Protein detection method and kit for implementing same
By generating signals on beads and immobilizing them in fibrin hydrogel for imaging, the problem of expensive and complex equipment in the existing technology is solved, and simplified, low-cost ultrasensitive protein detection is achieved, which is suitable for bedside diagnosis.
Patent Information
- Application Number
- CN202510903060.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-01
- Publication Date
- 2025-10-03
AI Technical Summary
Existing protein detection technologies are limited by expensive equipment and complex microfluidic systems in bedside diagnosis, making it difficult to achieve simplified, low-cost ultra-sensitive detection.
Tyramide signal amplification technology is used to generate signals on beads, and the beads are immobilized in fibrin hydrogels for imaging and single-molecule counting. Beads are directly labeled with tyramide-fluorophore conjugates, simplifying the steps and reducing equipment requirements.
It achieves ultra-sensitive protein detection, simplifies the operating process, reduces costs, is suitable for bedside diagnosis, and reduces equipment footprint and consumables usage.
Smart Images
Figure CN120741849A_ABST
Abstract
Description
Technical Field
[0001] The present application belongs to the field of biomedical detection technology, and specifically relates to a protein detection method and a kit for implementing the method, which are used for ultrasensitive detection of protein biomarkers in biological fluids. Background Art
[0002] In medical diagnosis, ultrasensitive detection of protein biomarkers is of great significance for early disease diagnosis, treatment monitoring, and disease recurrence monitoring. Technologies such as digital enzyme-linked immunosorbent assay (ELISA) and single-molecule array (Simoa) have been developed to accurately quantify protein concentrations as low as attomole levels. However, the instrument size and cost required for these technologies hinder their application in bedside diagnosis. Although the Simoa platform has high sensitivity, its complex microfluidic system and expensive equipment limit its widespread application in bedside diagnosis. Therefore, it is particularly important to develop a simplified, low-cost, and ultrasensitive protein detection method suitable for bedside diagnosis.
[0003] In view of this, there is an urgent need to provide a protein detection method and a kit for implementing the method to solve at least one of the above problems. Summary of the Invention
[0004] In view of this, the object of the present invention is to provide a protein detection method and a kit for implementing the method. The method generates signals on beads through tyramide signal amplification technology, and fixes the beads in fibrin hydrogel for imaging and single-molecule counting, thereby achieving ultrasensitive protein detection. The detection method is simple, low-cost, and suitable for bedside diagnosis.
[0005] According to the first aspect of the present application, a protein detection method is provided, comprising the following steps:
[0006] Antibody-coated capture beads are added to the sample to capture the target protein molecules;
[0007] Use biotinylated detection antibodies and streptavidin-polyHRP labeled captured target protein molecules to form a complete enzyme-labeled immune complex;
[0008] In a solution containing hydrogen peroxide and tyramide-fluorophore conjugate, HRP catalyzes the formation of free radical intermediates from tyramide, which are deposited on the beads to generate covalently linked fluorescent dyes.
[0009] The beads are immobilized in the fibrin hydrogel to form a fibrin hydrogel layer for bead immobilization;
[0010] Bright-field and fluorescence images of the bead arrays were captured and analyzed for single-molecule counting.
[0011] In some specific embodiments, the tyramide-fluorophore conjugate is tyramide-Alexa Fluor 488.
[0012] In some embodiments, the fibrin hydrogel is formed by enzymatic polymerization of fibrinogen by thrombin.
[0013] In some embodiments, the beads are paramagnetic beads with a diameter of 2.7 microns.
[0014] In some embodiments, the beads are immobilized in silicon isolation wells on a glass slide to form a fibrin hydrogel layer.
[0015] In some embodiments, bright field and fluorescence images of bead arrays are captured using an inverted fluorescence microscope.
[0016] In some embodiments, images are analyzed using a MATLAB algorithm for single molecule counting.
[0017] According to the second aspect of the present application, a kit is provided for implementing the method described in any one of the above embodiments, comprising:
[0018] antibody-coated capture beads;
[0019] biotinylated detection antibody;
[0020] Streptavidin-poly-HRP;
[0021] Tyramide-fluorophore conjugates;
[0022] thrombin and fibrinogen;
[0023] Buffer required for fibrin hydrogel formation.
[0024] In some specific embodiments, the antibody-coated capture beads are paramagnetic beads with a diameter of 2.7 microns.
[0025] In some specific embodiments, the tyramide-fluorophore conjugate is tyramide-Alexa Fluor 488.
[0026] The beneficial effects of the embodiments of the present application include at least:
[0027] This embodiment uses tyramide signal amplification for on-bead signal generation. By implementing the signal amplification step on the beads, where the fluorophore is directly bound to the beads, the need to separate the beads in a microwell array or microfluidic droplets is eliminated, reducing the need for expensive equipment and complex microfluidics or robotics. By combining the protein capture and antibody labeling steps into one step, and combining the steps in the TSA labeling technology by using a tyramide-fluorophore conjugate instead of a tyramide-biotin conjugate and fluorescently labeled streptavidin, the total number of steps in the immunoassay is reduced, thereby reducing the total assay time, allowing for faster results in a bedside setting, and reducing the number and volume of required reagents (such as wash buffer or certain binding reagents), reducing the equipment footprint and the number of consumables required for the assay. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.
[0029] Figure 1 A flowchart of a protein detection method provided in one embodiment of the present application;
[0030] Figure 2 This is a bright field image of hundreds of beads in a fibrin hydrogel provided in an embodiment of the present application;
[0031] Figure 3A This is a bright field image according to an embodiment of the present application. Figure 3B This is a fluorescent image according to an embodiment of the present application. DETAILED DESCRIPTION
[0032] Specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the described embodiments are merely some, and not all, of the embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the description of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.
[0033] In the description of the present invention, unless otherwise specified or limited, the terms "disposed," "installed," and "connected" should be understood broadly. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; and direct or indirect connections through an intermediary. Those skilled in the art will understand the specific meanings of these terms based on the specific circumstances.
[0034] The directions or positional relationships indicated by terms such as "upper", "lower", "left", "right", "front", "back", "top", "bottom", "inside" and "outside" are based on the directions or positional relationships shown in the accompanying drawings, or are the directions or positional relationships in which the inventive product is usually placed when in use. They are only for the convenience and simplification of description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operate in a specific direction. Therefore, they should not be understood as limiting the present invention.
[0035] The terms "first," "second," "third," etc. are merely used to distinguish elements of similar nature and do not indicate or imply relative importance or a particular order.
[0036] The terms "comprises," "comprising," or any other variations thereof, are intended to cover a non-exclusive inclusion of elements other than the listed elements and may also include additional elements not specifically listed.
[0037] Some embodiments of the present application provide a method for detecting protein. Figure 1 , protein detection methods include:
[0038] Step S1: Add antibody-coated capture beads to the sample to capture target protein molecules.
[0039] In some embodiments, the beads are paramagnetic beads with a diameter of 2.7 microns.
[0040] In this example, 200,000 antibody-coated capture beads were added to a sample volume of approximately 100 μL to capture the target protein molecules. Specifically, a greater number of beads than the number of target protein molecules was used to ensure that the assay followed a Poisson distribution, where the majority of beads did not bind to the target molecule and only a small number of beads bound to one target molecule.
[0041] Step S2: Use biotinylated detection antibodies and streptavidin-polyHRP to label the captured target protein molecules to form a complete enzyme-labeled immune complex.
[0042] In the examples of the present application, after protein capture, the target molecule is labeled with a biotinylated detection antibody and streptavidin-polyHRP (a streptavidin-coupled polymer containing multiple horseradish peroxidase molecules) to form a complete enzyme-labeled immune complex.
[0043] Step S3: In a solution containing hydrogen peroxide and a tyramide-fluorophore conjugate, HRP catalyzes tyramide to form a free radical intermediate, which is deposited on the beads to generate a covalently linked fluorescent dye.
[0044] In the examples of the present application, the beads are resuspended in a solution containing hydrogen peroxide and a tyramide-fluorophore conjugate to perform a signal generation step.
[0045] In some specific examples, the tyramide-fluorophore conjugate is tyramide-Alexa Fluor 488.
[0046] Specifically, in the presence of hydrogen peroxide, HRP catalyzes the formation of a free radical intermediate from tyramide. This tyramide radical forms a covalent bond with an aromatic ring near the HRP molecule, such as a tyrosine residue on a nearby protein and an antibody on the bead. At the end of this step, the beads with the intact immune complex are labeled with a large number of covalently linked fluorescent dyes. This completes the on-bead signal generation step and allows subsequent single molecule counting.
[0047] In some specific examples, no detectable cross-labeling between beads was observed after the tyramide labeling step: at low protein concentrations, only a small number of beads had a detectable fluorescent signal, which is expected in this assay format, which follows a Poisson distribution. Furthermore, during the tyramide labeling step, a dilute bead solution was used, making it unlikely that the tyramide radical would diffuse to another bead during the lifetime of the radical intermediate.
[0048] In the present application, the total number of steps in the immunoassay is reduced by combining the steps in the TSA labeling technique using a tyramide-fluorophore conjugate instead of a tyramide-biotin conjugate and fluorescently labeled streptavidin. This reduces the total assay time, allowing for faster results in a bedside setting; and simultaneously reduces the amount and volume of required reagents (such as wash buffer or certain binding reagents), reducing the equipment footprint and the number of consumables required for the assay.
[0049] Step S4: Immobilize the beads in the fibrin hydrogel to form a fibrin hydrogel layer for bead immobilization.
[0050] In some specific embodiments, the fibrin hydrogel is formed by enzymatic polymerization of fibrinogen by thrombin, which can be specifically described in the prior art and will not be elaborated here.
[0051] In the examples of the present application, since the amplified enzyme signal has been bound to the beads, there is no limitation of isolating the beads in microwells or droplets for enzyme amplification, and fibrin hydrogel can be used for bead immobilization.
[0052] Specifically, fibrin hydrogels are formed by enzymatically polymerizing fibrinogen through thrombin to form a fibrin hydrogel network. Synthetic fibrin hydrogels are commonly used in applications including cell encapsulation and tissue engineering and can be easily formed in situ. Bead encapsulation in fibrin hydrogels is a rapid and simple method for immobilizing beads for imaging. In addition, encapsulating beads in hydrogels allows the beads to remain in solution and allows subsequent in situ labeling experiments.
[0053] In some embodiments, the beads are immobilized in silicon isolation wells on a glass slide to form a fibrin hydrogel layer. In some embodiments, to immobilize the beads in the fibrin hydrogel, a bead solution is first added dropwise to a silicon isolation well (e.g., 7 x 7 x 2 mm) on a glass slide. Fibrinogen and thrombin solutions are then mixed and immediately added to the isolation wells. The hydrogel forms within approximately 15 minutes, with the beads trapped in the fibrin polymer network.
[0054] Step S5: Capture bright field and fluorescence images of the bead array, and analyze the images for single molecule counting.
[0055] In some specific embodiments, an inverted fluorescence microscope is used to capture bright field and fluorescence images of the bead array. Specific operations can be referred to the prior art and will not be described in detail here.
[0056] Figure 2 A bright-field image of several hundred beads within a fibrin hydrogel is shown. This image represents a small portion of a single isolated well within the entire fibrin hydrogel. The entire isolated well can be captured in the image at higher magnification. Because the beads are relatively large (2.7 μm in diameter) and quickly settle out of solution, they primarily reside in a single plane (the interface between the glass slide and the fibrin hydrogel). In addition to being fast and simple, encapsulating beads within a fibrin hydrogel is also a less expensive alternative to microwell arrays, as the cost of the hydrogel material, glass slide, and silicon isolated wells are all lower.
[0057] Take the small area of interest example, such as Figure 3A is a bright field image, Figure 3B For fluorescence images, bright-field images were used to locate each bead, and fluorescence images were used to identify beads with tyramide-Alexa Fluor 488 dye deposited from the signal amplification step.
[0058] Furthermore, the image was analyzed using a MATLAB algorithm to perform single-molecule counting. Specifically, the position of each bead was automatically determined by the MATLAB algorithm, and the fluorescence intensity corresponding to each bead was calculated. The specific operation can be referred to the existing technology and will not be described in detail here.
[0059] Some other embodiments of the present application provide a kit for implementing the protein detection method of the above embodiment, wherein the kit comprises:
[0060] antibody-coated capture beads;
[0061] biotinylated detection antibody;
[0062] Streptavidin-poly-HRP;
[0063] Tyramide-fluorophore conjugates;
[0064] thrombin and fibrinogen;
[0065] Buffer required for fibrin hydrogel formation.
[0066] In some specific embodiments, the antibody-coated capture beads are paramagnetic beads with a diameter of 2.7 microns.
[0067] In some specific embodiments, the tyramide-fluorophore conjugate is tyramide-Alexa Fluor 488.
[0068] In some embodiments of the present application, the scheme of the kit embodiment can be combined with the protein detection method embodiment. The implementation of the scheme of the kit embodiment can refer to the scheme of the protein detection method, which will not be repeated here.
[0069] This embodiment uses tyramide signal amplification for on-bead signal generation. By implementing the signal amplification step on the beads, where the fluorophore is directly bound to the beads, the need to separate the beads in a microwell array or microfluidic droplets is eliminated, reducing the need for expensive equipment and complex microfluidics or robotics. By combining the protein capture and antibody labeling steps into one step, and combining the steps in the TSA labeling technology by using a tyramide-fluorophore conjugate instead of a tyramide-biotin conjugate and fluorescently labeled streptavidin, the total number of steps in the immunoassay is reduced, thereby reducing the total assay time, allowing for faster results in a bedside setting, and reducing the number and volume of required reagents (such as wash buffer or certain binding reagents), reducing the equipment footprint and the number of consumables required for the assay.
[0070] The foregoing description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be readily conceived by a person skilled in the art within the technical scope disclosed herein are intended to be encompassed within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.
Claims
1. A protein detection method, characterized in that: The following steps are involved: Antibody-coated capture beads are added to the sample to capture the target protein molecules; Use biotinylated detection antibodies and streptavidin-polyHRP labeled captured target protein molecules to form a complete enzyme-labeled immune complex; In a solution containing hydrogen peroxide and tyramide-fluorophore conjugate, HRP catalyzes the formation of free radical intermediates from tyramide, which are deposited on the beads to generate covalently linked fluorescent dyes. The beads are immobilized in the fibrin hydrogel to form a fibrin hydrogel layer for bead immobilization; Bright-field and fluorescence images of the bead arrays were captured and analyzed for single-molecule counting.
2. The method according to claim 1, characterized in that The tyramide-fluorophore conjugate is tyramide-Alexa Fluor 488.
3. The method according to claim 1, characterized in that The fibrin hydrogel is formed by enzymatic polymerization of fibrinogen by thrombin.
4. The method according to claim 1, wherein The beads are paramagnetic beads with a diameter of 2.7 microns.
5. The method according to claim 1, wherein The beads were immobilized in silicon isolation wells on a glass slide to form a fibrin hydrogel layer.
6. The method according to claim 1, characterized in that Bright-field and fluorescence images of the bead arrays were captured using an inverted fluorescence microscope.
7. The method according to claim 1, characterized in that Images were analyzed using a MATLAB algorithm for single-molecule counting.
8. A kit for implementing the method according to any one of claims 1 to 7, characterized in that: include: antibody-coated capture beads; biotinylated detection antibody; Streptavidin-poly-HRP; Tyramide-fluorophore conjugates; thrombin and fibrinogen; Buffer required for fibrin hydrogel formation.
9. The kit according to claim 8, characterized in that The antibody-coated capture beads are paramagnetic beads with a diameter of 2.7 microns.
10. The kit according to claim 8, characterized in that The tyramide-fluorophore conjugate is tyramide-Alexa Fluor 488.