CN111748466B - Detection device based on digital micro-fluidic control, application and detection method thereof - Google Patents

Detection device based on digital micro-fluidic control, application and detection method thereof Download PDF

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CN111748466B
CN111748466B CN202010469052.9A CN202010469052A CN111748466B CN 111748466 B CN111748466 B CN 111748466B CN 202010469052 A CN202010469052 A CN 202010469052A CN 111748466 B CN111748466 B CN 111748466B
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detection
control unit
detection device
area
mixed solution
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CN111748466A (en
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张勇
王战涛
冯春生
刘水长
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Hunan University of Technology
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    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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Abstract

The invention discloses a detection device based on digital microfluidics and application and a detection method thereof, which are used for chemiluminescence immunodetection and PCR gene amplification and detection, wherein the detection device comprises a kit and a control platform, the kit comprises two-sided digital microfluidics chips which are arranged in parallel and oil which has sealing and lubricating effects on a system, electrowetting droplet brakes of a driving electrode array are distributed on the chips, and the control platform comprises a controller, a temperature control unit, a magnetic control unit and a detection control module, wherein the controller is connected with the electrowetting droplet brakes, the temperature control unit, the magnetic control unit and the detection control module. According to the invention, the sample and the reagent are allocated and transported through the liquid drop operation driven by the digital micro-flow control, and the reactants are rapidly and accurately controlled. The invention realizes multi-channel parallel detection, accelerates the reaction detection process and achieves the functional effect of 'one core and two purposes'.

Description

Detection device based on digital micro-fluidic control, application and detection method thereof
Technical Field
The invention relates to the technical field of biochemical inspection equipment, in particular to a digital microfluidic-based detection device and application and a detection method thereof.
Background
Immunoassay and PCR quantitative detection are two of the main means currently used for disease diagnosis. PCR is a molecular biological technique for amplifying specific DNA fragments in vitro, and can achieve amplification of more than a million-fold of a trace amount of DNA template within 1-2 hours. Thus, PCR plays a great role in archaeology, and medical diagnostics. Immunoassays are one of the most sensitive methods routinely used in clinical laboratories. Chemiluminescent immunoassays utilize the affinity and specificity between an antigen and its cognate antibody, in combination with the sensitivity of chemiluminescence, to detect and quantify the antigen or antibody in a sample matrix, and have been used today for detection assays of various antigens, antibodies, hormones, enzymes, fatty acid vitamins, drugs, and the like.
Existing large advanced laboratory immunoassays and PCR testers have good automation and throughput, but require a large number of samples and long analysis times for each test. The analyzers are large in size and expensive, often one instrument can only perform a test based on one detection principle, such as the existing PCR tester, can not perform immunoassay, and vice versa, and the instruments are low in integration degree, high in requirements on operators, long in detection period and easy to produce cross contamination and result deviation.
Disclosure of Invention
The invention aims to solve the technical problems of long immunoassay and PCR quantitative detection time and high operation requirement in the prior art, and provides a detection device based on digital microfluidics.
The invention aims to solve the other technical problem that the application method of the digital microfluidic detection device in chemiluminescence immune detection and/or PCR detection is based.
The aim of the invention is realized by the following technical scheme:
a detection device based on digital micro-fluidic, which comprises a digital micro-fluidic chip and a control platform,
the digital microfluidic chip comprises a first substrate and a second substrate, wherein the first substrate and the second substrate are separated in parallel to provide a liquid drop running space, the first substrate comprises a first bottom plate, a driving electrode, a dielectric layer and a hydrophobic layer, the driving electrode is arranged on the first bottom plate, and the dielectric layer with hydrophobicity is coated on the driving electrode; the second substrate comprises a second bottom plate, a grounding electrode and a hydrophobic layer, wherein the grounding electrode is arranged on the second bottom plate and is coated with the hydrophobic layer; the parallel separation space of the first substrate and the second substrate is divided into a storage area, a reaction area, a waste liquid area and a test area according to the arrangement of the driving electrodes.
The control platform comprises a controller, an electrowetting droplet brake composed of a driving electrode array, a temperature control unit, a magnetic control unit and a detection control module, wherein the controller is connected with the electrowetting droplet brake composed of the driving electrode array, the temperature control unit, the magnetic control unit and the detection control module. The temperature control unit and the magnetic control unit are arranged in a reaction area of the digital micro-fluidic chip, and the monitoring control module is arranged in a test area of the digital micro-fluidic chip.
Further, the dielectric layer having hydrophobicity includes being directly prepared from a hydrophobic material or coating a hydrophobic layer on the dielectric layer.
Furthermore, the filling medium is packaged in the parallel space between the first substrate and the second substrate, so that pollution problems such as aerosol and the like can be avoided. Preferably, the filling medium is silicone oil.
Further, the storage area comprises a plurality of storage areas, the storage areas are provided with filling holes, and reagents required by the test are respectively packaged. The reaction zone is divided into a plurality of reaction zones.
Further, an independent temperature control unit and/or a magnetic control unit are arranged below the reaction zone. The temperature control unit comprises a copper sheet and a heat transfer film for heating and cooling, and a temperature control system connected to the main control board for controlling the temperature. The magnetic control unit is a movable magnet or a permanent magnet with a motor.
Further, the detection monitoring module of the test area comprises an electrical and/or optical detection, wherein the electrical detection comprises a current detection, and the optical detection comprises one or more of absorbance, chemiluminescence, fluorescence detection.
Preferably, the optical detection module includes a PMT for immunoassay directly above the detection zone. A light emitting diode and a photodiode are mounted beside the PMT, and the photodiode is aligned with a specific area on the chip so that the PCR reaction can be detected in real time.
Further, the driving electrode is made of one or more of copper foil, chromium, ITO and conductive polymer; the dielectric layer is one or more of parylene, circuit board ink, photoresist and metal oxide; the hydrophobic layer is one or more of paraffin, polytetrafluoroethylene, cytop and octafluorocyclobutane; the ground electrode is a transparent conductive material.
The digital microfluidic-based detection device according to the above can be used for PCR detection and/or chemiluminescent immunoassay of body fluids, biological excretions and microorganisms.
The chemiluminescent immunity detection method comprises the following steps:
s1, respectively loading a sample to be detected, a reaction reagent, magnetic beads, a buffer solution, a detection reagent and a cleaning solution into each storage area in a liquid form, controlling to take a liquid drop of the sample to be detected, a liquid drop containing the magnetic beads, a liquid drop of the reaction reagent and a liquid drop of the buffer solution from the storage areas to mix in the reaction areas through a control platform, splitting and fusing the mixed liquid, and reacting to obtain a mixed liquid containing a binding component;
s2, fixing the magnetic beads by using a magnet, transporting unbound components in the mixed solution to a waste liquid area through electrowetting, and then controlling cleaning liquid drops to clean the magnetic beads bound with the product to obtain a pure product;
s3, mixing the detection reagent with the product, and controlling the mixture to be transported to a detection area for detection.
Further, the temperature of the incubation is 37 ℃ and the time is 6-10 min.
The digital microfluidic-based detection device can also be used for a PCR gene amplification method, and comprises the following steps:
s1, respectively loading a sample to be tested or an object to be tested containing the sample to be tested and a lysate, magnetic beads, an amplification reagent and a buffer solution into each storage area in a liquid drop mode, controlling the sample to be tested and the amplification reagent to be mixed into a first reaction area through a control platform, controlling the temperature of the first reaction area to be 92-98 ℃, and reacting to obtain a first mixed solution.
S2, transporting the mixed solution I of the S1 to a reaction zone II, controlling the temperature of the reaction zone II to be 52-58 ℃, controlling the primer liquid drops to be mixed with the mixed solution II to obtain mixed solution II, transporting the mixed solution II to the reaction zone II, controlling the temperature of the reaction zone III to be 70-75 ℃, and reacting to obtain mixed solution III; or directly transporting the mixed solution I of the S1 to a reaction zone III, controlling the temperature of the reaction zone III to be 70-75 ℃, and reacting to obtain the mixed solution III.
S3, carrying out thermal circulation on the mixed solution III in the first reaction zone to the third reaction zone to obtain a PCR amplification product.
S4, taking the liquid drop containing the coated probe, combining the PCR amplification product, and controlling the PCR amplification product to be transported to a detection area for detection.
Further, the thermal cycle conditions are preheating at 90 to 95 ℃ for 25 to 30 seconds to perform initial denaturation, then performing a cycle of denaturation at 93 to 95 ℃ for 30 to 50 times for 5 to 8 seconds, and performing an annealing/extension cycle at 50 to 55 ℃ and 70 to 75 ℃ for 8 to 10 seconds each time; the transfer rate of the PCR mixture droplets between 3 temperature zones is 18-22 electrodes/sec. Preferably, the thermal cycling conditions are pre-heated at 95 ℃ for 30 seconds to effect initial denaturation, followed by 40 cycles of 5 seconds denaturation at 95 ℃ and 8 seconds annealing/extension cycles at 55 ℃ and 72 ℃ each; the transfer rate of the PCR mixture droplets between 3 temperature zones was 20 electrodes/sec.
Further, the sample to be tested includes one or more of whole blood, serum, plasma, lymph, saliva, sputum, cerebral spinal fluid, amniotic fluid, semen, vaginal faeces, serum, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, faecal samples, bacteria, fungi and viruses.
Compared with the prior art, the beneficial effects are that:
the invention realizes the allocation and transportation of samples and reagents based on the liquid drop operation driven by digital micro-flow control, realizes the rapid and accurate control of reactants, and accelerates the reaction detection process. The device of the invention has the advantages of less consumption of reagents, high detection speed and high degree of automation, can complete the complete flow of immunoassay and PCR amplification on a chip, realizes multi-channel parallel detection, and achieves the functional effect of 'one-core double-purpose'. The detection device based on the digital microfluidic technology can shorten the time of traditional chemiluminescence detection and PCR quantitative analysis by 50%.
Drawings
Fig. 1 is a schematic design diagram of a microfluidic chip according to the present invention;
FIG. 2 is a control system architecture diagram of the test platform of the present invention;
FIG. 3 is a schematic view of a second structure of a substrate of the digital microfluidic chip according to the present invention;
fig. 4 is a schematic structural diagram of a substrate of the digital microfluidic chip according to the present invention.
The device comprises a PCR plate 1, a baseplate I, a driving electrode array coated with a dielectric layer 3, a hydrophobic layer I4, a hydrophobic layer II 5, a grounding electrode 6, a baseplate II 7, 8 liquid drops, 9 silicone oil, a storage area 10, a reaction area 11, a test area 12, a waste liquid area 13, a driving electrode array 14 and a filling hole 15.
Detailed Description
The present invention is further illustrated and described below with reference to examples, which are not intended to be limiting in any way. Unless otherwise indicated, the methods and apparatus used in the examples were conventional in the art and the starting materials used were all conventional commercially available.
Example 1
The embodiment provides a detection device based on digital micro-fluidic, which comprises a digital micro-fluidic chip and a control platform,
the digital micro-fluidic chip loaded on the PCR plate 1 comprises a first substrate and a second substrate, wherein the first substrate and the second substrate are separated in parallel to provide a liquid drop running space, and silicone oil 9 is packaged in the parallel space. The first substrate comprises a first bottom plate 2, a dielectric layer 3 containing a driving electrode array and a hydrophobic layer 4, wherein the dielectric layer 3 containing the driving electrode array is arranged on the first bottom plate 2, and the hydrophobic layer 4 is coated on the dielectric layer 3; the second substrate comprises a second bottom plate 7, a grounding electrode 6 and a second hydrophobic layer 5, wherein the grounding electrode 6 is arranged on the second bottom plate 7, and the second hydrophobic layer 5 is coated on the grounding electrode; the parallel separation space of the first and second substrates is divided into a storage area 10, a reaction area 11, a test area 12 and a waste liquid area 13 according to the arrangement of the driving electrodes.
The storage area 10 includes a plurality of storage areas, each of which encapsulates a reagent required for the test. The storage area 10 is provided with a filling hole 15, and the filling hole 15 can be used for supplementing a sample and a required reagent. The reaction zone 11 is divided into a plurality of reaction zones. An independent temperature control device and/or a magnetic control device are arranged below the reaction zone. The temperature control device comprises a copper sheet and a heat transfer film for heating and cooling, and a temperature control system connected to the main control board for controlling the temperature. The magnetic control device is a movable magnet or a permanent magnet with a motor.
The control platform comprises a controller, an electrowetting droplet brake composed of a driving electrode array, a temperature control unit, a magnetic control unit and a detection control module, wherein the controller is connected with the electrowetting droplet brake composed of the driving electrode array, the temperature control unit, the magnetic control unit and the detection control module. The temperature control unit and the magnetic control unit are arranged in a reaction area of the digital micro-fluidic chip, and the detection control module is arranged in a test area of the digital micro-fluidic chip. PMT for immunoassay directly above the test area was detected at a radius of about 10mm. A light emitting diode and a photodiode are mounted beside the PMT, and the photodiode is aligned with a specific area on the chip so that the PCR reaction can be detected in real time. The excitation wavelength of the PCR fluorometer is 495nm and the emission wavelength is about 525 nm. .
The driving electrode array 14 is made of one or more of copper foil, chromium, ITO and conductive polymer.
The dielectric layer 3 is one or more of parylene, circuit board ink, photoresist and metal oxide.
The hydrophobic layer one 4 or the hydrophobic layer two 5 is one or more of paraffin, polytetrafluoroethylene, cytop and octafluorocyclobutane.
The ground electrode 6 is made of transparent conductive material.
Example 2
The present example provides a detection method for detecting Alpha Fetoprotein (AFP) by using the digital microfluidic-based detection device described in example 1, wherein the reagents include serum, a reference substance, a quality control substance, magnetic beads, an AFP recognition antibody, an enzyme-labeled antibody buffer and a luminescent substrate. The reaction steps comprise:
s1, respectively loading serum to be detected, a reference substance and a quality control substance (the concentration of the quality control substance Q1 is 2.5-7.5ng/mL, the concentration of the quality control substance Q2 is 105-195 ng/mL), an AFP recognition monoclonal antibody, a horseradish peroxide marked monoclonal antibody, magnetic beads, a buffer solution and a luminous substrate reagent (4-methyl umbrella-shaped phosphate) into each storage area in a liquid drop mode, respectively taking the serum to be detected, the magnetic beads and the AFP recognition antibody from the storage areas by using a digital microfluidic operation through a control platform, mixing the 2uL to a reaction area, regulating the reaction temperature to about the body temperature, incubating for 6-10 min at 37 ℃, and binding and fixing the antibody and antigen which are specifically bound on the magnetic beads.
S2, fixing the magnetic beads by using a magnet, and separating the magnetic beads from liquid drop components without the magnetic beads (namely unbound antibodies and antigens) by using electrowetting; discharging liquid drops containing unbound substances to a waste liquid area, and then controlling buffer liquid drops to clean magnetic beads bound with the product to obtain a primary antibody-magnetic bead-antigen complex;
s3, mixing and incubating 2uL of horseradish peroxide-marked enzyme-labeled antibody droplets with the primary antibody-magnetic bead-antigen complex by utilizing digital microfluidic operation, and cleaning to obtain the primary antibody-antigen-secondary antibody complex.
S4, mixing and incubating luminescent substrate reagent (4-methyl umbrella-shaped snore phosphate) liquid drops and primary antibody-antigen-secondary antibody complex by utilizing digital microfluidic operation, and then transporting the mixture to a detection area for luminescent intensity test.
Example 3
The present embodiment provides a detection device based on digital microfluidic according to embodiment 1 for PCR gene amplification, comprising:
s1, respectively loading a whole blood sample, a lysate, DNA capture magnetic beads, an amplification mixed reagent and a buffer cleaning solution into each storage area in a liquid drop mode.
S2, taking the whole blood sample and the lysis buffer out of the storage area and uniformly mixing. The magnetic beads were immobilized using a magnet, then the lysed sample was transferred to DNA capture beads and the supernatant removed using droplet splitting. Buffer wash was allocated from the reservoir for removal of cell debris and the purified DNA bound to the magnetic beads was eluted.
S3, controlling the DNA and the reagent to be mixed to a first reaction zone through a control platform, regulating the temperature to 92-98 ℃ and reacting to obtain a first mixed solution. Transporting the mixed solution I to a reaction zone II, regulating the temperature to 52-58 ℃, controlling the primer droplets to be mixed with the primer droplets to obtain a mixed solution II, transporting the mixed solution II to the reaction zone II, and regulating the temperature to 70-75 ℃ for reaction to obtain a mixed solution III;
s3, carrying out thermal circulation on the mixed solution III in the first reaction area to the third reaction area to obtain the PCR gene amplification solution.
The thermal cycling conditions were preheated at 95 ℃ for 30 seconds to perform initial denaturation, followed by 40 cycles of 5 seconds denaturation at 95 ℃ and 8 seconds of annealing/extension cycles at 55 ℃ and 72 ℃; the transfer rate of the PCR mixture droplets between 3 temperature zones was 20 electrodes/sec.
S4, recording once by using a fluorescence photometer every time of amplification.
Example 4
The digital microfluidic-based detection device of example 1 of this embodiment is used for a nucleic acid detection method of a novel coronavirus (2019-nCoV), and adopts a 2019-nCoV-PCR-FAST kit provided by san-xiang biotechnology limited, henna, and includes a 2019-nCoV-PCR-FAST-reaction solution, a 2019-nCoV internal standard, a 2019-nCoV-PCR-FAST enzyme mixed solution, a buffer solution, and negative and positive controls quantitatively referencing a/B/C/D and 2019-nCoV-PCR-FAST. Wherein the 2019-nCoV-PCR-FAST quantitative reference and positive control are inactivated 2019-nCoV-PCR-FAST virus samples, and the negative control is an inactivated 2019-nCoV-PCR-FAST virus negative sample. The reaction steps comprise:
s1, two temperature areas are arranged on a chip, wherein the temperature areas are 55 ℃ and 95 ℃, and the temperature areas correspond to a first temperature area and a second temperature area respectively. And (3) annealing and extending the mixed solution of 2019-nCoV-PCR-FAST enzyme and DNA in the first temperature region, and activating Taq enzyme and denaturing the DNA in the second temperature region.
S2, taking a sample to be detected, negative control, positive control and quantitative reference A/B/C/D, wherein the total number of the sample to be detected is 1uL in 7 channels;
s3, 1uL of 2019-nCoV-PCR-FAST enzyme mixed solution is prepared corresponding to the 7 channels, and is respectively mixed with 7 to-be-detected objects in S1 and uniformly mixed;
s4, 5uL of 2019-nCoV-PCR-FAST-reaction solution is prepared corresponding to the 7 channels, and is mixed with the reactant in S2 and uniformly mixed;
s5, magnetically separating the supernatant by using a magnet;
s6, respectively preparing 5uL of buffer solution corresponding to the 7 channels, uniformly mixing the buffer solution with reactants in the S5, and promoting DNA elution by adopting magnetic separation;
s7, carrying out PCR temperature zone cycle fluorescence quantitative test, and recording the result of each cycle.
It is to be understood that the above examples of the present invention are provided by way of illustration only and not by way of limitation of the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Claims (7)

1. The detection device based on digital micro-fluidic is characterized by comprising a digital micro-fluidic chip and a control platform:
the digital microfluidic chip comprises a first substrate and a second substrate, wherein the first substrate and the second substrate are parallel and separated to provide a liquid drop running space, the first substrate comprises a first bottom plate and an electrowetting liquid drop brake formed by a driving electrode array, and a hydrophobic dielectric layer, the electrowetting liquid drop brake is arranged on the first bottom plate, and the hydrophobic dielectric layer is coated on the driving electrode; the second substrate comprises a second bottom plate, a grounding electrode and a hydrophobic layer, wherein the grounding electrode is arranged on the second bottom plate and is coated with the hydrophobic layer; dividing a parallel separation space of a first substrate and a second substrate into a storage area, a reaction area, a waste liquid area and a test area according to a driving electrode array, wherein the reaction area is divided into a plurality of reaction areas, each reaction area is provided with an independent temperature control unit and a magnetic control unit, and the magnetic control unit is a movable magnet or a permanent magnet with a motor;
the control platform comprises a controller, a temperature control unit, a magnetic control unit and a detection control module, wherein the controller is connected with pins of the electrowetting droplet brake, the temperature control unit, the magnetic control unit and the detection control module; the temperature control unit and the magnetic control unit are arranged in a reaction area of the digital micro-fluidic chip, and the detection control module is arranged in a test area of the digital micro-fluidic chip;
the PCR detection step by using the detection device based on digital microfluidics comprises the following steps:
s1, respectively loading a sample to be tested or an object to be tested containing the sample to be tested and a lysate, capturing magnetic beads, an amplification reagent and a buffer solution into each storage area in a liquid drop form, controlling the sample to be tested and the amplification reagent to be mixed into a first reaction area through a control platform, controlling the temperature of the first reaction area to be 92-98 ℃, and reacting to obtain a first mixed solution;
s2, transporting the mixed solution I of the S1 to a reaction zone II, controlling the temperature of the reaction zone II to be 52-58 ℃, controlling the primer liquid drops to be mixed with the mixed solution II to obtain mixed solution II, transporting the mixed solution II to a reaction zone III, controlling the temperature of the reaction zone III to be 70-75 ℃, and reacting to obtain mixed solution III; or directly transporting the mixed solution I of the S1 to a reaction zone III, controlling the temperature of the reaction zone III to be 70-75 ℃, and reacting to obtain a mixed solution III;
s3, carrying out thermal cycling on the mixed solution III in the first reaction zone to the third reaction zone to obtain a PCR amplification product;
s4, taking the liquid drop containing the coated probe, combining the PCR amplification product, and controlling the transportation of the PCR amplification product to a detection area for detection.
2. The digital microfluidic based detection device according to claim 1 wherein a filling medium is encapsulated in the parallel space between the first and second substrates.
3. The digital microfluidic based detection device according to claim 1 wherein the storage area comprises a plurality of storage areas provided with filling holes.
4. The digital microfluidic based detection device according to claim 1 wherein the detection monitoring module of the test zone comprises electrical and/or optical detection means, wherein the electrical detection comprises current detection means and the optical detection comprises one or more of absorbance, chemiluminescence, fluorescence detection means.
5. The digital microfluidic based detection device according to claim 4 wherein the optical detection device comprises a PMT, light emitting diode and photodiode for use in an immunoassay, the photodiode being aligned with a detection zone on the chip.
6. The digital microfluidic based detection device according to claim 1 wherein the material of the drive electrode is one or more of copper foil, chromium, ITO and conductive polymers; the dielectric layer is one or more of parylene, circuit board ink, photoresist and metal oxide; the hydrophobic layer is one or more of paraffin, polytetrafluoroethylene, cytop and octafluorocyclobutane; the ground electrode is a transparent conductive material.
7. The digital microfluidic based detection device according to claim 1, wherein the thermal cycle conditions are a cycle of preheating at 90-95 ℃ for 25-30 seconds to perform initial denaturation, then performing denaturation at 93-95 ℃ for 30-50 times for 5-8 seconds, and performing annealing/extension cycles at 50-55 ℃ and 70-75 ℃ for 8-10 seconds each time; the transfer rate of the PCR mixture droplets between 3 temperature zones is 18-22 electrodes/second.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113145187A (en) * 2021-03-06 2021-07-23 复旦大学 Intelligent liquid drop generating system based on ROS
CN113275052B (en) * 2021-06-04 2023-01-24 北京京东方传感技术有限公司 Micro-fluidic chip
CN115672417A (en) * 2021-07-28 2023-02-03 北京京东方传感技术有限公司 Digital microfluidic device and driving method thereof
CN114113010A (en) * 2021-10-28 2022-03-01 山东师范大学 Automatic bacteria detection system and method based on digital micro-fluidic
CN118076880A (en) * 2022-09-23 2024-05-24 京东方科技集团股份有限公司 Digital microfluidic device and detection method thereof
CN116376668A (en) * 2023-03-24 2023-07-04 中国科学院空间应用工程与技术中心 Fully integrated nucleic acid detection chip, laminated structure thereof and detection method

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1912625A (en) * 2006-08-25 2007-02-14 清华大学 Application of microdrop control in virus detection and detection method and chip
CN109536384A (en) * 2018-12-24 2019-03-29 中国科学院上海微系统与信息技术研究所 A kind of digital pcr system and its application for the quick absolute quantitation of nucleic acid
CN109557150A (en) * 2019-01-14 2019-04-02 大连大学 Digital microcurrent-controlled chip and pathogen immunologic detection method based on it
CN109580506A (en) * 2018-11-28 2019-04-05 天津瑞生物科技股份有限公司 A kind of fungal detection system and two Methods for Fungi Detection based on digital microfluidic technology
CN110295109A (en) * 2019-07-08 2019-10-01 中国科学院深圳先进技术研究院 Based on the digital pcr detection method of Microfluidic droplet print system and application
WO2019226919A1 (en) * 2018-05-23 2019-11-28 Miroculus Inc. Control of evaporation in digital microfluidics
CN110846388A (en) * 2018-08-21 2020-02-28 夏普生命科学（欧洲）有限公司 Microfluidic device and method for digital assays in biological assays

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI542879B (en) * 2015-03-16 2016-07-21 國立交通大學 Magnetic bead-based digital microfluidic immunoanalysis device and method thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1912625A (en) * 2006-08-25 2007-02-14 清华大学 Application of microdrop control in virus detection and detection method and chip
WO2019226919A1 (en) * 2018-05-23 2019-11-28 Miroculus Inc. Control of evaporation in digital microfluidics
CN110846388A (en) * 2018-08-21 2020-02-28 夏普生命科学（欧洲）有限公司 Microfluidic device and method for digital assays in biological assays
CN109580506A (en) * 2018-11-28 2019-04-05 天津瑞生物科技股份有限公司 A kind of fungal detection system and two Methods for Fungi Detection based on digital microfluidic technology
CN109536384A (en) * 2018-12-24 2019-03-29 中国科学院上海微系统与信息技术研究所 A kind of digital pcr system and its application for the quick absolute quantitation of nucleic acid
CN109557150A (en) * 2019-01-14 2019-04-02 大连大学 Digital microcurrent-controlled chip and pathogen immunologic detection method based on it
CN110295109A (en) * 2019-07-08 2019-10-01 中国科学院深圳先进技术研究院 Based on the digital pcr detection method of Microfluidic droplet print system and application

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