CN116351483A - Integrated microfluidic biological reaction/detection device - Google Patents

Abstract

The invention provides an integrated microfluidic biological reaction/detection device, which comprises: the device comprises a polystyrene tube body, wherein a sample inlet and a micro-channel communicated with the sample inlet are arranged on the polystyrene tube body; the interval section is arranged at one end of the polystyrene tube body far away from the sample inlet; the sample adding device is arranged at one end of the interval section and is communicated with the micro-flow channel, so that a sample enters into or exits from the micro-flow channel. The invention takes the polystyrene tube as a main body, the sample inlet and the micro-channel are arranged in the polystyrene tube, and meanwhile, the interval section and the sample adding device are integrated on the polystyrene tube, so that the polystyrene tube can be used as a device for sample collection, biological/biochemical reaction and molecular immunity detection, has simple structure and convenient use, and has smaller required sample quantity, higher reaction efficiency and no pollution to biological samples.

Description

Integrated microfluidic biological reaction/detection device

This application claims the priority of the Chinese patent application with the application number 202111629391.X and the invention title "Integrated Microfluidic Bioreaction/Detection Device" filed with the China Patent Office on December 28, 2021, the entire contents of which are incorporated by reference incorporated in this application.

technical field

The invention relates to the technical field of biological detection, in particular to an integrated microfluidic biological reaction/detection device.

Background technique

Immunoassay technology (Immunoassay) is one of the most important categories of biosensing technology, and it is also one of the most dependent detection technologies in current life science research and medical testing. Specifically, immunosensing technology refers to the detection technology that uses the specific binding of antibodies and antigens to convert the concentration of specific molecules in liquid samples into readable signals.

The most widely used immunosensing technology is solid-phase immunoassay. Competitive Immunoassay and Sandwich Immunoassay (Sandwich Immunoassay, also known as double-antibody sandwich method) are the most commonly used solid-phase immunoassays. The sandwich method is mainly used to detect larger biomolecules such as proteins, and is currently the most commonly used immunosensing technology in life science research and medical testing. The principle can be briefly described as the target protein in the liquid sample (such as serum, etc.) is specifically adsorbed to the solid surface of the sensor through the capture antibody (Capture Antibody), and then used with a certain label (such as enzyme, fluorescent probe, acridine Esters, or radioactive elements) The detection antibody (Detection Antibody) binds to the target protein molecule that is specifically adsorbed to the surface of the sensor, and then indirectly measures the concentration of the target protein in the sample by detecting the signal intensity brought by the biomarker molecule on the sensor .

At present, the most commonly used immunosensing technology is the enzyme-linked immunosorbent assay (ELISA) developed in the 1980s and 1990s, and the magnetic bead chemiluminescence assay (CLIA) popularized after 2010. Among them, ELISA is mainly used for laboratory testing, and magnetic bead CLIA is mainly used for clinical diagnosis. Enzyme-linked immunosorbent assay (ELISA) has been popularized in the field of life science research because of its high sensitivity, low cost, safety and no radiation risk. It was once popular in the field of medical testing. The types of samples that can be detected by ELISA include animal body fluids (serum, urine, cerebrospinal fluid, etc.), cell and tissue culture fluids, patient samples (such as serum), etc. Covers almost all aspects related to life sciences.

Due to the early development of ELISA technology, a large 96-well plate is still used as an immunoreactor, and the internal surface area to volume ratio of each well is relatively low. Moreover, the traditional 96-well plate ELISA has problems such as large sample volume, such as at least 100 μL, a long detection time, generally 4 to 6 hours, and many manual operations such as sample addition, sample change, and cleaning. These have greatly increased the difficulty for researchers to obtain key biological information in trace samples. CLIA technology uses automated mechanical devices to solve the problem of numerous manual operations, but it also fails to solve the problem of large sample volume (at least 100 μL).

Contents of the invention

In view of this, the technical problem to be solved by the present invention is to provide an integrated microfluidic bioreaction/detection device. The integrated microfluidic bioreaction/detection device provided by the present invention requires less sample volume (10 μL level and Below), and easy to use.

The invention provides a microfluidic biological reaction/detection device, comprising:

A polystyrene tube body, the polystyrene tube body is provided with a sample inlet and a micro flow channel communicated with the sample inlet;

A spacer set at the end of the polystyrene tube away from the injection port;

A sample loading device is arranged at one end of the spacer section, and the sample loading device is connected with the micro flow channel, so that the sample enters or exits the micro flow channel.

Specifically, the present invention provides an integrated microfluidic biological reaction/detection device, comprising:

A polystyrene tube body, the polystyrene tube body is provided with a sample inlet and a micro flow channel communicated with the sample inlet;

A spacer set at the end of the polystyrene tube away from the injection port;

a mechanical liquid pump arranged at one end of the spacer, and the mechanical liquid pump communicates with the micro-channel.

In one embodiment, the microchannel is a straight-through microchannel.

In one embodiment, the cross-section of the straight-through microchannel is circular, and the diameter of the straight-through microchannel is 200-1000 μm, preferably 400-800 μm.

In one embodiment, the cross-section of the straight-through microchannel is square or rectangular.

In one embodiment, the cross-section of the polystyrene tube is circular, and the diameter of the circular shape is 1-2 mm.

In one embodiment, the wall thickness of the polystyrene pipe body is 0.2-0.6 mm.

In one embodiment, the polystyrene pipe body has a rectangular or square cross section.

In one embodiment, the polystyrene pipe body has a height of 15-30 mm.

In one embodiment, the polystyrene pipe body comprises:

A tapered sample injection buffer with a sample inlet;

A reaction/detection area connected with the sample injection buffer, the micro-channel is set in the reaction/detection area.

In one embodiment, the mechanical liquid pump may be a piston type liquid pump, which may include:

a cavity disposed on the spacer;

a piston disposed in the cavity, one end of which can enter the spacer;

a piston pull rod arranged at one end of the piston;

The top end of the piston rod is provided with a driving device;

A limiting device arranged at the end of the cavity away from the spacer.

In one embodiment, the driving device may be a magnet block.

In other embodiments, the mechanical liquid pump may be a negative pressure air bag type liquid pump, which may include:

a cavity disposed on the spacer;

A negative pressure air bag covering the cavity.

In some embodiments, the function of the negative pressure airbag is to generate negative pressure through extrusion to allow the sample to enter or exit the microchannel. The present invention has no special limitation on the shape of the negative pressure airbag, which may be a balloon or an airbag of other shapes.

The invention provides an integrated microfluidic biological reaction/detection device, comprising: a polystyrene tube body, the polystyrene tube body is provided with a sample inlet and a microfluidic tube connected to the sample inlet channel; be arranged on the spacer at the end of the polystyrene pipe body away from the sample inlet; be arranged at the sampling device at one end of the spacer, and the sampler is connected with the micro flow channel, so that the sample enters or Drain the microfluidic channel. The present invention takes polystyrene tube as the main body, sets sample inlet and micro flow channel in the polystyrene tube, and integrates spacers and sample feeding devices on the polystyrene tube, such as mechanical liquid pump, to form an integrated structure, which can As a device for sample collection, biological reaction, and detection, the device has a simple structure, is convenient to use, requires a small sample size, and has high reaction efficiency without causing contamination of biological samples.

Description of drawings

Figure 1 is a schematic structural view of an integrated microfluidic bioreaction/detection device provided by an embodiment of the present invention;

Fig. 2 is a schematic diagram of the decomposition structure of the integrated microfluidic biological reaction/detection device provided by the embodiment of the present invention;

Fig. 3 is the schematic diagram of the longitudinal section structure of the polyethylene pipe body provided by the embodiment of the present invention;

Fig. 4 is the cross-sectional structure schematic diagram of the polyethylene pipe body that the embodiment of the present invention provides;

Fig. 5 is a cross-sectional schematic diagram of a polystyrene pipe body provided by other embodiments of the present invention;

Fig. 6 is a structural representation of a hole in a 96-orifice plate;

Fig. 7 is a cross-sectional schematic diagram of the device provided by the embodiment of the present invention;

Figure 8 is a schematic diagram of a detection process provided by the present invention;

Fig. 9 is a conceptual schematic diagram of chemiluminescent detection using the device provided by the present invention;

Figure 10 is a schematic diagram of the concept of enzymatic fluorescence detection using the device provided by the present invention;

Figure 11 is a conceptual schematic diagram of optical absorption detection using the device provided by the present invention;

Figure 12 is a schematic diagram of an enzymatic reaction process provided by the present invention;

Fig. 13 is a schematic structural diagram of a microfluidic biological reaction/detection device provided by an embodiment of the present invention.

Detailed ways

The invention provides an integrated microfluidic biological reaction/detection device, comprising:

A polystyrene tube body, the polystyrene tube body is provided with a sample inlet and a micro flow channel communicated with the sample inlet;

A spacer set at the end of the polystyrene tube away from the injection port;

A sample loading device is arranged at one end of the spacer section, and the sample loading device is connected with the micro flow channel, so that the sample enters or exits the micro flow channel.

Referring to Figure 1 and Figure 2, Figure 1 is a schematic structural diagram of the integrated microfluidic bioreaction/detection device provided by the embodiment of the present invention, and Figure 2 is a schematic diagram of the integrated microfluidic biological reaction/detection device provided by the embodiment of the present invention Schematic diagram of the decomposition structure, wherein 11 is a polystyrene tube body, 111 is a sample inlet, 113 is a sample buffer, 114 is a reaction/detection area, 12 is a spacer, and 13 is a mechanical liquid pump.

The microfluidic biological reaction detection device provided by the present invention uses polystyrene tube body 11 as the main structure. Polystyrene plastic has strong protein affinity and can be used for immunological detection without any additional chemical treatment.

Referring to Fig. 3 and Fig. 4, Fig. 3 is a schematic diagram of the vertical section structure of the polyethylene pipe body provided by the embodiment of the present invention, and Fig. 4 is a schematic diagram of the cross-sectional structure of the polyethylene pipe body provided by the embodiment of the present invention.

The polyethylene pipe body 11 is divided into a sample injection buffer 113 and a reaction/detection area 114. The sample injection buffer 113 is provided with a sample inlet 111. The sample injection buffer 113 has a tapered structure, which is convenient for sample injection.

The reaction/detection area 114 is provided with a micro-channel 112 for biological reaction of samples. 112 positions of the micro-channel are directly connected to the micro-channel, and the biological reaction is performed on the sample and detected.

The polyethylene pipe body 11 has a circular cross section, and the micro flow channel 112 has a circular cross section. In one embodiment, the diameter of the straight-through microchannel 112 is 200-1000 μm. In one embodiment, the diameter of the straight-through microchannel 112 is 400-800 μm, and the diameter of the polystyrene tube is 1-2 mm. In one embodiment, the diameter of the straight-through micro-channel 112 is 600 μm, and the diameter of the polystyrene tube is 1.4 mm.

In one embodiment, the wall thickness of the polystyrene pipe body is 0.2-0.6 mm. In one embodiment, the polystyrene tube body has a tube wall thickness of 0.4 mm.

In one embodiment, the polystyrene pipe body has a height of 15-30 mm. In one embodiment, the polystyrene pipe body has a height of 22 mm.

In other embodiments, the cross-section of the straight-through microchannel 112 can be a square or a rectangle, and the cross-section of the polystyrene tube body 11 can also be a rectangle or a square, as shown in FIG. The schematic diagram of the cross-sectional structure of the polystyrene tube body provided by other embodiments of the invention, wherein, Figure 5A shows that the cross-section of the polystyrene tube body is a square, and the cross-section of the straight-through microchannel is circular; Figure 5B is a polystyrene tube body The cross-section of the tube body is square, and the cross-section of the straight-through microchannel is square. Figure 5C shows that the cross-section of the polystyrene tube body is rectangular, and the cross-section of the straight-through microchannel is rectangular.

Compared with the 96-well plate, as shown in Figure 6, Figure 6 is a schematic diagram of the structure of a well in a 96-well plate. The diameter of a well is 6mm and the height is 10mm. After adding the sample, the sample can only be captured at the bottom of the well , its surface area and volume are relatively small, only 0.32mm ^-1 . And in the device provided by the present invention, see Fig. 7, Fig. 7 is the cross-sectional structure schematic diagram of the device provided by the embodiment of the present invention, the diameter of its straight-through micro flow channel is 0.6 mm, and the height is 22 mm, after adding the sample, the sample is in the micro channel The entire range of the flow channel is captured, with a large surface area to volume ratio, up to 5mm ^-1 , so that the average distance for molecules to move to the surface of the solid-phase immunosensor is greatly shortened, and the solid-phase immunoreaction process is also greatly accelerated s speed.

The microfluidic biological reaction detection device provided by the present invention also includes a spacer 12 arranged at the end of the polystyrene tube body 11 away from the sample inlet. The spacer 12 is used to separate the biological reaction/detection area and the mechanical liquid pump on the one hand. On the one hand, the automation of the detection device can be realized.

Specifically, the spacer section 12 is a hollow cavity, so that the mechanical liquid pump 13 communicates with the micro-channel, and the sample enters and exits the micro-channel under the suction and discharge of the mechanical liquid pump.

In one embodiment, the cross-sectional area of the spacer section 12 is larger than the cross-sectional area of the polystyrene tube body 11, that is, the spacer section 12 has a protruding outer surface relative to the polystyrene tube body 11, and can be used to communicate with the mechanical arm and the polystyrene tube body 11. The control system forms a mechanical connection, thereby realizing automatic detection.

In one embodiment of the present invention, the polystyrene pipe body 11 , the spacer section 12 and the mechanical fluid pump 13 (except the piston) can be cast and formed integrally with polystyrene material. In other embodiments, the polystyrene pipe body 11, the spacer section 12 and the mechanical liquid pump 13 (except the piston) can be processed separately, and then spliced into a whole to form an integrated structure.

The microfluidic biological reaction detection device provided by the present invention also includes a mechanical liquid pump 13 arranged at one end of the spacer section 12, and the mechanical liquid pump 13 communicates with the microfluidic channel 112 for sucking or discharging the sample into the microfluidic flow. road.

In one embodiment, the mechanical liquid pump 13 includes a cavity 131 disposed on the spacer section 12, a movable piston head 132 can be drawn in the cavity 131, and is disposed at one end of the cavity 131 for restricting the piston 132. The stroke limiting device 133 , the piston pull rod 134 connected to the piston head 132 and the magnet 135 arranged at the top of the piston pull rod 134 .

The main function of the cavity 131 is to accommodate the piston head 132 and provide a movable space for the piston head 132 , one end of which is connected to the spacer section 12 , and the other end is provided with a limiting device 133 . The function of the magnet 135 is that it can be used as a driving device to drive the piston to move, so as to realize the automation of reaction or detection. In other embodiments, the magnet can also be replaced by other driving devices.

In one embodiment, the cavity 131 , the spacer section 12 and the polystyrene tube body 11 can be cast and formed integrally with polystyrene.

The microfluidic biological reaction/detection device provided by the present invention integrates sample addition, biological reaction and detection, can use a smaller sample size for reaction and detection, is more convenient to use, and does not cause the problem of biological sample contamination.

The microfluidic biological reaction/detection device provided by the present invention can be used for enzyme-linked immunoassay, and the specific process is close to traditional ELISA. As shown in Figure 8, Figure 8 is a schematic diagram of a detection process provided by the present invention, taking the detection of protein molecules using the sandwich method as an example, the specific process is:

1. Aspirate the capture antibody solution into the microchannel, polystyrene has protein affinity, can coat the capture antibody on the inner surface of the microchannel, and use protein to block unbound sites;

2. Inhale the sample containing the target molecule into the microchannel coated with the antibody, so that the target molecule binds to the capture antibody;

3. Add the enzyme-linked detection antibody solution (the antibody is coupled to the enzyme molecule by chemical method in advance), so that it can fully react with the immune complex;

4. Add appropriate substrates to generate readable signals in the microchannel. There should be a corresponding washing process between each step to remove molecules that have not been stably bound to the surface of the microfluidic tube.

In addition, if a suitable antibody is used, the second step and the third step can be combined into one, that is, the sample is premixed with the enzyme-linked detection antibody and then sucked into the microfluidic reactor at the same time.

The microfluidic biological reaction/detection device provided by the present invention can be applied to chemiluminescent detection, as shown in Figure 9, which is a conceptual schematic diagram of chemiluminescent detection using the device provided by the present invention; it is also suitable for enzymatic fluorescence detection, As shown in Figure 10, Figure 10 is a schematic diagram of the concept of enzymatic fluorescence detection using the device provided by the present invention; it is also suitable for enzymatic chromogenic detection, as shown in Figure 11, and Figure 11 is an optical Schematic diagram of the concept of absorption detection. The present invention has no special limitation on the detection method, and it can be selected according to the selected detection method and the added detection substance. For example, chemiluminescence detection includes but not limited to luminol chemiluminescence and acridinium ester chemiluminescence; enzymatic chromogenic detection can measure absorbance at specific wavelengths, etc.

In addition, the microfluidic biological reaction/detection device provided by the present invention can also be used in the high-efficiency enzymatic synthesis/enzymatic preparation process of micro-sample, as long as the catalytic enzyme is immobilized on the inner surface of the reactor. Take the enzymatic proteolysis process as an example, see Figure 12, Figure 12 is a schematic diagram of an enzymatic reaction process provided by the present invention, and its specific process is:

1. Coating protease (such as papain; trypsin, etc.) on the inner surface of the microchannel;

2. Inhale the reaction raw material, that is, the aqueous solution containing protein. And wait for a period of time, so that the protease immobilized on the surface of the microchannel fully reacts with the protein solution;

3. Drain and collect the reaction product, ie the polypeptide solution.

The device can operate in parallel connection of multiple tubes (such as horizontal configuration or matrix configuration) in actual use. In addition, the device can also be directly combined with other biological culture systems (such as microfluidic organ chips, etc.), and used as a sample collection module.

In addition to the liquid pump with the above-mentioned piston and rod structure, the sampling device can also be a negative pressure air bag, such as a balloon, see Figure 13, Figure 13 is a schematic structural diagram of a microfluidic biological reaction/detection device provided by an embodiment of the present invention . Compared with the microfluidic biological reaction/detection device described above, the difference is that the balloon 23 is used instead of the piston and the pull rod for adding or discharging samples. In a specific implementation, the balloon 23 is sheathed on the spacer, and negative pressure is generated by extrusion to allow the sample to enter the microchannel. In a specific implementation manner, the polystyrene tube body, the spacer section and the balloon can be processed separately, and then spliced into a whole to form an integrated structure, which will not be repeated in the present invention.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto, any person familiar with the technical field within the technical scope disclosed in the present invention, according to the technical solution of the present invention Any equivalent replacement or change of the inventive concepts thereof shall fall within the protection scope of the present invention.

Claims (10)

1. A microfluidic biological reaction/detection device comprising:

the device comprises a polystyrene tube body, wherein a sample inlet and a micro-channel communicated with the sample inlet are arranged on the polystyrene tube body;

the interval section is arranged at one end of the polystyrene tube body far away from the sample inlet;

the sample adding device is arranged at one end of the interval section and is communicated with the micro-flow channel, so that a sample enters into or exits from the micro-flow channel.

2. The integrated microfluidic biological reaction/detection device of claim 1, wherein the sample addition device is a mechanical liquid pump.

3. The microfluidic biological reaction/detection device of claim 2 wherein the fluidic channel is a straight-through fluidic channel.

4. A microfluidic biological reaction/detection device according to claim 3, wherein the cross section of the through-flow microchannel is circular, the diameter of the through-flow microchannel being 200-1000 μm, preferably 400-800 μm.

5. A microfluidic biological reaction/detection device according to claim 3, wherein the cross section of the through-flow microchannel is square or rectangular.

6. The microfluidic biological reaction/detection device according to any one of claims 1 to 5, wherein the polystyrene tube has a circular cross section, and the diameter of the circular shape is 1 to 2mm.

7. The microfluidic biological reaction/detection device according to claim 6, wherein the polystyrene tube has a tube wall thickness of 0.2-0.6 mm.

8. The microfluidic biological reaction/detection device according to any one of claims 1 to 5, wherein the polystyrene tube has a rectangular or square cross section;

the height of the polystyrene tube body is 15-30 mm.

9. The microfluidic biological reaction/detection device according to any one of claims 1 to 5, wherein the polystyrene tube comprises:

a conical sample injection buffer area provided with a sample injection port;

and the reaction/detection area is communicated with the sample introduction buffer area, and the micro-channel is arranged in the reaction/detection area.

10. The microfluidic biological reaction/detection device of claim 2, wherein the mechanical liquid pump comprises:

a cavity disposed on the spacer;

the piston is arranged in the cavity, and one end of the piston can enter the interval section;

the piston pull rod is arranged at one end of the piston;

the top end of the piston pull rod is provided with a driving device;

the limiting device is arranged at one end of the cavity, which is far away from the spacing section;

or,

the mechanical liquid pump comprises:

a cavity disposed on the spacer;

and the negative pressure air bag is coated on the cavity.

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