CN119060821A

CN119060821A - A device for capturing foodborne pathogens by combining magnetic bead method with microfluidic chip

Info

Publication number: CN119060821A
Application number: CN202411262195.7A
Authority: CN
Inventors: 张文强; 张照轩; 许文涛; 朱龙佼; 周伟; 樊彧轩
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2024-09-09
Filing date: 2024-09-09
Publication date: 2024-12-03

Abstract

The invention belongs to the technical field of microfluidic chips, and particularly relates to a food-borne pathogenic bacteria capturing device combining a magnetic bead method and a microfluidic chip. The motor below the chip is utilized to drive the magnet to rotate to generate a changing magnetic field to drive the magnetic beads to actively move in the reaction cavity, so that the contact probability of the magnetic beads and the object to be detected is increased, the capturing time is shortened, and the capturing efficiency is improved. The steering engine is arranged on the microfluidic device, the steering engine rotates to drive the compression bar to press the runner to form the mechanical micro valve, the opening and the closing of the runner are controlled, the micro valve is opened, and the waste liquid is absorbed by the sponge sheet in the waste liquid pool under the action of gravity and capillary force. The devices are arranged on the bottom plate to be integrated, the devices can work simultaneously and automatically when being portable, the working efficiency is improved, and a novel mechanical device is provided for capturing and further detecting pathogenic bacteria by the magnetic beads in the microfluidic chip.

Description

Food-borne pathogenic bacteria capturing device combining magnetic bead method and microfluidic chip

Technical Field

The invention belongs to the technical field of microfluidic chips, and particularly relates to a food-borne pathogenic bacteria capturing device combining a magnetic bead method and a microfluidic chip.

Background

With the rise of living standard, more food sources and cooking modes certainly increase the ways of food-borne pathogenic bacteria infection, and global food-borne pathogenic bacteria infection is in an ascending state. There is a need for a highly efficient and reliable way of detecting food-borne pathogenic bacteria that prevents food poisoning events from occurring due to food-borne pathogenic bacteria contaminating the food.

The nucleic acid is used as a biological unique 'identity card', and the sensitive and specific detection of the food-borne pathogenic bacteria can be realized through the detection of the characteristic nucleic acid fragments of the food-borne pathogenic bacteria. The magnetic bead method is used as a classical nucleic acid detection technology, and mainly comprises the steps of operating magnetic nano particles with functional groups or specific probes modified on the surfaces in a magnetic field to separate, enrich and detect specific biological harmful substances or nucleic acids, and has the advantages of high detection sensitivity, good specificity and the like. However, the operation process of the conventional magnetic bead method is very complicated, and the application of the conventional magnetic bead method in on-site instant detection is restricted, so that the automation of the operation of the conventional magnetic bead method becomes a hot spot for research in the field.

With the development of microfluidic technology, many conventional experiments can be transferred to a microfluidic chip for reaction and measurement. Microfluidic control is a kind of accurate control and manipulation microscale fluid, and is to integrate basic operation units of sample preparation, reaction, separation, detection, etc. in biological, chemical and medical analysis processes onto a chip with micrometer scale, and automatically complete the whole analysis process. Due to the micro-scale structure, the fluid shows and produces special properties in the microfluidic chip that differ from the macro-scale, thus developing unique analytical properties. Meanwhile, the method has the advantages of light volume, small amount of used samples and reagents, low energy consumption, high reaction speed, capability of carrying out a large amount of parallel treatment, disposability and the like. The method gets rid of the constraint and limitation of the traditional large-scale experimental detection system, applies the microfluidic chip to the detection of food-borne pathogenic bacteria, realizes the operations of automatic capture, DNA extraction, purification, separation and the like of pathogenic bacteria in a micro-channel and a reaction cavity of the chip, can realize on-site instant detection, outputs results in a short time, and truly realizes the quick detection of the food-borne pathogenic bacteria, which is a difficult problem to be solved in a long time.

Based on the above, we designed an automatic capturing device for food-borne pathogenic bacteria combining a magnetic bead method and a microfluidic chip, and the micro-channels of the manufactured microfluidic chip are respectively subjected to physical and chemical hydrophilic treatment, so that multiple sample adding can be realized, the hydrophilic layer is not damaged, and a steering engine is utilized to rotate to drive a compression bar to serve as a mechanical valve, so that the micro-channels can be opened and closed, and the liquid flow can be controlled. The device is integrated and miniaturized, has a simple structure, can be applied to various biological experiments, and can be used for capturing and detecting various food-borne pathogenic bacteria.

Disclosure of Invention

The invention aims to provide a food-borne pathogenic bacteria capturing device combining a magnetic bead method and a microfluidic chip, which can realize multi-channel detection of one kind of bacteria by cooperative control of a plurality of devices, improve the accuracy of detection results, or capture and detect a plurality of bacteria simultaneously and realize on-site rapid detection of the food-borne pathogenic bacteria. The device comprises a microfluidic chip clamp, a base, a mechanical micro valve, a magnetic stirring module and a bottom plate.

The magnetic stirring module comprises an arduino control board, a stepping motor, a rotating table arranged on a central shaft of the motor, a magnet on the rotating table, a motor driver opposite to the stepping motor and a switching power supply box responsible for power supply. The revolving stage adopts 3D to print the method to make, and the revolving stage passes through the recess that has the inverse structure of motor center pin on the pole and is connected with the motor center pin, is equipped with two circular recesses of symmetry on the revolving stage mesa, and two circular magnets are placed in the recess, and fixed through the double faced adhesive tape, the magnet itself has the magnetic pole to divide, should ensure that two magnet up the face polarity opposite to simulate the inner structure of magnetic stirrer on the market, and have small and exquisite, portable advantage. After the mixed solution of the magnetic beads and the sample is injected into the reaction cavity, the switch is turned on, the magnet rotates to drive the magnetic beads in the reaction cavity to move, the capture and mixing speed of the magnetic beads to bacteria is accelerated, and the motor speed regulation is realized by changing the subdivision of the driver, so that the reaction is more complete.

The mechanical micro valve comprises a bus steering engine and a compression bar connected to the steering wheel, and in the control aspect, the bus steering engine sends an instruction in a serial port mode, so that the steering engine executes work according to a set speed target position. The steering wheel drives the compression bar to precisely rotate to reach a designated position so as to stop the flow passage. The bus steering engines on each device can be controlled in series, namely one steering engine is connected with one steering engine in series, and finally the steering engines are connected to the control board, so that independent control and cooperative control of each steering engine are realized through Python programming. The pressure lever is lifted, the valve is opened, the flow channel flows, the liquid flows into the waste liquid pool from the reaction cavity, the pressure lever is pressed down, the valve is closed, the flow channel is closed, and the liquid stops flowing.

The microfluidic chip is of a three-layer structure composed of a sample injection layer, a runner layer and a substrate layer, wherein the sample injection layer is made of PVC, the runner layer is made of 3M glue, the substrate layer is made of a glass slide commonly used in laboratories, and runner patterns on the runner layer and the sample injection layer are prepared by a laser cutting process. And the three-layer structure is bonded together through the transparent runner layer with double-sided adhesive in the middle, so that the assembly is completed. The whole chip structure consists of a sample adding cavity, a reaction cavity, a waste liquid cavity, a vent hole and a micro-channel connected between the cavities. The reaction cavity provides a cavity space for mixing the magnetic beads and the sample to be captured so as to improve the capturing efficiency of the magnetic beads to the sample to be captured. The reaction cavity is sealed by the upper PVC film, and only a vent hole with the diameter of 0.4mm is reserved, so that the liquid in the cavity is prevented from being polluted to the greatest extent, and the air pressure balance in the cavity is balanced when the liquid enters and exits. The waste liquid cavity is fixed with a sponge sheet for absorbing waste liquid.

The chip clamp and the motor base are manufactured by 3D printing, and are made of ABS (Acrylonitrile butadiene styrene) and have excellent performances such as wear resistance and impact resistance. The base is mainly used for installing the stepping motor and fixing a stepping motor driver, the base is provided with a groove matched with the size of the stepping motor, the driver is connected with an Arduino microcontroller outside, and the driver receives pulse signals from the Arduino and converts the pulses into proper current and voltage so as to control the operation of the stepping motor. By controlling the pulse frequency and the pulse number, different rotation speeds and rotation directions of the stepping motor can be realized. The top of the chip clamp is provided with an opening, the micro-fluidic chip can be conveniently taken and replaced, the plane on which the chip is placed on the clamp forms an included angle of 7 degrees with the horizontal plane, so that liquid can smoothly circulate under the action of gravity, and one side of the clamp is provided with a U-shaped groove for placing a steering engine and fixing the steering engine by using screws and nuts.

The invention has the beneficial effects that:

1. The invention uses the stepping motor to drive the magnet to rotate for stirring and drive the magnetic beads to move, does not need to put the device into a magnetic stirrer or a three-dimensional Helmholtz coil for driving, is miniaturized and portable, and is suitable for on-site instant detection.

2. Compared with the traditional PDMS chip, the manufacturing method is simple, the PDMS chip needs the processes of defoaming, heating and solidifying, turning over the mould, bonding and the like, the manufacturing process is complicated, and the cost is greatly reduced compared with the PDMS chip.

3. The bus steering engine is adopted to control the mechanical micro valve instead of the common steering engine, and the control over the plurality of steering engines can be realized only by one driving plate, so that the steering engines are conveniently connected in series to carry out the cooperative control of a plurality of devices, the complex and complicated operation process is simplified, and the operation is more convenient.

4. Through the whole open design of device to can conveniently with automatic application of sample device, temperature control device, fluorescence excitation device, image processing system etc. mutually support, realize the automation that food-borne pathogenic bacteria detected jointly.

Drawings

FIG. 1 is a schematic perspective view of a food-borne pathogenic bacteria capturing device according to the present invention;

FIG. 2 is a schematic view of a microfluidic partial explosion of a food-borne pathogenic bacteria capture device according to the present invention;

FIG. 3 is an exploded schematic view of a microfluidic chip structure;

FIG. 4 is a two-dimensional schematic diagram of a microfluidic chip sample injection layer and a flow channel layer cavity structure;

FIG. 5 is a schematic diagram of a magnetic stirring module structure;

FIG. 6 is a top view of the capture device base;

FIG. 7 is a schematic view of the structure of the bottom plate of the capturing device;

fig. 8 is a flow chart of the use of the capture device.

In the figure:

1. The micro-fluidic device comprises a micro-fluidic device part, a bus steering engine, 12, a compression bar, 13, a chip clamp, 14, a micro-fluidic chip, 15, a top clamping slider, 141, a chip sample injection layer, 142, a chip runner layer, 143, a chip substrate layer, 1411, a sample injection port, 1412, a vent hole, 1413, a waste liquid pool outlet, 1421, a sample injection cavity, 1422, a runner, 1423, a reaction cavity and 1424;

2. Magnetic stirring module, 21, step motor driver, 22, base, 23, step motor, 24, rotary table, 25, cylindrical magnet, 26, wedge-shaped platform, 221, lifting lug, 222, through hole, 223, protrusion, 3, bottom plate, 31, array screw hole.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the embodiments of the present invention, and it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Embodiment 1. The principle of operation of a food-borne pathogenic bacteria capturing device combining a magnetic bead method and a microfluidic chip.

The mixed liquid of the sample and the functionalized magnetic beads is added into a sample injection cavity 1421 through a sample injection port 1411 of a microfluidic chip of a liquid transfer device, the liquid flows into a reaction cavity 1423 under the dual action of gravity and capillary force, a stepping motor 23 positioned under the reaction cavity is started, the stepping motor 23 drives two cylindrical magnets 25 to rotate, the magnetic beads in the cavity move under the drive of the rotating magnets 25, the specific capture of bacteria in the sample is realized, a steering engine 11 is utilized to drive a pressure lever 12 to control the opening and closing of a micro-channel, the pressure lever 12 is lifted, a flow channel 1422 is opened, waste liquid flows into a waste liquid pool 1424 from the reaction cavity 1423, and the waste liquid is absorbed by a sponge sheet of the waste liquid pool 1424. Then adding buffer washing liquid to wash the magnetic beads combined with bacteria in the reaction cavity, turning on the stepping motor 23 again to make the washing more fully and uniformly, turning on the compression bar 12, and absorbing the washed waste liquid by the sponge sheet. The whole device is integrated and miniaturized, can realize the automatic mixing and capturing of the immunomagnetic beads and the objects to be detected, can also be matched with a heating device, a temperature control device, an ultraviolet excitation device and the like, designs different reaction systems, completes the representation and visual output of the captured objects, and is convenient for on-site instant detection.

The material of the chip sample injection layer 141 is polyvinyl chloride (PVC), and is cut and processed on a PVC film according to a pattern by a laser cutting machine, wherein the diameter of the sample injection port 1411 is 8mm, 30-40 mu l of liquid can be injected into the sample injection port, the requirement of a reaction system can be met, the sample injection port is used for injecting reagents such as magnetic beads, samples and buffer cleaning liquid, the diameter of the vent 1412 is 0.4mm, the air pressure balance in a cavity is balanced when the liquid enters and exits is ensured, the liquid smoothly flows down, the waste liquid pool 1424 is rectangular with the length of 20mm and the width of 15mm, the whole sample injection layer 141 is 75mm and the width of 25mm and is consistent with the size of the substrate layer 143, the material of the runner layer 142 is 3M glue with the thickness of 0.3mm, and the width of the runner 1422 is 0.6mm for better capillary force and siphon force effect.

The manufacturing process of the microfluidic chip comprises the steps of firstly placing a PVC film into a working panel of a laser cutting machine to perform patterning cutting, removing redundant waste materials to obtain a sample injection layer, uniformly spraying a release agent on a smooth and flat clean aluminum plate or iron plate, tearing off 3M glue, pasting the plate on the plate, flattening as much as possible without leaving bubbles, placing the plate into the laser cutting machine to perform cutting processing, taking out redundant waste materials with tweezers after the processing is finished to obtain a runner layer, aligning and pasting the sample injection layer which is processed in advance on the runner layer, then peeling off the glued sample injection layer and the runner layer from the plate together with tweezers, pasting the sample injection layer and the runner layer on a glass sheet which has been processed by a plasma cleaning machine, and pressing firmly to finish the manufacturing.

The laser cutting machine for manufacturing the chip is preferably model 3020 of Shandong Tornado corporation, the cutting power of the sample injection layer is preferably 7w, the cutting speed is 10mm/s, the cutting times are 1 time, the cutting power of the runner layer is preferably 7.8w, the cutting speed is 10mm/s, and the cutting times are 3 times.

For better capillary force, the substrate layer is subjected to hydrophilic treatment before the chip is manufactured, a glass sheet is firstly placed into a 1:1 mixed solution of acetone and ethanol for ultrasonic treatment for 10 minutes (removing surface oil stains), the glass sheet is taken out and washed by deionized water, a plasma washer (YZD 08-2C of Sailot technology Co., ltd.) is used after the glass sheet is dried by nitrogen or air, the surface of the substrate layer 143 is subjected to hydrophilic treatment by taking air as a medium and taking parameters of 100W and 5 minutes as power, the assembled microfluidic chip is subjected to secondary chemical hydrophilic treatment, namely, a hydrophilic reagent is mixed with isopropanol 7:3, vortex oscillation is carried out, the hydrophilic reagent solution is injected into a runner 1422 through a reaction cavity by a liquid shifter to soak the surface of the runner, the hydrophilic membrane is formed by the surface of the runner, and the hydrophilic reagent solution is discharged by a high-pressure air gun after one minute. In order to generate continuous capillary action of the microfluidic chip, a sponge sheet is attached to the waste liquid pool 1424 of the microfluidic chip.

The plane of the mounting chip forms an included angle of 7 degrees with the horizontal plane, and when liquid moves in a flow channel in the mounted microfluidic chip, the liquid is subjected to the action of capillary pressure and gravity at the same time, so that continuous sample injection of the liquid is ensured.

The sample layer 141 is used to maintain a relatively sealed environment for the reaction chamber 1423 of the microfluidic chip 14.

The device base 22 comprises a matrix groove 50mm long, 40mm wide and 2mm deep for placing the wedge-shaped mounting platform 26, and the base 22 is provided with a convex 223 part with a height of 3.5mm for limiting the freedom of the device in the horizontal plane direction.

The stepper motor 23 is placed on a wedge-shaped platform 26 with a square recess of 28mm x 28mm, the depth of the recess being 3mm, the angle between the plane and the horizontal being 7 deg., in order to ensure that the plane of rotation of the stepper motor 23 is parallel to the plane of the chip 14.

The wedge-shaped platform 26 is provided with four countersunk holes with an outer diameter of 5.2mm and an inner diameter of 2mm, and the wedge-shaped platform 26 and the device base 22 are fixed by bolts and nuts.

The cylindrical magnet 25 is a neodymium iron boron magnet with a diameter of 10mm and a thickness of 5mm, and is fixed in two circular grooves of the rotary table 24 by using a 3M double-sided tape, and the polarities of the two upward sides are opposite.

The motor in the magnetic stirring module 2 is preferably a miniature 28-step motor with the height of 28mm and the shaft diameter of 5mm, and the driver of the step motor is preferably a Racing technology model DM422S. During operation of the magnetic stirring module 2, the motor 23 preferably rotates at 60r/min.

The bus steering engine 11 is preferably a dual-axis serial bus digital robot steering engine of the femto company, and the model is SCS115. The rudder disk and the compression bar 12 are fixed by four M2 screws, the tail end of the compression bar 12 is a spherical contact, the steering engine 11 drives the compression bar 12 to rotate at an angle of 39-41 degrees, the spherical contact can completely stop the flow channel, the angle is overlarge, hydrophilic films on the inner wall of the flow channel are easily damaged or the flow channel is easily deformed, the chip fluxion is influenced, and the rotation angle is preferably 40 degrees.

The bottom plate 3 is used for the array of many detection device to be fixed, and board protruding portion height is 15mm, and protruding portion just contains diameter 4.2mm, and screw hole 31 that interval 40mm is used for fixing with device base 22 with the bolt and nut of M4, vibration when reducing motor 23 rotation makes the rotation more steady.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A foodborne pathogen capture device combining a magnetic bead method with a microfluidic chip, wherein the device comprises at least a microfluidic device (1), a magnetic stirring module (2) and a bottom plate (3); the microfluidic device (1) comprises a bus servo (11), a pressure rod (12), a chip fixture (13), a microfluidic chip (14), and a top clamping slider (15), characterized in that the microfluidic chip (14) is a three-layer structure, the bottom layer is a glass substrate, the top layer is a plastic film, and the middle layer is a double-sided adhesive, and the three-layer structure is tightly attached together by utilizing the double-sided adhesiveness of the middle layer; the microfluidic device (1) realizes opening and closing of the flow channel by driving the pressure rod through the rotation of the bus servo; the magnetic stirring module (2) comprises a stepping motor driver (21), a base (22), a stepping motor (23), a rotating table (24), a cylindrical magnet (25), and a wedge-shaped platform (26); the device drives the magnetic beads to be captured by the changing magnetic field generated by the rotation of the magnet driven by the motor.

2. The foodborne pathogen capture device combining a magnetic bead method and a microfluidic chip according to claim 1 is characterized in that the glass substrate is pre-hydrophilized, including treatment with a plasma cleaner and chemical hydrophilization with a hydrophilic reagent; the top plastic film is made of PVC or PET, the middle layer is 3M glue, and the top and middle layer materials are cut and patterned using a laser cutting method to complete the processing of the microfluidic chip structure.

3. The foodborne pathogen capture device combining the magnetic bead method and the microfluidic chip according to claim 1 is characterized in that the width of the microchannel is 600 μm, and the injection chamber (1421), the reaction chamber (1423), and the waste liquid pool (1424) are connected by a microchannel (1422).

4. The foodborne pathogen capture device combining the magnetic bead method and the microfluidic chip according to claim 1 is characterized in that the plane of the clamp forms a certain angle with the horizontal plane, and the chip is clamped by the top clamping slider (15) after being placed; one side of the clamp is provided with a U-shaped groove according to the outer dimensions of the steering gear (11), and a circular hole is opened on the groove for placing the steering gear, which is fixed with screws and nuts.

5. The foodborne pathogen capture device combining the magnetic bead method and the microfluidic chip according to claim 1 is characterized in that the bottom end thereof has a groove matching the shape of the motor (23) rotor, and the inner diameter of the groove is slightly larger than the diameter of the rotor to facilitate installation, and the shape is matched so that it rotates with the motor.

6. The foodborne pathogen capture device combining the magnetic bead method and the microfluidic chip according to claim 1 is characterized in that the upward surfaces of the two cylindrical magnets (25) have opposite polarities, i.e. one side has an N pole and the other side has an S pole, and are fixed on the rotating table (24) by 3M double-sided tape.

7. The foodborne pathogen capture device combining the magnetic bead method and the microfluidic chip according to claim 1, characterized in that an array of threaded holes (31) are punched on the bottom plate.