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

Abstract

The invention discloses a detection device based on digital microfluidics and its application and detection method, which is used for chemiluminescence immunoassay and PCR gene amplification and detection. The detection device includes a test kit and a control platform, and the test kit includes a parallel A double-panel digital microfluidic chip is arranged, as well as oil that seals and lubricates the system. Electrowetting droplet actuators that drive the electrode array are distributed on the chip. The control platform includes a controller, a temperature control unit, and a magnetic control unit. , detection control module, the controller is connected to the electrowetting droplet actuator, temperature control unit, magnetic control unit and detection control module. The invention realizes the preparation and transportation of samples and reagents through digital microfluidic-driven droplet operation, and quickly and accurately controls the reactants. The invention realizes multi-channel parallel detection, speeds up the reaction detection process, and achieves the functional effect of "one core for dual purposes".

Description

A detection device based on digital microfluidics and its application and detection method

Technical field

The present invention relates to the technical field of biochemical testing equipment, and more specifically, to a detection device based on digital microfluidics and its application and detection method.

Background technique

Immunoassays and PCR quantitative detection are the two main methods currently used for disease diagnosis. PCR is a molecular biology technology used to amplify specific DNA fragments in vitro. It can amplify trace amounts of DNA templates more than a million times in 1-2 hours. Therefore, PCR plays a huge role in paleontology, archeology and medical diagnosis. Immunoassays are among the most sensitive methods routinely used in clinical laboratories. Chemiluminescence immunoassay uses the affinity and specificity between the antigen and its cognate antibody, combined with the sensitivity of chemiluminescence, to detect and quantify the antigen or antibody in the sample matrix. It has been used for various antigens, antibodies, and hormones. Detection and analysis of enzymes, fatty acids, vitamins and drugs.

Existing large-scale advanced laboratory immunoanalyzers and PCR testers have good automation 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 perform tests based on one detection principle. For example, existing PCR testers cannot perform immune analysis, and vice versa. Moreover, the degree of integration of the instruments is low. It has high requirements on operators, long testing cycle, and is prone to cross-contamination and result deviation.

Contents of the invention

The technical problem to be solved by the present invention is to provide a detection device based on digital microfluidics in view of the existing technology's shortcomings in immunoassay and PCR quantitative detection of long time and high operational requirements.

Another technical problem to be solved by the present invention is an application method based on a digital microfluidic detection device in chemiluminescence immunoassay and/or PCR detection.

The purpose of the present invention is achieved through the following technical solutions:

A detection device based on digital microfluidics, the detection device includes a digital microfluidic chip and a control platform,

The digital microfluidic chip includes a substrate one and a substrate two. The substrate one and the substrate two are separated in parallel to provide a space for droplets to run. The substrate one includes a bottom plate, a driving electrode, a dielectric layer and a hydrophobic layer. The driving electrodes are arranged On the first base plate, a hydrophobic dielectric layer is coated on the driving electrode; the second base plate includes a second base plate, a ground electrode and a hydrophobic layer. The ground electrode is provided on the second base plate and is coated with a hydrophobic layer; according to The driving electrode arrangement divides the parallel separated space of substrate one and substrate two into a storage area, a reaction area, a waste liquid area and a test area.

The control platform includes 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. The controller is connected to the electrowetting droplet brake composed of a driving electrode array. , temperature control unit, magnetic control unit and detection control module. The temperature control unit and magnetic control unit are installed in the reaction area of the digital microfluidic chip, and the monitoring control module is installed in the test area of the digital microfluidic chip.

Further, the hydrophobic dielectric layer includes being directly prepared from hydrophobic materials or coating a hydrophobic layer on the dielectric layer.

Furthermore, a filling medium is encapsulated in the parallel space between the first substrate and the second substrate, which can avoid aerosol and other pollution problems. Preferably, the filling medium is silicone oil.

Further, the storage area includes multiple storage areas, and the storage areas are provided with filling holes to respectively package the reagents required for testing. The reaction zone is divided into multiple reaction zones.

Further, an independent temperature control unit and/or magnetic control unit is provided below the reaction partition. The temperature control unit includes copper sheets and heat transfer films for heating and cooling, and a temperature control system connected to the main control board for controlling temperature. The magnetic control unit is a movable magnet with a motor or a permanent magnet.

Further, the detection and monitoring module of the test area includes electrical and/or optical detection, where the electrical detection includes current detection, and the optical detection includes one or more of absorbance, chemiluminescence, and fluorescence detection.

Preferably, the optical detection module includes a PMT for immunoassay located directly above the detection area. A light-emitting diode and photodiode are mounted next to the PMT, and the photodiodes are aligned with specific areas on the chip to enable real-time detection of PCR reactions.

Further, the material of the driving electrode is 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. one or more; the hydrophobic layer is one or more of paraffin, polytetrafluoroethylene, cytop, and octafluorocyclobutane; the ground electrode is a transparent conductive material.

The above detection device based on digital microfluidics can be used for PCR detection and/or chemiluminescence immunodetection of body fluids, biological excretions and microorganisms.

The chemiluminescent immunoassay detection method includes:

S1. Load the sample to be tested, reaction reagents, magnetic beads, buffer, detection reagent and cleaning solution into each storage area in liquid form, and control the retrieval of sample droplets and magnetic beads containing the sample to be tested from the storage area through the control platform The droplets, reaction reagent droplets, and buffer droplets are mixed in the reaction area, and the mixed liquids are split and fused to obtain a mixed solution containing binding components;

S2. Use magnets to fix the magnetic beads, transport the unbound components in the mixed solution to the waste liquid area through electrowetting, and then control the cleaning droplets to clean the magnetic beads bound to the product to obtain pure products;

S3. Get the detection reagent and mix it with the product, and control its transportation to the detection area for detection.

Further, the incubation temperature is 37°C and the time is 6 to 10 minutes.

The detection device based on digital microfluidics can also be used in a PCR gene amplification method. The steps include:

S1. Load the sample to be tested or the substance to be tested, magnetic beads, amplification reagents and buffer containing the sample to be tested and lysate into each storage area in the form of droplets, and control the sample to be tested and amplification through the control platform The reagents are mixed into reaction zone one, the temperature of reaction zone one is controlled at 92-98°C, and the reaction produces mixed liquid one.

S2. Transport the mixed solution one of S1 to reaction zone two. The temperature of reaction zone two is controlled at 52-58°C. Control the primer droplets to mix with them to obtain mixed solution two. Then transport the mixed solution two to reaction zone two. The temperature of reaction zone three is Control it at 70-75°C, and the reaction will produce mixed liquid three; or directly transport the mixed liquid one of S1 to reaction zone three, control the temperature of reaction zone three at 70-75°C, and react to obtain mixed liquid three.

S3. Perform thermal cycling of mixed solution three from reaction zone one to reaction zone three to obtain PCR amplification products.

S4. Take the droplets containing the coated probe and combine the PCR amplification products, and control their transportation to the detection area for detection.

Further, the thermal cycle conditions are preheating at 90-95°C for 25-30 seconds for initial denaturation, and then performing 30-50 cycles of denaturation for 5-8 seconds at 93-95°C, and at 50 Perform annealing/extension cycles of 8 to 10 seconds each at ~55°C and 70~75°C; the transfer rate of PCR mixture droplets between the three temperature zones is 18 to 22 electrodes/second. Preferably, the thermal cycle conditions include preheating at 95°C for 30 seconds for initial denaturation, then 40 cycles of denaturation for 5 seconds at 95°C, and annealing at 55°C and 72°C for 8 seconds each. /extension cycle; the transfer rate of PCR mixture droplets between the 3 temperature zones is 20 electrodes/second.

Further, the samples to be tested include whole blood, serum, plasma, lymph, saliva, sputum, cerebrospinal fluid, amniotic fluid, semen, vaginal discharge, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, exudate Exudate, exudate, cystic fluid, bile, urine, gastric juice, intestinal juice, one or more of stool samples, bacteria, fungi and viruses.

Compared with existing technology, the beneficial effects are:

The invention realizes the preparation and transportation of samples and reagents based on digital microfluidic-driven droplet operation, realizes rapid and accurate control of reactants, and speeds up the reaction detection process. The device of the present invention requires less reagent consumption, has fast detection speed and a high degree of automation. It can complete the complete process of immune testing and PCR amplification on the chip, and realize multi-channel parallel detection, achieving "one core for dual purposes". Functional effects. Using the detection device based on digital microfluidic technology of the present invention, the time of traditional chemiluminescence detection and PCR quantitative analysis can be shortened by 50%.

Description of the drawings

Figure 1 is a schematic design diagram of the microfluidic chip involved in the present invention;

Figure 2 is a control system architecture diagram of the test platform of the present invention;

Figure 3 is a schematic structural diagram of the second substrate of the digital microfluidic chip of the present invention;

Figure 4 is a schematic structural diagram of the substrate of the digital microfluidic chip of the present invention.

Among them, 1 PCR plate, 2 bottom plate one, 3 driving electrode array coated with dielectric layer, 4 hydrophobic layer one, 5 hydrophobic layer two, 6 ground electrode, 7 bottom plate two, 8 droplets, 9 silicone oil, 10 storage area, 11 reaction area, 12 test area, 13 waste liquid area, 14 driving electrode array, 15 filling hole.

Detailed ways

The following further explains and illustrates the invention in conjunction with the examples, but the specific examples do not limit the invention in any way. Unless otherwise specified, the methods and equipment used in the examples are conventional methods and equipment in the art, and the raw materials used are all conventional commercially available raw materials.

Example 1

This embodiment provides a detection device based on digital microfluidics. The detection device includes a digital microfluidic chip and a control platform.

The digital microfluidic chip loaded on the PCR plate 1 includes a substrate 1 and a substrate 2. The substrate 1 and the substrate 2 are separated in parallel to provide a droplet running space, and silicone oil 9 is encapsulated in the parallel space. The substrate one includes a base plate 2, a dielectric layer 3 containing a driving electrode array, and a hydrophobic layer 4. The dielectric layer 3 containing the driving electrode array is on the base plate 2, and the hydrophobic layer 4 is coated on the dielectric layer. on layer 3; the substrate two includes a bottom plate 7, a ground electrode 6 and a hydrophobic layer 2 5. The ground electrode 6 is provided on the bottom plate 2 7 and is coated with a hydrophobic layer 2 5; according to the arrangement of the driving electrodes, the substrate one and The parallel separated space of the second substrate is divided into a storage area 10, a reaction area 11, a test area 12 and a waste liquid area 13.

The storage area 10 includes multiple storage areas, and the storage areas respectively contain reagents required for testing. The storage area 10 is provided with a filling hole 15 through which samples and required reagents can be replenished. The reaction zone 11 is divided into multiple reaction zones. There is an independent temperature control device and/or magnetic control device below the reaction partition. The temperature control device includes copper sheets and heat transfer films for heating and cooling, and a temperature control system connected to the main control board for controlling temperature. The magnetic control device is a movable magnet with a motor or a permanent magnet.

The control platform includes 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. The controller is connected to the electrowetting droplet brake composed of a driving electrode array. , temperature control unit, magnetic control unit and detection control module. The temperature control unit and the magnetic control unit are installed in the reaction area of the digital microfluidic chip, and the detection control module is installed in the test area of the digital microfluidic chip. The PMT used for immunoassays directly above the test area has a detection radius of approximately 10mm. A light-emitting diode and photodiode are mounted next to the PMT, and the photodiodes are aligned with specific areas on the chip to enable real-time detection of PCR reactions. The excitation wavelength of the PCR fluorometer is 495nm, and the emission wavelength is approximately 525nm. .

The material of the driving electrode array 14 is 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

This embodiment provides a detection method for detecting alpha-fetoprotein (AFP) using the chemiluminescence immunoassay based on the digital microfluidic detection device described in Example 1. The reagents used include serum, reference products, quality control products, and magnetic beads. AFP recognition antibody, enzyme-labeled antibody buffer and luminescent substrate. Reaction steps include:

S1. Combine the serum to be tested, reference material and quality control material (the concentration of quality control material Q1 should be 2.5-7.5ng/mL, quality control material Q2 should be 105-195ng/mL) AFP recognition monoclonal antibody, horseradish peroxidation Labeled monoclonal antibodies, magnetic beads, buffers, and luminescent substrate reagents (4-methylumbele phosphate) are loaded into each storage area in the form of droplets, and digital microfluidic operations are used through the control platform to Transfer 2uL each of serum to be tested, magnetic beads, and AFP-recognizing antibodies from the storage area to the reaction area to mix, adjust the reaction temperature to about body temperature, and incubate at 37°C for 6 to 10 minutes. The antibodies that specifically bind to the antigen are bound and fixed. on magnetic beads.

S2. Use a magnet to fix the magnetic beads, and use electrowetting to separate the magnetic beads from the droplet components that do not contain magnetic beads (i.e., unbound antibodies and antigens); discharge the droplets containing unbound substances to the waste area. The buffer droplets are then controlled to clean the magnetic beads bound to the product to obtain a primary antibody-magnetic bead-antigen complex;

S3. Use digital microfluidic operation to mix and incubate 2uL horseradish peroxide-labeled enzyme-labeled antibody droplets with the primary antibody-magnetic bead-antigen complex, and wash to obtain the primary antibody-antigen-secondary antibody complex.

S4. Use digital microfluidic operation to mix and incubate the luminescent substrate reagent (4-methylumbele phosphate) droplets and the primary antibody-antigen-secondary antibody complex, and then transport them to the detection area to measure the luminescence intensity. test.

Example 3

This embodiment provides the use of the detection device based on digital microfluidics described in Embodiment 1 for PCR gene amplification. The steps include:

S1. Load the whole blood sample, lysate, DNA capture magnetic beads, amplification mixed reagent and buffer cleaning solution into each storage area in the form of droplets.

S2. Remove the whole blood sample and lysis buffer from the storage area and mix them evenly. Magnets are used to immobilize magnetic beads, then the lysed sample is transferred to DNA capture beads and the supernatant is removed using droplet splitting. Dispense wash buffer from the storage area to remove cell debris and elute the purified DNA bound to the magnetic beads.

S3. Use the control platform to control the mixing of DNA and reagents into reaction zone one, adjust the temperature to 92~98°C and react to obtain mixed solution one. Transport mixed solution one to reaction zone two, adjust the temperature to 52-58°C, control the primer droplets to mix with them to obtain mixed solution two, then transport mixture two to reaction zone two, adjust the temperature to 70-75°C and react to obtain mixed solution three;

S3. Perform thermal cycling of mixed solution three from reaction zone one to reaction zone three to obtain a PCR gene amplification solution.

The thermal cycling conditions include a 30-second preheat at 95°C for initial denaturation, followed by 40 cycles of 5-second denaturation at 95°C, and 8-second annealing/extension cycles at 55°C and 72°C. ;The transfer rate of PCR mixture droplets between the three temperature zones is 20 electrodes/second.

S4. Use a fluorescence photometer to record each amplification.

Example 4

The digital microfluidic-based detection device described in Example 1 of this example is used for the nucleic acid detection method of the new coronavirus (2019-nCoV), using the 2019-nCoV-PCR-FAST kit provided by Hunan Shengxiang Biotechnology Co., Ltd. , including 2019-nCoV-PCR-FAST-reaction solution, 2019-nCoV internal standard, 2019-nCoV-PCR-FAST enzyme mixture, buffer, quantitative reference A/B/C/D and 2019-nCoV-PCR-FAST negative and positive controls. 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. Reaction steps include:

S1. Set two temperature zones on the chip, 55°C and 95°C, corresponding to temperature zone one and temperature zone two respectively. Warm zone one carries out 2019-nCoV-PCR-FAST enzyme mixture and DNA annealing and extension, while warm zone two carries out Taq enzyme activation and DNA denaturation.

S2. Get the sample to be tested, negative control, positive control, and quantitative reference A/B/C/D, a total of 7 channels, 1uL each;

S3. Correspond to the above 7 channels, take 1uL of 2019-nCoV-PCR-FAST enzyme mixture, mix it with the 7 analytes in S1 and mix thoroughly;

S4. Correspond to the above 7 channels, take 5uL of 2019-nCoV-PCR-FAST-reaction solution, mix it with the reactant in S2 and mix well;

S5. Use magnets for magnetic separation to remove the supernatant;

S6. Take 5uL of buffer corresponding to the above 7 channels, mix it with the reactants in S5, and use magnetic separation to promote DNA elution;

S7. Carry out PCR temperature zone cycle fluorescence quantitative test and record the results of each cycle.

Obviously, the above-mentioned embodiments of the present invention are only examples to clearly illustrate the present invention, and are not intended to limit the implementation of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the claims of the present invention.

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.