CN111748466A - Detection device based on digital microfluidic and application and detection method thereof - Google Patents
Detection device based on digital microfluidic and application and detection method thereof Download PDFInfo
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Abstract
The invention discloses a detection device based on digital microfluidics and an application and a detection method thereof, which are used for chemiluminescence immunoassay and PCR gene amplification and detection. The invention realizes the preparation and transportation of samples and reagents by the droplet operation driven by digital micro-fluidic, and quickly and accurately controls the reactants. The invention realizes multi-channel parallel detection, accelerates the reaction detection process and achieves the functional effect of 'one core and two purposes'.
Description
Technical Field
The invention relates to the technical field of biochemical test equipment, in particular to a detection device based on digital microfluidics and an application and a detection method thereof.
Background
Immunoassays and PCR quantitative detection are currently the two main approaches used for disease diagnosis. PCR is a molecular biology technique for amplifying specific DNA fragments in vitro, and can amplify a trace DNA template by more than one million times within 1-2 hours. Thus, PCR plays a great role in ancient biology, archaeology and medical diagnosis. Immunoassays are one of the most sensitive methods routinely used in clinical laboratories. Chemiluminescence immunoassay methods utilize the affinity and specificity between an antigen and its cognate antibody, and combine the sensitivity of chemiluminescence to detect and quantify the antigen or antibody in a sample matrix, and are currently used for detection and analysis of various antigens, antibodies, hormones, enzymes, fatty acid vitamins, drugs, and the like.
Existing large advanced laboratory immunoassays and PCR testers are well automated and throughput, but each test requires a large number of samples and a long analysis time. These analyzers are large in size and expensive, often one instrument can only execute tests based on one detection principle, for example, an existing PCR tester cannot perform immunoassay, and vice versa, and the instruments are low in integration degree, high in requirement on operators, long in detection period, and prone to cross contamination and result deviation.
Disclosure of Invention
The invention aims to solve the technical problems of long time and high operation requirement of immunoassay and PCR quantitative detection in the prior art, and provides a detection device based on digital microfluidics.
The invention also aims to solve the technical problem of an application method of the digital microfluidic detection device in chemiluminescence immunoassay and/or PCR detection.
The purpose of the invention is realized by the following technical scheme:
a detection device based on digital microfluidics comprises a digital microfluidic 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 parallelly separated to provide a droplet running space, the first substrate comprises a first bottom plate, a driving electrode, a dielectric layer and a first 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 the hydrophobic layer is coated on the grounding electrode; the parallel separated 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 liquid drop brake consisting 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 liquid drop brake consisting 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 microfluidic chip, and the monitoring control module is arranged in a test area of the digital microfluidic 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, filling media are packaged in the parallel space of the first substrate and the second substrate, so that the pollution problems of aerosol and the like can be avoided. Preferably, the filling medium is silicone oil.
Furthermore, the storage area comprises a plurality of storage areas, and the storage areas are provided with filling holes for respectively packaging reagents required by the test. The reaction zone is divided into a plurality of reaction zones.
Furthermore, an independent temperature control unit and/or a magnetic control unit are arranged below the reaction subarea. The temperature control unit comprises a copper sheet and a heat transfer rubber sheet which are used for heating and cooling, and a temperature control system which is connected to the main control board and used for controlling the temperature. The magnetic control unit is a movable magnet with a motor or a permanent magnet.
Further, the detection monitoring module of the test zone comprises electrical and/or optical detection, wherein the electrical detection comprises current detection, and the optical detection comprises one or more of absorbance, chemiluminescence and fluorescence detection.
Preferably, the optical detection module comprises a PMT for immunoassay located directly above the detection area. A light emitting diode and a photodiode are mounted beside the PMT and aligned with a specific region on the chip to enable real-time detection of the PCR reaction.
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 grounding electrode is made of transparent conductive materials.
The digital microfluidic-based detection device according to the above may be used for PCR detection and/or chemiluminescent immunoassay of body fluids, biological excretions and microorganisms.
The detection method of chemiluminescence immunity 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 the storage area to fetch a sample liquid drop to be detected, a liquid drop containing the magnetic beads, a reaction reagent liquid drop and a buffer liquid drop from the storage area through a control platform, mixing the sample liquid drop, the liquid drop containing the magnetic beads, the reaction reagent liquid drop and the buffer liquid drop into the reaction area, splitting and fusing the mixed liquid, and reacting to obtain a mixed liquid containing a binding;
s2, fixing magnetic beads by using a magnet, transporting unbound components in the mixed solution to a waste liquid area through electrowetting, and then controlling washing liquid drops to wash the magnetic beads bound with the product to obtain a pure product;
and S3, taking a detection reagent to mix with the product, and controlling the detection reagent to be transported to a detection area for detection.
Further, the incubation temperature is 37 ℃ and the incubation time is 6-10 min.
The detection device based on the digital microfluidics can also be used for a PCR gene amplification method, and comprises the following steps:
s1, respectively loading a sample to be detected or a substance to be detected containing the sample to be detected and a lysis solution, magnetic beads, an amplification reagent and a buffer solution into each storage area in a liquid drop mode, controlling the sample to be detected and the amplification reagent to be mixed to a first reaction area through an operation 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 obtained in the step 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 I to obtain a 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 a mixed solution III; or directly conveying the mixed solution I of S1 to a reaction zone III, controlling the temperature of the reaction zone III at 70-75 ℃, and reacting to obtain a mixed solution III.
And S3, performing thermal cycle on the mixed solution III from the first reaction zone to the third reaction zone to obtain a PCR amplification product.
S4, taking liquid drops containing the coated probes, combining PCR amplification products, and controlling the PCR amplification products to be transported to a detection area for detection.
Further, the thermal cycle conditions are preheating for 25-30 seconds at 90-95 ℃ for initial denaturation, then performing cycles of 5-8 seconds denaturation for 30-50 times at 93-95 ℃, and performing annealing/extension cycles for 8-10 seconds at 50-55 ℃ and 70-75 ℃ each time; the transmission rate of the PCR mixture liquid drops between the 3 temperature zones is 18-22 electrodes/second. Preferably, the thermocycling conditions are preheated at 95 ℃ for 30 seconds for initial denaturation, followed by 40 cycles of 5 seconds denaturation at 95 ℃ and 8 second annealing/extension cycles at 55 ℃ and 72 ℃; the transfer rate of the PCR mixture droplets between the 3 temperature zones was 20 electrodes/sec.
Further, the sample to be tested includes one or more of whole blood, serum, plasma, lymph fluid, saliva, sputum, cerebrospinal fluid, amniotic fluid, semen, vaginal discharge, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, exudate, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal sample, bacteria, fungi and viruses.
Compared with the prior art, the beneficial effects are:
the invention realizes the preparation and transportation of samples and reagents based on the droplet operation driven by the digital micro-fluidic, realizes the rapid and accurate control of reactants, and accelerates the reaction detection process. The device of the invention has the advantages of less reagent consumption, high detection speed and high automation degree, 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 dual-purpose'. The detection device based on the digital microfluidic technology can shorten the time of traditional chemiluminescence detection and PCR quantitative analysis by 50 percent.
Drawings
FIG. 1 is a schematic diagram of the design 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 diagram of a second substrate structure 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 PCR test device comprises a PCR plate 1, a base plate I2, a drive electrode array 3 coated with a dielectric layer, a hydrophobic layer I4, a hydrophobic layer II 5, a grounding electrode 6, a base plate II 7, liquid drops 8, silicone oil 9, a storage area 10, a reaction area 11, a test area 12, a waste liquid area 13, a drive electrode array 14 and filling holes 15.
Detailed Description
The following examples are further explained and illustrated, but the present invention is not limited in any way by the specific examples. Unless otherwise indicated, the methods and equipment used in the examples are conventional in the art and all materials used are conventional commercially available materials.
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 microfluidic chip loaded on the PCR plate 1 comprises a first substrate and a second substrate, wherein the first substrate and the second substrate are parallelly separated to provide a droplet running space, and silicone oil 9 is sealed in the parallel space. The substrate I comprises a bottom plate I2, a dielectric layer 3 containing a driving electrode array and a hydrophobic layer I4, wherein the dielectric layer 3 containing the driving electrode array is arranged on the bottom plate I2, and the hydrophobic layer I4 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 spaced-apart spaces of the first substrate and the second substrate are divided into a storage region 10, a reaction region 11, a test region 12, and a waste region 13 according to the arrangement of the driving electrodes.
The reservoir 10 includes a plurality of reservoirs, each of which encloses a reagent required for a test. The storage area 10 is provided with a filling hole 15, and the sample and the required reagent can be supplemented through the filling hole 15. 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 subarea. The temperature control device comprises a copper sheet and a heat transfer film which are used for heating and cooling, and a temperature control system which is connected to the main control board and used 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 liquid drop brake consisting 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 liquid drop brake consisting 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 microfluidic chip, and the detection control module is arranged in a test area of the digital microfluidic chip. The detection radius of the PMT for immunoassay, directly above the test area, was about 10 mm. A light emitting diode and a photodiode are mounted beside the PMT and aligned with a specific region on the chip to enable real-time detection of the PCR reaction. The PCR fluorometer has an excitation wavelength of 495nm and an emission wavelength of 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 I4 or the hydrophobic layer II 5 is one or more of paraffin, polytetrafluoroethylene, cytop and octafluorocyclobutane.
The grounding electrode 6 is made of transparent conductive material.
Example 2
This example provides a method for measuring alpha-fetoprotein (AFP) by chemiluminescence immunoassay using the digital microfluidic-based assay device described in example 1, wherein the reagents used include serum, reference and quality control substances, magnetic beads, AFP-recognizing antibodies, enzyme-labeled antibody buffer and luminescent substrate. The reaction steps comprise:
s1, respectively loading a 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, and the concentration of the quality control substance Q2 is 105-195ng/mL) AFP recognition monoclonal antibody, a horseradish peroxidase-labeled monoclonal antibody, magnetic beads, a buffer solution and a luminescent substrate reagent (4-methylumbelliferyl phosphate) into each storage area in a droplet form, 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 an operation platform, mixing the serum, the magnetic beads and the AFP recognition antibody to be detected by 2uL to a reaction area, adjusting the reaction temperature to be about body temperature, incubating for 6-10 min at 37 ℃, and binding and fixing the antibody and the antigen which are subjected to specific binding on the magnetic beads.
S2, fixing 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 the liquid drops containing the unbound substances to a waste liquid area, and then controlling the buffer liquid drops to clean the magnetic beads bound with the products to obtain an anti-magnetic bead-antigen complex;
s3, mixing and incubating the 2uL horse radish peroxide labeled enzyme-labeled antibody liquid drop and the anti-magnetic bead-antigen compound by using digital microfluidic operation, and cleaning to obtain the anti-antigen-secondary antibody compound.
S4, mixing and incubating a luminescent substrate reagent (4-methylumbelliferyl phosphate) droplet and a primary anti-antigen-secondary antibody compound by using digital microfluidic operation, and then transporting the mixture to a detection area for luminescent intensity test.
Example 3
This example provides the use of the digital microfluidic-based detection device of example 1 for PCR gene amplification, comprising the steps of:
s1, respectively loading the whole blood sample, the lysis solution, the DNA capture magnetic beads, the amplification mixed reagent and the buffer cleaning solution into each storage area in a liquid drop mode.
And S2, the whole blood sample and the lysis buffer solution are taken out from the storage area and mixed uniformly. The magnetic beads were immobilized using a magnet, and the lysed sample was then transferred to DNA capture beads and the supernatant was removed using droplet splitting. Buffer wash was dispensed from the reservoir for removal of cell debris and elution of purified DNA bound to the magnetic beads.
S3, controlling the DNA and the reagent to be mixed in the first reaction area through the control platform, and adjusting the temperature to 92-98 ℃ for reaction to obtain a first mixed solution. Transporting the mixed solution I to a reaction zone II, adjusting the temperature to 52-58 ℃, controlling the primer liquid drops to be mixed with the mixed solution I to obtain a mixed solution II, transporting the mixed solution II to the reaction zone II, adjusting the temperature to 70-75 ℃ and reacting to obtain a mixed solution III;
and S3, performing thermal cycle on the mixed solution III from the first reaction area to the third reaction area to obtain a PCR gene amplification solution.
The thermocycling conditions were preheated at 95 ℃ for 30 seconds for initial denaturation, followed by 40 cycles of 5 seconds denaturation at 95 ℃ and 8 second annealing/extension cycles at 55 ℃ and 72 ℃; the transfer rate of the PCR mixture droplets between the 3 temperature zones was 20 electrodes/sec.
And S4, recording once by using a fluorescence photometer after each amplification.
Example 4
In this example, the digital microfluidic-based detection device described in example 1 was used in a nucleic acid detection method for a novel coronavirus (2019-nCoV), and the 2019-nCoV-PCR-FAST kit provided by saint xiang biotechnology limited in Hunan was used, and included negative and positive controls of 2019-nCoV-PCR-FAST-reaction solution, 2019-nCoV internal standard, 2019-nCoV-PCR-FAST enzyme mixed solution, buffer solution, and quantitative reference a/B/C/D and 2019-nCoV-PCR-FAST. Wherein the 2019-nCoV-PCR-FAST quantitative reference and the positive control are inactivated 2019-nCoV-PCR-FAST virus samples, and the negative control is inactivated 2019-nCoV-PCR-FAST virus negative samples. The reaction steps comprise:
s1, two temperature zones are arranged on a chip, wherein the temperature of the temperature zone is 55 ℃ and the temperature of the temperature zone is 95 ℃, and the temperature zone corresponds to a first temperature zone and a second temperature zone respectively. Annealing and extending the 2019-nCoV-PCR-FAST enzyme mixed solution and DNA in the first temperature zone, and activating Taq enzyme and denaturing DNA in the second temperature zone.
S2, taking a sample to be detected, carrying out negative control, positive control and quantitative reference A/B/C/D, wherein 1uL of each of 7 channels is obtained;
s3, taking 2019-nCoV-PCR-FAST enzyme mixed liquor 1uL corresponding to the 7 channels, and respectively mixing and uniformly mixing the mixed liquor with 7 objects to be detected in S1;
s4, taking 2019-nCoV-PCR-FAST-reaction liquid 5uL corresponding to the 7 channels, mixing with the reactant in S2, and uniformly mixing;
s5, carrying out magnetic separation by using a magnet to remove the supernatant;
s6, respectively taking 5uL buffer solution corresponding to the 7 channels, uniformly mixing the buffer solution with the reactant in the S5, and promoting DNA elution by adopting magnetic separation;
and S7, performing PCR temperature zone circulating fluorescence quantitative test, and recording the result of each circulation.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. The detection device based on the digital microfluidics is characterized by comprising a digital microfluidic 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 parallelly separated to provide a droplet running space, the first substrate comprises a first bottom plate, an electrowetting droplet brake consisting of a driving electrode array and a hydrophobic dielectric layer, the electrowetting droplet 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 the hydrophobic layer is coated on the grounding electrode; dividing the parallel separated space of the first substrate and the second substrate into a storage area, a reaction area, a waste liquid area and a test area according to the driving electrode array;
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 a pin of the electrowetting liquid drop 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 microfluidic chip, and the detection control module is arranged in a test area of the digital microfluidic chip.
2. The digital microfluidic based detection device according to claim 1, wherein a filling medium is encapsulated in a parallel space of the first substrate and the second substrate; preferably, the filling medium is silicone oil.
3. The digital microfluidic based detection device according to claim 1, wherein the storage area comprises a plurality of storage areas, the storage areas being provided with filling holes; the reaction zone is divided into a plurality of reaction zones, and each reaction zone is provided with an independent temperature control unit and an independent magnetic control unit.
4. The digital microfluidic based detection device according to claim 1, wherein the detection monitoring module of the test zone comprises an electrical and/or optical detection device, wherein the electrical detection comprises an electrical current detection device, and the optical detection comprises one or more of an absorbance, chemiluminescence, and fluorescence detection device.
5. The digital microfluidic based detection device according to claim 4, wherein the optical detection device comprises PMT for immunoassay, light emitting diode and photodiode, and the photodiode is aligned with the detection region on the chip.
6. The digital microfluidic-based detection device according to claim 1, wherein 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 grounding electrode is made of transparent conductive materials.
7. Use of a digital microfluidic based detection device according to any one of claims 1 to 6 for chemiluminescent immunoassay and/or PCR assays of body fluids, biological excretions and microorganisms.
8. The use of the digital microfluidic based detection device of claim 7 for chemiluminescent immunoassay, wherein the step of performing chemiluminescent immunoassay with the digital microfluidic based detection device comprises:
s1, respectively loading a sample to be detected, a reaction reagent, magnetic beads, a buffer solution and a detection reagent into each storage area in a liquid form, transferring sample liquid drops to be detected, magnetic bead-containing liquid drops, reaction reagent liquid drops and buffer liquid drops from the storage areas to the reaction areas through control of an operation platform, mixing, adjusting to a proper temperature, splitting and fusing mixed liquid, and reacting to obtain a mixed liquid containing a binding component;
s2, fixing magnetic beads by using a magnet, utilizing electrowetting to divide liquid drops containing the magnetic beads and liquid drops containing unreacted combined components or separate the magnetic beads from the liquid drops, conveying sub-liquid drops containing the non-combined substances to a waste liquid area, repeating the steps until the liquid drops do not contain obvious non-combined substances any more, and then controlling a buffer solution to clean the magnetic beads of the combined products to obtain pure products;
and S3, taking a luminous detection reagent to mix with the reaction product, and controlling the luminous detection reagent to be transported to a detection area for detection.
9. The use of the digital microfluidic based detection device according to claim 7 for PCR detection, wherein the step of performing PCR detection using the digital microfluidic based detection device comprises:
s1, respectively loading a sample to be detected or a substance to be detected containing the sample to be detected and a lysis solution, a capture magnetic bead, an amplification reagent and a buffer solution into each storage area in a liquid drop form, controlling the sample to be detected and the amplification reagent to be mixed to a first reaction area through an operation 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 obtained in the step 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 I to obtain a mixed solution II, then 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 a mixed solution III; or directly transporting the mixed solution I of S1 to a reaction zone III, controlling the temperature of the reaction zone III at 70-75 ℃, and reacting to obtain a mixed solution III;
and S3, performing thermal cycle on the mixed solution III from the first reaction zone to the third reaction zone to obtain a PCR amplification product.
S4, taking liquid drops containing the coated probes, combining PCR amplification products, and controlling the PCR amplification products to be transported to a detection area for detection.
10. The use of the digital microfluidic-based detection device of claim 9 for PCR detection, wherein the thermal cycling conditions are preheating at 90-95 ℃ for 25-30 seconds for initial denaturation, then 30-50 cycles of denaturation at 93-95 ℃ for 5-8 seconds, and annealing/extension cycles at 50-55 ℃ and 70-75 ℃ for 8-10 seconds each; the transmission rate of the PCR mixture liquid drops between the 3 temperature zones is 18-22 electrodes/second.
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