Microelectrode for coronavirus nucleic acid detection and preparation method and application thereof

Abstract

The application relates to a microelectrode for detecting coronavirus nucleic acid and a preparation method and application thereof. The microelectrode comprises an insulating substrate, a microelectrode prepared on the insulating substrate and a wire, the microelectrode comprises a working electrode, a counter electrode and a reference electrode, graphene completely covers the surface of the working electrode, and the graphene on the surface of the working electrode is used for fixing a specially designed DNA probe; the DNA probe can be directly combined with a coronavirus nucleic acid sequence to be detected or with DNA obtained by reverse transcription of a characteristic sequence of coronavirus nucleic acid to be detected through base complementary pairing, due to repulsion effect, an electrochemical label at the top of the DNA probe contacts the graphene on the surface of the microelectrode, redox reaction occurs, and an electrochemical signal is generated. The application realizes the purpose of accurately and quantitatively detecting coronavirus nucleic acid, has short detection time, high sensitivity, good specificity and a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of sensor technology, and in particular relates to a microelectrode for coronavirus nucleic acid detection, its preparation method and application. Background Technology

[0002] Coronaviruses have a single-stranded RNA genetic material. Infection can be determined by detecting unique characteristic sequences within their RNA sequences. Currently, the gold standard methods for detecting coronavirus nucleic acid are conventional polymerase chain reaction (PCR) and quantitative real-time polymerase chain reaction (qPCR). However, these methods place high demands on instrumentation, operator expertise, and laboratory environment. They are also complex, time-consuming, and prone to false negatives. Therefore, there is an urgent need to develop simple, efficient, and highly specific methods for detecting coronavirus nucleic acid. Coronaviruses are highly contagious and spread rapidly. Because they are single-stranded RNA viruses, they mutate quickly, leading to less obvious symptoms and easier asymptomatic transmission, resulting in more cases. Therefore, establishing rapid diagnostic methods is crucial for blocking virus transmission and controlling the epidemic. Summary of the Invention

[0003] This invention provides a microelectrode for coronavirus nucleic acid detection, its preparation method, and its application, thereby overcoming the shortcomings of conventional polymerase chain reaction (PCR) and real-time PCR (qPCR) methods in coronavirus nucleic acid detection.

[0004] This invention achieves the goal of accurately and quantitatively detecting coronavirus nucleic acid. Compared with the conventional polymerase chain reaction and fluorescence quantitative polymerase chain reaction methods currently used for detecting viral nucleic acid, it has a shorter detection time, higher sensitivity, and better specificity, and has good application prospects.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] This invention provides a microelectrode for coronavirus nucleic acid detection. The microelectrode includes an insulating substrate, a microelectrode fabricated on the insulating substrate, and conductive wires. The microelectrode comprises a working electrode, a counter electrode, and a reference electrode. The working electrode surface is completely covered with graphene, and the graphene on the working electrode surface is modified to immobilize a DNA probe. The DNA probe can directly bind to the target coronavirus nucleic acid sequence or to DNA reverse-transcribed from the target coronavirus nucleic acid characteristic sequence through complementary base pairing. This allows the electrochemical tag on the DNA probe to contact the graphene on the microelectrode surface, resulting in a redox reaction and generating an electrochemical signal. The presence of coronavirus nucleic acid is detected based on the occurrence of this electrochemical signal.

[0007] In one embodiment of the present invention, the DNA probe is a single-stranded DNA or a three-dimensional nucleic acid nanostructure with one or more detection strands for complementary pairing with the coronavirus nucleic acid sequence and an electrochemical tag located at the top of the detection strand, wherein the three-dimensional nucleic acid nanostructure is self-assembled by complementary pairing of oligonucleotide strands.

[0008] In one embodiment of the present invention, the coronavirus nucleic acid characteristic sequence is one or more of the following: encoding a non-structural protein gene (ORF1ab), encoding a spike protein gene (S), encoding a nucleocapsid protein gene (N), or encoding an envelope protein gene (E).

[0009] In one embodiment of the present invention, the electrochemical tag is selected from one or more of methylene blue, ferrocene, or Prussian blue.

[0010] In one embodiment of the present invention, a single strand of nucleic acid that does not pair with the coronavirus nucleic acid sequence or the DNA reverse transcribed from the coronavirus nucleic acid sequence is added between the detection strand and the three-dimensional nucleic acid nanostructure to ensure that the detection strand can rotate in multiple degrees of freedom.

[0011] In one embodiment of the present invention, the DNA probe is covalently bonded to graphene on the surface of the working electrode.

[0012] In one embodiment of the present invention, the coronavirus is a virus belonging to the order Nematovirales, family Coronaviridae, and genus Coronavirus in systematic taxonomy, and its genome is a linear single-stranded positive-sense RNA.

[0013] In one embodiment of the present invention, the working electrode, the counter electrode, and the wire are selected from elemental metal materials, conductive silicides, carbides, or conductive polymer materials, wherein the elemental metal materials include gold, silver, copper, and titanium; and the reference electrode is silver / silver chloride.

[0014] In one embodiment of the present invention, the surface of the working electrode is completely covered with graphene, and the size of the working electrode is less than 800 micrometers.

[0015] In one embodiment of the present invention, the working electrode pattern is a centrally symmetrical shape, including a circle, a rounded square, and a rounded octagon, to ensure the uniformity of the electric field on the surface of the working electrode.

[0016] In one embodiment of the present invention, the insulating substrate is made of silicon dioxide.

[0017] In one embodiment of the present invention, the graphene is a single-layer graphene.

[0018] The present invention further provides a method for preparing a microelectrode for coronavirus nucleic acid detection, comprising the following steps:

[0019] Step 1: Fabricate the working electrode, reference electrode, counter electrode, and wires on the surface of an insulating substrate;

[0020] Step 2: Transfer graphene to the surface of the microelectrode and use photolithography to pattern the graphene so that the graphene completely covers the working electrode. Remove the graphene covering the surfaces of the counter electrode and the reference electrode to obtain the device to be modified.

[0021] Step 3: Add the linker molecule solution to the device to be modified for 0.5 to 2 hours to allow the linker molecule to adsorb onto the graphene surface, and synthesize the DNA probe at the same time.

[0022] Step 4: Fabricate a micro-liquid container on the microelectrode;

[0023] Step 5: Add DNA probe solution to the liquid container to allow the DNA probe to be covalently linked to the linker molecule, and let it stand for 2-10 hours (preferably 8-10 hours) to allow the DNA probe to be modified on the graphene surface.

[0024] Step 6: After modification, remove the DNA probe solution, add a buffer solution containing magnesium ions, and store the device at room temperature to obtain the microelectrode for coronavirus nucleic acid detection.

[0025] In one embodiment of the present invention, in step 4, the capacity of the micro-liquid container is 20 microliters to 1000 microliters, preferably 80 microliters to 100 microliters.

[0026] In one embodiment of the present invention, in step 6, the buffer solution may be selected as a TM buffer solution containing 12.5 mM magnesium ions.

[0027] Furthermore, for the microelectrode prepared for coronavirus nucleic acid detection, a negative voltage is applied to the working electrode before testing. During detection, the sample to be tested is added to a micro-liquid container, allowing it to contact and cover the graphene on the surface of the working electrode. High-sensitivity detection of coronavirus nucleic acid is achieved by detecting changes in electrochemical current. This invention also provides a method for applying the microelectrode for coronavirus nucleic acid detection to non-disease diagnostic and therapeutic purposes.

[0028] Before testing, the microelectrodes are electrically connected to the interface of the electrochemical testing device. By applying a negative voltage to the working electrode, during detection, the sample to be tested is added to a micro-liquid container so that it can contact and cover the graphene on the surface of the working electrode. The high-sensitivity detection of coronavirus nucleic acid is achieved by detecting changes in electrochemical current.

[0029] In one embodiment of the present invention, corresponding electrochemical detection parameters are set according to the redox potential of the DNA probe electrochemical tag, including a voltage scan start voltage, a stop voltage, a step voltage, and a pulse voltage; the start voltage and stop voltage are adjusted by 0.3 volts to 0.5 volts based on the redox potential of the electrochemical tag, the step voltage is 0.005 volts to 0.01 volts, and the pulse voltage is 0.03 volts to 0.08 volts. Before testing, a negative voltage of -0.9 volts to -0.1 volts is applied to the working electrode to keep the detection strand on the DNA probe and the electrochemical tag away from the graphene, thereby reducing noise.

[0030] In one embodiment of the present invention, during detection, a test solution is added to a micro-liquid container of an electrochemical microelectrode, such that the DNA probe modified on the electrochemical microelectrode binds to the coronavirus nucleic acid sequence or the DNA obtained by reverse transcription of coronavirus nucleic acid in the test solution in a complementary pairing.

[0031] After adding the test sample and incubating for 5 to 30 minutes, the magnitude of the negative voltage is controlled and the strength of the applied repulsive electric field is adjusted to distinguish between the test strands that have been bound to the coronavirus nucleic acid and those that have not. This keeps the top of the test strands that have not been bound to the coronavirus nucleic acid away from the graphene, while keeping the top of the test strands that have been bound to the coronavirus nucleic acid closer to the graphene, thereby improving the detection sensitivity and accuracy.

[0032] The electrochemical response during the detection process serves as a signal for nucleic acid detection. The presence of an oxidation-reduction current peak near the oxidation-reduction potential of the electrochemical tag indicates a positive nucleic acid result, while the absence of such a peak indicates a negative result.

[0033] Finally, by detecting changes in redox current in real time, the concentration of coronavirus nucleic acid was calculated using a concentration formula.

[0034] In one embodiment of the invention, the occurrence of a redox current peak is defined as being greater than 120% of the average current value corresponding to the electrochemically labeled redox potential ±0.2 volts, including 120%.

[0035] In one embodiment of the present invention, the absence of a redox current peak is defined as less than 120% (excluding 120%) of the average current corresponding to the electrochemically labeled redox potential ±0.2 volts.

[0036] In one embodiment of the present invention, the sample to be tested added to the micro-liquid container is a coronavirus nucleic acid sample. The coronavirus nucleic acid sample is extracted from the sampled virus specimen after processing. Specifically, the method is as follows: an appropriate amount of virus specimen is drawn from the virus sample collection tube, placed into a nucleic acid extraction kit, and then the kit is placed into an automatic or semi-automatic nucleic acid extractor to extract the coronavirus nucleic acid sample from the kit.

[0037] The DNA sequence obtained by reverse transcription of the coronavirus nucleic acid sequence to be tested is as follows: the extracted coronavirus nucleic acid is reverse transcribed using a reverse transcription kit. The treatment method is as follows: after treatment with the reverse transcription kit, the sample is heated and annealed at 20-30 degrees Celsius for 5-20 minutes, then heated at 35-45 degrees Celsius for 1-2.5 hours, and finally heated at 80-90 degrees Celsius for 2-10 minutes to obtain the reverse transcribed DNA sequence.

[0038] The microelectrode in this invention is a sensor device that detects changes in electrochemical current signals and has advantages such as high sensitivity, high selectivity, and real-time detection.

[0039] Compared with existing technologies, the advantages of this invention are as follows: It constructs a microelectrode for coronavirus nucleic acid detection, which is a new method for detecting coronavirus nucleic acid. The principle is to achieve real-time detection by designing and synthesizing DNA probes with different structures to hybridize with the coronavirus nucleic acid sequence or the reverse transcribed DNA sequence of the coronavirus nucleic acid gene to induce changes in electrochemical current. By controlling the magnitude of the negative voltage and adjusting the strength of the applied repulsive electric field, the sensitivity and accuracy can be improved. It also has the advantages of simple operation, good specificity, short response time, integrability, and low cost. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the surface of the electrochemical microelectrode in Embodiment 1 of the present invention and a schematic diagram of its connection with the electrochemical testing device;

[0041] Figure 2 This is the differential pulse voltammetry current response curve of the novel coronavirus SARS-CoV-2 nucleic acid extracted from the sample in Example 1 of this invention;

[0042] Figure 3 This is the square wave voltammetry current response curve of the novel coronavirus SARS-CoV-2 nucleic acid extracted from the sample in Example 5 of this invention. Detailed Implementation

[0043] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0044] Example 1

[0045] This embodiment provides a specific microelectrode for coronavirus nucleic acid detection, its preparation method, and its application for non-disease diagnosis and treatment purposes.

[0046] First, chromium / gold (5 / 40 nm) wires, a working electrode (circular, 200 μm radius), a counter electrode (surrounding the working electrode), and a silver (40 nm) electrode (located to the left of the working electrode) were prepared by thermal evaporation. Then, a silver / silver chloride reference electrode was obtained on the silver electrode by drop coating, and finally, an electrochemical microelectrode was obtained.

[0047] Monolayer graphene was prepared on a 25-micrometer-thick copper foil using chemical vapor deposition, and the prepared graphene was transferred to the surface of an electrochemical microelectrode using chemical etching.

[0048] Patterned electrodes were fabricated using ultraviolet lithography and oxygen plasma etching. Then, the graphene electrochemical microelectrode was immersed in a solution of N-hydroxysuccinimide benzoate for 0.5 to 2 hours and rinsed with ultrapure water. The pyrene group at one end of the molecule was adsorbed onto the surface of the electrochemical microelectrode through π-π stacking.

[0049] A micro-liquid container is fabricated and placed on the graphene working electrode. The capacity of the micro-liquid container is approximately 400-500 microliters.

[0050] A probe based on a DNA tetrahedral nanostructure was designed. This DNA tetrahedral nanostructure has a detection strand at its tip for binding to the nucleic acid of the novel coronavirus SARS-CoV-2. The tip of the detection strand is methylene blue. A single-stranded nucleic acid that does not pair with the coronavirus nucleic acid sequence is added between the detection strand and the three-dimensional nucleic acid nanostructure, ensuring that the detection strand can rotate in multiple degrees of freedom. The probe is self-assembled from four DNA single strands through complementary base pairing. The method for synthesizing this DNA probe involves heating the DNA mixture to 95°C and holding it for 5 minutes, then uniformly cooling it to 15°C within 1 minute, and finally storing it at 4°C. Subsequently, the DNA probe solution is placed in a microvolume container. The amino groups attached to the DNA probe base can bind to N-hydroxysuccinimide groups, immobilizing the DNA probe on the interface of a graphene electrochemical microelectrode for approximately 8-10 hours, followed by rinsing with ultrapure water.

[0051] The samples were processed to extract viral nucleic acid. The extraction method was to draw 200 microliters of sample from the sample collection tube, put it into the nucleic acid extraction kit, and then put the kit into an automatic or semi-automatic nucleic acid extractor to extract the novel coronavirus SARS-CoV-2 nucleic acid, which was then stored at 4 degrees Celsius.

[0052] Before testing, a voltage of -0.7 volts was applied to the working electrode to keep the detection strand on the DNA probe and the electrochemical tag away from the graphene, reducing noise. Electrochemical testing parameters were set to induce a methylene blue reaction: initial voltage -0.6 volts, termination voltage -0.2 volts, step voltage 0.005 volts, and pulse voltage 0.05 volts. 80 μL of test solution was added, allowing the DNA probe modified on the electrochemical microelectrode to complementary pair with the coronavirus nucleic acid sequence in the test solution. After adding the test sample and incubating for 30 minutes, the magnitude of the negative voltage was controlled to adjust the strength of the applied repulsive electric field, distinguishing between detection strands bound to coronavirus nucleic acid and those not bound. This kept the tips of the unbound detection strands away from the graphene, while bringing the tips of the bound detection strands closer to the graphene, improving detection sensitivity and accuracy. The minimum detection concentration was 5 copies / μL.

[0053] Figure 1 This is a schematic diagram of the surface of the electrochemical microelectrode in Example 1 and a schematic diagram of its connection with the electrochemical testing device.

[0054] Figure 1 In this setup, the electrochemical testing device 1 is connected to a reference electrode 3, a working electrode 4, and a counter electrode 5 via wires 2. Wires 2, reference electrode 3, working electrode 4, and counter electrode 5 are all fabricated on an insulating substrate 6. The surface of the working electrode 4 is completely covered with graphene, and the graphene on the surface of the working electrode 4 is modified to immobilize a DNA probe 7. The DNA probe 7 consists of a three-dimensional nucleic acid nanostructure with multiple detection strands 8 for complementary pairing with coronavirus nucleic acid sequences and an electrochemical tag 9 at the top of the detection strands 8. The three-dimensional nucleic acid nanostructure is self-assembled through complementary pairing of oligonucleotide chains. A micro-liquid container 10 with a capacity of approximately 80-100 microliters is placed on the graphene working electrode 4.

[0055] Figure 2 This is the differential pulse voltammetry current response curve of the novel coronavirus SARS-CoV-2 nucleic acid extracted from the sample in Example 1.

[0056] Example 2

[0057] This embodiment provides a specific microelectrode for coronavirus nucleic acid detection, its preparation method, and its application for non-disease diagnosis and treatment purposes.

[0058] First, chromium / gold (5 / 40 nm) wires, working electrode (rounded square, 300 μm long and wide), counter electrode (surrounding the working electrode), and silver (40 nm) electrode (located to the left of the working electrode) were prepared by thermal evaporation. Then, a silver / silver chloride reference electrode was obtained on the silver electrode by drop coating, and finally, an electrochemical microelectrode was obtained.

[0059] Monolayer graphene was prepared on a 25-micrometer-thick copper foil using chemical vapor deposition, and the prepared graphene was transferred to the surface of an electrochemical microelectrode using chemical etching and exfoliation.

[0060] Patterned electrodes were fabricated using ultraviolet lithography and oxygen plasma etching. Then, the graphene electrochemical microelectrode was immersed in a solution of N-hydroxysuccinimide benzoate for 0.5 to 2 hours and rinsed with ultrapure water. The pyrene group at one end of the molecule was adsorbed onto the surface of the electrochemical microelectrode through π-π stacking.

[0061] A micro-liquid container is fabricated and placed on the graphene working electrode. The capacity of the micro-liquid container is approximately 100-200 microliters.

[0062] A DNA prism-based nanostructure was designed, with detection strands at the three apexes of the DNA prism. These strands bind to reverse-transcribed DNA encoding non-structural protein genes (ORF1ab), spike protein genes (S), and nucleocapsid protein genes (N) for SARS virus nucleic acid detection. The tips of the detection strands are modified with methylene blue. A single-stranded nucleic acid that does not pair with the reverse-transcribed DNA of the coronavirus nucleic acid sequence is added between the detection strands and the three-dimensional nucleic acid nanostructure, ensuring that the detection strands can rotate in multiple degrees of freedom. The probe is self-assembled from nine DNA single strands through complementary base pairing. The synthesis method involves heating the DNA mixture to 95°C for 5 minutes, then uniformly cooling it to 15°C within 1 minute, and finally storing it at 4°C. The DNA probe solution is then placed in a microvolume container. The amino groups attached to the DNA probe base can bind to N-hydroxysuccinimide groups, immobilizing the DNA probe on the interface of a graphene electrochemical microelectrode for approximately 8-10 hours, followed by rinsing with ultrapure water.

[0063] The sample was processed to extract viral nucleic acid. The extracted SARS virus nucleic acid was reverse transcribed using a reverse transcription kit. The processing method was as follows: after reverse transcription, the sample was heated and annealed at 20-30 degrees Celsius for 5-20 minutes, then heated at 35-45 degrees Celsius for 1-2.5 hours, and finally heated at 80-90 degrees Celsius for 2-10 minutes. The DNA sequence obtained after reverse transcription of the extracted SARS virus nucleic acid was stored at 4 degrees Celsius.

[0064] Before testing, a voltage of -0.8 volts was applied to the working electrode to keep the detection strand on the DNA probe and the electrochemical tag away from the graphene, reducing noise. Electrochemical testing parameters that would induce a methylene blue reaction were set using square wave voltammetry: initial voltage -0.7 volts, termination voltage -0.1 volts, step voltage 0.005 volts, and pulse voltage 0.05 volts. 150 μL of the test solution was added, allowing the DNA probe modified on the electrochemical microelectrode to bind complementary to the DNA obtained after reverse transcription of the coronavirus nucleic acid sequence in the test solution. After adding the test sample and incubating for 30 minutes, the magnitude of the negative voltage was controlled to adjust the strength of the applied repulsive electric field, distinguishing between the detection strands that had bound to the reverse-transcribed DNA and those that had not. This ensured that the tip of the detection strand not bound to the reverse-transcribed DNA was moved away from the graphene, while the tip of the detection strand bound to the reverse-transcribed DNA was moved closer to the graphene, improving detection sensitivity and accuracy. Ultimately, this microelectrode demonstrated high sensitivity to SARS virus nucleic acid extracted from the sample, with a detection limit of 0.5 copies / µL.

[0065] Example 3

[0066] This embodiment provides a specific microelectrode for coronavirus nucleic acid detection, its preparation method, and its application for non-disease diagnosis and treatment purposes.

[0067] First, chromium / gold (5 / 40 nm) wires, working electrode (rounded octagon, 400 μm in length and width), counter electrode (surrounding the working electrode), and silver (40 nm) electrode (located to the left of the working electrode) are prepared by thermal evaporation. Then, a silver / silver chloride reference electrode is obtained on the silver electrode by drop coating, and finally an electrochemical microelectrode is obtained.

[0068] Monolayer graphene was prepared on a 25-micrometer-thick copper foil using chemical vapor deposition, and the prepared graphene was transferred to the surface of an electrochemical microelectrode using an electrochemical exfoliation method.

[0069] Patterned electrodes were fabricated using ultraviolet lithography and oxygen plasma etching. Then, the graphene electrochemical microelectrode was immersed in a solution of N-hydroxysuccinimide benzoate for 0.5 to 2 hours and rinsed with ultrapure water. The pyrene group at one end of the molecule was adsorbed onto the surface of the electrochemical microelectrode through π-π stacking.

[0070] A micro-liquid container is fabricated and placed on the graphene working electrode. The capacity of the micro-liquid container is approximately 500-1000 microliters.

[0071] A detection strand based on a DNA cube structure was designed, with four vertices containing genes encoding non-structural proteins (ORF1ab), envelope proteins (E), spike proteins (S), and nucleocapsid proteins (N) for detecting Middle East Respiratory Syndrome (MERS) virus. The detection strands are modified with ferrocene at their tips. A single-stranded nucleic acid that does not pair with the reverse-transcribed DNA from the coronavirus nucleic acid sequence is added between the detection strand and the three-dimensional nucleic acid nanostructure, ensuring the detection strand can rotate in multiple degrees of freedom. This probe is self-assembled from twelve DNA single strands through complementary base pairing. The synthesis method involves heating the DNA mixture to 95°C for 5 minutes, then uniformly cooling it to 15°C over 1 minute, and finally storing it at 4°C. The DNA probe solution is then placed in a microvolume container. The amino groups attached to the DNA probe base bind to N-hydroxysuccinimide groups, immobilizing the DNA probe on the interface of a graphene electrochemical microelectrode for approximately 8-10 hours, followed by rinsing with ultrapure water.

[0072] The samples were processed to extract viral nucleic acid. The extraction method involved drawing 200 μL of sample from a sample collection tube, placing it into a nucleic acid extraction kit, and then placing the kit into an automated or semi-automated nucleic acid extractor to extract Middle East Respiratory Syndrome Virus (MERS-CoV) nucleic acid. The extracted coronavirus nucleic acid was then reverse transcribed using a reverse transcription kit. The reverse transcription process involved heating and annealing at 20–30°C for 5–20 minutes, followed by heating at 35–45°C for 1–2.5 hours, and finally at 80–90°C for 2–10 minutes. The DNA sequence obtained after reverse transcription of the MERS-CoV nucleic acid was stored at 4°C.

[0073] Before testing, a voltage of -0.4 volts was applied to the working electrode to keep the detection strand on the DNA probe and the electrochemical tag away from the graphene, reducing noise. Electrochemical testing parameters that induce a ferrocene reaction were set using differential pulse voltammetry: initial voltage 0 volts, termination voltage 0.6 volts, step voltage 0.005 volts, and pulse voltage 0.05 volts. 800 μL of the test solution was added, allowing the DNA probe modified on the electrochemical microelectrode to bind complementary to the DNA obtained from the reverse transcription of coronavirus nucleic acid in the test solution. After adding the test sample and incubating for 30 minutes, the magnitude of the negative voltage was controlled to adjust the strength of the applied repulsive electric field, distinguishing between detection strands bound to and unbound from the reverse-transcribed DNA. This ensured that the tip of the unbound detection strand was moved away from the graphene, while the tip of the bound detection strand was moved closer to the graphene, improving detection sensitivity and accuracy. Ultimately, the microelectrode demonstrated high sensitivity to the Middle East Respiratory Syndrome Virus nucleic acid extracted from the sample, with a detection limit of 0.1 copies / µL.

[0074] Example 4

[0075] This embodiment provides a specific microelectrode for coronavirus nucleic acid detection, its preparation method, and its application for non-disease diagnosis and treatment purposes.

[0076] First, gold (1000 nm) wires, a working electrode (circular with a radius of 400 μm), a counter electrode (surrounding the working electrode), and a silver / silver chloride reference electrode were prepared using inkjet printing technology to obtain an electrochemical microelectrode.

[0077] Monolayer graphene was prepared on a 25-micrometer-thick copper foil using chemical vapor deposition, and the prepared graphene was transferred to the surface of an electrochemical microelectrode using chemical etching.

[0078] Patterned electrodes were fabricated using ultraviolet lithography and oxygen plasma etching. Then, the graphene electrochemical microelectrode was immersed in a solution of N-hydroxysuccinimide benzoate for 0.5 to 2 hours and rinsed with ultrapure water. The pyrene group at one end of the molecule was adsorbed onto the surface of the electrochemical microelectrode through π-π stacking.

[0079] A micro-liquid container is fabricated and placed on the graphene working electrode. The capacity of the micro-liquid container is approximately 10-50 microliters.

[0080] A detection strand based on a DNA dodecahedral structure was designed. At the four vertices of the DNA cube, there are detection strands encoding genes for non-structural proteins (ORF1ab), envelope proteins (E), spike proteins (S), and nucleocapsid proteins (N) for SARS virus nucleic acid detection. The top of the detection strand is modified with Prussian blue. A single-stranded nucleic acid that does not pair with the coronavirus nucleic acid sequence is added between the detection strand and the three-dimensional nucleic acid nanostructure, ensuring that the detection strand can rotate in multiple degrees of freedom. This probe is self-assembled from twelve DNA single strands through complementary base pairing. The method for synthesizing this DNA probe involves heating the DNA mixture to 95°C and holding it for 5 minutes, then uniformly cooling it to 15°C within 1 minute, and finally storing it at 4°C. Then, the DNA probe solution is placed in a microvolume container. The amino groups attached to the DNA probe base can bind to N-hydroxysuccinimide groups to immobilize the DNA probe on the interface of the graphene electrochemical microelectrode surface, which takes approximately 8-10 hours. Afterwards, it is rinsed thoroughly with ultrapure water.

[0081] The samples were processed to extract viral nucleic acid. The extraction method involved drawing 200 μL of sample from a collection tube, placing it into a nucleic acid extraction kit, and then placing the kit into an automated or semi-automated nucleic acid extractor. The extracted SARS viral nucleic acid was stored at 4°C. Before testing, a voltage of -0.5 volts was applied to the working electrode to keep the detection strand on the DNA probe and the electrochemical tag away from the graphene, reducing noise. Electrochemical testing parameters were set to induce a Prussian blue reaction, using square wave voltammetry with an initial voltage of 0 volts, a termination voltage of 0.6 volts, a step voltage of 0.005 volts, and a pulse voltage of 0.05 volts. 40 μL of the test solution was added, allowing the DNA probe modified on the electrochemical microelectrode to bind complementary to the coronavirus nucleic acid sequence in the test solution. After adding the sample and incubating for 30 minutes, the magnitude of the negative voltage and the strength of the applied repulsive electric field were controlled to distinguish between the detection strands bound to coronavirus nucleic acid and those not bound. This resulted in the tops of the non-bound detection strands being moved away from the graphene, while the tops of the bound strands were brought closer to the graphene, thus improving detection sensitivity and accuracy. Ultimately, this microelectrode demonstrated high sensitivity for SARS virus nucleic acid extracted from the sample, with a detection limit of 0.1 copies / µL.

[0082] Example 5

[0083] This embodiment provides a specific microelectrode for coronavirus nucleic acid detection, its preparation method, and its application for non-disease diagnosis and treatment purposes.

[0084] First, chromium / gold (5 / 40 nm) wires, interdigitated electrodes (1000 μm long and 20 μm wide), and silver / silver chloride reference electrodes (located below the interdigitated electrodes) were prepared using inkjet printing technology to obtain an electrochemical microelectrode.

[0085] Monolayer graphene was prepared on a 25-micrometer-thick copper foil using chemical vapor deposition, and the prepared graphene was transferred to the surface of an electrochemical microelectrode using chemical etching.

[0086] Patterned electrodes were fabricated using ultraviolet lithography and oxygen plasma etching. Then, the graphene electrochemical microelectrode was immersed in a solution of N-hydroxysuccinimide benzoate for 0.5 to 2 hours and rinsed with ultrapure water. The pyrene group at one end of the molecule was adsorbed onto the surface of the electrochemical microelectrode through π-π stacking.

[0087] A micro-liquid container is fabricated and placed on the graphene working electrode. The capacity of the micro-liquid container is approximately 80-100 microliters.

[0088] A probe based on a DNA tetrahedral nanostructure was designed. This DNA tetrahedral nanostructure has a detection strand at its tip for binding to the nucleic acid of the novel coronavirus SARS-CoV-2. The tip of the detection strand is methylene blue. A single-stranded nucleic acid that does not pair with the coronavirus nucleic acid sequence is added between the detection strand and the three-dimensional nucleic acid nanostructure, ensuring that the detection strand can rotate in multiple degrees of freedom. The probe is self-assembled from four DNA single strands through complementary base pairing. The method for synthesizing this DNA probe involves heating the DNA mixture to 95°C and holding it for 5 minutes, then uniformly cooling it to 15°C within 1 minute, and finally storing it at 4°C. Subsequently, the DNA probe solution is placed in a microvolume container. The amino groups attached to the DNA probe base can bind to N-hydroxysuccinimide groups, immobilizing the DNA probe on the interface of a graphene electrochemical microelectrode for approximately 8-10 hours, followed by rinsing with ultrapure water.

[0089] The samples were processed to extract viral nucleic acid. The extraction method was to draw 200 microliters of sample from the sample collection tube, put it into the nucleic acid extraction kit, and then put the kit into an automatic or semi-automatic nucleic acid extractor to extract the novel coronavirus SARS-CoV-2 nucleic acid, which was then stored at 4 degrees Celsius.

[0090] Before testing, a voltage of -0.7 volts was applied to the working electrode to keep the detection strand on the DNA probe and the electrochemical tag away from the graphene, reducing noise. Electrochemical testing parameters that would induce a methylene blue reaction were set using square wave voltammetry: initial voltage -0.7 volts, termination voltage -0.1 volts, step voltage 0.005 volts, and pulse voltage 0.05 volts. 80 μL of the test solution was added, allowing the DNA probe modified on the electrochemical microelectrode to complementary pair with the coronavirus nucleic acid sequence in the test solution. After adding the test sample and incubating for 30 minutes, the magnitude of the negative voltage was controlled to adjust the strength of the applied repulsive electric field, distinguishing between the detection strands bound to coronavirus nucleic acid and those not bound. This kept the tips of the unbound detection strands away from the graphene, while bringing the tips of the bound detection strands closer to the graphene, improving detection sensitivity and accuracy. Ultimately, this microelectrode demonstrated high sensitivity to the novel coronavirus SARS-CoV-2 nucleic acid extracted from the sample, with a detection limit of 0.05 copies / µL.

[0091] Figure 3 This is the square wave voltammetry current response curve of the novel coronavirus SARS-CoV-2 nucleic acid extracted from the sample in Example 5.

[0092] Example 6

[0093] This embodiment provides a specific microelectrode for coronavirus nucleic acid detection, its preparation method, and its application for non-disease diagnosis and treatment purposes.

[0094] First, chromium / gold (5 / 40 nm) wires, interdigitated electrodes (1000 μm long and 20 μm wide), and silver / silver chloride reference electrodes (located below the interdigitated electrodes) were prepared using inkjet printing technology to obtain an electrochemical microelectrode.

[0095] Monolayer graphene was prepared on a 25-micrometer-thick copper foil using chemical vapor deposition, and the prepared graphene was transferred to the surface of an electrochemical microelectrode using chemical etching.

[0096] Patterned electrodes were fabricated using ultraviolet lithography and oxygen plasma etching. Then, the graphene electrochemical microelectrode was immersed in a solution of N-hydroxysuccinimide benzoate for 0.5 to 2 hours and rinsed with ultrapure water. The pyrene group at one end of the molecule was adsorbed onto the surface of the electrochemical microelectrode through π-π stacking.

[0097] A micro-liquid container is fabricated and placed on the graphene working electrode. The capacity of the micro-liquid container is approximately 200-300 microliters.

[0098] A probe based on a DNA tetrahedral nanostructure was designed. The top of this DNA tetrahedral nanostructure features a detection strand for binding to the nucleic acid of Middle East Respiratory Syndrome (MERS-CoV). The detection strand is topped with methylene blue. A single-stranded nucleic acid that does not pair with the coronavirus nucleic acid sequence is added between the detection strand and the three-dimensional nucleic acid nanostructure, ensuring that the detection strand can rotate in multiple degrees of freedom. The probe is self-assembled from four DNA single strands through complementary base pairing. The synthesis method involves heating a DNA mixture to 95°C and holding it for 5 minutes, then uniformly cooling it to 15°C over 1 minute, and finally storing it at 4°C. Subsequently, the DNA probe solution is placed in a microvolume container. The amino groups attached to the DNA probe base can bind to N-hydroxysuccinimide groups, immobilizing the DNA probe on the interface of a graphene electrochemical microelectrode for approximately 8-10 hours, followed by rinsing with ultrapure water.

[0099] The samples were processed to extract viral nucleic acid. The extraction method was to draw 200 microliters of sample from the sample collection tube, put it into a nucleic acid extraction kit, and then put the kit into an automatic or semi-automatic nucleic acid extractor. The extracted Middle East Respiratory Syndrome virus nucleic acid was stored at 4 degrees Celsius.

[0100] Before testing, a voltage of -0.7 volts was applied to the working electrode to keep the detection strand on the DNA probe and the electrochemical tag away from the graphene, reducing noise. Electrochemical testing parameters that would trigger a methylene blue reaction were set using square wave voltammetry: initial voltage -0.7 volts, termination voltage -0.1 volts, step voltage 0.005 volts, and pulse voltage 0.05 volts. 200 μL of the test solution was added, allowing the DNA probe modified on the electrochemical microelectrode to bind complementary to the coronavirus nucleic acid sequence in the test solution. After adding the test sample and incubating for 30 minutes, the magnitude of the negative voltage was controlled to adjust the strength of the applied repulsive electric field, distinguishing between the detection strands bound to coronavirus nucleic acid and those not bound. This kept the tips of the unbound detection strands away from the graphene, while bringing the tips of the bound detection strands closer to the graphene, improving detection sensitivity and accuracy. Ultimately, the microelectrode demonstrated high sensitivity to the Middle East Respiratory Syndrome Virus nucleic acid extracted from the sample, with a detection limit of 0.05 copies / µL.

[0101] The viruses detected based on the above Examples 1-6 and the qualitative results of the detection are shown in Table 1.

[0102] Table 1. Viruses detected and qualitative results of schemes 1-6 in Examples 1-6.

[0103]

[0104] The above embodiments demonstrate that the present invention can accurately quantify coronavirus nucleic acid. Compared with the conventional polymerase chain reaction and fluorescence quantitative polymerase chain reaction methods currently used for detecting viral nucleic acid, the technical solution provided by the present invention has a shorter detection time, higher sensitivity, and better specificity, and has good application prospects.

[0105] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A microelectrode device for coronavirus nucleic acid detection, characterized in that, The microelectrode device includes an insulating substrate, a microelectrode fabricated on the insulating substrate, and wires. The microelectrode includes a working electrode, a counter electrode, and a reference electrode. The surface of the working electrode is completely covered with graphene, and the graphene on the surface of the working electrode is modified and immobilized with a DNA probe. The DNA probe can bind to the DNA after reverse transcription of the characteristic sequence of the coronavirus nucleic acid to be tested through complementary base pairing. The repulsion effect caused by the binding allows the electrochemical tag on the DNA probe to contact the graphene on the surface of the microelectrode, resulting in a redox reaction and generating an electrochemical signal. The DNA probe is a three-dimensional nucleic acid nanostructure based on a DNA prism. At the three apex of the DNA prism are detection chains that bind to reverse-transcribed DNA for the detection of coronavirus nucleic acid characteristic sequences, namely the gene encoding the non-structural protein ORF1ab, the gene encoding the spike protein, and the gene encoding the nucleocapsid protein, as well as an electrochemical tag located at the top of the detection chain. The coronavirus nucleic acid characteristic sequence is one or more of the following: a gene encoding a non-structural protein, a gene encoding a spike protein, or a gene encoding a nucleocapsid protein. A single strand of nucleic acid that does not pair with the DNA reverse-transcribed from the coronavirus nucleic acid characteristic sequence is added between the detection strand and the three-dimensional nucleic acid nanostructure to ensure that the detection strand can rotate in multiple degrees of freedom.

2. The microelectrode device for coronavirus nucleic acid detection according to claim 1, characterized in that, The working electrode pattern is a centrally symmetrical shape; the insulating substrate is made of silicon dioxide; and the graphene is a single layer of graphene.

3. The method for preparing the microelectrode device for coronavirus nucleic acid detection according to any one of claims 1-2, characterized in that, Includes the following steps: Step 1: Fabricate the working electrode, reference electrode, counter electrode, and wires on the surface of an insulating substrate; Step 2: Transfer graphene to the surface of the microelectrode and use photolithography to pattern the graphene so that the graphene completely covers the working electrode. Remove the graphene covering the surfaces of the counter electrode and the reference electrode to obtain the device to be modified. Step 3: Add the linker molecule solution to the device to be modified for 0.5 to 2 hours to allow the linker molecule to adsorb onto the graphene surface, and synthesize the DNA probe at the same time. Step 4: Fabricate a micro-liquid container on the microelectrode; Step 5: Add DNA probe solution to the liquid container to allow the DNA probe to be covalently linked to the linker molecule. Allow the reaction to stand for 2–10 hours to allow the DNA probe to be modified onto the graphene surface. Step 6: After modification, remove the DNA probe solution, add a buffer solution containing magnesium ions, and store the device at room temperature to obtain the microelectrode device for coronavirus nucleic acid detection.

4. The method of using the microelectrode device for coronavirus nucleic acid detection as described in any one of claims 1-2 for purposes other than disease diagnosis and treatment, characterized in that, Includes the following steps: Before testing, the microelectrode device is electrically connected to the interface of the electrochemical testing device. By applying a negative voltage to the working electrode, during detection, the sample to be tested is added to a micro-liquid container so that it can contact and cover the graphene on the surface of the working electrode. High-sensitivity detection of coronavirus nucleic acid is achieved by detecting changes in electrochemical current. The corresponding electrochemical detection parameters are set according to the redox potential of the DNA probe electrochemical tag, including voltage scan start voltage, stop voltage, step voltage, and pulse voltage; the start voltage and stop voltage are adjusted by 0.3 volts to 0.5 volts respectively based on the redox potential of the electrochemical tag, the step voltage is 0.005 volts to 0.01 volts, and the pulse voltage is 0.01 volts to 0.1 volts; before testing, a negative voltage of −0.9 volts to −0.1 volts is applied to the working electrode to keep the detection strand on the DNA probe and the electrochemical tag away from the graphene, thereby reducing noise.

5. The application method according to claim 4, characterized in that, During detection, the test solution is added to a micro-liquid container of the electrochemical microelectrode, so that the DNA probe modified on the electrochemical microelectrode can bind complementary to the DNA obtained by reverse transcription of the coronavirus nucleic acid characteristic sequence in the test solution. After adding the test sample and incubating for 5 to 30 minutes, the magnitude of the negative voltage is controlled to adjust the strength of the applied repulsive electric field, thereby distinguishing between the test strands that have been bound to the DNA obtained by reverse transcription of the coronavirus nucleic acid characteristic sequence and the test strands that have not been bound to the DNA obtained by reverse transcription of the coronavirus nucleic acid characteristic sequence. This keeps the top of the test strands that have not been bound to the DNA obtained by reverse transcription of the coronavirus nucleic acid characteristic sequence away from the graphene, while keeping the top of the test strands that have been bound to the DNA obtained by reverse transcription of the coronavirus nucleic acid characteristic sequence closer to the graphene, thus improving the detection sensitivity and accuracy. The electrochemical response during the detection process serves as a signal for nucleic acid detection. The presence of an oxidation-reduction current peak near the oxidation-reduction potential of the electrochemical tag indicates a positive result for nucleic acid detection, while the absence of such a peak indicates a negative result. Finally, by detecting changes in redox current in real time, the concentration of coronavirus nucleic acid was calculated using a concentration formula.

6. The application method according to claim 5, characterized in that, The appearance of a redox current peak is defined as 120% or more of the average current corresponding to the electrochemically labeled redox potential ±0.2 volts. The absence of a redox current peak is indicated by being below 120% (excluding 120%) of the average current corresponding to the electrochemical label redox potential ±0.2 volts.

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