Microfluidics-based control system and method for multi-channel treatment of biological sample

Abstract

The invention discloses a control system and method for multi-channel treatment of a biological sample. A capture antibody layer of an optical fiber SPR sensor and a real-time detection biological solution can be prepared; comprising a microfluidic module, a sensing module and a computer, the sensing module is used for monitoring resonance wavelength information of a biological solution flowing through a sensing area of the optical fiber SPR sensor in real time; the micro-fluidic module is used for enabling the solution in the corresponding liquid storage tank to flow through the outer surface of the optical fiber SPR sensor in the micro-fluidic chip through the micro-fluidic channel under the set pressure; and the computer is used for detecting the corresponding content of the biological solution in the current liquid storage tank by measuring the resonant wavelength and comparing the resonant wavelength with pre-stored data based on the received electric signal of the spectrum. According to the control system, a micro-fluidic chip is used as a container for accommodating a sample to be detected or prepared, and a biological solution slowly flows into the micro-fluidic chip through a micro-fluid channel so as to realize preparation of a capture antibody layer or real-time detection of the biological solution.

Description

Technical Field

[0001] The invention belongs to the field of biomedical optical detection, and in particular relates to a control system and method for multi-channel processing of biological samples based on microfluidics technology and optical fiber SPR sensors. Background Art

[0002] Microfluidics is an emerging technology that controls and configures the flow of a small amount of fluid in a microscale channel. It was originally developed in the Mathies Laboratory at the University of California, Berkeley (UCBerkeley) (US20040209354A1), and can control the fluid flow rate in an operating volume from 20nL to 10L. In microfluidics, as the size of the fluid is reduced to the micron level, the specific surface area of ​​the fluid increases, making it show the advantage of viscous force over inertial force, more efficient mass transfer and heat transfer behavior, and significant surface effects relative to macroscopic fluids.

[0003] The surface plasmon resonance (SPR) phenomenon refers to a collective resonance phenomenon caused by electromagnetic waves exciting free electrons in materials. SPR is very sensitive to changes in the dielectric constant of the local environment, so it is often used to detect changes in liquid concentration and real-time monitoring of the adsorption and binding of biomolecules. The fiber-based SPR effect can be regarded as a nonlinear multiple integral of the prism-based SPR effect at the incident angle and incident plane. Fiber-optic SPR sensors have the advantages of anti-electromagnetic interference, corrosion resistance, small size, and strong flexibility. They can be easily formed into sensor networks through cascading or other methods, thereby enabling real-time, online, remote and multi-parameter detection of target analytes. Therefore, the research on fiber-optic SPR sensors has made significant progress in the field of biochemical sensing.

[0004] The fiber optic surface plasmon resonance sensing technology based on microfluidic technology uses microfluidic technology to reduce the amount of liquid required and allow it to flow into the sensing area stably. Compared with traditional static liquid detection, this technology has a faster detection speed. In addition, microfluidics can continuously pass a variety of liquids, which can achieve real-time, rapid and continuous monitoring of the liquid being tested. In addition, part of the production process of the fiber optic sensor can be integrated into the operation of the microfluidic system, so that the sensor preparation and the test liquid detection can be integrated, making the experiment more continuous and rapid. Summary of the invention

[0005] The present invention combines microfluidics with optical fiber SPR sensors to provide a control system and method for multi-channel biological sample processing based on microfluidics. The control system includes a microfluidics chip and its flow fluid channels and gas channels, so that the biological sample flows through the optical fiber SPR sensor through the microchannels; the control system can be used to prepare the capture antibody layer of the optical fiber SPR sensor and detect biological solutions in real time.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] A control system for multi-channel processing of biological samples, capable of preparing a capture antibody layer of an optical fiber SPR sensor and detecting biological solutions in real time; comprising a microfluidic module, a sensor module and a computer;

[0008] The sensing module includes an optical fiber SPR sensor, a light source and a spectrum analyzer, two ends of the optical fiber SPR sensor are respectively connected to the light source and the spectrum analyzer through optical fiber jumpers, and the spectrum analyzer is electrically connected to a computer;

[0009] The microfluidic module includes a microfluidic channel, a gas channel, a microfluidic mainboard and a microfluidic chip; one end of the microfluidic channel is connected to a liquid reservoir, and the other end is connected to a microfluidic chip, and a conversion valve is provided on the microfluidic channel, which is controlled by the microfluidic mainboard to open the channel between the corresponding liquid reservoir and the microfluidic chip;

[0010] The microfluidic chip comprises a groove, a liquid inlet and a liquid outlet, wherein the liquid inlet and the liquid outlet are connected to the groove, and the groove is used to accommodate the optical fiber SPR sensor and the biological solution flowing therethrough; the liquid inlet is connected to a microfluidic channel, and the liquid outlet is connected to a waste liquid tank through a pipeline;

[0011] The gas channel is provided with a pressure pump and a gas distributor, wherein the gas distributor is a multi-channel distributor, one end of which is connected to the pressure pump, and the multi-channels at the other end are respectively connected to the liquid reservoirs, and are used to input gas into the liquid reservoirs according to the pressure pump; the pressure pump is electrically connected to the microfluidic mainboard, and is controlled by the microfluidic mainboard, and the flow rate of the microfluidic channel can be controlled by the pressure pump;

[0012] The sensing module is used to monitor the resonance wavelength information of the biological solution flowing through the sensing area of ​​the optical fiber SPR sensor in real time;

[0013] The microfluidic module is used to make the solution in the corresponding liquid reservoir flow through the outer surface of the optical fiber SPR sensor in the microfluidic chip through the microfluidic channel under a set pressure;

[0014] The computer is used to detect the corresponding content in the biological solution in the current storage tank based on the received spectral electrical signal by measuring the resonance wavelength and comparing it with pre-stored data.

[0015] Furthermore, when the control system is used to prepare a capture antibody layer, an antibody solution is placed in at least one of the liquid reservoirs, and at least a portion of the outer surface of the optical fiber SPR sensor is a noble metal layer; the antibody solution flows from the liquid reservoir into the groove of the microfluidic chip in the microfluidic channel at a speed of 0.1-2000ul / min, so that the optical fiber SPR sensor is placed in the antibody solution as a whole for a period of time, thereby depositing a capture antibody layer on the upper surface of the noble metal layer of the optical fiber SPR sensor;

[0016] When the control system is used for real-time detection of biological solutions, biological solutions of different concentrations are placed in each of the liquid reservoirs, and at least part of the outer surface of the optical fiber SPR sensor is a capture antibody layer, and the capture antibody layer is combined with the antigen of the biological solution to be detected; the biological solution in the fluid channel flows from the liquid reservoir to the microfluidic chip at a speed of 0.1-2000ul / min, and contacts with the capture antibody layer of the optical fiber SPR sensor, and the spectrum analyzer receives the light signal and converts it into an electrical signal, and then transmits the spectrum signal to the computer; when the resonance valley of the spectrum signal is stable, the computer stores the data of the resonance valley, and detects the corresponding content in the biological solution in the current liquid reservoir by measuring the resonance wavelength and comparing it with the pre-stored data.

[0017] In one embodiment, the optical fiber SPR sensor uses a single-mode optical fiber as a substrate, and a noble metal layer is plated on the outer surface of the single-mode optical fiber.

[0018] In one embodiment, the microfluidic channel is further provided with a flow meter between the conversion valve and the microfluidic chip.

[0019] Furthermore, an air pressure regulating valve is provided on the gas channel, the air inlet end of the air pressure regulating valve is connected to the air source, and the air outlet end is connected to the pressure pump, which is used to adjust the pressure in the gas channel so that the air pressure in the gas channel is stabilized at 1000mbar-1200mbar.

[0020] In one embodiment, the microfluidic channel includes a conversion valve, a microfluidic chip and a waste liquid pool connected in sequence through pipelines; the microfluidic chip is used to install a fiber optic SPR sensor; the conversion valve is a multi-channel conversion valve, which is controlled by a microfluidic mainboard to open the channel between the corresponding liquid storage tank and the microfluidic chip; the microfluidic mainboard is used to control the conversion valve and the pressure pump according to the instructions of the computer; and the microfluidic mainboard is powered by an external power supply.

[0021] In one embodiment, the two ends of the fiber optic SPR sensor are respectively connected to a light source and a spectrum analyzer through fiber optic jumpers, and the spectrum analyzer is electrically connected to a computer, and the computer processes the received spectral signal to detect the antigen solution in the reservoir.

[0022] In one embodiment, the gas channel includes a gas source, a gas pressure regulating valve, a pressure pump, a gas distributor and a liquid storage tank which are sequentially connected through a gas path, and the gas distributor is a multi-channel distributor, whose channels are respectively connected to each liquid storage tank, and is used to input the gas from the pressure pump into each liquid storage tank;

[0023] The pressure pump is used to form pressure in the microfluidic channel in a direction away from the liquid reservoir, so that the solution in the liquid reservoir flows toward the microfluidic chip under the action of pressure.

[0024] In one embodiment, a method for preparing a capture antibody layer of an optical fiber SPR sensor using the control system comprises:

[0025] S1: Multiple reservoirs contain different biological solutions, one of which is empty, and the fiber-optic SPR sensor to be prepared is installed in the microfluidic chip;

[0026] S2: The microfluidic mainboard controls the conversion valve and the pressure pump to control the opening of the channel of the liquid reservoir corresponding to the required biological solution, and the biological solution flows into the microfluidic chip equipped with the optical fiber SPR sensor through the microfluidic channel at a set constant flow rate / constant pressure;

[0027] S3: allowing the biological solution in the microfluidic chip to cover the surface of the optical fiber SPR sensor, and incubating at 3-6° C. for 6 to 8 hours, so that the capture antibody layer is deposited on the upper surface of the noble metal layer of the optical fiber SPR sensor;

[0028] The conversion valve is turned to open the microfluidic channel between the empty liquid storage tank and the microfluidic chip, and the pressure pump is turned on to introduce nitrogen into the microfluidic chip, so that the solution in the microfluidic chip flows into the waste liquid tank;

[0029] S4: Then, according to the set order, the conversion valve is controlled to open the pipelines between other liquid storage tanks and the microfluidic chip in turn, and steps S2 and S3 are executed, so that the remaining organisms are coated with the optical fiber SPR sensor in turn until the preparation of the required capture antibody layer is completed; S5: After taking out the optical fiber SPR sensor, the unfixed biological solution is rinsed with phosphate buffer solution, and it is dried for later use to form a capture antibody layer.

[0030] In one embodiment, a method for real-time detection of a biological solution using the control system includes:

[0031] S1: The liquid reservoirs contain biological solutions and detergent solutions of different concentrations, and one of the liquid reservoirs is empty; the optical fiber SPR sensor is installed in the microfluidic chip, and the capture antibody layer of the optical fiber SPR sensor can be combined with the biological solution to be detected;

[0032] S2: The microfluidic mainboard controls the conversion valve and the pressure pump to control the opening of the channel of the liquid reservoir corresponding to the required biological solution, and the biological solution flows into the microfluidic chip equipped with the optical fiber SPR sensor through the microfluidic channel at a set constant flow rate / constant pressure;

[0033] S3: The biological solution in the microfluidic chip covers the outer surface of the optical fiber SPR sensor, and the biological solution flows from the liquid reservoir through the microfluidic channel at a speed of 0.1-2000ul / min through the surface of the optical fiber SPR sensor on the microfluidic chip, and the spectrum analyzer receives light, converts the optical signal into an electrical signal, and transmits the spectrum signal to the computer; when the resonance valley of the spectrum signal is stable, the computer stores the data of the resonance valley, and detects the corresponding content in the biological solution in the current liquid reservoir by measuring the resonance wavelength and comparing it with the pre-stored data;

[0034] S4: Then, according to the set sequence, the conversion valve is controlled to open the pipelines between other liquid storage tanks and the microfluidic chip in turn, and steps S2 and S3 are executed, so as to obtain the detection of the content of biological solutions with different concentrations respectively.

[0035] Furthermore, the obtained spectral data is post-processed, including zero-phase filtering of the spectral data and fitting of the smoothed data using an asymmetric double S function; the computer calculates the antigen concentration of the current biological solution based on the linear relationship between the fitted resonance wavelength and the antigen concentration.

[0036] Furthermore, after the biological solution covers the outer surface of the optical fiber SPR sensor in step S3 of the method for real-time detection of the biological solution using the control system, the method further includes:

[0037] The conversion valve is turned to open the microfluidic channel between the empty liquid storage tank and the microfluidic chip, and the pressure pump is turned on to introduce nitrogen into the microfluidic chip, so that the solution in the microfluidic chip flows into the waste liquid tank;

[0038] The switching valve is turned to open the microfluidic channel between the liquid storage tank filled with detergent solution and the microfluidic chip to clean the microfluidic channel and the microfluidic chip. Then, the switching valve is turned again to open the pressure pump to introduce nitrogen into the microfluidic chip, so that the detergent solution in the microfluidic chip flows into the waste liquid tank.

[0039] Furthermore, the height of the groove in the microfluidic chip is higher than the height of the optical fiber SPR sensor, so that the optical fiber SPR sensor can be immersed in the biological solution.

[0040] The beneficial effects of the present invention are:

[0041] The control system uses a microfluidic chip as a container for holding samples to be tested or prepared, and introduces a pressurized biological solution through a microfluidic channel, so that the biological solution remains in the microfluidic chip or slowly flows through the microfluidic chip, so that samples of extremely small volumes can be effectively tested;

[0042] At the same time, the control system described in the present invention can also realize the two functions of preparing the capture antibody layer of the optical fiber SPR sensor and real-time detection of the biological solution; it can prepare the required capture antibody layer on the surface of the existing optical fiber SPR sensor with a precious metal layer on the surface, and can also use the optical fiber SPR sensor with a capture antibody layer coated on the surface to realize continuous injection of different liquids and continuous monitoring of the resonance wavelength. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a schematic diagram of the structure of the control system described in Example 1 of the present invention. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solution, beneficial effects and significant progress of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in conjunction with the drawings provided in the examples of the present invention. Obviously, all the described embodiments are only partial embodiments of the present invention, rather than all embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0045] In the description of this application, unless otherwise clearly specified and limited, the terms "first", "second", and "third" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance; the term "plurality" refers to two or more; unless otherwise specified or explained, the terms "connected", "fixed", etc. should be understood in a broad sense, for example, "connected" can be a fixed connection, a detachable connection, an integral connection, or an electrical connection; "connected" can be a direct connection or an indirect connection through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0046] Example 1

[0047] like Figure 1 As shown, a control system for multi-channel processing of biological samples based on microfluidic technology can prepare the capture antibody layer of the optical fiber SPR sensor; it includes a microfluidic module, a sensor module, an electrical module and an inlet and outlet liquid module.

[0048] The inlet and outlet liquid module is composed of ten 15ml liquid storage tanks and one 50ml waste liquid tank. The waste liquid tank is connected to the microfluidic chip of the sensor module to receive waste liquid from the preparation of the optical fiber SPR sensor; each of the liquid storage tanks contains different biological solutions for preparing the capture antibody layer. The ten liquid storage tanks are respectively connected to the microfluidic subsystem and the gas subsystem to ensure the control of the inlet and outlet liquids by the pressure pump.

[0049] The sensing module includes a light source, a spectrum analyzer, a fiber optic SPR sensor to be prepared and a Y-type fiber optic patch cord; the light source is a wide-spectrum halogen lamp light source with a wavelength range of 360nm-2400nm; the wavelength range of the spectrum analyzer is: 165nm-1100nm, and the spectral resolution is: 0.75nm-1.12nm; the upper surface of the fiber optic SPR sensor is plated with a precious metal layer, and the bare fiber core diameter of the fiber optic SPR sensor is 600um.

[0050] Specifically, referring to the near-waveguide fiber SPR probe structure disclosed in CN114441481A, the optical fiber SPR sensor includes a second precious metal layer, a dielectric layer and a first precious metal layer in sequence from the core end face of the multimode optical fiber to the outside, the dielectric layer is uniformly doped with nano-metal particles, the first precious metal layer is solidified on the outer surface of the dielectric layer, and can stimulate the SPR effect; and the first precious metal layer is a gold film.

[0051] The microfluidic module specifically includes a gas subsystem and a microfluidic subsystem.

[0052] The microfluidic subsystem includes a microfluidic channel, a pressure pump, a conversion valve, a flow meter, a microfluidic chip and a microfluidic mainboard. The microfluidic subsystem supports two modes of driving: constant flow rate and constant air pressure, and its operating range is 0-2000ul / min and 0-1000mbar.

[0053] The pressure pump has an optional pressure range of -800mbar to 1000mbar, and can realize both positive and negative pressure inlet and outlet of liquid. When using the negative pressure method, it can prevent the problem of unsmooth positive pressure injection caused by residual air in the liquid inlet pipe; pressure resolution: 0.03% of the range; pressure stability: 0.1%; number of channels: 10; response time: <40ms; working temperature: 5-40℃. Moreover, the pressure pump can realize two injection methods: 0-2000ul / min constant flow and -800mbar to 1000mbar constant pressure.

[0054] Switching valve: 10-channel electric valve; switching time: <400ms; dead volume: 0;

[0055] Flowmeter: sensor inner diameter: 430um; maximum pressure resistance: 100bar; range: 0±500uL / min; accuracy: flow rate at 2.4uL / min-25uL / min: 5%, flow rate above 25uL / min: 20%, and the detection range of the flowmeter is 0-2ml. The flowmeter can feed back the specific flow rate to the microfluidic mainboard in real time, so as to achieve stable control and real-time monitoring of the flow rate.

[0056] The microfluidic chip includes a groove, a liquid inlet and a liquid outlet, the liquid inlet and the liquid outlet are connected to the groove, and the groove is used to accommodate the optical fiber SPR sensor and the biological solution flowing through, and the height of the groove in the microfluidic chip is higher than the height of the optical fiber SPR sensor, so that the optical fiber SPR sensor can be immersed in the biological solution. The liquid inlet is connected to the microfluidic channel, and the liquid outlet is connected to the waste liquid pool through a pipeline. The internal dimensions of the chip: the inner diameter of the reaction pool: 1.5mm; the length of the reaction pool: 15mm; the thread specification of the optical fiber / inlet and outlet liquid interface: 1 / 4-28UNF; the diameter of the liquid pipeline: 1 / 16 inch; the external dimensions of the chip: length: 39mm; width: 39mm; height: 50mm.

[0057] Fluid channel: a conversion valve, a flow meter, a microfluidic chip, a fiber optic SPR sensor and a waste liquid tank connected in sequence through pipelines, the liquid storage tank is connected to the conversion valve through a pipeline, the other end of the conversion valve is connected to the liquid inlet of the microfluidic chip through a pipeline, and the fiber optic SPR sensor is placed in the microfluidic chip, the liquid outlet of the microfluidic chip is connected to the waste liquid tank by a pipeline, and the waste liquid is transported to the waste liquid tank; and a flow meter is arranged on the pipeline between the conversion valve and the microfluidic chip.

[0058] After the optical fiber SPR sensor is connected to the microfluidic chip, under the condition of stable air pressure and good air tightness, the microfluidic channel connected to the conversion valve is controlled by the microfluidic mainboard, and the biological solution in the liquid reservoir passes through the conversion valve at a set flow rate / under a set pressure and enters the flow meter to detect the flow rate of the biological solution in real time. Finally, the biological solution enters the microfluidic chip and contacts the optical fiber sensor, causing the loss valley of the receiving spectrum to shift, and finally flows into the waste liquid pool.

[0059] The gas subsystem comprises a gas channel, a gas source, a gas pressure regulating valve and a gas path distributor, and the gas distributor is a ten-channel distributor.

[0060] The electrical module includes a power supply, a keyboard, a display and a computer. The power supply supplies power to the computer, the microfluidic mainboard in the microfluidic subsystem, and the light source in the sensor module to ensure stable power supply for the entire instrument. The keyboard and the display are electrically connected to the computer respectively. After the computer performs wavelet transform denoising and polynomial fitting on the received spectral signal, the processed signal is presented through the display.

[0061] Gas channel: The outlet of the gas source is connected to one end of the gas pressure regulating valve through a pipeline, the other end of the gas pressure regulating valve is connected to the pressure pump, the other end of the pressure pump is connected to the gas distributor, and the gas distributor is connected to ten liquid storage tanks respectively; the nitrogen from the gas source is input into the pressure pump after the pressure is adjusted to 1000-1200mbar by the gas pressure regulating valve, and then the gas is changed from one gas path to ten gas paths through the gas path distributor, and then connected to the ten liquid storage tanks respectively, and the gas is input into each liquid storage tank.

[0062] The control system also includes circuits and microfluidic instructions.

[0063] Circuit and microfluidic instructions: The keyboard and display are electrically connected to the computer respectively, the computer is electrically connected to the microfluidic mainboard and the spectrometer respectively, and the microfluidic mainboard is electrically connected to the conversion valve and the pressure pump respectively; the user uses the keyboard to issue instructions, and the computer transmits the instructions to the microfluidic mainboard, thereby controlling the conversion valve and the pressure pump to control the flow of the liquid and the pressure in the microfluidic channel.

[0064] The method for preparing a capture antibody layer of an optical fiber SPR sensor using the control system comprises:

[0065] S1. Place nine biological solutions in nine reservoirs respectively, leaving one empty reservoir, and install the fiber optic SPR sensor to be prepared inside the microfluidic chip; turn on the power, gas source and light source switches, turn the air pressure regulating valve and observe the change in the air pressure value until the air pressure stabilizes at 1000mbar-1200mbar and then stop turning the air pressure regulating valve; wherein, the ten antibody solutions include: dopamine solution prepared by Tris (2mg mL-1, pH=8.5); dopamine capture antibody (Cab) solution with a concentration of 2μgmL-1 prepared by PBS; phosphate buffered saline (PBS) solution; 1mg mL-1 bovine serum albumin (BSA) solution;

[0066] Controlling the liquid level in the microfluidic chip to cover the upper surface of the optical fiber SPR sensor;

[0067] S2. Using the driver of the microfluidic subsystem, manually or programmatically brake the conversion valve and the pressure pump in real time to control the opening of the channel of the liquid reservoir corresponding to the required biological solution, so that the required biological solution flows from its liquid reservoir through the pipeline into the microfluidic chip equipped with the optical fiber SPR sensor according to the set constant pressure, and contacts with the optical fiber SPR sensor; at this time, the flow meter monitors the flow rate flowing into the chip on the pipeline, and the flow meter feeds back the real-time flow rate to the microfluidic main board, so as to obtain the real-time flow rate and air pressure value;

[0068] The microfluidic mainboard controls the conversion valve to open the channel between the liquid reservoir containing the dopamine solution and the microfluidic chip, and starts the pressure pump to allow nitrogen to flow into the liquid reservoir through the gas path distributor. When the liquid flows all over the microfluidic chip, the pressure pump is turned off, and the optical fiber SPR sensor is immersed in the dopamine solution (2 mg mL-1, pH=8.5) prepared by Tris for 2 hours. Under alkaline conditions, the self-polymerization of dopamine can form a polydopamine (PDA) layer with catechol quinone active functional groups;

[0069] The conversion valve is turned to open the microfluidic channel between the empty liquid storage tank filled with nitrogen and without liquid and the microfluidic chip, and the pressure pump is turned on to introduce nitrogen into the microfluidic chip through the microfluidic channel, so that the solution in the microfluidic chip flows into the waste liquid tank;

[0070] According to the above working process, the switching valve is continuously controlled to switch the channel, so that the channel between the reservoir containing the CAb solution and the microfluidic chip is opened, the pressure pump is turned on, and the CAb solution with a concentration of 2 μg mL-1 prepared by PBS is introduced until the liquid fills the microfluidic chip, the pressure pump is turned off, and it is left to stand at 4°C for 24 hours, so that the capture antibody is deposited on the surface of the dopamine layer of the optical fiber SPR sensor; then the switching valve is turned to open the microfluidic channel between the empty reservoir and the microfluidic chip, and the pressure pump is turned on to introduce nitrogen into the microfluidic chip, so that the solution in the microfluidic chip flows into the waste liquid tank;

[0071] Afterwards, the switching valve was controlled to switch the channel, and PBS solution was introduced for 60 seconds to flush and remove the free Cab, and then the switching valve was turned to open the microfluidic channel between the empty liquid reservoir and the microfluidic chip, and the pressure pump was turned on to introduce nitrogen into the microfluidic chip, so that the solution in the microfluidic chip flowed into the waste liquid tank;

[0072] Then control the switching valve to switch the channel and pass the concentration of 1 mg mL -1 BSA solution was added for 2 minutes to block nonspecific binding sites; then the conversion valve was turned to open the microfluidic channel between the empty liquid reservoir and the microfluidic chip, and the pressure pump was turned on to introduce nitrogen into the microfluidic chip, so that the solution in the microfluidic chip flowed into the waste liquid pool.

[0073] After taking out the optical fiber SPR sensor, the unfixed solution was washed away with a phosphate buffer solution, and the sensor was dried for later use, thereby forming an optical fiber SPR sensor coated with a capture antibody layer.

[0074] Example 2

[0075] This embodiment discloses a control system for multi-channel processing of biological samples based on microfluidic technology. The control system is basically similar to Example 1, and the same contents are not repeated here. The only difference is that each of the liquid reservoirs contains antigen solutions of different concentrations, and the upper surface of the optical fiber SPR sensor in the sensing module is coated with a capture antibody layer, and the capture antibody layer is used to detect the antigen solution in the liquid reservoir in real time; the control system also includes an optical path, an electric circuit and microfluidic instructions.

[0076] Optical path: The light source is connected to the input end of the optical fiber SPR sensor through a Y-type optical fiber jumper, and the output end of the optical fiber SPR sensor is connected to a spectrum analyzer through an optical fiber jumper to transmit the optical signal to the spectrum analyzer. The spectrum analyzer is connected to an electrical module to transmit data to the computer of the electrical module for data analysis.

[0077] Circuit and microfluidic instructions: the keyboard and the display are electrically connected to the computer respectively, the computer is electrically connected to the microfluidic mainboard and the spectrum analyzer respectively, and the microfluidic mainboard is electrically connected to the conversion valve and the pressure pump respectively; the user uses the keyboard to issue instructions, and the computer transmits the instructions to the microfluidic mainboard, thereby controlling the conversion valve and the pressure pump to control the flow of the liquid; the computer performs wavelet transform denoising and polynomial fitting on the spectral signal from the spectrum analyzer to obtain an image of the dynamic change of the resonance valley, and presents the processed image through the display to achieve real-time detection of the liquid to be tested.

[0078] Specific control methods include:

[0079] S1. Place eight different concentrations of human alpha-fetoprotein solutions and one alpha-fetoprotein detergent solution in nine reservoirs, leaving one empty reservoir, and wait for the fiber optic SPR sensor to be installed in the microfluidic chip; turn on the power, gas source and light source switches, adjust the air pressure regulating valve to stabilize the air pressure at 1000mbar-1200mbar, and stop turning the air pressure regulating valve;

[0080] S2. Using the driver of the microfluidic subsystem, manually or programmatically brake the conversion valve and the pressure pump in real time, control the opening of the channel of the first liquid reservoir, turn on the pressure pump, and allow the human alpha-fetoprotein solution in the first liquid reservoir to slowly flow from the liquid reservoir through the microfluidic channel into the microfluidic chip equipped with the optical fiber SPR sensor at a set constant flow rate, and contact the optical fiber SPR sensor; at this time, the flow meter monitors the flow rate flowing into the chip on the pipeline, and the flow meter feeds back the real-time flow rate to the microfluidic main board, so that the real-time flow rate and air pressure value can be obtained;

[0081] Under the action of pressure, the biological solution slowly flows through the capture antibody layer of the optical fiber SPR sensor, the optical fiber SPR sensor is placed in the biological solution, and the pressure pump is controlled to stop working;

[0082] After 1 hour, the switching valve is turned to open the microfluidic channel between the empty liquid reservoir and the microfluidic chip, and the pressure pump is turned on to introduce nitrogen into the microfluidic chip, so that the solution in the microfluidic chip flows into the waste liquid pool;

[0083] The switching valve is turned to open the microfluidic channel between the liquid reservoir containing the detergent solution and the microfluidic chip to clean the microfluidic channel and the microfluidic chip, and then the switching valve is turned again to open the pressure pump to introduce nitrogen into the microfluidic chip, so that the detergent solution in the microfluidic chip flows into the waste liquid tank;

[0084] S3. After the microfluidic subsystem is started, the spectrum analyzer is connected to an electrical module to transmit data to the computer of the electrical module for data analysis; the light of the light source is coupled into the optical fiber SPR sensor via one side of the optical fiber, and the resonance wavelength of the surface plasmon resonance (SPR) of the optical fiber SPR sensor is closely related to the dielectric constant and thickness of the metal film layer and the nanomaterial layer, the refractive index of the sensor surface surrounding environment and other parameters; therefore, the optical fiber SPR sensor is located in a chip where the liquid to be tested flows slowly, and the refractive index will change continuously during the liquid renewal process, or the liquid will cause the refractive index to change over time due to its own reaction, and the resonance wavelength will change, the spectrum analyzer receives the data and transmits it to the computer, when the resonance valley of a spectrum signal is stable, the computer stores the data of the resonance valley, and by measuring the resonance wavelength and comparing it with the data previously calibrated with the standard refractive index solution, the corresponding content in the solution in the first liquid storage tank can be detected.

[0085] S4. Then, according to the set sequence, the conversion valve is controlled to open the pipelines between other liquid storage tanks and the microfluidic chip in turn, and steps S2 and S3 are executed, so as to obtain the detection of the content of human alpha-fetoprotein solutions with different concentrations respectively.

[0086] S5. Post-process the resonance valley data obtained in S3 and S4, specifically including:

[0087] S51: The acquired spectral data includes wavelength and corresponding transmission coefficient; the data is subjected to zero-phase filtering to obtain a signal without phase distortion;

[0088] Since ordinary filters will introduce phase differences, if the signal is processed by zero-phase filtering, a signal without phase distortion can be obtained. The zero-phase filter is an offline filter. First, the signal needs to be passed through the filter in the forward direction, and then the obtained signal is passed through the filter in the reverse direction. This processing of the signal will produce a double amplitude attenuation, but no phase difference will be generated. The principle is as follows.

[0089] First, the signal is passed through a filter

[0090] y1(n)=x(n)·h(n)

[0091] The obtained signal y1(n) is inverted

[0092] y2(n)=y1(N-1-n)

[0093] The obtained signal y2(n) is then passed through the filter

[0094] y3(n)=y2(n)·h(n)

[0095] The obtained signal y3(n) is reversed and then output to obtain an output signal without phase difference.

[0096] y4(n)=y3(N-1-n)

[0097] Where y(n) is the zero-phase output signal, x(n) is the input signal, and h(n) is the digital filter impulse response sequence.

[0098] The above discusses the specific operation steps for obtaining the zero-phase filtered signal sequence. The following will explain the principles of the filtering process from a frequency domain perspective.

[0099] Y1(e jω )=X(e jω )H(e jω )

[0100] Y2(e jω )=e -jω(N-1) Y1(e -jω )

[0101] Y3(e jω )=Y2(e jω )H(e jω )

[0102] Y(e jω )=e -jω(N-1) Y3(e-jω )

[0103] In summary

[0104] Y(e jω )=e jω(N-1) ·H(e -jω )·e -jω(N-1) ·X(e jω )·H(e jω )

[0105] Y(e jω )=X(e jω )·H(e -jω )·H(e jω )

[0106] Y(e jω )=X(e jω )|H(e jω )| 2

[0107] That is, the output Y(e jω ) and input X(e jω ), there is no additional phase between them, that is, zero-phase filtering is achieved.

[0108] S52: Since the two sides of the fiber SPR resonance valley are usually asymmetric, the asymmetric double S function (Asymmetric Double Sigmoidal, Asym2sig) is used to fit the smoothed data until convergence. The accuracy of the resonance valley after fitting and the detection reliability of the wavelength are improved.

[0109] The asymmetric double S function is a commonly used function for parallel multi-step process signal deconvolution in dynamic mechanical analysis (DMA). The six parameters of the Asym2sig function describe the asymmetric curve. The algebraic expression of the Asym2sig function is written as:

[0110]

[0111] Where x is the independent variable; y0 is the constant term, which is the initial value of the Asym2sig function at x0; A is the peak intensity, which is the maximum amplitude of the curve, i.e. the maximum value; x p is the peak center; ω1 is the half-maximum full width; ω2 and ω3 are shape parameters, which are the low-energy side variance and high-energy side variance, respectively.

[0112] S6. The computer determines the severity of the disease corresponding to the human alpha-fetoprotein solution in each channel based on the preset antigen and disease correspondence standards.

[0113] The computer calculates the antigen concentration of the sample in the experiment based on the linear relationship between the resonant wavelength calibrated by the SPR sensor and the antigen concentration and the resonant wavelength during the experiment. The degree of liver cancer corresponding to the human alpha-fetoprotein solution in each channel is judged based on the alpha-fetoprotein concentration in clinical medicine and the corresponding standards for liver cancer judgment.

[0114] After the experiment is completed, the control system needs to be operated subsequently, including:

[0115] S7. After the experiment is completed, store the experimental data and pause the microfluidic subsystem. Replace the ten-way liquid reservoir with an empty liquid reservoir and install it. Manually control the microfluidic driver to blow air in, turn the conversion valve in turn, and pass gas into each channel at a constant pressure of 500mbar for 20s, so that the liquid in each channel is completely discharged into the waste liquid tank. Then pause the driver and remove the optical fiber sensor from the chip;

[0116] S8. Install ten reservoirs filled with enzyme cleaning agents into the inlet and outlet liquid modules, manually control and adjust the switching valves in sequence, and pass the cleaning liquid into each channel at a constant pressure of 500 mbar for 20 seconds, so that each channel is completely cleaned, and then the driver is suspended;

[0117] S9. ten liquid reservoirs filled with isopropyl tone are replaced in the liquid inlet and outlet modules, and the switching valves are adjusted in sequence by manual control. Isopropyl tone is introduced into each channel at a constant pressure of 500 mbar for 20 seconds, so that each channel is completely flushed, and then the driver is suspended;

[0118] S10. Replace ten empty liquid storage tanks into the liquid inlet and outlet modules, manually control and adjust the conversion valves in sequence, and introduce nitrogen into each channel at a constant pressure of 500 mbar for 20 seconds to make each channel dry, and finally end the driver program.

[0119] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode includes only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in the embodiments may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A control system for multi-channel processing of biological samples, characterized in that: It includes a microfluidic module, a sensor module and a computer; The sensing module includes an optical fiber SPR sensor, a light source and a spectrum analyzer, two ends of the optical fiber SPR sensor are respectively connected to the light source and the spectrum analyzer through optical fiber jumpers, and the spectrum analyzer is electrically connected to a computer; The microfluidic module includes a microfluidic channel, a gas channel, a microfluidic mainboard and a microfluidic chip; one end of the microfluidic channel is connected to a liquid reservoir, and the other end is connected to a microfluidic chip, and a conversion valve is provided on the microfluidic channel, which is controlled by the microfluidic mainboard to open the channel between the corresponding liquid reservoir and the microfluidic chip; The microfluidic chip comprises a groove, a liquid inlet and a liquid outlet, wherein the liquid inlet and the liquid outlet are connected to the groove, and the groove is used to accommodate the optical fiber SPR sensor and the biological solution flowing therethrough; the liquid inlet is connected to a microfluidic channel, and the liquid outlet is connected to a waste liquid tank through a pipeline; The gas channel is provided with a pressure pump and a gas distributor, wherein the gas distributor is a multi-channel distributor, one end of which is connected to the pressure pump, and the multi-channels at the other end are respectively connected to the liquid reservoirs, and are used to input gas into the liquid reservoirs according to the pressure pump; the pressure pump is electrically connected to the microfluidic mainboard, and is controlled by the microfluidic mainboard, and the flow rate of the microfluidic channel can be controlled by the pressure pump; The sensing module is used to monitor the resonance wavelength information of the biological solution flowing through the sensing area of ​​the optical fiber SPR sensor in real time; The microfluidic module is used to make the solution in the corresponding liquid reservoir flow through the outer surface of the optical fiber SPR sensor in the microfluidic chip through the microfluidic channel under a set pressure; The computer is used to detect the corresponding content in the biological solution in the current storage tank based on the received spectral electrical signal by measuring the resonance wavelength and comparing it with pre-stored data.

2. The control system for multiplex processing of biological samples according to claim 1, characterized in that: When the control system is used to prepare a capture antibody layer, an antibody solution is placed in at least one of the liquid reservoirs, and at least a portion of the outer surface of the optical fiber SPR sensor is a noble metal layer; the antibody solution flows from the liquid reservoir into the groove of the microfluidic chip in the microfluidic channel at a speed of 0.1-2000ul / min, so that the optical fiber SPR sensor is placed in the antibody solution for a period of time, thereby depositing a capture antibody layer on the upper surface of the noble metal layer of the optical fiber SPR sensor; When the control system is used for real-time detection of antigen solutions, biological solutions of different concentrations are placed in each of the liquid reservoirs, and at least part of the outer surface of the optical fiber SPR sensor is a capture antibody layer, and the capture antibody layer is combined with the antigen of the biological solution to be detected; the biological solution in the fluid channel flows from the liquid reservoir to the microfluidic chip at a speed of 0.1-2000ul / min, and contacts with the capture antibody layer of the optical fiber SPR sensor, and the spectrum analyzer receives the light signal and converts it into an electrical signal, and then transmits the spectrum signal to the computer; when the resonance valley of the spectrum signal is stable, the computer stores the data of the resonance valley, and detects the corresponding content in the biological solution in the current liquid reservoir by measuring the resonance wavelength and comparing it with the pre-stored data.

3. The control system for multiplex processing of biological samples according to claim 2, characterized in that: When the control system is used to prepare a capture antibody layer, the optical fiber SPR sensor uses a single-mode optical fiber as a substrate, and a noble metal layer is plated on the outer surface of the single-mode optical fiber.

4. The control system for multiplex processing of biological samples according to claim 1, characterized in that: The microfluidic channel is also provided with a flow meter between the conversion valve and the microfluidic chip.

5. The control system for multiplex processing of biological samples according to claim 1, characterized in that: The gas channel is also provided with an air pressure regulating valve, the air inlet end of the air pressure regulating valve is connected to the air source, and the air outlet end is connected to the pressure pump, which is used to adjust the pressure in the gas channel so that the air pressure in the gas channel is stabilized at 1000mbar-1200mbar.

6. A method for preparing a capture antibody layer of an optical fiber SPR sensor, characterized in that: The control system for multiplex processing of biological samples according to claim 1 comprises: S1: Multiple reservoirs contain different biological solutions, one of which is empty, and the fiber-optic SPR sensor to be prepared is installed in the microfluidic chip; S2: The microfluidic mainboard controls the conversion valve and the pressure pump to control the opening of the channel of the liquid reservoir corresponding to the required biological solution, and the biological solution flows into the microfluidic chip equipped with the optical fiber SPR sensor through the microfluidic channel at a set constant flow rate / constant pressure; S3: allowing the biological solution in the microfluidic chip to cover the surface of the optical fiber SPR sensor, and incubating at 3-6° C. for 6 to 8 hours, so that the capture antibody layer is deposited on the upper surface of the noble metal layer of the optical fiber SPR sensor; The conversion valve is turned to open the microfluidic channel between the empty liquid storage tank and the microfluidic chip, and the pressure pump is turned on to introduce nitrogen into the microfluidic chip, so that the solution in the microfluidic chip flows into the waste liquid tank; S4: Then, according to the set order, the conversion valve is controlled to open the pipelines between other liquid storage tanks and the microfluidic chip in turn, and steps S2 and S3 are executed, so that the remaining organisms are coated with the optical fiber SPR sensor in turn until the preparation of the required capture antibody layer is completed; S5: After taking out the optical fiber SPR sensor, the unfixed biological solution is rinsed with phosphate buffer solution, and it is dried for later use to form a capture antibody layer.

7. A method for real-time detection of biological solutions, characterized in that: The control system for multiplex processing of biological samples according to claim 1 comprises: S1: The liquid reservoirs contain biological solutions and detergent solutions of different concentrations, and one of the liquid reservoirs is empty; the optical fiber SPR sensor is installed in the microfluidic chip, and the capture antibody layer of the optical fiber SPR sensor can be combined with the biological solution to be detected; S2: The microfluidic mainboard controls the conversion valve and the pressure pump to control the opening of the channel of the liquid reservoir corresponding to the required biological solution, and the biological solution flows into the microfluidic chip equipped with the optical fiber SPR sensor through the microfluidic channel at a set constant flow rate / constant pressure; S3: The biological solution in the microfluidic chip covers the outer surface of the optical fiber SPR sensor, and the biological solution flows from the liquid reservoir through the microfluidic channel at a speed of 0.1-2000ul / min through the capture antibody layer of the optical fiber SPR sensor on the microfluidic chip, and the spectrum analyzer receives light, converts the optical signal into an electrical signal, and transmits the spectrum signal to the computer; when the resonance valley of the spectrum signal is stable, the computer stores the data of the resonance valley, and detects the corresponding content in the biological solution in the current liquid reservoir by measuring the resonance wavelength and comparing it with the pre-stored data; S4: Then, according to the set sequence, the conversion valve is controlled to open the pipelines between other liquid storage tanks and the microfluidic chip in turn, and steps S2 and S3 are executed, so as to obtain the detection of the content of biological solutions with different concentrations respectively.

8. The method for real-time detection of biological solution according to claim 7, characterized in that: After the biological solution covers the outer surface of the optical fiber SPR sensor in step S3, the method further comprises: The conversion valve is turned to open the microfluidic channel between the empty liquid storage tank and the microfluidic chip, and the pressure pump is turned on to introduce nitrogen into the microfluidic chip, so that the solution in the microfluidic chip flows into the waste liquid tank; The switching valve is turned to open the microfluidic channel between the liquid storage tank filled with detergent solution and the microfluidic chip to clean the microfluidic channel and the microfluidic chip. Then, the switching valve is turned again to open the pressure pump to introduce nitrogen into the microfluidic chip, so that the detergent solution in the microfluidic chip flows into the waste liquid tank.

9. The method for real-time detection of biological solution according to claim 7, characterized in that: The obtained spectral data are post-processed, including zero-phase filtering of the spectral data and fitting of the smoothed data using an asymmetric double S function; the computer calculates the antigen concentration of the current biological solution based on the linear relationship between the fitted resonance wavelength and the antigen concentration.

Images