CN113720818B - Fluorescent immunodetection system - Google Patents

Abstract

The invention provides a fluorescence immunoassay system. The system comprises a microfluidic chip and portable analysis equipment; the protein marker substrate is preprocessed to form a multi-index biological array; the chip shell is provided with a sample pool, a driving piston, a piston driving pool, a reaction pool, a cleaning pool and a detection area respectively; the analysis equipment has the function of assisting the substrate to complete biological reaction in the microfluidic chip and collecting signals and analyzing results. The bottom end of the piston driving pool is connected with the top end of the sample pool, the sample liquid enters the reaction pool and reacts with the substrate through the microfluidic structure under the movement of the piston, the instrument controls the substrate to finish cleaning and fluorescent marker capturing in the chip, and the fluorescent signal of the substrate is collected in the detection area. The analysis equipment realizes the automatic realization of the instrument functions and the instrument structure integration 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.

Description

A fluorescent immunoassay system

Technical field

The invention belongs to the field of instant diagnosis and also relates to a microfluidic chip technology, specifically a fluorescence immunodetection system suitable for joint rapid detection of multiple indicators.

Background technique

Microfluidics chip technology integrates basic operating units such as sample preparation, reaction, separation, and detection of biological, chemical, and medical analysis processes into a micron-scale biochip. By accurately controlling and manipulating micro-scale fluids, Modern technology for analysis by detecting electrical, magnetic or optical signals on a chip. With the deepening of research, microfluidic chips have developed into a new research field that intersects various fields such as biology, chemistry, medicine, and mechanics, showing great potential in many fields such as biochemistry, environmental monitoring, and health and epidemic prevention.

Microfluidic chips have the characteristics of controllable liquid flow, relatively closed environment, small sample consumption, fast reaction speed, and easy integration. They have unique advantages compared with current detection technologies in the fields of medical diagnosis and analysis. At present, microfluidic chip technology has made great progress in disease diagnosis such as biomarker immune analysis, nucleic acid sequencing, cell sorting and identification, especially with the current medical field's focus on infectious diseases, tumor markers, sex hormones, There is a demand for early diagnosis products for thyroid and other related diseases. Compared with the current traditional instrument detection, microfluidic chips have shown great potential in the field of portability, convenience and sensitive detection technology.

Most of the relatively mature detection technologies based on microfluidic chips on the market today use electrical signals or magnetic signals. However, this type of biochip has extremely high requirements for equipment and chips. The chip design is complex, the equipment is large and the detection cost is high. High and difficult to promote and use. In recent years, fluorescence, as a relatively direct marker with mature detection principles, has been increasingly used in the field of microfluidic chip development to develop more convenient detection systems. For example, BorFuh et al. (A magneto-microfluidic platform for fluorescence immunosensing using quantum dotnanoparticles. BorFuh et al. Nanotechnology, 2019, 30) proposed that functional magnetic nanoparticles labeled with antibodies are pre-deposited on the microfluid, and the antigens interact with the antibodies through the deposition area. The reaction is carried out, and fluorescent nanoparticles labeled with antibodies are used to detect and confirm the antigens in the immune complex, and a fluorescence reading device is used to collect the signal. At the same time, Fang Xuen and others (Efficient Microfluidic-Based Air Sampling/MonitoringPlatform for Detection of Aerosol SARS-CoV-2On-site, X Fang et al. Analytical Chemistry, 2021, 93, 9) proposed to construct a small-volume rotating microfluid The fluorescent chip integrates the aerosol SARA-CoV-2 sampling system to meet the needs of rapid on-site sample collection and detection within the microfluidic chip. In summary, the above technology divides the microfluidic chip into modules and functions and preprocesses to ensure the needs and progress of biological reactions, and then uses equipment or sensors to collect and process the signals of fluorescent markers. Therefore, the microfluidic chip is The design, material (transmittance, polarization, etc.) of the chip and even the excitation-emission angle between the device and the microfluidic chip, the accuracy of microfluidic chip processing, and the instrument structure design all have strict requirements, making it difficult to achieve quantitative results. production and large-scale application.

Contents of the invention

The purpose of this invention is to solve the problems of current multi-index fluorescence automated analysis equipment based on microfluidic technology, such as difficulty in processing, high cost, complex and large instrument size, etc., by combining microfluidic technology with an embedded control system. The protein marker substrate is a biological reaction carrier. By controlling the interaction between the substrate and the microfluidic chip, the control and signal acquisition modules are simplified and integrated to achieve high-precision, multi-index rapid automated detection of biomolecules.

The object of the present invention is achieved by at least one of the following technical solutions.

A fluorescence immunoassay system, including a microfluidic chip and analysis equipment, the analysis equipment is used to collect fluorescence signals and analyze results of the coated sites after reaction in the detection area;

The microfluidic chip includes a chip shell and a protein marker substrate. The protein marker substrate includes a coating carrier and a plurality of coating sites provided on the coating carrier, serving as a reaction substrate for multi-index joint detection;

The chip housing is provided with a sample pool, a driving piston and a piston driving pool, a reaction pool, a cleaning pool and a detection area in sequence. The upper end of the sample pool is connected to the piston driving pool, and the lower end is connected to the reaction pool through a microfluidic structure. ; The driving piston can move up and down in the piston driving pool; the cleaning pool is used to clean the residual reagents after the reaction; the analysis equipment includes a signal acquisition module and an embedded control module, the signal acquisition module is connected to the embedded control module, and the The signal acquisition module is used to collect fluorescence signals from the coated sites after reaction in the detection area. The embedded control module is used to control the work of the signal acquisition module, process the signals collected by the signal acquisition module, and calculate the samples to be detected. The concentration of the antigen to be detected.

By driving the piston, the piston drives the air in the pool into the sample pool. The sample is driven by pressure and enters the reaction pool through the microfluidic structure. After the fluorescently labeled detection antibody is captured, the protein marker substrate is collected by the signal of the analysis device in the detection area. The module collects fluorescence signals.

Furthermore, the coating sites of the protein marker substrate are evenly distributed on the coating carrier, and each coating site is pre-coated with antibodies that can specifically bind to different detection markers.

Further, the sample pool includes a sample cover and a cavity. The sample cover is located above the sample pool and is connected to the bottom of the piston driving pool through the first microfluidic channel. The bottom end of the cavity is connected to the reaction pool through a microfluidic structure. The sample enters the reaction cell through the microfluidic structure driven by pressure.

Further, the microfluidic structure includes a second microfluidic channel and a filter tank connected to the second microfluidic channel, one end of the second microfluidic channel is connected to the bottom end of the cavity, the filter tank is connected to the reaction tank, and A blood filtration membrane is provided in the filter tank. Larger proteins and other impurities are filtered out by setting up a hemofiltration membrane.

Further, the reaction pool includes a first reaction pool and a second reaction pool. The first reaction pool is used to place a buffer solution. The second reaction pool is used to place a fluorescently labeled detection antibody solution. In the first reaction pool and the second reaction pool, , a cleaning pool is provided between the second reaction pool and the detection area. The sample is mixed with the buffer in the first reaction pool. After the pre-coated coating antibody on the coating carrier reacts with the sample mixed with the buffer, the residual reagent is washed in the cleaning pool and then enters the second reaction pool. To improve detection accuracy, the cleaned protein marker substrate enters the second reaction pool to complete the capture of fluorescently labeled detection antibodies, then continues into the cleaning pool for cleaning, and then enters the detection area for collection of fluorescent signals.

Further, the signal acquisition module includes a signal acquisition unit, including a fluorescence excitation module and a signal detection module. The fluorescence excitation module is used to excite fluorescent molecules, and the signal detection module is used to receive excitation light.

The signal acquisition module also includes a signal acquisition motor. The signal acquisition motor controls the signal acquisition unit to approach a certain coating site. The signal acquisition unit provides excitation light with a smaller spot to excite fluorescent molecules in the area. The excitation light is absorbed by the signal acquisition unit. The signal detection module receives and completes the conversion and collection of the optical-electrical-digital signal of the fluorescence of the coating site, and then completes the signal collection of each coating site in sequence.

Further, the analysis equipment also includes an instrument housing, a mechanical skeleton, an electronic touch panel, an embedded control module, a power module and an operation module,

The mechanical skeleton is set inside the instrument casing;

The electronic touch panel, embedded control module and operating module are all set on the mechanical skeleton, and the electronic touch panel, operating module and signal acquisition module are all connected to the power module through the embedded control module;

The operation module includes a substrate movement sub-module, a piston drive sub-module and a clamp sub-module. The substrate movement sub-module is used to manipulate the movement of the protein marker substrate. The piston drive sub-module is used to control the up and down movement of the drive piston to move the sample cell through pressure changes. The sample is sent into the reaction tank, and the card clip module is used to fix the microfluidic chip.

Further, the substrate moving sub-module includes a moving motor and guide rail, a gripper motor and a mechanical gripper controlled by the motor. The three sets of mobile motors are used to control the movement of the gripper and the grasping action of the protein marker substrate.

Further, in the base moving sub-module, the output shaft of the gripper motor is connected to the gripper and fixed to the output shaft of the moving motor through a nut.

Further, the clamp sub-module is located below the base moving sub-module and the piston driving sub-module, and includes a fixed card holder and a temperature control device. The fixed card holder is connected to the external card inlet of the instrument housing through a slide rail. The microfluidic chip slides along with the card holder on the slide rail from the card inlet to the bottom of the base moving sub-module.

Further, the piston drive sub-module is located above the inside of the bayonet outside the instrument housing, and includes a push rod of the piston and a set of motors and guide rails. The motor output shaft and the pull rod are connected by a nut, which can drive the pull rod to move up and down.

The present invention combines a microfluidic chip and an embedded control system, uses a protein marker substrate as a carrier for fluorescence immunoassay biological reactions to form a biological microarray, and develops a portable multi-index fluorescence immunoassay system based on microfluidic technology. and methods. Compared with the prior art, the beneficial effects that the present invention can achieve are at least as follows:

1) The chip processing technology is low-difficulty and the cost is controllable. Combining microfluidic technology with embedded control modules, the biological reaction automation is realized through the interaction between the system control substrate and the microfluidic chip, and the signal acquisition module is used to sequentially excite and collect the coating sites on the substrate, which is the same as what is currently available on the market. Compared with commonly used chips, it avoids the cumbersome need for chip processing, reduces processing difficulty and costs;

2) The instrument is small in size and highly integrated. By using the piston driving sub-module as the driving force of the microfluid, the present invention integrates a series of automated control devices such as the piston driving sub-module and the embedded control module into the mechanical skeleton, thereby realizing the steps from sample processing to The automation from biological reaction to signal detection and analysis has the characteristics of portability, simple operation and rapid detection.

3) The present invention is suitable for the fields of clinical diagnosis and disease screening, especially early screening and self-examination of a series of diseases such as infectious diseases, tumor markers, sex hormones, and thyroid.

Description of the drawings

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

Figure 2 is a schematic diagram of the structure of a protein marker substrate in an embodiment of the present invention.

Figure 3 is a schematic diagram of the bottom structure of a microfluidic chip in an embodiment of the present invention.

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

Figure 5 is a schematic structural diagram of the portable analysis equipment.

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

Figure 7 is a schematic structural diagram of the base moving sub-module.

Figure 8 is a schematic structural diagram of the card clip module.

Figure 9 is a schematic structural diagram of the piston drive submodule.

Detailed ways

In order to make the purpose, technical solutions and characteristics of the embodiments of the present invention clearer, the present invention will be described in further detail below in conjunction with the accompanying drawings and examples. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than All embodiments; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

Please refer to Figures 1-6. The present invention provides a fluorescence immunoassay system, including a microfluidic chip and a portable analysis device. The role of portable analysis equipment is to assist the microfluidic chip in completing biological reactions, and to complete signal collection and result analysis.

Please refer to Figures 1 to 4. The microfluidic chip includes a chip housing 1 and a protein marker substrate 2. The protein marker substrate 2 includes a coating carrier 2-1 and a multi-layer substrate disposed on the coating carrier 2-1. A coating site 2-2 serves as the reaction base for multi-index joint detection.

In the present invention, the chip housing 1 is provided with a sample pool 3, a driving piston 4-1 and a piston driving pool 4-2, a reaction pool 5, a cleaning pool 6 and a detection area 7 in sequence. The upper end of the sample pool 3 is connected to the piston. The driving pool 4-2 is connected, and the lower end is connected to the reaction pool 5 through a microfluidic structure; the cleaning pool 6 is used to clean the residual reagents after the reaction; the analysis equipment includes a signal acquisition module and an embedded control module. The signal acquisition module Connected to the embedded control module, the signal acquisition module is used to collect fluorescence signals from the reacted coating sites 2-2 in the detection area 7. The embedded control module is used to control the work of the signal acquisition module 13, process the signals collected by the signal acquisition module 13, and calculate the concentration of the antigen to be detected in the sample to be detected.

In some embodiments of the present invention, please refer to Figure 1. A substrate carrying cavity 12 is also provided in the chip housing 1, and the protein marker substrate 2 is arranged on the substrate carrying cavity 12 to facilitate ensuring that the protein marker substrate 2 and the microfluidic The storage and transportation of chips also facilitate the automated operation of the instrument’s detection process.

In some embodiments of the present invention, as shown in Figure 2, the coating sites 2-2 of the protein marker substrate 2 are distributed on the coating carrier 2-1. Coating with antibodies that can specifically bind to different types of markers to prepare a multi-index joint detection bioarray.

In some embodiments of the present invention, the coating sites 2-2 are square or circular, and each coating site is evenly distributed and neatly arranged. The signal acquisition module analyzes each coating site 2-2 site by site. Excite and collect signals in the middle area to ensure that each site responds fully and the signal intensity is balanced.

In some embodiments of the present invention, please refer to Figures 1, 3, and 4. The sample pool 3 is a relatively sealed structure composed of a sample cover 3-1 and a cavity. Opening the sample cover 3-1 can facilitate the injection of samples into the cavity. . The sample cover 3-1 is located at the upper end of the sample pool 3. The sample cover 3-1 is provided with a mounting hole. A first microfluidic channel 11 that can communicate with the cavity is provided in the mounting hole. The free end of the first microfluidic channel 11 The bottom of the cavity is connected with the bottom of the piston driving pool 4-2, and the bottom end of the cavity is connected with the reaction pool 5 through the microfluidic structure.

In some embodiments of the present invention, as shown in Figures 1 and 3, the lower end of the piston driving pool 4-2 is externally connected to the small hole at the bottom end of the chip housing 1 through the microchannel 10, and passes through the soft first microcirculation The channel 11 is connected with the mounting hole on the sample cover 3-1, which provides power for the sample in the sample pool 3 to pass through the microfluidic structure between the sample pool 3 and the reaction pool 5, and also facilitates the processing of the chip housing.

In some embodiments of the present invention, as shown in Figure 3, the microfluidic structure includes a second microfluidic channel 8 and a filter pool 9 connected to the second microfluidic channel 8. One end of the second microfluidic channel 8 is connected to the sample. The bottom end of the cavity of the pool 3 is connected, the filter pool 9 is connected to the reaction pool 5, and the filter pool 9 is provided with a blood filtration membrane. By setting up a blood filtration membrane to block larger proteins and other impurities, the impact on the test results can be avoided and the test accuracy can be improved.

In some embodiments of the present invention, the reaction pool 5 includes a first reaction pool 5-1 and a second reaction pool 5-2. The first reaction pool 5-1 is used to place buffer solution, and the second reaction pool 5-2 is used to place buffer solution. In order to place the fluorescently labeled detection antibody solution, a cleaning pool 6 is provided between the first reaction pool 5-1 and the second reaction pool 5-2, and between the second reaction pool 5-2 and the detection area 7, wherein, The filter pool 9 is connected with the first reaction pool 5-1. Preferably, two independent cleaning pools are provided behind the first reaction pool 5-1, defined as the first cleaning pool 6-1 and the second cleaning pool 6-2. The same is true after the second reaction pool 5-2. Set up two independent cleaning pools, defined as the third cleaning pool 6-3 and the fourth cleaning pool 6-4. The two adjacent cleaning pools have the same function. The purpose is to ensure that there is too much reaction in the previous reaction pool. Residual reagents are completely cleaned.

When working, the driving piston 4-1 on the microfluidic chip is driven, and the air in the piston driving pool 4-2 enters the sample pool 3. The sample is driven by the pressure and enters the first reaction pool 5-1 through the microfluidic structure, and The buffer therein is mixed, and then the protein marker substrate 2 is washed in the first cleaning pool 6-1 and the second cleaning pool 6-2 in sequence, and the fluorescently labeled detection antibody is captured in the second reaction pool 5-2. Then, the residual reagent is washed away in the third cleaning pool 6-3 and the fourth cleaning pool 6-4 and then enters the detection area 7, where the analysis equipment completes the fluorescence signal collection of the protein marker substrate 2.

In this embodiment of the present invention, the initial state of the microfluidic chip is:

The coating site 2-2 on the protein marker substrate 2 is pre-coated with the detection marker antibody;

The first reaction tank 5-1 is pre-loaded with buffer preparation solution, and the first cleaning tank 6-1 and the second cleaning tank 6-2 are pre-loaded with cleaning solution 1;

The second reaction pool 5-2 is pre-loaded with a fluorescently labeled detection antibody solution;

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

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

In some embodiments of the present invention, please refer to Figure 6. The signal acquisition module 13 controls a smaller signal acquisition unit to approach the coating site through the signal acquisition motor 13-1. The signal acquisition unit is provided with a smaller light spot. The excitation light excites the fluorescent molecules in the area, and the excitation light is received by the signal detection module of the signal acquisition unit to complete the conversion and collection of optical-electrical-digital signals. Specifically, the signal acquisition module includes a signal acquisition motor 13-1, a signal acquisition guide rail 13-2 and a signal acquisition unit. 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 signal can be adjusted. The position of the acquisition unit can then be used to collect signals from coating sites located at different heights.

The signal acquisition unit includes a fluorescence excitation module 13-3-1 and a signal detection module 13-3-2. The fluorescence excitation module 13-3-1 is used to excite fluorescent molecules, and the signal detection module 13-3-2 used to receive excitation light. In some embodiments of the present invention, the fluorescence excitation module 13-3-1 includes three sets of ultraviolet LED light sources and ultraviolet filters, which can provide excitation light of specific wavelengths with relatively concentrated energy. The excitation light passes through the ultraviolet filters. Converging and irradiating on the middle area of the coating site, the fluorescent molecules in the area are excited, and the excited light is received by the signal detection module 13-3-2 to complete the conversion and collection of the optical-electrical-digital signal of the site fluorescence. , and then complete the signal collection of each coating site in sequence. Preferably, the signal detection module 13-3-2 includes a half-mirror, a narrow-band filter and a signal detector. 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 respectively 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 photoelectric detectors can be selected as needed.

In some embodiments of the present invention, the acquisition method of the signal acquisition module 13 is to scan the biological array frame by frame to ensure the accuracy of the fluorescence intensity value in a single reaction area.

In the present invention, please refer to Figure 5. The analysis equipment also includes an instrument housing, a mechanical skeleton 14, an electronic touch panel, an embedded control module, a power module and an operation module. The mechanical skeleton 14 is arranged in the analysis housing; the electronic touch panel, The embedded control module and the operating module are both arranged on the mechanical skeleton 14, and the electronic touch panel, operating module and signal acquisition module are all connected to the power module through the embedded control module; the embedded control module includes a main control board and a control module. The circuit is used to control the coordinated movement of the electronic touch panel and the operating module. It is also used to carry the operating software and perform data transmission and processing. The operation module includes a substrate movement sub-module 15-1, a piston drive sub-module 15-2 and a clamp sub-module 15-3. The substrate movement sub-module 15-1 is used to manipulate the movement of the protein marker substrate 2, and the piston drive sub-module 15- 2 is used to control the up and down movement of the driving piston 4-1 to send the sample liquid in the sample pool 3 into the reaction pool 5 through pressure changes, and the card clip module 15-3 is used to fix the microfluidic chip.

In the present invention, the base moving sub-module 15-1 includes an up and down movement motor 15-1-1, a left and right movement motor 15-1-2, a gripper motor 15-1-3, a mechanical gripper 15-1-4 and Guide rail, the mechanical gripper 15-1-4 is driven by the gripper motor 15-1-3 to grasp the protein marker substrate 2, and the up and down movement motor 15-1-1 and the left and right movement motor 15-1-2 are used for control respectively. The mechanical gripper 15-1-4 moves in the up and down and left and right directions.

In some embodiments of the present invention, please refer to Figure 7. The output shaft of the gripper motor 15-1-3 is connected to the mechanical gripper 15-1-4 and connected to the up and down movement motor 15-1-1 and the left and right movement motor through a nut. The output shaft of 15-1-2 is fixed.

The output shafts of the up and down movement motor 15-1-1 and the left and right movement motor 15-1-2 are respectively connected to a module composed of a gripper motor 15-1-3 and a mechanical gripper 15-1-4 for controlling the module. Movement to realize the movement of the gripper on the chip and the cleaning and reaction actions. The gripper motor is used to control the clamping piece of the mechanical gripper to complete the gripping action of the protein marker substrate 2. Its output shaft is connected to the gears on the mechanical gripper 15-1-4 respectively, and the mechanical gripper 15- is controlled through the gears. 1-4 moves on the guide rail to grab and fix.

In some embodiments of the present invention, please refer to Figure 8. The clamp module 15-3 is used to fix the position of the microfluidic chip, including a clamp motor 15-3-3 and a clamp motor 15-3-3. The driven clamp guide rail 15-3-2, the fixed card holder 15-3-1 and the temperature control device. The fixed card holder 15-3-1 is located at the bottom of the mechanical skeleton 14, and the temperature control device is used to control the environment during the reaction process. temperature, the fixed card holder 15-3-1 is connected to the external card inlet of the analysis housing through the clamp guide rail 15-3-2. Driven by the clamp motor 15-3-3, the fixed card holder 15-3-1 equipped with the microfluidic chip is moved to the preset position, and then the subsequent steps are performed.

The piston drive sub-module 15-2 is located above the inside of the external bayonet of the analysis housing, and includes a push rod 15-2-1, a push motor 15-2-2, a push guide rail 15-2-3, and a push motor 15-2 The output shaft of -2 is connected to the push rod 15-2-1 by a nut, which can drive the push rod 15-2-1 to move up and down. The driving piston 4-1 is connected to the push rod 15-2-1, and the push rod 15- The up and down movement of 2-1 can drive the piston 4-1 to move up and down in the piston driving pool 4-2.

In some embodiments of the present invention, please refer to Figure 5, the substrate moving sub-module 15-1 is located above the card clip sub-module 15-3, and the piston driving sub-module 15-2 is located above the inside of the card inlet. When the microfluidic control The chip is moved to a fixed position through the card clip module 15-3. The piston driving sub-module 15-2 takes the lead in operating 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 above three sub-modules are connected to independent motors to provide driving force for the operation of the module and achieve precise motion control. They are also connected to the electronic touch panel through the main control board and the power module to control the coordinated operation of each module. .

In some embodiments of the present invention, operators can complete human-computer interaction functions, instruction transmission, data analysis, result presentation, data storage and other functions through the main control board, and realize data processing through built-in GRPS, Bluetooth, LAN, USB and other ports. input and output.

When using the fluorescence immunoassay system provided in the previous embodiment for detection, the following steps are included:

S1. Add 200uL sample into the cavity of sample pool 3, cover the sample cover 3-1, put the microfluidic chip into the fixed card holder 15-3-1, start the equipment, and the microfluidic chip enters the fixed position;

S2. Sample injection and dilution: The analysis equipment presses the driving piston 4-1 on the microfluidic chip. The sample in the sample pool 3 passes through the filter pool 9, enters the first reaction pool 5-1, and is mixed with the buffer therein. uniform;

S3. Marker capture: The analysis equipment moves the coated carrier into the first reaction pool 5-1, and the coated antibody pre-coated on the coated carrier reacts with the sample in the reaction pool, incubates for 5 minutes, and stops the reaction;

S4. Cleaning: The analysis equipment moves the coated carrier into the first cleaning tank 6-1 and the second cleaning tank 6-2 in sequence, and washes away the residual reagents on the surface;

S5. Fluorescent marker capture: The analysis equipment moves the coated carrier into the second reaction pool 5-2, and the coated carrier is coated with the antibody-antigen complex to be detected or with the fluorescently labeled detection antibody in the reaction pool 5-2 or Antigen reacts;

S6. Cleaning: The analysis equipment moves the coated carrier into the third cleaning tank 6-3 and the fourth cleaning tank 6-4 in sequence, and washes away the residual reagent;

S7. Signal collection: The analysis equipment moves the coated carrier into the detection area 7, and the detection instrument excites the coated carrier - the coated antibody on the coating site - the antigen to be detected - the fluorescently labeled antibody, and captures the excited fluorescence signal;

S8. Result analysis: 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 in the above embodiments, the sandwich method among the immunoassay methods was selected. In other embodiments of the present invention, the sandwich method, competition method or other analysis methods based on fluorescence immunoassay can be selected and used according to the molecular characteristics of the markers to be tested. .

In some embodiments of the present invention, the control software of the instrument includes embedded software and host computer software. The embedded software is used to independently control each part of the instrument, and the host computer software is used to complete the embedding of the instrument through a computer. The control of the system and the writing of instrument data are connected through wireless or wired connections.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features can be equivalently replaced; and these modifications or substitutions do not deviate from the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention. scope.

Claims (9)

1. A fluorescence immunoassay system, characterized by comprising a microfluidic chip and analysis equipment; The microfluidic chip includes a chip housing (1) and a protein marker substrate (2). The protein marker substrate (2) includes a coating carrier (2-1) and a coating carrier (2-1). ) on multiple coating sites (2-2) as the reaction substrate for multi-index joint detection; The chip housing (1) is sequentially provided with a sample pool (3), a driving piston (4-1), a piston driving pool (4-2), a reaction pool (5), a cleaning pool (6) and a detection area ( 7), the upper end of the sample pool (3) is connected to the piston driving pool (4-2), and the lower end is connected to the reaction pool (5) through the microfluidic structure; the driving piston (4-1) can be in the piston driving pool (4-2). 4-2) moves up and down; the cleaning pool (6) is used to clean the residual reagents after the reaction; the analysis equipment includes a signal acquisition module (13) and an embedded control module, and the signal acquisition module (13) and the embedded The control module is connected, the signal acquisition module is used to collect fluorescence signals from the coated site (2-2) after reaction in the detection area (7), and the embedded control module is used to control the signal acquisition module (13) The work is to process the signals collected by the signal acquisition module (13) and calculate the concentration of the antigen to be detected in the sample to be detected; The analysis equipment also includes an instrument shell, a mechanical skeleton (14), an electronic touch panel, a power module and an operation module, The mechanical skeleton (14) is arranged in the instrument casing; The electronic touch panel, embedded control module and operating module are all arranged on the mechanical skeleton (14), and the electronic touch panel, operating module and signal acquisition module (13) are all connected to the power module through the embedded control module; The operation module includes the substrate movement submodule (15-1), the piston drive submodule (15-2) and the clamp submodule (15-3). The substrate movement submodule (15-1) is used to manipulate the protein marker substrate (2 ), the piston drive sub-module (15-2) is used to control the up and down movement of the drive piston (4-1) to send the sample in the sample pool (3) into the reaction pool (5) through pressure changes, and the clamp sub-module (15-3) is used to fix the microfluidic chip. 2. A fluorescence immunoassay system according to claim 1, characterized in that the coating sites (2-2) of the protein marker substrate (2) are evenly distributed on the coating carrier (2-1) Each coating site (2-2) is pre-coated with antibodies that can specifically bind to different detection markers. 3. A fluorescence immunoassay system according to claim 1, characterized in that the sample pool (3) includes a sample cover (3-1) and a cavity, The sample cover (3-1) is located above the sample cell and is connected to the bottom of the piston driving cell (4-2) through the first microfluidic channel (11). The bottom end of the cavity is connected to the reaction cell (5) through the microfluidic structure. . 4. A fluorescence immunoassay system according to claim 1, characterized in that the microfluidic structure includes a second microfluidic channel (8) and a filter pool (8) connected to the second microfluidic channel (8). 9), one end of the second microfluidic channel (8) is connected to the bottom end of the cavity, the filter pool (9) is connected to the reaction pool (5), and a blood filtration membrane is provided in the filter pool (9). 5. A fluorescence immunoassay system according to claim 1, characterized in that the reaction pool (5) includes a first reaction pool (5-1) and a second reaction pool (5-2), and the first reaction pool (5-1) is used to place the buffer solution, and the second reaction pool (5-2) is used to place the fluorescently labeled detection antibody solution in the first reaction pool (5-1) and the second reaction pool (5-2) , a cleaning pool (6) is provided between the second reaction pool (5-2) and the detection area (7). 6. A fluorescence immunoassay system according to claim 1, characterized in that the signal acquisition module (13) includes a signal acquisition unit, and the signal acquisition unit includes a fluorescence excitation module (13-3-1) and signal detection module (13-3-2), the fluorescence excitation module (13-3-1) is used to excite fluorescent molecules, and the signal detection module (13-3-2) is used to receive excitation light. 7. A fluorescence immunoassay system according to claim 1, characterized in that the substrate moving sub-module includes a moving motor and a guide rail, a gripper motor and a mechanical gripper controlled by the gripper motor for controlling the machinery. The gripper moves up and down and grasps the protein marker substrate. 8. A fluorescence immunoassay system according to claim 1, characterized in that the piston driving sub-module includes a push motor (15-2-2) and is driven up and down by the push motor (15-2-2). The moving push rod (15-2-1), the driving piston (4-1) is connected with the push rod (15-2-1). 9. A fluorescence immunoassay system according to claim 1, characterized in that the clamp sub-module is located below the base moving sub-module and includes a fixed card holder, a clamp motor and a clamp guide rail, and the fixed card holder passes through the card. The clamp guide rail is connected to the external bayonet opening of the analysis housing.