Rapid immunohistochemical staining system based on microfluidic technology and application thereof

Abstract

The invention provides a rapid immunohistochemical staining system based on a microfluidic technology and application of the rapid immunohistochemical staining system, and belongs to the technical field of biomedical detection. According to the system provided by the invention, the micro-fluidic chip, the temperature control device and the pump system are integrated, so that the full-process automation of dyeing is realized; according to the micro-fluidic chip, a distributed channel network and a square reaction chamber are adopted, and a V-shaped passive mixing structure is combined, so that laminar flow is destroyed, radial mixing is enhanced, and the antigen-antibody binding efficiency is remarkably improved. When the system is used for immunohistochemical staining, the staining step can be automatically executed through a preset program, the total consumed time is less than 15 minutes and is shortened by more than 80% compared with the traditional method, and the system also has the advantages of small reagent dosage, high result consistency and the like; the invention provides an efficient, low-cost and high-repeatability solution for pathological routine and freezing rapid diagnosis and scientific research, and has a wide clinical pathological application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical detection, and in particular to a rapid immunohistochemical staining system based on microfluidics and its application in immunohistochemical detection of tissue samples. Background Art

[0002] Immunohistochemistry (IHC) is a core technique in pathological diagnosis and biomedical research. It uses specific antibodies to label target proteins in tissues, aiding in disease diagnosis and treatment assessment. However, traditional IHC techniques have the following limitations: 1) cumbersome operation: multiple manual steps, including dewaxing, antigen retrieval, antibody incubation, and washing, are time-consuming (typically 6-8 hours for a single-color sample and 2 days for a multicolor sample); 2) high cost: the complex procedures make it difficult for unskilled researchers to produce usable stained sections; 3) poor consistency of results: manual operation can easily introduce errors, leading to significant differences in results between batches and laboratories; and 4) low automation: existing automated equipment is expensive and lacks flexibility, making it difficult to widely use.

[0003] The antigen-antibody interaction is a specific bimolecular reaction. Antigen and antibody molecules must first diffuse into each other and collide before binding occurs. Therefore, the three main factors influencing the rate of the immune reaction are diffusion, mass transfer, and the affinity of the antigen and antibody. In traditional immunohistochemistry, an antibody solution is applied to the surface of a tissue sample and incubated for a specified period of time to allow for a full immune reaction. This static method inevitably faces limitations in reaction kinetics.

[0004] Microfluidics is a technology that precisely manipulates minute volumes of fluid (nanoliter to microliter volumes) through micron-scale channels (typically tens to hundreds of micrometers). Its core technology leverages the unique physical properties of fluids at microscale (such as laminar flow, surface tension, and high diffusion efficiency) to enable efficient integration and automation of processes such as chemical reactions and biological detection. Since its emergence in the 1990s, microfluidics has demonstrated revolutionary potential in fields such as biomedicine, chemical analysis, and environmental monitoring, and has spawned the "Lab-on-a-Chip" concept, which aims to condense traditional laboratory functions onto centimeter-scale chips. Its key advantages include miniaturization and high throughput. Microchannel networks enable parallel processing of multiple samples or reactions, significantly improving detection efficiency. Nanoliter-scale fluid control significantly reduces reagent usage, lowering costs and reducing waste. Furthermore, microfluidics utilizes microvalves, pumps, and temperature control modules to precisely control reaction conditions (such as temperature, flow rate, and concentration gradients). Combined with the shortened diffusion distances at the microscale, this technology accelerates mass transfer and reaction rates, providing a highly efficient platform for complex biological detection.

[0005] In the biomedical field, the application of microfluidics has made significant progress. For example, liquid biopsy enables early diagnosis of cancer by capturing circulating tumor cells (CTCs) or exosomes; single-cell analysis uses microwell or droplet technology to study cellular heterogeneity; point-of-care (POCT) diagnostics develop portable devices for pathogen detection (such as COVID-19 antigen detection); and organ-on-a-chip simulates the microenvironment of human organs to evaluate drug toxicity or disease mechanisms.

[0006] Based on this, the present invention develops a rapid immunohistochemical staining system based on microfluidic technology to solve the problems of low efficiency and poor consistency of results of traditional methods. Summary of the Invention

[0007] In response to the above shortcomings of the existing technology, the present invention provides a rapid immunohistochemical staining system based on microfluidic technology. Through an integrated design, it realizes the automation and acceleration of the staining process, significantly shortens the detection time and improves the repeatability of the results. It can be used to realize efficient and automated immunohistochemical detection of tissue samples.

[0008] To achieve the above object, the specific technical solutions of the present invention are as follows:

[0009] In a first aspect, the present invention provides a rapid immunohistochemical staining system based on microfluidic technology, comprising: a microfluidic chip, a temperature control device, and a pump system;

[0010] Wherein, the microfluidic chip includes a distributed channel network, multiple independent reaction chambers and a passive mixing structure;

[0011] The temperature control device is used to regulate the temperature of the reaction chamber;

[0012] The pump system is used to control reagent injection, circulation and cleaning, reducing mass transfer limitations.

[0013] Furthermore, the microfluidic chip is placed on a temperature control device and connected to a pump system.

[0014] In the rapid immunohistochemical staining system based on microfluidic technology of the present invention, a distributed channel network is designed in the microfluidic chip to ensure that the reagents evenly cover the entire tissue sample; multiple independent square reaction chambers with a side length of 18-25 mm are designed to cover most tissue sections and support parallel processing of multiple samples; the reaction chamber height is 150μm, which has a high specific surface area to promote reagent diffusion; and a pump system is used to control the rapid fluid flow in the chip to effectively reduce mass transfer restrictions. The fluid flow in the microfluidic chip is usually laminar flow, and there are still certain restrictions on the diffusion of antibodies to the antigen surface. The present invention, by combining the passive mixing technology of the microfluidic chip, designs a V-shaped structure with a height of 10 μm, a width of 0.5 mm, and an angle of 90 degrees inside the chip. Through its unique geometric characteristics, it can effectively destroy the laminar state of the fluid traveling in the chip, induce complex secondary flow and vortex effects at the fluid mechanics level, thereby changing the streamline distribution of the fluid and enhancing radial mixing. Compared with the microfluidic chip lacking a passive mixing structure, the microfluidic chip containing a passive mixing structure of the present invention can significantly increase the collision frequency and binding probability between antigen and antibody molecules, and improve the consistency of the staining results. At the same time, the reaction rate is regulated in combination with the heating stage temperature control module to achieve efficient regulation of reaction kinetics, providing a stable and controllable microenvironment platform for antigen-antibody reaction.

[0015] Furthermore, the rapid immunohistochemical staining system based on microfluidic technology also includes a control system, which includes an integrated terminal and a programmed operation interface, and a standard staining process is preset according to needs.

[0016] Furthermore, the reaction chamber is a square structure with a side length of 18-25 mm and a height of 150 μm.

[0017] Furthermore, the passive mixing structure has a height of 10 μm, a width of 0.5 mm, and an angle of 90 o The V-shaped passive mixing structure induces secondary flow and vortex effects, disrupts laminar flow and enhances radial mixing, thereby improving antigen-antibody binding efficiency.

[0018] Furthermore, the pump system controls the fluid by reciprocating push-pull operations, the number of reciprocating times is 5-20 times, the reciprocating flow rate is 10-50 μL / s, and the single reciprocating volume is 50-100 μL.

[0019] Furthermore, the temperature control device includes but is not limited to a heating platform.

[0020] Furthermore, the temperature control device regulates the temperature in the range of 25-45°C.

[0021] Furthermore, the temperature control device adjusts the temperature to 37°C.

[0022] In a second aspect, the present invention provides the application of the rapid immunohistochemical staining system based on microfluidics technology in immunohistochemical detection.

[0023] Furthermore, the immunohistochemical detection includes detection of CK or Ki-67.

[0024] In a third aspect, the present invention provides a device for immunohistochemical staining, comprising the rapid immunohistochemical staining system based on microfluidics technology.

[0025] In a third aspect, the present invention provides a method for rapid immunohistochemical staining using the device, comprising the following steps:

[0026] Place the sample to be tested into the microfluidic chip, and automatically perform primary antibody incubation, secondary antibody incubation, color development and washing through the preset program, and then output it.

[0027] Furthermore, before the sample to be tested is placed into the microfluidic chip, the paraffin-embedded sample is pretreated including dewaxing, hydration and antigen retrieval; if the sample to be tested is a fresh surgical specimen, no pretreatment is required.

[0028] Furthermore, the dewaxing is performed using a dewaxing agent three times, each time for 8-10 minutes; the hydration is performed using gradient alcohol (e.g., 70%, 85%, 95%), each gradient for 3-5 minutes; the antigen retrieval is performed using a microwave treatment using EDTA buffer at pH 9.0 (preheated to boiling point: 100% power for 3 minutes, 30%-50% power to maintain boiling point for 10-12 minutes).

[0029] Furthermore, the primary antibody incubation time is 1-5 min, the secondary antibody incubation time is 1-3 min, and the color development time is 0.5-2 min; PBS buffer is used for washing, and the single washing time is 10-30 s.

[0030] Furthermore, the method is applicable to the detection of CK or Ki-67 in paraffin-embedded and fresh surgical tissue sections of breast cancer, with the primary antibody dilution ratio being 1:50 (CK detection) or 1:200 (Ki-67 detection).

[0031] Furthermore, the preset program is controlled by an integrated terminal, supporting user-defined flow rate, volume, temperature and reaction time parameters.

[0032] Compared with the prior art, the present invention is beneficial in that:

[0033] The present invention provides a rapid immunohistochemical staining system based on microfluidic technology, which solves the problems of traditional immunohistochemical technology, such as cumbersome operation, long time consumption (6-8 hours for a single indicator), poor result consistency and insufficient automation. The system realizes the automation of the whole staining process by integrating microfluidic chips, temperature control devices and pump systems. The microfluidic chips use a distributed channel network and a square reaction chamber with a side length of 18-25 mm and a height of 150 μm, combined with a height of 10 μm, a width of 0.5 mm and a 90° angle. o The V-shaped passive mixing structure disrupts laminar flow and enhances radial mixing, significantly improving the efficiency of antigen-antibody binding. The temperature control device precisely regulates the reaction temperature, and the pump system reduces mass transfer limitations through bidirectional fluid circulation. The staining steps of the present invention (primary antibody, secondary antibody, color development, etc.) are automatically executed through a preset program, with a total time of less than 15 minutes, which is more than 80% shorter than traditional methods. It also has the advantages of low reagent usage and high consistency of results. The present invention provides an efficient, low-cost, and highly reproducible solution for routine pathology and rapid frozen diagnosis and scientific research, and has broad application prospects in clinical pathology. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the microfluidic chip structure;

[0035] Figure 2 A diagram of a device for rapid immunohistochemical staining;

[0036] Figure 3 The immunohistochemical staining results of Example 1 are shown;

[0037] Figure 4 The immunohistochemical staining results of Example 1 and Comparative Example 1 are shown;

[0038] Figure 5 The immunohistochemical staining scores of CK in serial sections were detected using the system of the present invention. DETAILED DESCRIPTION

[0039] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0040] The present invention provides a rapid immunohistochemical staining system based on microfluidic technology, comprising: a microfluidic chip, a temperature control device and a pump system;

[0041] Wherein, the microfluidic chip (schematic diagram see Figure 1) includes a distributed channel network, multiple independent reaction chambers and a passive mixing structure;

[0042] The temperature control device is used to regulate the temperature of the reaction chamber;

[0043] The pump system is used to control reagent injection, circulation and cleaning, reducing mass transfer limitations.

[0044] In the following specific examples, the microfluidic chip is placed on a temperature control device and connected to a pump system.

[0045] In the following specific example, the rapid immunohistochemistry staining system based on microfluidic technology also includes a control system, which includes an integrated terminal and a programmed operation interface, and presets a standard staining process according to needs.

[0046] In some examples, the reaction chamber is a square structure with a side length of 18-25 mm and a height of 150 μm; the passive mixing structure is a square structure with a height of 10 μm, a width of 0.5 mm, and an angle of 90 o The V-shaped passive mixing structure induces secondary flow and vortex effects, disrupts laminar flow and enhances radial mixing, thereby improving antigen-antibody binding efficiency.

[0047] In the following specific examples, the temperature control device is a heating stage.

[0048] In some examples, the pump system controls the fluid by a reciprocating push-pull operation, the number of reciprocations is 5-20 times, the reciprocating flow rate is 10-50 μL / s, and the single reciprocating volume is 50-100 μL.

[0049] Example 1 Immunohistochemical staining of paraffin-embedded breast cancer tissue sections

[0050] 1. Material collection and preliminary processing

[0051] Paraffin tissue sections were prepared by fixing, dehydrating, clearing, wax-impregnating, embedding, and sectioning fresh tissue. Tissue sections were first oven-baked at 65°C for 60 minutes to ensure firm adhesion to the slide. Deparaffinization was then performed three times with a dewaxing agent, each for 8 minutes, to ensure complete removal of the paraffin. Dewaxed sections were then rehydrated with a gradient of 95%, 85%, and 70% alcohol, each for 5 minutes. Antigen retrieval was performed using either EDTA (pH 9.0) or citrate buffer (pH 6.0) using a microwave retrieval method: heating at 100% power for 3 minutes, followed by heating at 30% power for an additional 12 minutes. After retrieval, sections were returned to room temperature and incubated in 3% hydrogen peroxide solution for 10 minutes at room temperature to block endogenous peroxidase activity and reduce nonspecific background staining.

[0052] 2. Microfluidic Immunohistochemical Staining

[0053] Figure 2 Shown is a diagram of the apparatus used for rapid immunohistochemical staining.

[0054] The microfluidic chip is reversibly bonded to the fixture, placed on a heating platform at 37°C, and connected to a pump. CK and Ki-67 primary antibody working solutions are prepared at dilution ratios of 1:50 and 1:200, respectively. The prepared secondary antibody working solution, linker reagent, DAB staining solution, and PBS buffer are placed. Reaction parameters are set according to the preset program. Click "Run" and the system automatically completes the antibody incubation, washing, staining, and other processes. The reaction reagents are injected through the pump system and repeatedly pushed and pulled to produce a bidirectional flow of "intake" and "extraction." This recycles the unreacted antibodies in the microchannel, significantly enhancing the efficiency of the antigen-antibody reaction.

[0055] The reaction parameters set in this embodiment are as follows:

[0056]

[0057] According to the set reaction parameters, the primary antibody incubation time was 4 minutes, the linker incubation time was 2 minutes, the secondary antibody incubation time was 2 minutes, the DAB incubation time was 1 minute, and the washing time was 20 seconds each time, for a total of 11 minutes.

[0058] 3. Effect verification

[0059] Staining results are shown in Figure 3 Figures A and C show the staining results of breast cancer paraffin sections for CK and Ki-67 using the automated system in this embodiment, respectively, with a process time of 11 minutes; Figures B and D show the staining results of breast cancer paraffin sections for CK and Ki-67 using conventional immunohistochemical staining techniques under the same time conditions. From the staining results, it can be seen that the immunohistochemical staining effect using the system of the present invention is more sensitive than that using conventional immunohistochemical staining techniques, and the system of the present invention also has the advantages of being fast, efficient, and requiring less reagents.

[0060] Comparative Example 1

[0061] The immunohistochemical staining system used in this comparative example is basically the same as that in Example 1, except that the passive mixing structure is a wavy structure with a height of 10 μm and a width of 0.5 mm. The staining steps using this system are the same as those in Example 1.

[0062] Staining results are shown in Figure 4 From the staining results, it can be seen that the immunohistochemical staining using the system of the present invention has a better staining effect and a stronger positive signal.

[0063] In addition, the rapid immunohistochemical staining system based on microfluidic technology of the present invention was used to detect CK in serial sections, and the immunohistochemical staining scores obtained by quantitative analysis (H score measured by InForm analysis software) showed no significant difference (such as Figure 5 ), indicating that the immunohistochemical staining using the system of the present invention has the advantage of high consistency of results.

[0064] In summary, the present invention has constructed a rapid immunohistochemical staining system based on microfluidic technology. The system realizes the automation of the entire staining process by integrating a microfluidic chip, a temperature control device and a pump system. The microfluidic chip adopts a distributed channel network and a square reaction chamber, and combines a V-shaped passive mixing structure to destroy the laminar flow and enhance radial mixing, significantly improving the antigen-antibody binding efficiency. When using this system for immunohistochemical staining, the staining step can be automatically executed by a preset program, and the total time is less than 15 minutes, which is more than 80% shorter than the traditional method. It also has the advantages of low reagent usage and high result consistency. The present invention provides an efficient, low-cost, and highly repeatable solution for routine pathology and rapid frozen diagnosis and scientific research, and has a broad prospect for clinical pathology application.

[0065] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A rapid immunohistochemical staining system based on microfluidics technology, characterized in that: include: Microfluidic chips, temperature control devices, and pump systems; Wherein, the microfluidic chip includes a distributed channel network, multiple independent reaction chambers and a passive mixing structure; The temperature control device is used to regulate the temperature of the reaction chamber; The pump system is used to control reagent injection, circulation and cleaning.

2. The rapid immunohistochemical staining system based on microfluidic technology according to claim 1, characterized in that: It also includes a control system, which includes an integrated terminal and a programmed operation interface, and a standard dyeing process is preset according to needs.

3. The rapid immunohistochemical staining system based on microfluidic technology according to claim 1, characterized in that: The passive mixing structure has a height of 10 μm, a width of 0.5 mm, and an angle of 90 o V-shaped structure.

4. The rapid immunohistochemical staining system based on microfluidic technology according to claim 1, characterized in that: The reaction chamber is a square structure with a side length of 18-25 mm and a height of 150 μm.

5. The rapid immunohistochemical staining system based on microfluidic technology according to claim 1, characterized in that: The pump system controls the fluid by reciprocating push-pull operations, wherein the number of reciprocating times is 5-20 times, the reciprocating flow rate is 10-50 μL / s, and the single reciprocating volume is 50-100 μL.

6. The rapid immunohistochemical staining system based on microfluidic technology according to claim 1, characterized in that: The temperature control device includes but is not limited to a heating stage.

7. Use of the rapid immunohistochemical staining system based on microfluidic technology according to any one of claims 1 to 6 in immunohistochemical detection.

8. Use of the rapid immunohistochemical staining system based on microfluidics technology in immunohistochemical detection according to claim 7, characterized in that: The immunohistochemical detection includes detection of CK or Ki-67.

9. A device for immunohistochemical staining, characterized in that: The invention comprises the rapid immunohistochemical staining system based on microfluidic technology according to any one of claims 1 to 6.

10. A method for rapid immunohistochemical staining using the device according to claim 9, characterized in that: The following steps are involved: Place the sample to be tested into the microfluidic chip, and automatically perform primary antibody incubation, secondary antibody incubation, color development and washing through the preset program, and then output it.

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