CN110940820B - High-flux single-worm analysis device based on centrifugal microfluidic technology and application method thereof - Google Patents
High-flux single-worm analysis device based on centrifugal microfluidic technology and application method thereof Download PDFInfo
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Abstract
The invention discloses a high-flux single-worm analysis device based on a centrifugal microfluidic technology and a use method thereof, and belongs to the fields of centrifugal microfluidic technologies and single-worm analysis. The device comprises a disc type single-worm analysis microfluidic chip and a portable centrifugal analysis device; the microfluidic chip comprises a plurality of nematode analysis units which are uniformly distributed along the circle center; the nematode analysis unit comprises a liquid storage tank, a single-worm analysis channel and a nematode removal structure, and a waste liquid tank; the single-worm analysis channel consists of a nematode sample injection structure, a nematode culture pond, a nematode capturing structure and a channel outlet; the nematode removal structure includes a nematode removal channel and an integrated microvalve; the portable centrifugal analysis device comprises: comprises a centrifugal control module and a nematode detection module. The method is simple, convenient, quick and low in cost, is particularly suitable for long-term culture and observation of the single-thread worm with high flux, automation and portability, and provides a feasible solution for deep research and wide application of model organism nematodes.
Description
Technical Field
The invention belongs to the fields of centrifugal microfluidic technology and single-worm analysis, and particularly relates to a high-flux single-worm analysis device based on the centrifugal microfluidic technology and a use method thereof.
Background
Caenorhabditis elegans is an excellent model organism and has been widely used in life span, development, exercise and other studies involving nematode culture and detection. Conventional nematode culture is mainly carried out on liquid or agar plates, and a culture medium needs to be periodically replaced in the experimental process; when the nematodes are researched, the single nematodes are required to be cleaned, picked, anesthetized, fixed, recorded, photographed and the like, the steps are complicated, the accurate tracking and observation of the single nematodes cannot be realized, the parents and the filial generations of the nematodes are difficult to separate, and the nerve and the behavior of the nematodes are likely to be caused by fixation with glue or anesthetic. In order to realize long-term culture and research of nematodes, the technical problems of single nematode tracking, experimental operation under physiological conditions and the like are solved. Meanwhile, in order to ensure the reliability and statistical significance of data, it is necessary to perform high-throughput analysis of single nematodes. The micro-fluidic technology is a new technology for precisely controlling nano-liter to picoliter fluid in a micrometer-scale flow channel, and has the outstanding characteristics of microminiaturization, integration, high flux, automation and the like. The microfluidic Chip (Lab on a Chip) can be used as a practical technical platform for controlling micro-scale mode biological nematodes, has various single-worm control and analysis technologies, and is widely applied to different nematode research fields. However, most of the existing microfluidic nematode analysis systems adopt a pressure driving mode, and the general microfluidic chip has large design and processing difficulty and high cost, and often requires complex flow path interfaces and additional fluid driving control equipment, so that the microfluidic nematode analysis system has poor portability and low automation degree, simultaneously increases the complexity of operation and control, has a plurality of inconveniences in practical application, and limits the application range of the microfluidic nematode analysis system.
Disclosure of Invention
In order to solve the problems, the invention provides a high-flux single-worm analysis device based on a centrifugal microfluidic technology and a use method thereof, and the method is simple, convenient, quick and low in cost, is particularly suitable for long-term culture and observation of single-worm with high flux, automation and portability, and provides a feasible solution for deep research and wide application of model organism nematodes.
In order to achieve the above object, the present invention provides a high-throughput single-worm analysis device based on centrifugal microfluidic technology, comprising a disc-type single-worm analysis microfluidic chip and a portable centrifugal analysis device, wherein:
the disc type single-worm analysis microfluidic chip comprises: comprises a plurality of nematode analysis units which are uniformly distributed along the circle center; the nematode analysis unit sequentially comprises a liquid storage tank, a plurality of single-worm analysis channels and nematode removal structures which are radially arranged from the center of the circle outwards, and a waste liquid tank; the single-worm analysis channel consists of a nematode sample injection structure, a nematode culture pond, a nematode capturing structure and a channel outlet from one side connected with the liquid storage pond; the nematode removing structure comprises a nematode removing channel and an integrated micro valve;
the portable centrifugal analysis device comprises: comprises a centrifugal control module and a nematode detection module; the centrifugal control module consists of a rotating shaft and a rotating motor, can be matched with the disc-type microfluidic chip, and can accurately control the rotating speed, time and rotating direction of the microfluidic chip; the nematode detection module is used for sequentially carrying out video recording on the single worms in the plurality of culture ponds or carrying out microscopic imaging on the single worms fixed at the nematode capturing structure.
Further, in the above technical scheme, the microfluidic chip is composed of an upper microchannel structure layer and a lower bottom plate layer, the two layers are tightly bonded, and the upper microchannel layer is made of an air-permeable elastic silicone rubber material, and comprises polydimethylsiloxane and polydimethylsiloxane PDMS; the bottom plate layer is made of light-transmitting materials including glass, polymethyl methacrylate (PMMA) and Polycarbonate (PC).
Further, in the above technical scheme, in the single-worm analysis channel, the nematode sampling structure is composed of a conical structure channel and a strip-shaped structure channel, the conical bottom of the conical structure channel faces the liquid storage tank, a strip-shaped structure channel is arranged between the conical top and the nematode culture tank, the width of the strip-shaped structure channel is smaller than the diameter of the nematode sample, and the depth is equivalent to the diameter of the nematode sample. The nematode trapping structure is a conical structure channel, the conical bottom of the conical structure channel faces the nematode culture pond, the conical top of the conical structure channel is connected with the channel outlet, the width of the conical structure channel top is smaller than the diameter of the nematode sample, and the depth of the conical structure channel is equal to the diameter of the nematode sample.
Further, in the above technical scheme, the nematode culture pond in the single-worm analysis channel is elliptical or circular, the diameter of the nematode culture pond is greater than the length of the nematode sample, and the depth is greater than the diameter of the nematode sample.
Further, in the above technical solution, in the nematode removing structure, the width and depth of the channel are both larger than the diameter of the nematode sample; the integrated micro valve is an automatically controlled electromagnetic valve or a mechanical micro valve and is positioned above the nematode removing channel and used for controlling the opening and closing states of the nematode removing channel. .
Further, in the above technical solution, two ends of the liquid storage tank are provided with liquid adding holes, and two ends of the liquid storage tank are connected with a liquid storage tank exhaust channel; and two ends of the waste liquid pool are connected with waste liquid pool exhaust channels.
Further, in the above technical scheme, the centrifugal control module is composed of a rotating shaft and a rotating motor, wherein the rotating shaft and the rotating motor are matched with the disc-type microfluidic chip.
Furthermore, in the above technical scheme, the nematode detection module can record video of the single-thread worm in the nematode culture tank or perform microscopic imaging on the single-thread worm fixed at the nematode capturing structure.
The invention also provides a using method of the high-flux single-worm analysis device based on the centrifugal microfluidic technology, which comprises the following steps:
(1) preparing a microfluidic chip: closing an integrated micro valve on the nematode removing channel, adding a nematode sample and a culture solution into a liquid storage tank from a liquid adding hole together, closing the liquid adding hole, and fixing a micro-fluidic chip on a rotating shaft of a centrifugal control module;
(2) nematode sample addition: starting a rotating motor to drive the microfluidic chip to rotate, so that nematodes enter the nematode sampling structure of the single-worm analysis channel and are fixed;
(3) residual nematode removal: starting a nematode detection module, and observing the fixation condition of nematodes in real time; after single nematodes are fixed at all nematode sampling structures, opening an integrated micro valve, and simultaneously continuing rotating the micro-fluidic chip to enable the remaining nematodes in the liquid storage tank to enter the waste liquid tank from the nematode removing channel;
(4) nematode sampling: closing the integrated micro valve, rotating the micro-fluidic chip, flushing nematodes fixed at the nematode sampling structure into a nematode culture pond, and at the moment, replacing the culture solution in the single nematode analysis channel by periodically replacing the culture solution in the liquid storage pond, removing waste liquid in the waste liquid pond and rotating the micro-fluidic chip so as to keep the normal growth of the nematodes and recording the motion video of the single nematodes in sequence;
(5) single-thread worm fixing in the culture pond: the microfluidic chip is enabled to rotate continuously, nematodes in the nematode culture pond enter and are fixed at the nematode capturing structure, and at the moment, imaging detection of single nematodes is carried out by using the nematode detecting module;
(6) fixed single worm release: closing the rotating motor, and gradually stopping rotating the microfluidic chip; after the culture solution in the microfluidic chip stops flowing, the immobilized single-worm gradually returns to the nematode culture pond from the nematode capturing structure, so that the single-worm can be reversibly immobilized and released.
The invention has the following beneficial effects:
1. the invention designs the disc type single-worm analysis microfluidic chip and is matched with the portable centrifugal analysis device, so that the high-flux single-worm automatic control, long-term culture and observation can be realized, and the practicability of the microfluidic single-worm research method is effectively improved.
2. The high-flux single-worm analysis method provided by the invention is simple, convenient, quick and low in cost, does not need pump valve control and flow path interfaces, and provides a feasible solution for deep research and wide application of model organism nematodes.
Drawings
FIG. 1 is a schematic diagram of a microfluidic chip structure;
FIG. 2 is a schematic diagram of a single-worm analysis channel structure;
FIG. 3 is a schematic diagram of a nematode removal structure;
fig. 4 is a schematic structural diagram of a high-throughput single-worm analysis device based on a centrifugal microfluidic technology;
FIG. 5 is a schematic diagram of a disc microfluidic chip containing four nematode analysis units;
in the figure: 1. the microfluidic chip 11, the nematode analysis unit 111, the liquid storage tank 112, the single-wire worm analysis channel 1121, the nematode sampling structure 1122, the nematode culture tank 1123, the nematode capturing structure 1124 and the channel outlet; 113. a nematode removal structure 1131, a nematode removal channel 1132, and an integrated microvalve; 114. the waste liquid pool, 115, the liquid adding hole, 116, the liquid pool exhaust channel, 117 and the waste liquid pool exhaust channel; 12. a chip fixing hole; 2. the centrifugal control module 21, the rotating shaft 22 and the rotating motor; 3. a nematode detection module.
Detailed Description
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way. In the schematic diagrams of the microfluidic chip and the device, the structural dimensions are not marked, the structural configuration is mainly embodied, and the structural proportion and the dimensions can be adjusted according to the requirements in the actual production and use process.
Example 1
The invention is further described below with reference to the accompanying drawings. FIG. 1 shows a schematic diagram of the structure of a microfluidic chip of the present invention; as can be seen from the figure, the single-worm analysis microfluidic chip 1 of the invention is a disc type, the microfluidic chip 1 is composed of a chip bottom plate and a micro-channel structure layer, the chip bottom plate is circular, a chip fixing hole 12 is arranged in the middle, and the micro-channel structure layer is composed of a plurality of fan-shaped nematode analysis units 11 tightly bonded on the chip bottom plate; the nematode analysis units 11 are uniformly distributed along the circle center at a certain angle, one side of the nematode analysis units 11, which is close to the chip fixing hole 12, is provided with a liquid storage pool 111, the liquid storage pool 111 is in a fan ring shape, liquid adding holes 115 are respectively formed in the positions, close to the circle center, of the two ends of the liquid storage pool 111, the liquid adding holes are used for storing nematode sample solutions during sample injection, and nematode culture solutions are used for storing nematode culture solutions during culture so as to realize periodic updating. A waste liquid pool 114 is arranged at one side far away from the chip fixing hole 12, the waste liquid pool 114 is in a fan ring shape, a plurality of single nematode analysis channels 112 and nematode removing structures 113 are connected between the liquid pool 111 and the waste liquid pool 114, and the nematode removing structures 113 are inserted between the single nematode analysis channels 112 in a symmetrical distribution mode. The waste liquid pool 114 is used for storing the generated waste liquid and collecting nematode samples removed during sample introduction and nematode eggs during culture.
As shown in fig. 2, a schematic diagram of a single-worm analysis channel structure is shown, where the single-worm analysis channel 112 includes, from the center of the circle to the outside, a nematode sampling structure 1121, a nematode culture tank 1122, a nematode capturing structure 1123, and a channel outlet 1124. The joint of the nematode sampling structure 1121 and the liquid storage tank 111 is a conical channel, the conical bottom is connected with the liquid storage tank 111, one end of the nematode culture tank 1122 is connected with the nematode sampling structure 1121 through a thin channel, the width of the thin channel is smaller than the diameter of a nematode sample, the design is favorable for nematodes to enter the open end at a lower rotating speed and be temporarily fixed at the thin channel, at the moment, the flowing resistance of the channel where the nematodes are located is obviously increased, so that the follow-up nematodes are prevented from entering, and the separation of single worms is ensured. The size of the nematode culture pond 1122 is designed according to the size of the adult nematode, so that the culture pond provides enough space for nematode activity and can observe the movement of the nematode in the whole culture pond; meanwhile, the nematode capturing structure 1123 of the conical channel at the other end of the nematode culture tank 1122 faces the nematode culture tank 1122, the conical bottom of the nematode capturing structure 1123 is smaller than the diameter of the nematode sample, and single-worm entering the nematode culture tank 1122 can be limited in the single-worm analysis channel 112, so that single-worm control and long-term tracking are realized. The channel outlet 1124 is connected to the waste reservoir 114.
As shown in fig. 3, which is a schematic diagram of a nematode removal structure, the nematode removal structure 113 is composed of an integrated micro valve 1132 and a nematode removal channel 1131 sequentially from the center to the outside. The width and depth of the nematode removal channel 1131 are both greater than the maximum diameter of the nematode sample, and all nematodes remaining in the reservoir 111 can be flushed into the waste reservoir 114 for removal. The integrated micro valve 1132 is an automatically controlled electromagnetic valve or a mechanical micro valve, and can control the opening and closing states of the nematode cleaning channel 1131 according to a flow design.
FIG. 4 is a schematic structural diagram of a high-throughput single-worm analysis device based on a centrifugal microfluidic technology; as can be seen from the figure, the portable centrifugal analysis device: comprising a centrifugal control module 2 and a nematode detection module 3. The centrifugal control module 2 consists of a rotating shaft 21 and a rotating motor 22; the disc-type microfluidic chip 1 is fixed on a rotating shaft 21, and the rotating speed, time and rotating direction of the microfluidic chip 1 are precisely controlled by a rotating motor 22, so that the flow of the culture solution and the movement state of nematodes are controlled. The detection field of the nematode detection module 3 may cover the whole nematode culture tank 1122 or the nematode fixing structure 1123, and is used for sequentially recording video of single-thread worms in the nematode culture tanks 1122 or microscopic imaging of single-thread worms fixed at the nematode capturing structure 1123.
Example 2
The disc microfluidic chip used in this example contains four nematode analysis units, each nematode analysis unit includes 17 single-wire worm analysis channels and 2 nematode removal structures (see fig. 5), and the diameter of the microfluidic chip is 100mm; the minimum channel width of the nematode feeding structure and the capturing structure is 22 mu m, the depth is 50 mu m, and feeding and separation of caenorhabditis elegans L4 stage larvae can be realized; the nematode culture pond is of an elliptic structure, has the diameter of 1.5mm multiplied by 1.35mm, and can meet the normal activity requirement of single caenorhabditis elegans adults. The dimension of the nematode cleaning channel is 7.4mm multiplied by 100 mu m multiplied by 50 mu m, and the integrated micro valve adopts a self-holding electromagnetic valve.
Example 3
The invention relates to a use method of a high-flux single-worm analysis device based on a centrifugal microfluidic technology, which comprises the following steps:
(1) preparing a microfluidic chip: closing an integrated micro valve on the nematode removing channel, adding a nematode sample and a culture solution into a liquid storage tank from a liquid adding hole together, closing the liquid adding hole, and fixing a micro-fluidic chip on a rotating shaft of a centrifugal control module;
(2) nematode sample addition: starting a rotating motor to drive the microfluidic chip to rotate, so that nematodes enter the nematode sampling structure of the single-worm analysis channel and are fixed;
(3) residual nematode removal: starting a nematode detection module, and observing the fixation condition of nematodes in real time; after single nematodes are fixed at all nematode sampling structures, opening an integrated micro valve, and simultaneously continuing rotating the micro-fluidic chip to enable the remaining nematodes in the liquid storage tank to enter the waste liquid tank from the nematode removing channel;
(4) nematode sampling: closing the integrated micro valve, rotating the micro-fluidic chip, flushing nematodes fixed at the nematode sampling structure into a nematode culture pond, and at the moment, replacing the culture solution in the single nematode analysis channel by periodically replacing the culture solution in the liquid storage pond, removing waste liquid in the waste liquid pond and rotating the micro-fluidic chip so as to keep the normal growth of the nematodes and recording the motion video of the single nematodes in sequence;
(5) single-thread worm fixing in the culture pond: the microfluidic chip is enabled to rotate continuously, nematodes in the nematode culture pond enter and are fixed at the nematode capturing structure, and at the moment, imaging detection of single nematodes is carried out by using the nematode detecting module;
(6) fixed single worm release: closing the rotating motor, and gradually stopping rotating the microfluidic chip; after the culture solution in the microfluidic chip stops flowing, the immobilized single-worm gradually returns to the nematode culture pond from the nematode capturing structure, so that the single-worm can be reversibly immobilized and released.
Claims (8)
1. A high-flux single-worm analysis device based on a centrifugal microfluidic technology is characterized in that: comprises a disc-type micro-fluidic chip and a portable centrifugal analysis device, wherein:
the disc type micro-fluidic chip comprises: comprises a plurality of nematode analysis units which are uniformly distributed along the circle center; the nematode analysis unit sequentially comprises a liquid storage tank, a plurality of single-worm analysis channels and nematode removal structures which are radially arranged from the center of the circle outwards, and a waste liquid tank; the single-worm analysis channel consists of a nematode sample injection structure, a nematode culture pond, a nematode capturing structure and a channel outlet from one side connected with the liquid storage pond; the nematode cleaning structure comprises a nematode cleaning channel and an integrated micro valve, wherein the integrated micro valve is positioned above the nematode cleaning channel;
the portable centrifugal analysis device comprises: comprises a centrifugal control module and a nematode detection module;
the nematode sample injection structure consists of a conical structure channel and a strip-shaped structure channel, wherein the conical bottom of the conical structure channel faces the liquid storage tank, a strip-shaped structure channel is arranged between the conical top and the nematode culture tank, the width of the strip-shaped structure channel is smaller than the diameter of a nematode sample, and the depth of the strip-shaped structure channel is equal to the diameter of the nematode sample;
the nematode trapping structure is a conical structure channel, the conical bottom of the conical structure channel faces the nematode culture pond, the conical top of the conical structure channel is connected with the channel outlet, the width of the top of the conical structure channel is smaller than the diameter of the nematode sample, and the depth of the conical structure channel is equal to the diameter of the nematode sample;
the liquid storage tank is in a fan shape, and the waste liquid tank is in a fan shape.
2. The centrifugal microfluidic technology-based high-throughput single-worm analysis device according to claim 1, wherein: the micro-fluidic chip consists of an upper micro-channel structure layer and a lower bottom plate layer, wherein the two layers are tightly bonded, and the upper micro-channel structure layer is made of an air-permeable elastic silicone rubber material and comprises polydimethylsiloxane and polydimethylsiloxane PDMS; the bottom plate layer is made of light-transmitting materials including glass, polymethyl methacrylate (PMMA) and Polycarbonate (PC).
3. The centrifugal microfluidic technology-based high-throughput single-worm analysis device according to claim 1, wherein: the nematode culture pond in the single-worm analysis channel is elliptical or circular, the diameter of the nematode culture pond is larger than the length of the nematode sample, and the depth of the nematode culture pond is larger than the diameter of the nematode sample.
4. The centrifugal microfluidic technology-based high-throughput single-worm analysis device according to claim 1, wherein: the width and the depth of the nematode removal channel are both larger than the diameter of the nematode sample; the integrated micro valve is an automatically controlled electromagnetic valve or a mechanical micro valve and is positioned above the nematode removing channel.
5. The centrifugal microfluidic technology-based high-throughput single-worm analysis device according to claim 1, wherein: liquid adding holes are formed at two ends of the liquid storage tank, and two ends of the liquid storage tank are connected with liquid storage tank exhaust channels; and two ends of the waste liquid pool are connected with waste liquid pool exhaust channels.
6. The centrifugal microfluidic technology-based high-throughput single-worm analysis device according to claim 1, wherein: the centrifugal control module consists of a rotating shaft and a rotating motor, wherein the rotating shaft is matched with the disc-type microfluidic chip.
7. The centrifugal microfluidic technology-based high-throughput single-worm analysis device according to claim 1, wherein: the nematode detection module can record videos of the single worms in the nematode culture tank or perform microscopic imaging on the single worms fixed at the nematode capturing structure.
8. The method of using a centrifugal microfluidic technology-based high throughput single-worm analysis device according to any one of claims 1 to 7, characterized in that: the method comprises the following operation steps:
(1) preparing a microfluidic chip: closing an integrated micro valve on the nematode removing channel, adding a nematode sample and a culture solution into a liquid storage tank from a liquid adding hole together, closing the liquid adding hole, and fixing a micro-fluidic chip on a rotating shaft of a centrifugal control module;
(2) nematode sample addition: starting a rotating motor to drive the microfluidic chip to rotate, so that nematodes enter the nematode sampling structure of the single-worm analysis channel and are fixed;
(3) residual nematode removal: starting a nematode detection module, and observing the fixation condition of nematodes in real time; after single nematodes are fixed at all nematode sampling structures, opening an integrated micro valve, and simultaneously continuing rotating the micro-fluidic chip to enable the remaining nematodes in the liquid storage tank to enter the waste liquid tank from the nematode removing channel;
(4) nematode sampling: closing the integrated micro valve, rotating the micro-fluidic chip, flushing nematodes fixed at the nematode sampling structure into a nematode culture pond, and at the moment, replacing the culture solution in the single nematode analysis channel by periodically replacing the culture solution in the liquid storage pond, removing waste liquid in the waste liquid pond and rotating the micro-fluidic chip so as to keep the normal growth of the nematodes and recording the motion video of the single nematodes in sequence;
(5) single-thread worm fixing in the culture pond: the microfluidic chip is enabled to rotate continuously, nematodes in the nematode culture pond enter and are fixed at the nematode capturing structure, and at the moment, imaging detection of single nematodes is carried out by using the nematode detecting module;
(6) fixed single worm release: closing the rotating motor, and gradually stopping rotating the microfluidic chip; after the culture solution in the microfluidic chip stops flowing, the immobilized single-worm gradually returns to the nematode culture pond from the nematode capturing structure, so that the single-worm can be reversibly immobilized and released.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105707010A (en) * | 2014-12-05 | 2016-06-29 | 中国科学院大连化学物理研究所 | Micro-valve-based dual-detection microfluidic chip and diabetic model construction method |
| CN105886386A (en) * | 2016-04-06 | 2016-08-24 | 苏州汶颢芯片科技有限公司 | High-flux bacterial colony testing chip, system and method |
| CN106053859A (en) * | 2016-08-02 | 2016-10-26 | 杭州霆科生物科技有限公司 | Centrifugal type glycosylated hemoglobin detection micro-fluidic chip |
| CN106525724A (en) * | 2016-10-26 | 2017-03-22 | 杭州霆科生物科技有限公司 | Centrifugal fully integrated detection system of methanal and pH value |
| CN107570246A (en) * | 2017-10-23 | 2018-01-12 | 广东第二师范学院 | A kind of nematode chip and its microfluidic methods for selection of antibacterial |
| CN207287462U (en) * | 2017-04-07 | 2018-05-01 | 杭州霆科生物科技有限公司 | A kind of centrifugal type microfludic chip for the detection of textile triclosan |
| CN207586245U (en) * | 2017-09-29 | 2018-07-06 | 深圳国际旅行卫生保健中心 | Centrifugal type microfludic chip |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7951333B2 (en) * | 2006-09-05 | 2011-05-31 | Samsung Electronics Co., Ltd. | Centrifugal force-based microfluidic device for protein detection and microfluidic system including the same |
| CN107398307B (en) * | 2016-05-18 | 2023-07-25 | 博奥生物集团有限公司 | Integrated micro-fluidic chip |
-
2019
- 2019-12-13 CN CN201911285621.8A patent/CN110940820B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105707010A (en) * | 2014-12-05 | 2016-06-29 | 中国科学院大连化学物理研究所 | Micro-valve-based dual-detection microfluidic chip and diabetic model construction method |
| CN105886386A (en) * | 2016-04-06 | 2016-08-24 | 苏州汶颢芯片科技有限公司 | High-flux bacterial colony testing chip, system and method |
| CN106053859A (en) * | 2016-08-02 | 2016-10-26 | 杭州霆科生物科技有限公司 | Centrifugal type glycosylated hemoglobin detection micro-fluidic chip |
| CN106525724A (en) * | 2016-10-26 | 2017-03-22 | 杭州霆科生物科技有限公司 | Centrifugal fully integrated detection system of methanal and pH value |
| CN207287462U (en) * | 2017-04-07 | 2018-05-01 | 杭州霆科生物科技有限公司 | A kind of centrifugal type microfludic chip for the detection of textile triclosan |
| CN207586245U (en) * | 2017-09-29 | 2018-07-06 | 深圳国际旅行卫生保健中心 | Centrifugal type microfludic chip |
| CN107570246A (en) * | 2017-10-23 | 2018-01-12 | 广东第二师范学院 | A kind of nematode chip and its microfluidic methods for selection of antibacterial |
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