low-cost, and low-error protein detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-10
- Publication Date
- 2026-03-31
AI Technical Summary
Traditional Western blot techniques are cumbersome to operate, have limited throughput, poor reproducibility, and high cost. Existing automated equipment cannot be widely adopted, and the high hydrophobicity of membrane proteins reduces sensitivity.
A fully automated multi-cycle detection device was designed, integrating antibody denaturing solution and chemiluminescence detection. It includes a liquid handling module, an incubation reaction module, a signal capture module, and a program control module. It achieves antibody residue removal and signal crosstalk standardization, and realizes high-sensitivity detection through chemiluminescent substrate and antibody denaturing solution.
It achieves high-throughput detection of multiple targets on a single membrane, reduces human error, improves detection sensitivity, saves costs, and the equipment's self-cleaning system requires no manual maintenance.
Smart Images
Figure CN224066807U_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological detection technology, specifically to a fully automated multi-round Western blot device and method based on antibody denaturing solution and chemiluminescence detection, for achieving high-throughput continuous detection of multiple target proteins on a single transfer membrane. Background Technology
[0002] Traditional Western blot technology has three major technical bottlenecks:
[0003] The process is cumbersome: a single test requires 10-15 steps, including electrophoresis, membrane transfer, blocking, incubation with primary / secondary antibodies, and color development, taking 8-12 hours.
[0004] Throughput limitations: A single assay typically only analyzes 2-3 target proteins, and repeated membrane cutting is required;
[0005] Poor repeatability: Manual operation leads to a batch-to-batch coefficient of variation (CV) > 20%.
[0006] While existing automated equipment (such as capillary electrophoresis Western spectroscopy systems) reduces operation time, it has the following drawbacks:
[0007] The equipment is expensive and cannot be widely adopted.
[0008] The need for ultra-high-speed centrifugation (100,000×g) results in high costs for the supporting equipment;
[0009] The high hydrophobicity of membrane proteins makes it impossible to achieve high-quality separation.
[0010] The sample loading amount is only 1 / 50 of that of the traditional method, and the sensitivity is reduced by about two orders of magnitude.
[0011] The purpose of this invention is to provide a fully automated Western blot device that integrates antibody denaturation and regeneration with high-sensitivity chemiluminescence detection, enabling continuous detection of multiple targets on a single membrane, while simultaneously solving the problems of antibody residue removal, signal crosstalk, and standardized operation. Utility Model Content
[0012] This utility model provides a fully automated multi-round cyclic detection device for Western blot, comprising:
[0013] The liquid processing module is equipped with at least a chemiluminescent substrate storage unit and an antibody denaturing solution storage unit, as well as a liquid dispensing system. The antibody denaturing solution is used to remove antibody residues in the membrane and tubing, and supports multiple rounds of detection cycle operation.
[0014] An incubation reaction module comprising at least two reaction chambers, wherein the reaction chambers are configured with an oscillation mechanism;
[0015] The signal acquisition module includes a transmitting light source, a CCD or CMOS sensor, and a dark box structure;
[0016] The program control module controls the coordinated operation of each module and supports setting machine operating parameters through a touch screen interface.
[0017] Preferably, the luminescent substrate is an enzymatic luminescent substrate of horseradish peroxidase or alkaline phosphatase, and these substrate components contain at least one of luminol, isoluminol, AMPPD, and CSPD.
[0018] Preferably, the antibody denaturing solution is selected from at least one of the following: an acidic solution with pH ≤ 2, an alkaline solution with pH ≥ 12, a 6-8M urea solution, a 4-6M guanidine salt solution, and a 5-7M thiocyanate solution.
[0019] Preferably, the wavelength range of the emitting light source is 300nm to 700nm.
[0020] Preferably, the signal acquisition module consists of a CCD or CMOS sensor and an optical lens.
[0021] Preferably, the liquid processing module includes, in addition to the chemiluminescent substrate unit and the antibody denaturing solution unit, a rinsing solution unit, a blocking solution unit, and a deionized water unit.
[0022] Preferably, the reaction chamber of the incubation reaction module is driven by a motor to achieve cyclic oscillation motion.
[0023] Preferably, the program control module supports setting incubation time, number of rinses, and image transmission functions.
[0024] Preferably, the liquid distribution system is driven by a liquid pump and delivers the liquid through plastic pipes.
[0025] Preferably, the contact area between the door and the body of the dark box structure is provided with a concave-convex sealing structure to prevent external light from seeping in.
[0026] Antibody denaturing solution: The antibody denaturing solution removes primary / secondary antibody residues from the membrane and tubing, thereby enabling 20 rounds of detection cycles. At the same time, the antibody denaturing solution removes contamination from the previous round of detection for the next round of detection.
[0027] Chemiluminescence system: Chemiluminescence is formed by substrate catalysis. Labeled secondary antibodies are protein components that can be effectively denatured by protein denaturants. In contrast, fluorescently labeled secondary antibodies are labeled with macromolecular compounds on the outside of antibody proteins. Protein denaturants alone cannot act on them, thus preventing antibody dissociation.
[0028] Self-cleaning system: Cleans by circulating denaturing liquid, rinsing liquid and deionized water, requiring no manual maintenance. Beneficial effects
[0029] Increased throughput: Multi-membrane parallel operation eliminates the need for sample preparation, electrophoresis, and membrane transfer; one membrane can be repeatedly used for up to 20 detections.
[0030] Standardization: No need to cut the film, multiple subsequent operations can be completed with one click, without human intervention, reducing human error.
[0031] High sensitivity: Current chemiluminescent liquids have broken through the femtogram level of sensitivity, while traditional colorimetric or fluorescence detection methods are above 10 pg.
[0032] Cost savings: Automated operation eliminates a significant amount of electrophoresis transfer, staining, and membrane cutting, with no human intervention throughout the entire process. The self-cleaning system uses a circulating cycle of denaturing solution, rinsing solution, and deionized water for cleaning, requiring no manual maintenance. Attached Figure Description
[0033] Figure 1 Overall structure diagram of the equipment
[0034] Figure 2 Schematic diagram of the right side of the equipment
[0035] Figure 3 Determination of damage to membrane antigens by multiple rounds of testing
[0036] Figure 4 Determination of the effect of antibody denaturing solution on antibody removal from membranes
[0037] The reference numerals in the attached diagrams are explained as follows: 1-6, liquid storage tanks (1 sealing solution, 2 rinsing solution, 3 luminescent solution A, 4 luminescent solution B, 5 antibody denaturing solution, 6 deionized water); 7, multi-channel pump control module; 8, darkroom door; 9, touch screen controller; 10, CMOS sensor; 11, antibody pre-storage area; 12, white LED light source; 13, darkroom housing; 14, sample dispensing head; 15, waste liquid discharge port; 16, antibody incubation module. Detailed Implementation
[0038] Fill the storage tanks (1-6) with the following solutions: 1. Blocking solution, 2. Rinsing solution, 3. Luminescent solution A, 4. Luminescent solution B, 5. Antibody denaturing solution, 6. Deionized water.
[0039] Check or modify machine operating parameters, such as antibody incubation time, rinsing time and number of rinses, using the touch screen controller (9).
[0040] Open the dark box door (8) and place the transferred film inside the reaction chamber.
[0041] The primary and secondary antibodies to be tested in each round are placed in the antibody pre-storage area (11) in a fixed position.
[0042] Click the "Start" button on the touch screen controller (9). The system will perform a self-check based on the feedback data from the sensor. If there is an error, an error will be reported. If there is no error, the process will continue.
[0043] According to the system's set program, complete the following steps in sequence: Step 1, Blocking operation: Add blocking solution (1), incubate with blocking solution, drain blocking solution; Step 2, Primary antibody incubation and rinsing: Add primary antibody, incubate primary antibody, drain primary antibody, rinse with rinsing solution (2) multiple times; Step 3, Secondary antibody incubation and rinsing: Add secondary antibody, incubate secondary antibody, drain secondary antibody, rinse with rinsing solution multiple times; Step 4, Signal detection operation: Add luminescent solution A (3), add luminescent solution B (4), mix luminescent solutions, drain luminescent solution, turn on CMOS sensor (10), turn on LED light (12), autofocus CMOS sensor, take bright field photos, turn off LED light, take dark field photos with CMOS sensor, turn off CMOS sensor; Step 5, Antibody stripping operation: Add deionized water (6), rinse repeatedly with deionized water, add antibody denaturing solution (5), treat antibody denaturing solution repeatedly, drain denaturing solution, add deionized water, rinse repeatedly with deionized water; Step 6, Cycling operation: Add blocking solution again and start the cycle for the next round of detection.
[0044] Figure 3 Bright-field images of the same transfer membrane after 0, 3, 6, and 9 rounds of detection are shown. From left to right, the four images represent no stripping, 3 stripping cycles, 6 stripping cycles, and 9 stripping cycles. The leftmost lane on the membrane represents the pre-stained marker, and the other two lanes represent the test samples. Changes in the pre-stained marker can indicate the protein loss on the membrane after multiple rounds of detection. Brief operating parameters for each round of Western blotting: blocking for 5 minutes, primary antibody incubation at room temperature for 4 hours, secondary antibody incubation for 1 hour, followed by 4 washes with TBST buffer for 5 minutes each, luminescence detection for 3 minutes, rapid rinsing with deionized water twice before and after stripping, and stripping with stripping buffer once for 10 minutes. Image J analysis of the pre-stained marker band grayscale values showed that after 9 rounds of detection, the attenuation of marker proteins from 10kD to 170kD was less than 5%. The antibody denaturing solution used in this example was selected from Willget Bio's commercially available NC membrane antibody high-efficiency stripping solution, and the blocking and antibody dilution solutions were selected from Willget Bio's commercially available Western blotting and sensitizing dilution solution.
[0045] Figure 4BSA protein was spotted onto two NC membranes, starting at 10 ng and decreasing fourfold from left to right. Western spectroscopy was then performed using rabbit anti-BSA serum and goat anti-rabbit HRP secondary antibody. For the first membrane, antibody denaturing solution was added after detection to remove the antibody; thereafter, no primary antibody was added, and secondary antibody was added directly for detection. If the primary antibody was not completely removed, the newly added secondary antibody would react with it to form a signal; if neither the primary nor secondary antibody was removed, the new secondary antibody would compete with the original secondary antibody for signal formation during incubation. Comparison of the two membranes showed that the antibody denaturing solution completely removed the original primary antibody. The antibody denaturing solution used in this example was selected from Willget Bio's commercially available NC membrane antibody high-efficiency stripping solution.
Claims
1. A fully automated multi-round cyclic detection device for Western blot, characterized in that, include: The liquid processing module is equipped with at least a chemiluminescent substrate storage unit and an antibody denaturing solution storage unit, as well as a liquid dispensing system. The antibody denaturing solution is used to remove antibody residues from the membrane and tubing, supporting multi-round detection cycles. The incubation reaction module includes at least two reaction chambers, each equipped with an oscillation mechanism. The signal acquisition module includes an emission light source, a CCD or CMOS sensor, and a dark box structure. The program control module controls the coordinated operation of each module and supports setting machine operating parameters via a touch screen interface.
2. The fully automated multi-round cyclic detection device for Western blot according to claim 1, characterized in that: The luminescent substrate is an enzymatic luminescent substrate of horseradish peroxidase or alkaline phosphatase, and these substrate components contain at least one of luminol, isoluminol, AMPPD, and CSPD.
3. The fully automated multi-round cyclic detection device for Western blot according to claim 1, characterized in that: The antibody denaturing solution is selected from at least one of the following: an acidic solution with pH ≤ 2, an alkaline solution with pH ≥ 12, a 6-8M urea solution, a 4-6M guanidine salt solution, and a 5-7M thiocyanate solution.
4. The fully automated multi-round cyclic detection device for Western blot according to claim 1, characterized in that: The wavelength range of the emission light source is 300nm to 700nm.
5. The fully automated multi-round cyclic detection device for Western blot according to claim 1, characterized in that: The signal acquisition module consists of a CCD or CMOS sensor and an optical lens.
6. The fully automated multi-round cyclic detection device for Western blot according to claim 1, characterized in that: In addition to the chemiluminescent substrate unit and the antibody denaturing solution unit, the liquid processing module also includes a rinsing solution unit, a blocking solution unit, and a deionized water unit.
7. The fully automated multi-round cyclic detection device for Western blot according to claim 1, characterized in that: The reaction chamber of the incubation reaction module is driven by a motor to achieve cyclic oscillation motion.
8. The fully automated multi-round cyclic detection device for Western blot according to claim 1, characterized in that: The program control module supports setting incubation time, number of rinses, and image transmission functions.
9. The fully automated multi-round cyclic detection device for Western blot according to claim 1, characterized in that: The liquid distribution system is driven by a liquid pump and delivers liquid through plastic pipes.
10. The fully automated multi-round cyclic detection device for Western blot according to claim 1, characterized in that: The door of the dark box structure has a concave-convex sealing structure at the contact point with the box body to prevent external light from seeping in.