A microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection, and a preparation method and application thereof

Abstract

The application discloses a micro-fluidic chip integrating nucleic acid extraction and high-throughput ECL detection, and a preparation method and application thereof. The micro-fluidic chip comprises a cover sheet, an intermediate layer and a substrate from top to bottom. The intermediate layer is provided with a nucleic acid extraction reagent storage bin, an electrochemical luminescence reagent storage bin, an electrochemical luminescence reaction bin and a waste liquid bin. The substrate is provided with a gold electrode corresponding to the electrochemical luminescence reaction bin. The preparation method comprises the following steps: substrate cleaning, nano-gold preparation, substrate gold electrode preparation, PDMS glue preparation and micro-fluidic chip construction. The application has the advantages that the operation process of nucleic acid extraction reduces external pollution, improves nucleic acid detection throughput, shortens nucleic acid extraction and detection time, reduces detection cost, and greatly reduces professional requirements for nucleic acid extraction and detection.

Description

A microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection, its preparation method and application Technical Field

[0001] This invention relates to the field of microfluidic chip technology, and more particularly to microfluidic chip technology related to nucleic acid extraction and high-throughput electrochemiluminescence. Background Technology

[0002] Many major emerging infectious diseases are caused by viral infections, which can easily trigger global public health crises. Molecular diagnostics is currently an effective tool for disease screening and diagnosis, with nucleic acid testing becoming the gold standard for many diseases. However, rapid and sensitive nucleic acid testing still faces the following two challenges: 1) The nucleic acid extraction process is complex and cumbersome, requiring specialized equipment and laboratories; 2) The concentration of target nucleic acid in samples is usually very low, necessitating amplification techniques to achieve highly sensitive detection.

[0003] With the increasing demand for point-of-care testing (POCT), microfluidic technology has gradually gained attention in the field of biochemical analysis due to its advantages such as miniaturization, automation, low reagent and sample consumption, low pollution, and high throughput. Currently, some automated microfluidic chips for nucleic acid extraction and detection have been developed, but most of their detection methods rely on enzyme-catalyzed amplification fluorescence detection, which prolongs detection time, increases detection costs, and can only detect one target with low throughput. Electrochemiluminescence (ECL), on the other hand, is a rapid, low-cost, low-background, high-signal, and wide-linearity analytical method. Improving ECL luminescence efficiency holds promise for achieving amplification-free, highly sensitive, and rapid detection. Therefore, developing a microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection has significant scientific research value for the rapid screening and diagnosis of emerging infectious diseases. Summary of the Invention

[0004] The purpose of this invention is to provide a microfluidic chip that integrates nucleic acid extraction and high-throughput ECL detection, as well as its preparation method and application, to solve the problems of long detection time, high detection cost and low detection throughput of existing microfluidic chips.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection includes a chip body and a matching magnet. The chip body, from top to bottom, includes a cover plate, an intermediate layer, and a substrate. The cover plate is used to seal the top of the intermediate layer, and the intermediate layer and substrate are sealed together.

[0007] The cover plate is made of a deformable material;

[0008] The intermediate layer is provided with a nucleic acid extraction reagent storage chamber, an electrochemiluminescence reagent storage chamber, an electrochemiluminescence reaction chamber, and a waste liquid chamber. The electrochemiluminescence reagent storage chamber is connected to the nucleic acid extraction reagent storage chamber and the electrochemiluminescence reaction chamber through a flow channel. The nucleic acid extraction reagent storage chamber is connected to the electrochemiluminescence reaction chamber through a flow channel. The electrochemiluminescence reaction chamber is connected to the waste liquid chamber through a flow channel.

[0009] The substrate is provided with a bipolar gold electrode corresponding to the electrochemiluminescence reaction chamber, and the bipolar gold electrode is fixed with a capture probe modified with the target nucleic acid.

[0010] Furthermore, the nucleic acid extraction reagent storage chamber includes a lysis buffer and magnetic bead storage chamber, a protein washing buffer storage chamber, one or more washing buffer storage chambers connected in series, and an elution buffer storage chamber. The lysis buffer and magnetic bead storage chamber, the protein washing buffer storage chamber, the washing buffer storage chamber, and the elution buffer storage chamber are connected in sequence through a flow channel. The elution buffer storage chamber is connected to the electrochemiluminescence reaction chamber through a flow channel.

[0011] The electrochemiluminescence reagent storage compartment includes an electrochemiluminescence probe storage compartment, a NaCl solution storage compartment, and a TPA storage compartment.

[0012] The electrochemiluminescence probe storage chamber is connected to the eluent storage chamber via a flow channel, and the NaCl solution storage chamber and TPA storage chamber are connected to the electrochemiluminescence reaction chamber via flow channels. Alternatively, the electrochemiluminescence probe storage chamber, NaCl solution storage chamber, and TPA storage chamber are all connected to the eluent storage chamber via flow channels.

[0013] The electrochemiluminescence reaction chamber has one or more chambers, and when there are two or more chambers, they are arranged in parallel.

[0014] Furthermore, the top of the lysis buffer and magnetic bead storage chamber, the protein washing buffer storage chamber, the washing buffer storage chamber, and the elution buffer storage chamber are all provided with pre-loaded reagent holes;

[0015] The lysis solution and magnetic bead storage chamber are equipped with pre-loaded reagent holes at both the beginning and end of the top.

[0016] The top of the electrochemiluminescence reaction chamber is equipped with a capture probe addition hole;

[0017] The flow channel is located on the bottom surface of the intermediate layer;

[0018] The top of the electrochemiluminescence reagent storage chamber is deformable;

[0019] The intermediate layer has a cutout at the electrical interface of the corresponding bipolar gold electrode, or the area of ​​the intermediate layer is smaller than that of the substrate, and the electrical interface of the gold electrode is exposed after the two are sealed together, so as to facilitate the connection of the electrode.

[0020] Furthermore, the cover sheet is a 3M film;

[0021] The intermediate layer is PDMS adhesive, and the top of the electrochemiluminescence reagent storage chamber is a PDMS film.

[0022] The substrate is a glass sheet.

[0023] This invention also provides a method for preparing the microfluidic chip that integrates nucleic acid extraction and high-throughput ECL detection, the process comprising:

[0024] (1) The substrate is cleaned and then silanized;

[0025] (2) Preparation of nano-gold solution;

[0026] (3) Preparation of gold electrode on substrate: According to the predetermined pattern of bipolar gold electrode, a nano-gold solution is dropped onto the substrate for nano-gold incubation; the nano-gold solution that becomes transparent on the substrate surface is removed and new nano-gold solution is added, and the dropping is repeated until a nano-gold sub-monolayer is formed. Then the substrate is immersed in Au. 3+ In NH2OH aqueous solution, nano-gold sub-monolayers and Au 3+ The Au thin film formed by the NH2OH aqueous solution completes the growth and infiltration, forming the substrate gold electrode;

[0027] (4) PDMS adhesive preparation: PDMS and initiator are stirred and mixed; the mixed PDMS is poured into a mold; the PDMS adhesive is heated and cured to form a PDMS adhesive and then peeled off. The peeled PDMS adhesive is used to make holes in the top of the nucleic acid extraction reagent storage chamber and the electrochemiluminescence reaction chamber for reagent preloading.

[0028] (5) Microfluidic chip construction: Place the substrate obtained in step (3) with the PDMS channel face up and the surface smooth and dust-free into the plasma processor; calibrate and seal the gold electrode and PDMS glue on the plasma-treated substrate; bake the sealed chip, then fix and modify the capture probe, preload the required reagents and nucleic acid samples to be tested, and seal with a cover plate to form a solid and complete chip.

[0029] Further, the process of step (1) includes: washing the substrate surface with water and drying the surface moisture; cleaning the substrate surface with methanol and then ultrasonically cleaning it in methanol for 5-15 minutes, preferably 10 minutes; immersing the substrate in 30%-60% APTES-MeOH treatment, preferably 50% APTES-MeOH treatment, for 1 hour, so that its surface is positively charged; then thoroughly cleaning the substrate with methanol and ultrasonically cleaning it in methanol to remove excess APTES, for 10 minutes; and placing the cleaned substrate in a ventilated place to air dry.

[0030] Further, step (2) includes: preparing 50 mL of 1 mM chloroauric acid and 15 mL of 38.8 mM sodium citrate solution; adding chloroauric acid and a magnetic stir bar into a reaction vessel and placing it on a magnetic stirrer, reflux heating until boiling at 130°C, adding sodium citrate solution to react, and continuing to heat and reflux for 15-20 min after reaching a deep red color; stopping heating, continuously stirring and refluxing to room temperature to obtain a nano gold solution for later use.

[0031] Further, step (3) includes: covering a protective film on a substrate, the protective film having cutouts designed according to a predetermined pattern of bipolar gold electrodes in the area of ​​the substrate where bipolar gold electrodes need to be designed; dropping a nano-gold solution onto the exposed substrate through the cutouts in the protective film for nano-gold incubation; every 2 hours, removing the nano-gold solution that has become transparent on the substrate surface and adding new nano-gold solution, repeating the dropping until the negatively charged nano-gold combines with the positively charged substrate surface to form a nano-gold sub-monolayer; immersing the substrate with the nano-gold sub-monolayer in Au. 3+ Au with a concentration of 0.1 mM to 0.5 mM and an NH2OH concentration of 0.1 mM to 0.5 mM 3+ In NH2OH aqueous solution, Au 3+ The preferred concentration is 0.3 mM, the preferred concentration of NH2OH is 0.3 mM, and the nano-gold sub-monolayer with Au... 3+ The Au thin film formed by the NH2OH aqueous solution completes the growth and infiltration, forming the substrate gold electrode.

[0032] Further, the process of step (4) includes: adding 1g of PDMS to 1g of initiator and stirring to remove air bubbles, stirring for 15min; pouring the mixed PDMS into a mold and removing air bubbles; heating at 100℃~150℃ for 10min~15min to cure and peel off the PDMS adhesive, preferably heating at 120℃ for 12min; then punching holes in the top of the nucleic acid extraction reagent storage chamber and the electrochemiluminescence reaction chamber for reagent preloading.

[0033] Further, the process of step (5) includes: placing the substrate obtained in step (3) with the PDMS glue channel face up and the surface smooth and dust-free into a plasma processor, setting the equilibration time to 60s and the discharge time to 20s; plasma treatment of the surface characteristics of the substrate gold electrode and PDMS glue, and calibration and sealing; baking the sealed chip at 70℃~90℃ for 15-30min, treating the capture probe with TCEP at room temperature for 1~3h, preferably 2h, reducing the thiol-modified capture probe, capture probe:TCEP = 1:5~1:50, preferably 1:10, and then taking 20μL of the capture probe and TCEP mixture and dropping it onto the gold electrode in (3), and incubating it for 8~24h, preferably 12h; fixing and modifying the capture probe onto the substrate gold electrode by forming Au-S bonds, washing it with water 3 times, and then preloading the required reagents and nucleic acid samples to be tested, and sealing it with a 3M membrane as a cover to preserve the reagents and avoid external contamination to form a firm and complete chip.

[0034] The use of microfluidic chips for simple and rapid nucleic acid extraction and ECL luminescence detection includes the following steps:

[0035] (1) First, inject the electrochemiluminescence probe, 0.1M to 3M NaCl solution, and tripropylamine (TPA) auxiliary solution into the electrochemiluminescence probe storage chamber, NaCl solution storage chamber, and TPA storage chamber in sequence using a syringe; the concentration of NaCl solution is 0.1M to 3M, preferably 0.3M;

[0036] (2) 70 μL to 100 μL, 30 μL to 40 μL, 30 μL to 40 μL, 30 μL to 40 μL, 30 μL to 40 μL, and 10 μL to 20 μL of silicone oil are added sequentially to the lysis buffer and magnetic bead storage chamber, the protein washing buffer storage chamber, the two series-connected washing buffer storage chambers, and the elution buffer storage chamber. Preferably, 90 μL, 38 μL, 38 μL, 38 μL, and 18 μL are added. Then, 250 μL of lysis buffer, magnetic beads, 150 μL of protein washing buffer, 150 μL of washing buffer, 150 μL of washing buffer, and 150 μL of elution buffer are added sequentially.

[0037] (3) Collect virus samples;

[0038] (4) Add 50 μL to 150 μL of virus sample, preferably 100 μL, to the lysis buffer and magnetic bead storage chamber, seal the middle layer with a cover, and lyse at 70°C for 2 min.

[0039] (5) Use a magnet to move the magnetic beads back and forth in the lysis buffer and the magnetic bead storage chamber to mix them for 2 to 5 minutes, preferably 3 minutes, so as to adsorb viral nucleic acid;

[0040] (6) The magnet continues to drive the magnetic beads back and forth in the protein washing solution storage chamber to remove the protein;

[0041] (7) The magnet continues to drive the magnetic beads through two washing liquid storage chambers in sequence to remove other impurities;

[0042] (8) Finally, the magnet drives the magnetic beads into the eluent storage chamber, and heats them at 60-80°C for 2-5 minutes, preferably at 70°C for 3 minutes, to elute viral nucleic acid.

[0043] (9) First, press down the cover of the electrochemiluminescence probe storage chamber to drive the extracted nucleic acid to be evenly distributed in the four electrochemiluminescence reaction chambers, and based on the principle of complementary base matching, the capture probe on the gold electrode binds to the target viral nucleic acid; continue to press down the cover of the electrochemiluminescence probe storage chamber to make the electrochemiluminescence probe, the target viral nucleic acid and the capture probe form a sandwich structure.

[0044] (10) Press down the cover of the NaCl solution storage compartment to wash away unbound nucleic acids and electrochemiluminescent probes;

[0045] (11) Finally, press down the cover of the TPA storage chamber to fill the electrochemiluminescence reaction chamber with TPA auxiliary liquid, connect the power supply, measure the ECL intensity in each electrochemiluminescence reaction chamber, and evaluate the level of target viral nucleic acid content based on the ECL intensity.

[0046] The advantages of this invention include:

[0047] (1) Bipolar gold electrodes for electrochemiluminescence with predetermined patterns are prepared by in-situ growth of gold nanoparticles via chemical deposition.

[0048] (2) This invention simplifies the nucleic acid extraction process, shortens the nucleic acid extraction time, and reduces external contamination;

[0049] (3) Nucleic acid extraction using this device requires less reagent and is highly efficient;

[0050] (4) This invention breaks the limitation that nucleic acid extraction needs to be carried out in a professional laboratory, and provides a new tool for POCT detection;

[0051] (5) The present invention has a high nucleic acid detection throughput, providing a reference for the simultaneous detection of multiple targets. Attached Figure Description

[0052] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings:

[0053] Figure 1 is a schematic diagram of the microfluidic chip structure;

[0054] Figure 2 is a schematic diagram of the PDMS adhesive structure;

[0055] Figure 3 is a schematic diagram of the ECL detection results in four electrochemiluminescence reaction chambers.

[0056] 1. Lysis buffer and magnetic bead storage chamber; 11. Pre-loaded reagent well; 2. Protein washing buffer storage chamber; 3-4. Washing buffer storage chamber; 5. Elution buffer storage chamber; 6. Electrochemiluminescence probe storage chamber; 7. NaCl solution storage chamber; 8. TPA storage chamber; 12. Electrochemiluminescence reaction chamber; 121. Capture probe addition well; 16. Waste liquid chamber; 17. Cover plate; 18. Intermediate layer; 181. Hole cutout at the corresponding electrical interface of the intermediate layer; 19. Substrate; 191. Gold electrode; 192. Electrical interface of the gold electrode; 20-23. ECL detection result curve of the electrochemiluminescence reaction chamber. Detailed Implementation

[0057] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0058] APTES: Triethoxysilane, 3-aminopropyltriethoxysilane

[0059] PDMS: polydimethylsiloxane

[0060] TPA: tripropylamine

[0061] TCEP: Tris(2-carboxyethyl)phosphine

[0062] Example 1: Microfluidic Chip

[0063] This embodiment provides a microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection, as shown in Figure 1. It includes a chip body and a matching magnet. The chip body includes a cover plate 17, an intermediate layer 18, and a substrate 19 from top to bottom. The cover plate 17 is used to seal the top of the intermediate layer 18, and the intermediate layer 18 and the substrate 19 are sealed together. The cover plate 17 is a 3M film; the intermediate layer 18 is PDMS adhesive; and the substrate 19 is a glass sheet.

[0064] The intermediate layer 18 is provided with a nucleic acid extraction reagent storage chamber, an electrochemiluminescence reagent storage chamber, an electrochemiluminescence reaction chamber 12, and a waste liquid chamber 16. The electrochemiluminescence reagent storage chamber is connected to the nucleic acid extraction reagent storage chamber and the electrochemiluminescence reaction chamber 12 through a flow channel. The nucleic acid extraction reagent storage chamber is connected to the electrochemiluminescence reaction chamber 12 through a flow channel. The electrochemiluminescence reaction chamber 12 is connected to the waste liquid chamber 16 through a flow channel.

[0065] The substrate 19 is provided with a bipolar gold electrode 191 corresponding to the electrochemiluminescence reaction chamber 12, and the bipolar gold electrode 191 is fixed with a capture probe modified with the target nucleic acid.

[0066] The nucleic acid extraction reagent storage chamber includes a lysis buffer and magnetic bead storage chamber 1, a protein washing buffer storage chamber 2, two washing buffer storage chambers 3 and 4 connected in series, and an elution buffer storage chamber 5; the lysis buffer and magnetic bead storage chamber 1, the protein washing buffer storage chamber 2, the washing buffer storage chambers 3 and 4, and the elution buffer storage chamber 5 are connected sequentially through a flow channel; the elution buffer storage chamber 5 is connected to the electrochemiluminescence reaction chamber 12 through a flow channel;

[0067] The electrochemiluminescence reagent storage chamber includes an electrochemiluminescence probe storage chamber 6, a NaCl solution storage chamber 7, and a TPA storage chamber 8.

[0068] The electrochemiluminescence probe storage chamber 6 is connected to the eluent storage chamber 5 through a flow channel, and the NaCl solution storage chamber 7 and the TPA storage chamber 8 are connected to the electrochemiluminescence reaction chamber 12 through a flow channel. Alternatively, as shown in Figure 2, the electrochemiluminescence probe storage chamber 6, the NaCl solution storage chamber 7, and the TPA storage chamber 8 are all connected to the eluent storage chamber 5 through a flow channel.

[0069] There may be one or more electrochemiluminescence reaction chambers 12, and when there are two or more electrochemiluminescence reaction chambers 12, they are arranged in parallel.

[0070] The top of the lysis buffer and magnetic bead storage chamber 1, the protein washing buffer storage chamber 2, the washing buffer storage chamber, and the elution buffer storage chamber 5 are all provided with pre-loaded reagent holes 11;

[0071] The lysis solution and magnetic bead storage chamber 1 are provided with pre-loaded reagent holes 11 at both the first and last ends of the top;

[0072] The top of the electrochemiluminescence reaction chamber 12 is provided with a capture probe addition hole 121;

[0073] The flow channel is located on the bottom surface of the intermediate layer 18;

[0074] The top of the electrochemiluminescence reagent storage chamber is deformable;

[0075] The area of ​​the intermediate layer 18 is smaller than that of the substrate 19. After the two are sealed together, the electrical interface 192 of the gold electrode is exposed. Alternatively, as shown in Figure 2, the intermediate layer 18 has a cutout 181 at the electrical interface 192 of the corresponding bipolar gold electrode to facilitate electrode connection.

[0076] Example 2: Fabrication of Microfluidic Chips

[0077] 1. Glass slide cleaning

[0078] (1) Clean the glass slide surface and dry the surface moisture;

[0079] (2) Clean the glass slide with methanol, and then sonicate it in methanol for 10 minutes.

[0080] (3) Immerse in 50% APTES-MeOH for 1 hour;

[0081] (4) Clean the glass slide thoroughly with methanol and then ultrasonically clean it in methanol to remove excess APTES. The ultrasonic cleaning time is 10 min.

[0082] (5) Place the cleaned glass slide in a ventilated place to air dry for 30 minutes.

[0083] 2. Preparation of nano-gold solution

[0084] (1) Prepare 50 mL of 1 mM chloroauric acid and 15 mL of 38.8 mM sodium citrate solution;

[0085] (2) Add 50 mL of chloroauric acid and magnetic stirrer to a two-necked round-bottom flask, and place the two-necked round-bottom flask on a magnetic stirrer.

[0086] (3) Attach a condenser and a stopper to the two openings of the two round-bottom flasks respectively, and let flowing water flow back through;

[0087] (4) Set the temperature of the magnetic stirrer to 150°C;

[0088] (5) Heat until boiling. Use a 10ml disposable syringe to add sodium citrate solution. The boiling temperature is about 130℃.

[0089] (6) After reaching a deep red color, continue heating and reflux for 15-20 minutes.

[0090] (7) After the time has elapsed, stop heating, but continue stirring and refluxing to cool it to room temperature to obtain a nano gold solution for later use.

[0091] 3. Preparation of glass-gold electrodes

[0092] (1) Cover a glass slide with a protective film. The protective film has cutouts in the area of ​​the glass slide where bipolar gold electrodes need to be designed according to the predetermined pattern of the bipolar gold electrodes. Take a nano gold solution and drop it onto the glass slide exposed in the cutouts of the protective film to incubate the nano gold.

[0093] (2) Every 2 hours, remove the transparent gold nanoparticle solution from the glass surface and add a new gold nanoparticle solution. Repeat this process until a gold nanoparticle sub-monolayer (AuNPs sub-monolayer) is formed.

[0094] (3) Immerse the glass slides obtained in (2) in Au solution with a concentration of 0.3 mM.3+ In NH2OH aqueous solution, nano-gold sub-monolayers and Au 3+ The Au thin film formed by the NH2OH aqueous solution completes growth and infiltration within approximately 200 seconds, forming a glass-gold electrode. Gold nanosheets are then grown in situ on the glass material via chemical deposition to prepare a bipolar gold electrode with a predetermined pattern for ECL detection.

[0095] 4. Preparation of PDMS adhesive

[0096] (1) Add 1g of initiator to 10g of PDMS and stir until well mixed. Remove air bubbles by vacuuming. Stir for about 15 minutes.

[0097] (2) Pour the mixed PDMS into an Al mold processed by CNC precision machining, and remove air bubbles by vacuuming.

[0098] (3) Place the mold on the baking tray and heat it to solidify it into PDMS adhesive and then peel it off. The heating temperature is 120℃ and the time is 12min.

[0099] (4) The stripped PDMS glue is used to make holes in the top of the nucleic acid extraction reagent storage chamber and the electrochemiluminescence reaction chamber for reagent preloading.

[0100] 5. Microfluidic chip construction

[0101] (1) Place the PDMS channel face up and the smooth, dust-free glass plate together into the plasma processor, set the equilibration time to 60s and the discharge time to 20s;

[0102] (2) The plasma-treated glass gold electrode and PDMS adhesive were calibrated and sealed;

[0103] (3) Bake the sealed chip at 80℃ for 15-30 minutes;

[0104] (4) With a molar ratio of capture probe:TCEP = 1:10, the capture probe was treated with TCEP at room temperature for 1 hour. Then, 20 μL of the capture probe and TCEP mixture was dropped onto the gold electrode in (3), and incubated overnight. The mixture was then washed with water 3 times and set aside.

[0105] (5) Preload the required reagents and viral nucleic acid samples, and seal them with a 3M membrane as a cover to form a solid and complete chip.

[0106] Example 3

[0107] 1. RNA extraction and ECL detection of mouse hepatitis virus (MHV)

[0108] (1) After sealing the intermediate layer and substrate and fixing the modified capture probe according to Examples 1 and 2, the electrochemiluminescence probe, 0.3M NaCl solution and TPA auxiliary solution were injected into the electrochemiluminescence probe storage chamber, NaCl solution storage chamber and TPA storage chamber in sequence using a syringe.

[0109] (2) Silicone oil was added sequentially through the pre-loaded reagent holes at the front end of the lysis buffer and magnetic bead storage chamber, the pre-loaded reagent hole of the protein washing buffer storage chamber, the pre-loaded reagent hole of the first washing buffer storage chamber, the pre-loaded reagent hole of the second washing buffer storage chamber, and the pre-loaded reagent hole of the elution buffer storage chamber, in amounts of 90 μL, 38 μL, 38 μL, 38 μL, and 18 μL, respectively; then, 250 μL of lysis buffer, 12.5 μL of magnetic beads, 150 μL of protein washing buffer, 150 μL of washing buffer, 150 μL of washing buffer, and 150 μL of elution buffer were added sequentially through the pre-loaded reagent holes at the front end of the lysis buffer and magnetic bead storage chamber, the pre-loaded reagent hole of the protein washing buffer storage chamber, the pre-loaded reagent hole of the first washing buffer storage chamber, the pre-loaded reagent hole of the second washing buffer storage chamber, and the pre-loaded reagent hole of the elution buffer storage chamber.

[0110] (3) Collect MHV virus samples;

[0111] (4) Add 100 μL MHV virus sample to the lysis buffer and magnetic bead storage chamber through the pre-loaded reagent hole at the end of the lysis buffer and magnetic bead storage chamber, seal the middle layer with a cover, and lyse at 70°C for 2 min.

[0112] (5) Use a magnet to move the magnetic beads back and forth in the pyrolysis solution and the magnetic bead storage chamber to mix them. The time is 3 minutes.

[0113] (6) The magnet continues to drive the magnetic beads back and forth in the protein washing solution storage chamber to remove the protein;

[0114] (7) The magnet continues to drive the magnetic beads through the first washing liquid storage chamber and the second washing liquid storage chamber in sequence to remove other impurities;

[0115] (8) Finally, the magnet drives the magnetic beads into the elution buffer storage chamber, and heats it to 70°C for 3 minutes to elute the viral nucleic acid;

[0116] (9) First, press down the cover of the electrochemiluminescence probe storage chamber to drive the extracted nucleic acid to be evenly distributed in the four electrochemiluminescence reaction chambers, and based on the principle of complementary base matching, the capture probe on the gold electrode binds to the target viral nucleic acid; continue to press down the cover of the electrochemiluminescence probe storage chamber to make the electrochemiluminescence probe, the target viral nucleic acid and the capture probe form a sandwich structure.

[0117] (10) Press down the cover of the NaCl solution storage compartment to wash away unbound nucleic acids and electrochemiluminescent probes;

[0118] (11) Finally, press down the cover of the TPA storage compartment to fill the electrochemiluminescence reaction compartment with TPA auxiliary liquid, connect the power supply, measure the ECL intensity in each electrochemiluminescence reaction compartment, and evaluate the level of MHV virus nucleic acid content based on the ECL intensity.

[0119] The detection results are shown in curves 20-23 in Figure 3. The experimental results demonstrate that this method can be used for nucleic acid extraction and ECL detection.

[0120] In this embodiment, capture probes and signal probes are designed based on specific fragments of MHV viral RNA;

[0121] The capture probe sequence is: 5'-AGTTTAGATTAGATTTAAACTACAAGAGTT TTA-SH-3';

[0122] The electrochemiluminescence probe sequence is: 5'-SH-AGAGGGTACGTACGGACGCCAATC ACTCTTATA-3'.

[0123] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection, characterized in that: The device includes a chip body and a matching magnet. The chip body, from top to bottom, comprises a cover plate, an intermediate layer, and a substrate. The cover plate seals the top of the intermediate layer, and the intermediate layer and substrate are sealed together. The cover plate is made of a deformable material. The intermediate layer has a nucleic acid extraction reagent storage chamber, an electrochemiluminescence reagent storage chamber, an electrochemiluminescence reaction chamber, and a waste liquid chamber. The electrochemiluminescence reagent storage chamber is connected to the nucleic acid extraction reagent storage chamber and the electrochemiluminescence reaction chamber via a flow channel. The electrochemiluminescence reaction chamber is also connected to the waste liquid chamber via a flow channel. The nucleic acid extraction reagent storage chamber includes a lysis buffer and magnetic bead storage chamber, a protein washing buffer storage chamber, one or more washing buffer storage chambers connected in series, and an elution buffer storage chamber. The lysis buffer and magnetic bead storage chamber... The protein washing solution storage chamber, washing solution storage chamber, and elution solution storage chamber are sequentially connected via flow channels. The elution solution storage chamber is connected to the electrochemiluminescence reaction chamber via a flow channel. The top of the electrochemiluminescence reagent storage chamber is deformable. The electrochemiluminescence reagent storage chamber includes an electrochemiluminescence probe storage chamber, a NaCl solution storage chamber, and a TPA storage chamber. The electrochemiluminescence probe storage chamber is connected to the elution solution storage chamber via a flow channel, and the NaCl solution storage chamber and the TPA storage chamber are connected to the electrochemiluminescence reaction chamber via flow channels. Alternatively, the electrochemiluminescence probe storage chamber, the NaCl solution storage chamber, and the TPA storage chamber are all connected to the elution solution storage chamber via flow channels. The substrate is provided with a bipolar gold electrode corresponding to the electrochemiluminescence reaction chamber, and a capture probe modified with the target nucleic acid is fixed on the bipolar gold electrode.

2. The microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection according to claim 1, characterized in that: The electrochemiluminescence reaction chamber has one or more chambers, and when there are two or more chambers, they are arranged in parallel.

3. The microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection according to claim 2, characterized in that: The top of the lysis buffer and magnetic bead storage chamber, the protein washing buffer storage chamber, the washing buffer storage chamber, and the elution buffer storage chamber are all provided with pre-loaded reagent holes; the top of the lysis buffer and magnetic bead storage chamber is provided with pre-loaded reagent holes at both the beginning and end; the top of the electrochemiluminescence reaction chamber is provided with a capture probe addition hole; the flow channel is located on the bottom surface of the intermediate layer; the intermediate layer has a cutout at the electrical interface corresponding to the bipolar gold electrode, or the area of ​​the intermediate layer is smaller than that of the substrate, and the electrical interface of the gold electrode is exposed after the two are sealed together, so as to facilitate the connection of the electrode.

4. A microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection according to any one of claims 2-3, characterized in that: The cover is a 3M film; the intermediate layer is PDMS adhesive; the top of the electrochemiluminescence reagent storage compartment is a PDMS film; and the substrate is a glass sheet.

5. A method for preparing a microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection as described in any one of claims 4, characterized in that: The process includes: The substrate is cleaned and then silanized. Preparation of nano-gold solution; Substrate gold electrode fabrication: Following the predetermined pattern of the bipolar gold electrode, a nano-gold solution was dropped onto the substrate for nano-gold incubation. The nano-gold solution that had become transparent on the substrate surface was removed, and fresh nano-gold solution was added repeatedly until a nano-gold sub-monolayer was formed. The substrate was then immersed in Au. 3+ In NH2OH aqueous solution, nano-gold sub-monolayers and Au 3+ The Au film formed by the NH2OH aqueous solution completes the growth and infiltration, forming the substrate gold electrode; PDMS adhesive preparation: PDMS and initiator are stirred and mixed; the mixed PDMS is poured into the mold; the PDMS adhesive is heated and cured to form a PDMS adhesive and then peeled off. The peeled PDMS adhesive is used to make holes in the top of the nucleic acid extraction reagent storage chamber and the electrochemiluminescence reaction chamber for reagent preloading; microfluidic chip construction: the substrate obtained in step (3) with the PDMS channel face up and the surface smooth and dust-free is placed in the plasma processor; the plasma-treated substrate gold electrode and PDMS adhesive are calibrated and sealed; the sealed chip is baked, and then the modified capture probe is fixed, the required reagents and the nucleic acid sample to be tested are preloaded, and the chip is sealed with a cover to form a solid and complete chip.

6. The method for preparing a microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection according to claim 5, characterized in that: The process of step (1) includes: washing the substrate surface with water and drying the surface moisture; cleaning the substrate surface with methanol and then ultrasonically cleaning it in methanol for 5-15 minutes; immersing the substrate in 30%-60% APTES-MeOH for 1 hour; thoroughly cleaning the substrate with methanol and ultrasonically cleaning it in methanol to remove excess APTES for 10 minutes; and placing the cleaned substrate in a ventilated place to air dry.

7. The method for preparing a microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection according to claim 5, characterized in that: The process of step (2) includes: preparing 50 mL of 1 mM chloroauric acid and 15 mL of 38.8 mM sodium citrate solution; adding chloroauric acid and a magnetic stir bar into the reaction vessel and placing it on a magnetic stirrer, reflux heating until boiling at 130°C, adding sodium citrate solution to react, and continuing to heat and reflux for 15-20 min after reaching a deep red color; stopping heating, continuing to stir and reflux to room temperature to obtain a nano gold solution for later use.

8. The method for preparing a microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection according to claim 5, characterized in that: The process of step (3) includes: covering a protective film on the substrate, the protective film having cutouts designed according to the predetermined pattern of the bipolar gold electrodes in the area of ​​the substrate where bipolar gold electrodes need to be designed; dropping a nano-gold solution onto the exposed substrate through the cutouts of the protective film for nano-gold incubation; every 2 hours, removing the nano-gold solution that has become transparent on the surface of the substrate and dropping in a new nano-gold solution, repeating the dropping until a nano-gold sub-monolayer is formed; immersing the substrate with the nano-gold sub-monolayer in Au. 3+ Au with a concentration of 0.1 mM to 0.5 mM and an NH2OH concentration of 0.1 mM to 0.5 mM 3+ In NH2OH aqueous solution, nano-gold sub-monolayers and Au 3+ The Au thin film formed by the NH2OH aqueous solution completes the growth and infiltration, forming the substrate gold electrode.

9. The method for preparing a microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection according to claim 5, characterized in that: The process of step (4) includes: adding 10 g of PDMS to 1 g of initiator and stirring to remove air bubbles, stirring for 15 min; pouring the mixed PDMS into a mold and removing air bubbles; heating at 100℃~150℃ for 10 min~15 min to cure and peel off the PDMS adhesive, and then punching holes in the top of the nucleic acid extraction reagent storage chamber and the electrochemiluminescence reaction chamber for reagent preloading.

10. The method for preparing a microfluidic chip integrating nucleic acid extraction and high-throughput ECL detection according to claim 5, characterized in that: The process of step (5) includes: placing the substrate obtained in step (3) with the PDMS glue channel side facing up and the surface smooth and dust-free into the plasma processor, setting the equilibration time to 60 s and the discharge time to 20 s; calibrating and sealing the gold electrode and PDMS glue on the plasma-treated substrate; baking the sealed chip at 70℃~90℃ for 15-30 min, and treating the capture probe with TCEP at room temperature for 1-3 h with a molar ratio of capture probe:TCEP=1:5~1:50, and then taking 20 µL of the capture probe and TCEP mixture and dropping it onto the gold electrode in (3), incubating it for 8-24 h, washing it with water 3 times, and then preloading the required reagents and nucleic acid samples to be tested, and sealing it with a cover plate to form a solid and complete chip.

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