阅读说明：本技术 一种荧光免疫检测系统 (Fluorescence immunoassay system ) 是由 唐本忠 贾红青 刘勇 王志明 于 2021-08-27 设计创作，主要内容包括：本发明提供了一种荧光免疫检测系统。所述系统包括微流控芯片及便携式的分析设备；所述蛋白质标志物基底预处理形成多指标生物阵列；所述芯片壳体分别设置样本池、驱动活塞及活塞驱动池、反应池、清洗池、检测区；所述分析设备的作用是辅助基底在微流控芯片中完成生物反应,并进行信号采集与结果分析。所述活塞驱动池底端与样本池顶端连接,样本液在活塞运动下经微流控结构进入反应池和基底反应,仪器操纵基底在芯片中完成清洗及荧光标记物捕获,在检测区完成基底荧光信号的采集。分析设备通过嵌入式控制模块实现上述仪器功能的自动化实现和仪器结构集成。本发明具有低成本、便于加工、便携等特点,属于即时诊断领域。(The invention provides a fluorescence immunoassay system. The system comprises a microfluidic chip and portable analysis equipment; pretreating the protein marker substrate to form a multi-index biological array; the chip shell is respectively provided with a sample cell, a driving piston, a piston driving cell, a reaction cell, a cleaning cell and a detection zone; the analysis equipment is used for assisting the substrate to complete biological reaction in the microfluidic chip, and performing signal acquisition and result analysis. The bottom end of the piston driving pool is connected with the top end of the sample pool, sample liquid enters the reaction pool through the microfluidic structure under the movement of the piston and reacts with the substrate, the substrate is operated by an instrument to complete cleaning and fluorescent marker capture in the chip, and the acquisition of a substrate fluorescent signal is completed in the detection area. The analysis equipment realizes the automatic realization of the functions of the instrument and the integration of the instrument structure through the embedded control module. The invention has the characteristics of low cost, convenient processing, portability and the like, and belongs to the field of instant diagnosis.)

1. A fluorescence immunoassay system is characterized by comprising a microfluidic chip and an analysis device;

the microfluidic chip comprises a chip shell (1) and a protein marker substrate (2), wherein the protein marker substrate (2) comprises a coating carrier (2-1) and a plurality of coating sites (2-2) arranged on the coating carrier (2-1) and is used as a reaction substrate for multi-index joint detection;

the chip shell (1) is sequentially provided with a sample cell (3), a driving piston (4-1), a piston driving cell (4-2), a reaction cell (5), a cleaning cell (6) and a detection zone (7), wherein the upper end of the sample cell (3) is connected with the piston driving cell (4-2), and the lower end of the sample cell is communicated with the reaction cell (5) through a microfluidic structure; the driving piston (4-1) can move up and down in the piston driving pool (4-2); the cleaning pool (6) is used for cleaning residual reagents after reaction; the analysis equipment comprises a signal acquisition module (13) and an embedded control module, wherein the signal acquisition module (13) is connected with the embedded control module, the signal acquisition module is used for acquiring fluorescent signals of the reacted coating sites (2-2) in a detection area (7), the embedded control module is used for controlling the work of the signal acquisition module (13), processing the signals acquired by the signal acquisition module (13), and calculating the concentration of the antigen to be detected in the sample to be detected.

2. The immunofluorescent assay system according to claim 1, wherein said coating sites (2-2) of said protein marker substrate (2) are uniformly distributed on said coating support (2-1), each coating site (2-2) being pre-coated with an antibody capable of specifically binding to a different detection marker.

3. The immunofluorescent assay system according to claim 1, wherein said sample cell (3) comprises a sample cover (3-1) and a chamber,

the sample cover (3-1) is positioned above the sample cell and is connected with the lower part of the piston driving cell (4-2) through a first micro-flow channel (11), and the bottom end of the cavity is connected with the reaction cell (5) through a micro-flow control structure.

4. The fluorescence immunoassay system of claim 1, wherein the microfluidic structure comprises a second microfluidic channel (8) and a filter tank (9) communicated with the second microfluidic channel (8), one end of the second microfluidic channel (8) is connected with the bottom end of the cavity, the filter tank (9) is connected with the reaction tank (5), and a blood filtering membrane is arranged in the filter tank (9).

5. The immunofluorescence detection system according to claim 1, wherein, the reaction tank (5) comprises a first reaction tank (5-1) and a second reaction tank (5-2), the first reaction tank (5-1) is used for placing buffer solution, the second reaction tank (5-2) is used for placing detection antibody solution of fluorescent label, and a cleaning tank (6) is arranged between the first reaction tank (5-1) and the second reaction tank (5-2), the second reaction tank (5-2) and the detection zone (7).

6. The immunofluorescent detection system according to claim 1, wherein the signal acquisition module (13) comprises a signal acquisition unit, the signal acquisition unit comprises a fluorescence excitation module (13-3-1) and a signal detection module (13-3-2), the fluorescence excitation module (13-3-1) is used for exciting fluorescent molecules, and the signal detection module (13-3-2) is used for receiving excitation light.

7. The immunofluorescent detection system according to any one of claims 1 to 6, wherein the analytical device further comprises an instrument housing, a mechanical skeleton (14), an electronic touch pad, a power supply module and an operation module,

the mechanical skeleton (14) is arranged in the instrument shell;

the electronic touch panel, the embedded control module and the operation module are all arranged on the mechanical framework (14), and the electronic touch panel, the operation module and the signal acquisition module (13) are all connected with the power supply module through the embedded control module;

the operation module comprises a substrate moving submodule (15-1), a piston driving submodule (15-2) and a clamping submodule (15-3), wherein the substrate moving submodule (15-1) is used for controlling the movement of the protein marker substrate (2), the piston driving submodule ((15-2) is used for controlling the driving piston (4-1) to move up and down so as to send the sample in the sample pool (3) into the reaction pool (5) through pressure change, and the clamping submodule ((15-3)) is used for fixing the microfluidic chip.

8. The immunofluorescence detection system according to claim 7, wherein the substrate moving sub-module comprises a moving motor and a guide rail, a gripper motor and a mechanical gripper controlled by the gripper motor, and is used for controlling the up-and-down movement of the mechanical gripper and the gripping action of the protein marker substrate.

9. The immunofluorescent detection system according to claim 7, wherein the piston driving submodule comprises a pushing motor (15-2-2) and a pushing pull rod (15-2-1) driven by the pushing motor (15-2-2) to move up and down, and the driving piston (4-1) is connected with the pushing pull rod (15-2-1).

10. The immunofluorescence detection system according to claim 7, wherein the clip submodule is located below the base moving submodule and comprises a fixed clip seat, a clip motor and a clip guide rail, and the fixed clip seat is connected with an external inlet bayonet of the analysis housing through the clip guide rail.

Technical Field

The invention belongs to the field of instant diagnosis, and also relates to a microfluidic chip technology, in particular to a fluorescence immunoassay system which is suitable for multi-index combined rapid detection.

Background

The microfluidic chip technology is a modern technology that integrates basic operation units of sample preparation, reaction, separation, detection and the like in the processes of biological, chemical and medical analysis on a micron-scale biochip, and analyzes by accurately controlling and controlling a micro-scale fluid and detecting electric, magnetic or optical signals on the biochip. With the development of the research, the micro-fluidic chip has been developed into a new research field crossing various fields such as biology, chemistry, medicine and machinery, and has great potential in numerous fields such as biochemistry, environmental monitoring and epidemic prevention.

The microfluidic chip has the characteristics of controllable liquid flow, relatively closed environment, small sample consumption, high reaction speed, easy integration and the like, and has unique advantages compared with the detection technology in the field of medical diagnosis and analysis at present. At present, the microfluidic chip technology has been developed greatly in the aspects of disease diagnosis such as biomarker immunoassay, nucleic acid sequencing, cell sorting and identification, and particularly, along with the demand of early diagnosis products for related diseases such as infectious diseases, tumor markers, sex hormones, thyroid gland and the like in the current medical field, the microfluidic chip shows great potential in the technical field of portability, convenience and sensitivity detection compared with the current traditional instrument detection.

Most of the mature detection technologies established on the basis of the microfluidic chip in the market at present adopt electric signals or magnetic signals, but the requirements of the biochip on equipment and chips are extremely high, the chip design is complex, the equipment is large in size and high in detection cost, and the biochip is difficult to popularize and use. In recent years, fluorescence is increasingly applied to the field of microfluidic chip development as a more direct marker with a mature detection principle, so as to develop a more convenient detection system. For example, BorFuh et al (A magnetic-microfluidic platform for fluorescence amplification using quantum dot nanoparticles, BorFuh et al nanotechnology,2019,30) propose that functional magnetic nanoparticles labeled with antibodies are pre-deposited on a microfluidic, the antigens are reacted with the antibodies through a deposition region, and then the fluorescent nanoparticles labeled with the antibodies are used to detect and confirm the antigens in the immune complexes, and the signals are collected using a fluorescent reader. Meanwhile, Sjohn et al (effective Microfluidic-Based Air Sampling/Monitoring Platform for Detection of Aerosol SARS-CoV-2On-site, X Fang et al analytical Chemistry,2021,93, 9) propose to integrate an Aerosol SARA-CoV-2 Sampling system by constructing a small volume rotary Microfluidic fluorescent chip to realize the On-site rapid sample collection and the Detection requirement in the Microfluidic chip. In summary, the above technologies perform module and function division and pretreatment on the microfluidic chip to ensure the need and progress of biological reactions, and then use equipment or sensors to collect and process signals of the fluorescent markers, so that strict requirements are imposed on the design and material (light transmittance, polarization degree, etc.) of the microfluidic chip, even the excitation-emission angle between the equipment and the microfluidic chip, the processing accuracy of the microfluidic chip, and the instrument structure design, and mass production and large-scale application are difficult to achieve.

Disclosure of Invention

The invention aims to solve the problems of high processing difficulty, high cost, complex instrument, large volume and the like of the conventional multi-index fluorescence automatic analysis equipment based on the microfluidic technology, combines the microfluidic technology with an embedded control system, takes a protein marker substrate as a biological reaction carrier, simplifies and integrates a control and signal acquisition module by controlling the interaction between the substrate and a microfluidic chip, and realizes the high-precision and multi-index rapid automatic detection of biomolecules.

The purpose of the invention is realized by at least one of the following technical solutions.

A fluorescence immunoassay system comprises a microfluidic chip and an analysis device, wherein the analysis device is used for carrying out fluorescence signal acquisition and result analysis on a reacted coating site in a detection area;

the microfluidic chip comprises a chip shell and a protein marker substrate, wherein the protein marker substrate comprises a coating carrier and a plurality of coating sites arranged on the coating carrier and is used as a reaction substrate for multi-index joint detection;

the chip shell is sequentially provided with a sample cell, a driving piston, a piston driving cell, a reaction cell, a cleaning cell and a detection zone, wherein the upper end of the sample cell is connected with the piston driving cell, and the lower end of the sample cell is communicated with the reaction cell through a microfluidic structure; the driving piston can move up and down in the piston driving pool; the cleaning pool is used for cleaning residual reagents after reaction; the analysis equipment comprises a signal acquisition module and an embedded control module, wherein the signal acquisition module is connected with the embedded control module, the signal acquisition module is used for acquiring fluorescent signals of the reacted coating sites in a detection area, and the embedded control module is used for controlling the signal acquisition module to work, processing the signals acquired by the signal acquisition module and calculating the concentration of the antigen to be detected in the sample to be detected.

The piston is driven to drive air in the pool to enter the sample pool, a sample enters the reaction pool through the microfluidic structure under the drive of pressure, and after the fluorescent labeling detection antibody is captured, the fluorescent signal acquisition is carried out on the protein marker substrate in the detection area by a signal acquisition module of the analysis equipment.

Further, the coating sites of the protein marker substrate are uniformly distributed on the coating carrier, and each coating site is pre-coated with an antibody capable of being specifically combined with different detection markers.

Furthermore, the sample cell comprises a sample cover and a cavity, the sample cover is positioned above the sample cell and is connected with the lower part of the piston driving cell through a first micro-flow channel, and the bottom end of the cavity is connected with the reaction cell through a micro-flow control structure. The sample enters the reaction cell through the microfluidic structure under the pressure driving.

Furthermore, the microfluidic structure comprises a second microfluidic channel and a filtering tank communicated with the second microfluidic channel, one end of the second microfluidic channel is connected with the bottom end of the cavity, the filtering tank is connected with the reaction tank, and a blood filtering membrane is arranged in the filtering tank. Larger proteins and other impurities are filtered out by providing a blood filter membrane.

Further, the reaction tank comprises a first reaction tank and a second reaction tank, the first reaction tank is used for placing buffer solution, the second reaction tank is used for placing fluorescence-labeled detection antibody solution, and a cleaning tank is arranged between the first reaction tank and the second reaction tank, and between the second reaction tank and the detection area. The sample is uniformly mixed with the buffer solution of the first reaction tank, the coating antibody coated on the coating carrier in advance reacts with the sample uniformly mixed with the buffer solution, the residual reagent is cleaned in the cleaning tank and then enters the second reaction tank to improve the detection precision, the cleaned protein marker substrate enters the second reaction tank to finish the capture of the fluorescence labeling detection antibody, then the protein marker substrate continues to enter the cleaning tank to be cleaned, and then the protein marker substrate enters the detection zone to collect a fluorescence signal.

Further, the signal acquisition module includes the signal acquisition unit, arouses the module and the signal detection module including fluorescence, and fluorescence arouses the module and is used for arousing fluorescence molecule, and the signal detection module is used for receiving the excitation light.

The signal acquisition module also comprises a signal acquisition motor, the signal acquisition motor controls the signal acquisition unit to approach a certain coating site, exciting light with small light spots is provided in the signal acquisition unit to excite fluorescent molecules in the region, the exciting light is received by the signal detection module of the signal acquisition unit to complete conversion and acquisition of the optical-electrical-digital signals of the fluorescence of the coating site, and then signal acquisition of each coating site is sequentially completed.

Furthermore, the analysis equipment also comprises an instrument shell, a mechanical framework, an electronic touch panel, an embedded control module, a power supply module and an operation module,

the mechanical framework is arranged in the instrument shell;

the electronic touch panel, the embedded control module and the operation module are all arranged on the mechanical framework, and the electronic touch panel, the operation module and the signal acquisition module are all connected with the power supply module through the embedded control module;

the operation module comprises a substrate moving submodule, a piston driving submodule and a clamping submodule, the substrate moving submodule is used for controlling the movement of the protein marker substrate, the piston driving submodule is used for controlling the driving piston to move up and down so as to send a sample in the sample pool into the reaction pool through pressure variation, and the clamping submodule is used for fixing the microfluidic chip.

Further, the substrate moving submodule comprises moving motors and guide rails, a gripper motor and a mechanical gripper controlled by the motors, and the three groups of moving motors are respectively used for controlling the movement of the gripper and the gripping action of the protein marker substrate.

Further, in the substrate moving submodule, an output shaft of a gripper motor is connected with the gripper and fixed with an output shaft of a moving motor through a nut.

Furthermore, the clamping submodule is positioned below the substrate moving submodule and the piston driving submodule and comprises a fixed clamping seat and a temperature control device, and the fixed clamping seat is connected with an external inlet bayonet of the instrument shell through a sliding rail. The microfluidic chip slides to the lower part of the substrate moving sub-module from the card inlet along with the card seat on the slide rail.

Furthermore, the piston driving submodule is located above the inner portion of the access opening outside the instrument shell and comprises a pushing pull rod of the piston, a group of motors and a guide rail, and an output shaft of each motor is connected with the corresponding pull rod through a nut and can drive the corresponding pull rod to move up and down.

The invention combines a micro-fluidic chip and an embedded control system, constructs a biological microarray by taking a protein marker substrate as a carrier for fluorescence immunoassay biological reaction, develops a portable multi-index fluorescence immunoassay system and a method based on the micro-fluidic technology, and has the following beneficial effects compared with the prior art:

1) the chip processing technology has low difficulty and controllable cost. The micro-fluidic technology is combined with the embedded control module, biological reaction automation is realized through interaction between the system control substrate and the micro-fluidic chip, and the signal acquisition module is used for sequentially exciting and acquiring the encapsulated sites on the substrate;

2) the instrument is small in size and high in integration degree. According to the invention, the piston driving submodule is used as the driving force of the microfluid, and a series of automatic control devices such as the piston driving submodule and the embedded control module are integrated in the mechanical skeleton, so that the automation from sample processing, biological reaction to signal detection and analysis in an instrument with a small volume is realized, and the device has the characteristics of convenience in carrying, simplicity in operation and quickness in detection.

3) The invention is suitable for the fields of clinical diagnosis and disease screening, in particular to the early primary screening and self-checking of a series of diseases such as infectious diseases, tumor markers, sex hormones, thyroid and the like.

Drawings

Fig. 1 is a schematic structural diagram of a microfluidic chip in an embodiment of the present invention.

FIG. 2 is a schematic diagram of the structure of a protein marker substrate according to an embodiment of the present invention.

Fig. 3 is a schematic view of the bottom structure of the microfluidic chip in the embodiment of the present invention.

Fig. 4 is a schematic diagram of the internal structure of the microfluidic chip in the embodiment of the present invention.

Fig. 5 is a schematic diagram of a portable analysis device.

Fig. 6 is a schematic structural diagram of a fluorescence signal acquisition module.

FIG. 7 is a schematic diagram of a substrate moving submodule structure.

FIG. 8 is a schematic diagram of a clip submodule structure.

Fig. 9 is a schematic structural diagram of the piston driving sub-module.

Detailed Description

In order to make the objects, technical solutions and features of the embodiments of the present invention clearer, the present invention will be described in further detail with reference to the accompanying drawings and the embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1-6, the present invention provides a fluorescence immunoassay system, which includes a microfluidic chip and a portable analysis device. The portable analysis equipment is used for assisting the micro-fluidic chip to complete biological reaction and complete signal acquisition and result analysis.

Referring to fig. 1-4, the microfluidic chip includes a chip housing 1 and a protein marker substrate 2, wherein the protein marker substrate 2 includes a coating carrier 2-1 and a plurality of coating sites 2-2 disposed on the coating carrier 2-1, which serve as a reaction substrate for multi-index joint inspection.

In the invention, a sample cell 3, a driving piston 4-1, a piston driving cell 4-2, a reaction cell 5, a cleaning cell 6 and a detection zone 7 are sequentially arranged in a chip shell 1, the upper end of the sample cell 3 is connected with the piston driving cell 4-2, and the lower end is communicated with the reaction cell 5 through a microfluidic structure; the cleaning pool 6 is used for cleaning residual reagents after reaction; the analysis equipment comprises a signal acquisition module and an embedded control module, wherein the signal acquisition module is connected with the embedded control module, and the signal acquisition module is used for acquiring fluorescent signals of the reacted coating sites 2-2 in a detection area 7. The embedded control module is used for controlling the work of the signal acquisition module 13, processing the signal acquired by the signal acquisition module 13 and calculating the concentration of the antigen to be detected in the sample to be detected.

In some embodiments of the present invention, referring to fig. 1, a substrate carrying cavity 12 is further disposed in the chip housing 1, and the protein marker substrate 2 is disposed on the substrate carrying cavity 12, so as to ensure the storage and transportation of the protein marker substrate 2 and the microfluidic chip, and facilitate the automated operation of the instrument on the detection process.

In some embodiments of the present invention, as shown in fig. 2, the coating sites 2-2 of the protein marker substrate 2 are distributed on the coating carrier 2-1, and the multi-index combined detection bioarray is prepared by pre-coating antibodies that can specifically bind to different kinds of markers at the coating sites 2-2.

In some embodiments of the present invention, the coating sites 2-2 are square or circular, each coating site is uniformly distributed and regularly arranged, and the signal acquisition module excites and acquires signals in the middle area of each coating site 2-2 bit by bit to ensure that each bit reacts sufficiently and the signal intensity is balanced.

In some embodiments of the present invention, referring to fig. 1, 3 and 4, the sample cell 3 is a relative sealing structure formed by the sample cover 3-1 and the cavity, and the sample can be conveniently injected into the cavity by opening the sample cover 3-1. The sample cover 3-1 is positioned at the upper end of the sample cell 3, the sample cover 3-1 is provided with a mounting hole, a first micro-flow channel 11 which can be communicated with the cavity is arranged in the mounting hole, the free end part of the first micro-flow channel 11 is communicated with the lower part of the piston driving cell 4-2, and the bottom end of the cavity is communicated with the reaction cell 5 through a micro-flow control structure.

In some embodiments of the present invention, as shown in fig. 1 and 3, the lower end of the piston driving cell 4-2 is externally connected to the small hole at the bottom end inside the chip housing 1 through the micro channel 10, and is communicated with the mounting hole on the sample cap 3-1 through the soft first micro channel 11, so as to provide power for the sample in the sample cell 3 to pass through the micro-fluidic structure between the sample cell 3 and the reaction cell 5, and also facilitate the processing of the chip housing.

In some embodiments of the present invention, as shown in fig. 3, the microfluidic structure includes a second microfluidic channel 8 and a filter cell 9 communicated with the second microfluidic channel 8, one end of the second microfluidic channel 8 is connected to the bottom end of the cavity of the sample cell 3, the filter cell 9 is connected to the reaction cell 5, and a blood filtration membrane is disposed in the filter cell 9. The blood filtering membrane is arranged to block larger protein and other impurities, so that the influence on the detection result can be avoided, and the detection accuracy is improved.

In some embodiments of the present invention, the reaction tank 5 includes a first reaction tank 5-1 and a second reaction tank 5-2, the first reaction tank 5-1 is used for placing a buffer solution, the second reaction tank 5-2 is used for placing a fluorescence labeled detection antibody solution, a cleaning tank 6 is disposed between the first reaction tank 5-1 and the second reaction tank 5-2 and between the second reaction tank 5-2 and the detection zone 7, and the filter tank 9 is communicated with the first reaction tank 5-1. Preferably, two mutually independent cleaning pools, namely a first cleaning pool 6-1 and a second cleaning pool 6-2, are arranged behind the first reaction pool 5-1, and two mutually independent cleaning pools, namely a third cleaning pool 6-3 and a fourth cleaning pool 6-4, are also arranged behind the second reaction pool 5-2, and the action of the two adjacent cleaning pools is consistent, so that excessive residual reagents reacted in the previous reaction pool are completely cleaned.

When the device works, a driving piston 4-1 on a micro-fluidic chip is driven, air in a piston driving pool 4-2 enters a sample pool 3, a sample enters a first reaction pool 5-1 through a micro-fluidic structure under the driving of pressure intensity and is uniformly mixed with a buffer solution in the first reaction pool, then a protein marker substrate 2 is sequentially cleaned in a first cleaning pool 6-1 and a second cleaning pool 6-2, fluorescent labeling detection antibody capture is completed in the second reaction pool 5-2, then residual reagents are cleaned in a third cleaning pool 6-3 and a fourth cleaning pool 6-4 and then enter a detection area 7, and an analysis device finishes fluorescent signal acquisition on the protein marker substrate 2.

In the embodiment of the invention, the initial state of the microfluidic chip is as follows:

a detection marker antibody is coated on a coating site 2-2 on the protein marker substrate 2 in advance;

the first reaction tank 5-1 is pre-filled with buffer blending liquid, and the first cleaning tank 6-1 and the second cleaning tank 6-2 are pre-filled with cleaning liquid 1;

a detection antibody solution labeled by fluorescence is preloaded in the second reaction tank 5-2;

the third cleaning pool 6-3 and the fourth cleaning pool 6-4 are pre-filled with cleaning liquid 2.

The cleaning solution 1 and the cleaning solution 2 are selected according to the experimental reaction conditions of the project.

In some embodiments of the present invention, referring to fig. 6, the signal collection module 13 controls the smaller signal collection unit to approach the coating site through the signal collection motor 13-1, the excitation light with smaller light spot is provided in the signal collection unit to excite the fluorescent molecule in the region, and the excitation light is received by the signal detection module of the signal collection unit to complete the conversion and collection of the optical-electrical-digital signal. Specifically, the signal acquisition module comprises a signal acquisition motor 13-1, a signal acquisition guide rail 13-2 and a signal acquisition unit, wherein the signal acquisition motor 13-1 drives the signal acquisition unit to move up and down along the signal acquisition guide rail 13-2, so that the position of the signal acquisition unit can be adjusted, and signals at coating sites with different heights can be acquired.

The signal acquisition unit comprises a fluorescence excitation module 13-3-1 and a signal detection module 13-3-2, wherein the fluorescence excitation module 13-3-1 is used for exciting fluorescent molecules, and the signal detection module 13-3-2 is used for receiving exciting light. In some embodiments of the present invention, the fluorescence excitation module 13-3-1 includes 3 sets of uv LED light sources and uv filters, and can provide excitation light with concentrated energy and specific wavelength, the excitation light is converged and irradiated to the middle region of the coated sites through the uv filters, fluorescence molecules in the region are excited, the excited light is received by the signal detection module 13-3-2, and the conversion and collection of optical-electrical-digital signals of site fluorescence are completed, and then the signal collection of each coated site is sequentially completed. Preferably, the signal detection module 13-3-2 includes a half mirror, a narrow band filter, and a signal detector, where the half mirror is used to shield stray light and excitation light, and the signal detector completes fluorescence reception and signal conversion.

In some embodiments of the present invention, the fluorescence excitation module 13-3-1 uses 2-4 sets of excitation wavelengths to separately excite different labeled fluorescence on the protein marker substrate.

In some embodiments of the present invention, the selected signal detector is a CCD detector, and in other embodiments of the present invention, other photodetectors may be selected as desired.

In some embodiments of the present invention, the signal acquisition module 13 performs a grid-by-grid scan on the biological array to ensure the accuracy of the fluorescence intensity values in the individual reaction regions.

In the present invention, referring to fig. 5, the analysis device further includes an instrument housing, a mechanical skeleton 14, an electronic touch pad, an embedded control module, a power module, and an operation module, wherein the mechanical skeleton 14 is disposed in the analysis housing; the electronic touch panel, the embedded control module and the operation module are all arranged on the mechanical framework 14, and the electronic touch panel, the operation module and the signal acquisition module are all connected with the power supply module through the embedded control module; the embedded control module comprises a main control board and a control circuit, is used for controlling the electronic touch control board and the operation module to move coordinately, and is also used for carrying operation software and carrying out data transmission and processing. The operation module comprises a substrate moving submodule 15-1, a piston driving submodule 15-2 and a clamping submodule 15-3, the substrate moving submodule 15-1 is used for controlling the movement of the protein marker substrate 2, the piston driving submodule 15-2 is used for controlling the driving piston 4-1 to move up and down so as to send the sample liquid in the sample pool 3 into the reaction pool 5 through pressure variation, and the clamping submodule 15-3 is used for fixing the microfluidic chip.

In the invention, the substrate moving submodule 15-1 comprises an up-down moving motor 15-1-1, a left-right moving motor 15-1-2, a gripper motor 15-1-3, a mechanical gripper 15-1-4 and a guide rail, wherein the mechanical gripper 15-1-4 is driven by the gripper motor 15-1-3 to grip the protein marker substrate 2, and the up-down moving motor 15-1-1 and the left-right moving motor 15-1-2 are respectively used for controlling the mechanical gripper 15-1-4 to move in the up-down direction and the left-right direction.

In some embodiments of the present invention, referring to fig. 7, the output shaft of the gripper motor 15-1-3 is fixed to the output shaft of the mechanical gripper 15-1-4 and the output shaft of the up-down moving motor 15-1-1 and the left-right moving motor 15-1-2 through nuts.

The output shafts of the up-down moving motor 15-1-1 and the left-right moving motor 15-1-2 are respectively connected with a module consisting of a gripper motor 15-1-3 and a mechanical gripper 15-1-4 to control the module to move, so that the movement, cleaning and reaction of the gripper on the chip are realized. The gripper motor is used for controlling the clamping piece of the mechanical gripper to complete the gripping action on the protein marker substrate 2, the output shaft of the gripper motor is respectively connected with the gears on the mechanical grippers 15-1-4, and the mechanical grippers 15-1-4 are controlled by the gears to move on the guide rail to perform the gripping and fixing action.

In some embodiments of the present invention, referring to fig. 8, the clip submodule 15-3 is used to fix the position of the microfluidic chip, and includes a clip motor 15-3-3, a clip guide 15-3-2 driven by the clip motor 15-3-3, a fixed clip seat 15-3-1 and a temperature control device, the fixed clip seat 15-3-1 is located at the bottom end of the mechanical skeleton 14, the temperature control device is used to control the temperature of the environment in the reaction process, and the fixed clip seat 15-3-1 is connected with the external inlet bayonet of the analysis housing through the clip guide 15-3-2. The fixed clamping seat 15-3-1 provided with the micro-fluidic chip is moved to a preset position by the driving of the clamping motor 15-3-3, and then the subsequent steps are carried out.

The piston driving submodule 15-2 is located above the inner portion of the card inlet outside the analysis shell and comprises a push pull rod 15-2-1, a push motor 15-2-2 and a push guide rail 15-2-3, an output shaft of the push motor 15-2-2 is connected with the push pull rod 15-2-1 through a nut and can drive the push pull rod 15-2-1 to move up and down, a driving piston 4-1 is connected with the push pull rod 15-2-1, and the piston 4-1 can be driven to move up and down in a piston driving pool 4-2 by the up-and-down movement of the push pull rod 15-2-1.

In some embodiments of the present invention, referring to fig. 5, the substrate moving sub-module 15-1 is located above the card holder sub-module 15-3, the piston driving sub-module 15-2 is located above the inside of the card inlet, when the microfluidic chip moves to a fixed position through the card holder sub-module 15-3, the piston driving sub-module 15-2 operates first to push the driving piston 4-1 to the bottom, and then the substrate moving sub-module 15-1 operates the protein marker substrate 2. The three sub-modules are connected with independent motors respectively to provide driving force for operation of the modules and realize control of accurate movement, and are connected with the electronic touch control board through the main control board and the power supply module to control coordinated operation among the modules.

In some embodiments of the present invention, an operator may complete functions such as a human-computer interaction function, instruction transmission, data analysis, result presentation, and data storage through the main control board, and input and output of data may be implemented through ports such as a built-in GRPS, bluetooth, LAN, and USB.

When the fluorescence immunoassay system provided by the previous embodiment is used for detection, the method comprises the following steps:

s1, adding 200uL of sample into the cavity of the sample cell 3, covering the sample cover 3-1, placing the microfluidic chip into the fixed clamping seat 15-3-1, starting the device, and enabling the microfluidic chip to enter a fixed position;

s2, sample injection and dilution: the analytical equipment presses a driving piston 4-1 on the microfluidic chip, and a sample in the sample pool 3 passes through a filter pool 9, enters a first reaction pool 5-1 and is uniformly mixed with a buffer solution in the first reaction pool;

s3, marker capture: the analysis equipment moves the coating carrier into the first reaction pool 5-1, the coating antibody coated on the coating carrier in advance reacts with the sample in the reaction pool, and the reaction is stopped after incubation for 5 minutes;

s4, cleaning: the analytical equipment sequentially transfers the coating carrier into a first cleaning pool 6-1 and a second cleaning pool 6-2, and washes away residual reagent on the surface;

s5, fluorescent marker capture: the analysis equipment moves the coating carrier into a second reaction tank 5-2, and the coating carrier is coated with an antibody-antigen complex to be detected or reacts with a fluorescence-labeled detection antibody or antigen in the reaction tank 5-2;

s6, cleaning: the analytical equipment sequentially moves the coating carrier into a third cleaning pool 6-3 and a fourth cleaning pool 6-4, and residual reagents are washed away;

s7, signal acquisition: the analytical equipment moves the coating carrier into the detection zone 7, and the detection instrument excites the coating carrier, the coating antibody on the coating site, the antigen to be detected and the fluorescence labeled antibody to capture the excited fluorescence signal;

s8, result analysis: and the analysis equipment calculates the concentration of the antigen to be detected in the sample to be detected, takes out the microfluidic detection chip and closes the analysis equipment.

It should be noted that, the sandwich method in the immunoassay method is selected in the above embodiments, and in other embodiments of the present invention, the sandwich method, the competition method or other fluorescent immune-based assay methods can be selected according to the molecular characteristics of the marker to be detected.

In some embodiments of the present invention, the control software of the instrument includes embedded software and upper computer software, the embedded software is used to independently complete control of each part of the instrument, the upper computer software is used to complete control of an embedded system of the instrument and writing of instrument data through a computer, and the embedded software and the upper computer software are connected wirelessly or through wires.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.