CN111748466A - A detection device based on digital microfluidics and its application and detection method - Google Patents

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

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 particularly, to a detection device based on digital microfluidics and its application and detection method.

Background technique

Immunoassay and PCR quantitative detection are the two main methods currently used for disease diagnosis. PCR is a molecular biology technique used to amplify specific DNA fragments in vitro, which can achieve a million-fold amplification of trace DNA templates within 1-2 hours. Therefore, PCR has played a huge role in paleontology, archaeology and medical diagnosis. Immunoassays are among the most sensitive methods routinely used in clinical laboratories. Chemiluminescence immunoassay utilizes the affinity and specificity between antigens and their cognate antibodies, combined with the sensitivity of chemiluminescence, to detect and quantify antigens or antibodies in sample matrices, and has been used for various antigens, antibodies, hormones , enzymes, fatty acids, vitamins and drugs detection and analysis.

Existing large and advanced laboratory immunoanalyzers and PCR testers have good automation and throughput, but each test requires a large number of samples and long analysis time. These analyzers are large in size and expensive. Often, an instrument can only perform tests based on one detection principle. For example, existing PCR testers cannot perform immunoassays, and vice versa, and the degree of integration of the instruments is low. High requirements for operators, long detection cycle, prone to cross-contamination and deviation of results.

SUMMARY 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 shortcomings of the prior art for immunoassay and PCR quantitative detection with long time and high operation requirements.

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

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

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

The digital microfluidic chip 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. The substrate 1 includes a base plate 1, a driving electrode, a dielectric layer and a hydrophobic layer. The driving electrodes are arranged On the base plate 1, a hydrophobic dielectric layer is coated on the driving electrodes; the base plate 2 comprises a base plate 2, a ground electrode and a hydrophobic layer, and the ground electrode is arranged on the base plate 2, and the hydrophobic layer is coated thereon; The arrangement of the driving electrodes divides the parallel space between the first substrate and the second substrate 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 magnetron unit, and a detection control module, and the controller is connected to the electrowetting droplet brake composed of the 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 monitoring and control module is installed in the test area of the digital microfluidic chip.

Further, the dielectric layer having hydrophobicity includes directly preparing from a hydrophobic material or coating a hydrophobic layer on the dielectric layer.

Further, a filling medium is encapsulated in the parallel space of the first substrate and the second substrate, which can avoid pollution problems such as aerosol. Preferably, the filling medium is silicone oil.

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

Further, an independent temperature control unit and/or a magnetic control unit is provided below the reaction zone. 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 temperature control. The magnetic control unit is a movable magnet with a motor or a permanent magnet.

Further, the detection monitoring module of the test area includes electrical and/or optical detection, wherein 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 photodiode is aligned with a specific area 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 digital microfluidic-based detection device according to the above can be used for PCR detection and/or chemiluminescence immunodetection of body fluids, biological excrement and microorganisms.

The detection method for chemiluminescence immunity, the steps include:

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

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

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

Further, the temperature of the incubation is 37°C, and the time is 6-10 min.

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

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

S2. The mixed solution 1 of S1 is transported to the reaction zone 2, and the temperature of the reaction zone 2 is controlled at 52-58 ° C, and the primer droplets are controlled to be mixed with it to obtain the mixed solution 2, and then the mixture 2 is transported to the reaction zone 2, and the temperature of the reaction zone 3 is Controlling at 70～75 ℃, the reaction obtains mixed solution 3; or directly transporting the mixed solution 1 of S1 to reaction zone 3, the temperature of reaction zone 3 is controlled at 70～75 ℃, and the reaction obtains mixed solution 3.

S3. The mixed solution 3 is thermally cycled in the reaction zone 1 to the reaction zone 3 to obtain a PCR amplification product.

S4. Take the droplet containing the coated probe and combine the PCR amplification product, and control its transport to the detection area for detection.

Further, the thermal cycling conditions are preheating at 90-95° C. for 25-30 seconds for initial denaturation, then performing 30-50 cycles of denaturation at 93-95° C. for 5-8 seconds, and at 50° C. Annealing/extension cycles of 8-10 sec each were performed at ~55°C and 70-75°C; the transport rate of PCR mixture droplets between the 3 temperature zones was 18-22 electrodes/sec. Preferably, the thermal cycling conditions are preheated at 95°C for 30 seconds for initial denaturation, followed by 40 cycles of 5 second denaturation at 95°C and 8 seconds each annealing at 55°C and 72°C /extension cycle; PCR mix droplets were transported at a rate of 20 electrodes/sec between the 3 temperature zones.

Further, the samples to be tested include whole blood, serum, plasma, lymph, saliva, sputum, cerebrospinal fluid, amniotic fluid, semen, vaginal excrement, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, osmotic fluid One or more of exudates, exudates, cystic fluids, bile, urine, gastric juices, intestinal fluids, stool samples, 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 digital microfluidics, realizes rapid and precise manipulation of reactants, and speeds up the reaction detection process. The device of the invention requires less reagent consumption, fast detection speed and high degree of automation, can complete the complete process of immune testing and PCR amplification on the chip, and realize multi-channel parallel detection, so as to achieve "one core and two uses". functional effect. By using the digital microfluidic technology-based detection device of the present invention, the time of traditional chemiluminescence detection and PCR quantitative analysis can be shortened by 50%.

Description of drawings

Fig. 1 is the design principle diagram of the microfluidic chip involved in the present invention;

Fig. 2 is the control system frame diagram of the test platform of the present invention;

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

FIG. 4 is a schematic structural diagram of a 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 zone, 12 test zone, 13 waste liquid zone, 14 drive electrode array, 15 filling hole.

Detailed ways

The following is further explained and illustrated in conjunction with the examples, but the specific examples do not limit the present invention in any form. 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 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 board 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 a silicone oil 9 is encapsulated in the parallel space. The substrate 1 includes a bottom 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 bottom plate 2, and the hydrophobic layer 4 is coated on the dielectric layer. layer 3; the substrate two includes a bottom plate 7, a ground electrode 6 and a hydrophobic layer 2 5, and the ground electrode 6 is arranged on the bottom plate two 7, and is coated with a hydrophobic layer 2 5; The parallel spaced 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 a plurality of storage areas, and the storage areas respectively encapsulate the reagents required for the test. The storage area 10 is provided with a filling hole 15 through which the sample and required reagents can be replenished. The reaction zone 11 is divided into a plurality of reaction zones. An independent temperature control device and/or a magnetic control device is provided below the reaction zone. 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 temperature control. The magnetic control device is a movable magnet or a permanent magnet with a motor.

The control platform includes a controller, an electrowetting droplet brake composed of a driving electrode array, a temperature control unit, a magnetron unit, and a detection control module, and the controller is connected to the electrowetting droplet brake composed of the 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 for immunoassay just above the test area has a detection radius of about 10 mm. A light-emitting diode and photodiode are mounted next to the PMT, and the photodiode is aligned with a specific area on the chip to enable real-time detection of PCR reactions. The excitation wavelength of the PCR fluorometer is 495 nm, and the emission wavelength is about 525 nm. .

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 a transparent conductive material.

Example 2

This example provides a detection method for the determination of alpha-fetoprotein (AFP) by chemiluminescence immunoassay using the digital microfluidic-based detection device described in Example 1. The reagents used include serum, reference substance and quality control substance, magnetic beads, AFP recognizes antibodies, enzyme-labeled antibody buffers and luminescent substrates. The reaction steps include:

S1. The serum to be tested, the reference product and the quality control product (the concentration of the quality control product Q1 should be 2.5-7.5ng/mL, the quality control product Q2 should be 105-195ng/mL) AFP recognition monoclonal antibody, horseradish peroxidation The labeled monoclonal antibodies, magnetic beads, buffers, and luminescent substrate reagents (4-methylumbelliferate monoclonal phosphate) were loaded into each storage area in the form of droplets, and the digital microfluidic operation was used to control the platform. Transfer 2uL of serum to be tested, magnetic beads, and AFP-recognizing antibody in the storage area to mix in the reaction area, adjust the reaction temperature to about body temperature, and incubate at 37°C for 6-10 minutes. The specific binding antibody and antigen are combined and fixed. on the magnetic beads.

S2. Use a magnet to immobilize the magnetic beads, and use electrowetting to separate the magnetic beads from the droplet components that do not contain magnetic beads (ie, unbound antibodies and antigens); discharge the droplets containing unbound substances to the waste liquid area, Then control the buffer droplet to wash the magnetic beads bound to the product to obtain the primary antibody-magnetic bead-antigen complex;

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

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

Example 3

This embodiment provides the use of the digital microfluidic-based detection device described in Embodiment 1 for PCR gene amplification, and the steps include:

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

S2. Transfer whole blood sample and lysis buffer from storage area and mix. 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. A buffer wash solution is dispensed from the storage area to remove cellular debris and the purified DNA bound to the magnetic beads is eluted.

S3. Control the mixing of DNA and reagents to reaction zone 1 by controlling the platform, and adjust the temperature to 92-98° C. for reaction to obtain mixed solution 1. The mixed solution 1 is transported to the reaction zone 2, the temperature is adjusted to 52-58 °C, the primer droplets are controlled to be mixed with it to obtain the mixed solution 2, and then the mixture 2 is transported to the reaction zone 2, and the temperature is adjusted to 70-75 °C to react to obtain the mixed solution. three;

S3. The mixed solution 3 is thermally cycled in the reaction zone 1 to the reaction zone 3 to obtain a PCR gene amplification solution.

The thermal cycling conditions described were a 30 s preheat at 95°C for initial denaturation, followed by 40 cycles of 5 s denaturation at 95°C and 8 s annealing/extension cycles at 55°C and 72°C each ; The transfer rate of PCR mixture droplets between the 3 temperature zones is 20 electrodes/sec.

S4. Use a fluorometer to record once 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 mix, 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 the inactivated 2019-nCoV-PCR-FAST virus negative sample. The reaction steps include:

S1. Set two temperature zones on the chip, 55°C and 95°C, corresponding to temperature zone 1 and temperature zone 2 respectively. In temperature zone 1, the 2019-nCoV-PCR-FAST enzyme mixture and DNA annealing and extension were performed, and in temperature zone 2, Taq enzyme activation and DNA denaturation were performed.

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

S3. Take 1uL of the 2019-nCoV-PCR-FAST enzyme mixture corresponding to the above 7 channels, mix it with the 7 test substances in S1 and mix well;

S4. Take 5uL of the 2019-nCoV-PCR-FAST-reaction solution corresponding to the above 7 channels, mix with the reactants in S2 and mix well;

S5. Use a magnet to perform magnetic separation to remove the supernatant;

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

S7. Carry out the 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 for clearly illustrating the present invention, rather than limiting the embodiments of the present invention. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. Any modification, equivalent replacement and improvement made within the spirit and principle of the present invention shall be included within 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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