CN115343343A - Microelectrode for coronavirus nucleic acid detection and preparation method and application thereof - Google Patents
Microelectrode for coronavirus nucleic acid detection and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a microelectrode for detecting coronavirus nucleic acid and a preparation method and application thereof. The microelectrode comprises an insulating substrate, a microelectrode and a lead, wherein the microelectrode is prepared on the insulating substrate and comprises 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 used for modifying and fixing a specially designed DNA probe; the DNA probe can be directly combined with a nucleic acid sequence of the coronavirus to be detected or DNA subjected to reverse transcription with a nucleic acid characteristic sequence of the coronavirus to be detected through base complementary binding, and due to a repulsion effect, an electrochemical label at the top end of the DNA probe is contacted with graphene on the surface of the microelectrode to generate an oxidation-reduction reaction and generate an electrochemical signal. The invention realizes the purpose of accurately and quantitatively detecting the coronavirus nucleic acid, has short detection time, high sensitivity and good specificity and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of sensors, and particularly relates to a microelectrode for coronavirus nucleic acid detection and a preparation method and application thereof.
Background
Coronavirus genetic material is a single RNA chain, and whether coronavirus infection belongs to can be determined by detecting unique characteristic sequences in RNA sequences. At present, the detection method of coronavirus nucleic acid is mainly used as a common polymerase chain reaction and a fluorescent quantitative polymerase chain reaction of a gold standard, but the detection methods have high requirements on the specialities of instruments and operators and a laboratory environment, and have complex operation process, long detection time and easy occurrence of false negative results, so that the development of a simple, efficient and high-specificity detection method of coronavirus nucleic acid is urgently needed. The coronavirus has strong infectivity and high transmission speed. As the coronavirus is a single-stranded RNA virus, the variation speed is high, so that the symptoms are less obvious, latent transmission is easier to form, and more people are ill. Therefore, it is necessary to establish a rapid detection and diagnosis method which has important effects on blocking virus transmission and controlling epidemic situations.
Disclosure of Invention
The invention provides a microelectrode for detecting coronavirus nucleic acid, a preparation method and application thereof, thereby overcoming the defects of the common polymerase chain reaction and fluorescent quantitative polymerase chain reaction methods in the aspect of detection of coronavirus nucleic acid.
The invention realizes the purpose of accurately and quantitatively detecting the coronavirus nucleic acid, and compared with the common polymerase chain reaction and fluorescent quantitative polymerase chain reaction methods used for detecting the virus nucleic acid at present, the detection method has the advantages of short detection time, high sensitivity, good specificity and good application prospect.
The purpose of the invention can be realized by the following technical scheme:
the invention provides a microelectrode for detecting coronavirus nucleic acid, which comprises an insulating substrate, a microelectrode and a lead, wherein the microelectrode is prepared on the insulating substrate and comprises 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 modifies and fixes a DNA probe; the DNA probe can be directly combined with a nucleic acid sequence of the coronavirus to be detected or DNA subjected to reverse transcription with a nucleic acid characteristic sequence of the coronavirus to be detected through base complementary combination, so that an electrochemical label on the DNA probe is contacted with graphene on the surface of the microelectrode to generate an oxidation-reduction reaction, and an electrochemical signal is generated. The detection of the presence of coronavirus nucleic acid is achieved based on the presence or absence of an electrochemical signal.
In one embodiment of the invention, the DNA probe is obtained by adding one or more detection strands for complementary pairing with a coronavirus nucleic acid sequence and an electrochemical tag at the top of the detection strand to a DNA single strand or a three-dimensional nucleic acid nanostructure, wherein the three-dimensional nucleic acid nanostructure is self-assembled through oligonucleotide strand complementary pairing.
In one embodiment of the invention, the coronavirus nucleic acid signature sequence is one or more of a gene encoding a non-structural protein (ORF 1 ab), a gene encoding a spike protein (S), a gene encoding a nucleocapsid protein (N), or a gene encoding an envelope protein (E).
In one embodiment of the invention, the electrochemical tag is selected from one or more of methylene blue, ferrocene or prussian blue.
In one embodiment of the present invention, a single nucleic acid strand that does not pair with the coronavirus nucleic acid sequence or the DNA after reverse transcription of 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 with multiple degrees of freedom.
In one embodiment of the present invention, the DNA probe is linked to graphene on the surface of the working electrode through a covalent bond.
In one embodiment of the invention, the coronavirus is a virus of the genus coronavirus, family coronaviridae, belonging to the phylogenetic group of the order capsuloviridae, the genome of which is a linear single-stranded positive-stranded RNA.
In one embodiment of the present invention, the working electrode, the counter electrode and the conducting 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; the reference electrode is silver/silver chloride.
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.
In one embodiment of the invention, the working electrode pattern is a centrosymmetric pattern, including a circle, a rounded square and a rounded octagon, so as to ensure the uniformity of the electric field on the surface of the working electrode.
In one embodiment of the present invention, the insulating substrate is made of silicon dioxide.
In one embodiment of the present invention, the graphene is a single-layer graphene.
The invention further provides a preparation method of the microelectrode for detecting the coronavirus nucleic acid, which comprises the following steps:
step 1: preparing a working electrode, a reference electrode, a counter electrode and a lead on the surface of an insulating substrate;
step 2: transferring graphene to the surface of a microelectrode, patterning the graphene by utilizing a photoetching technology to enable the graphene to completely cover a working electrode, and removing the graphene covering the surfaces of a counter electrode and a reference electrode to obtain a device to be modified;
and step 3: adding a device to be modified into a connecting molecule solution for 0.5-2 hours to enable the connecting molecules to be adsorbed to the surface of the graphene, and simultaneously synthesizing a DNA probe;
and 4, step 4: manufacturing a micro-liquid container on the micro-electrode;
and 5: adding a DNA probe solution into a liquid container, connecting the DNA probe with a connecting molecule through a covalent bond, and standing for 2-10 (preferably 8-10) hours to modify the DNA probe on the surface of graphene;
step 6: and after finishing modification, taking out the DNA probe solution, adding a buffer solution containing magnesium ions, and storing the device at normal temperature to obtain the microelectrode for detecting the coronavirus nucleic acid.
In one embodiment of the present invention, in step 4, the volume of the micro liquid container is 20 microliters to 1000 microliters, preferably 80 microliters to 100 microliters.
In one embodiment of the present invention, in step 6, the buffer may be selected as a TM buffer containing magnesium ions at a concentration of 12.5 mM.
In addition, before testing, negative voltage is applied to the working electrode, when in detection, a sample to be detected is added into the micro liquid container, so that the sample can contact and cover graphene on the surface of the working electrode, and high-sensitivity detection of the coronavirus nucleic acid is realized by detecting electrochemical current change. The invention also provides an application method of the microelectrode for the detection of coronavirus nucleic acid for the purpose of non-disease diagnosis and treatment,
before testing, the microelectrode is respectively electrically connected with an interface of an electrochemical testing device, negative voltage is applied to the working electrode, during detection, a sample to be tested is added into the micro liquid container, so that the sample can contact and cover graphene on the surface of the working electrode, and high-sensitivity detection of coronavirus nucleic acid is realized by detecting electrochemical current change.
In one embodiment of the invention, the corresponding electrochemical detection parameters are set according to the oxidation-reduction potential of the DNA probe electrochemical label, and comprise a voltage scanning starting voltage, a termination voltage, a stepping voltage and a pulse voltage; the initial voltage and the final voltage are respectively plus or minus 0.3 volt to 0.5 volt according to the oxidation-reduction potential of the electrochemical tag, the stepping voltage is 0.005 volt to 0.01 volt, and the pulse voltage is 0.03 volt to 0.08 volt. Before testing, the detection chain on the DNA probe and the electrochemical label are far away from the graphene by applying a negative voltage of-0.9 volt to-0.1 volt on the working electrode, so that the noise is reduced.
In one embodiment of the invention, during detection, a solution to be detected is added into a micro liquid container of an electrochemical microelectrode, so that a DNA probe modified on the electrochemical microelectrode is combined with a coronavirus nucleic acid sequence in the solution to be detected or DNA complementary pairing obtained after the coronavirus nucleic acid is subjected to reverse transcription;
after adding a detection sample and incubating for 5-30 minutes, regulating the intensity of the applied repulsive electric field by controlling the magnitude of negative voltage, and distinguishing the detection chain combined with the coronavirus nucleic acid from the detection chain not combined with the coronavirus nucleic acid, so that the top end of the detection chain not combined with the coronavirus nucleic acid is far away from graphene, and the top end of the detection chain combined with the coronavirus nucleic acid is close to the graphene, thereby improving the detection sensitivity and precision;
the electrochemical response in the detection process is used as a signal for nucleic acid detection, namely, the nucleic acid can be judged to be positive if an oxidation-reduction current peak appears near the oxidation-reduction potential of the electrochemical tag, and the nucleic acid can be judged to be negative if the oxidation-reduction current peak does not appear near the oxidation-reduction potential of the electrochemical tag;
and finally, calculating the concentration of the coronavirus nucleic acid by detecting the change of the redox current in real time and a concentration formula.
In one embodiment of the invention, the occurrence of a redox current peak is marked by greater than 120%, including 120%, of the average of the current corresponding to the electrochemical tag redox potential ± 0.2 volts.
In one embodiment of the invention, the absence of a redox current peak is indicated by less than 120%, excluding 120%, of the average current corresponding to the electrochemical tag redox potential ± 0.2 volts.
In one embodiment of the present invention, the sample to be tested added to the micro-volume liquid container is a coronavirus nucleic acid sample. The coronavirus nucleic acid sample is extracted after being processed by a sampled virus specimen, and the specific method comprises the following steps: and (2) extracting a proper amount of virus specimen from a virus sample collection tube, putting the virus specimen into a nucleic acid extraction kit, putting the kit into an automatic or semi-automatic nucleic acid extractor, and extracting the coronavirus nucleic acid sample from the kit.
The method for detecting the DNA sequence obtained after the reverse transcription of the coronavirus nucleic acid sequence comprises the following steps: and carrying out reverse transcription treatment on the extracted coronavirus nucleic acid by using a reverse transcription kit, wherein the treatment method comprises the steps of heating and annealing after the treatment by using the reverse transcription kit, keeping the heating temperature at 20-30 ℃ for 5-20 minutes, keeping the heating temperature at 35-45 ℃ for 1-2.5 hours, and keeping the heating temperature at 80-90 ℃ for 2-10 minutes to obtain a reverse transcribed DNA sequence.
The microelectrode of the invention is a sensing device for detecting the change of electrochemical current signals, and has the advantages of high sensitivity, high selectivity, real-time detection and the like.
Compared with the prior art, the invention has the advantages that: a microelectrode for detecting nucleic acid of coronavirus is constructed as a new method for detecting nucleic acid of coronavirus, and the principle is that the real-time detection is realized by the change of electrochemical current caused by complementary hybridization of DNA probes with various structures and nucleic acid sequences of coronavirus or reverse transcription DNA sequences of nucleic acid genes of coronavirus through design and synthesis, the intensity of applied repulsive electric field is adjusted by controlling the magnitude of negative voltage, the sensitivity and the accuracy are improved, and the microelectrode has the advantages of simplicity and convenience in operation, good specificity, short response time, integration, low cost and the like.
Drawings
FIG. 1 is a view showing the surface of an electrochemical micro-electrode in example 1 of the present invention schematic diagram and connection schematic diagram with electrochemical testing device;
FIG. 2 is a differential pulse voltammetry current response curve of novel coronavirus SARS-CoV-2 nucleic acid extracted from a specimen in example 1 of the present invention;
FIG. 3 is a square wave voltammetry current response curve of the novel coronavirus SARS-CoV-2 nucleic acid extracted from a sample in example 5 of the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1
This example provides a specific microelectrode for detecting coronavirus nucleic acid, its preparation method and application for non-disease diagnosis and treatment
Firstly, preparing a chromium/gold (5/40 nanometer) lead, a working electrode (circular and 200 micrometer in radius), a counter electrode (surrounding the working electrode) and a silver (40 nanometer) electrode (positioned on the left side of the working electrode) by a thermal evaporation technology, and then obtaining a silver/silver chloride reference electrode on the silver electrode by a dropping coating method to finally obtain the electrochemical microelectrode.
Preparing single-layer graphene on a copper foil with the thickness of 25 microns by using a chemical vapor deposition method, and transferring the prepared graphene to the surface of an electrochemical microelectrode by using a chemical etching method.
Preparing a patterned electrode by adopting an ultraviolet lithography method and an oxygen plasma etching technology, then soaking the graphene electrochemical microelectrode in a benzoic acid N-hydroxysuccinimide ester solution for 0.5-2 hours, washing the graphene electrochemical microelectrode with ultrapure water, and adsorbing a pyrenyl group at one end of the molecule to the surface of the electrochemical microelectrode through pi-pi stacking.
And manufacturing a micro-liquid container, and placing the micro-liquid container on the graphene working electrode, wherein the volume of the micro-liquid container is about 400-500 microliters.
Designing a probe based on a DNA tetrahedral nano structure, wherein the top end of the DNA tetrahedral nano structure is provided with a detection chain for combining with novel coronavirus SARS-CoV-2 nucleic acid, the top end of the detection chain is methylene blue, and a section of nucleic acid single chain which is not matched with a coronavirus nucleic acid sequence is added between the detection chain and the three-dimensional nucleic acid nano structure, so that the detection chain can rotate in multiple degrees of freedom. The probe is formed by self-assembling four DNA single strands through base complementary pairing, and the method for synthesizing the DNA probe is to heat DNA mixed liquor to 95 ℃ for 5 minutes, then uniformly cool the mixed liquor to 15 ℃ within 1 minute, and finally store the mixed liquor at 4 ℃. Then, the DNA probe solution is placed in a micro-liquid container, amino groups connected on a DNA probe base can be combined with N-hydroxysuccinimide groups to fix the DNA probe on an interface on the surface of the graphene electrochemical microelectrode, the required time is about 8-10 hours, and then the DNA probe solution is washed clean by using ultra-pure water.
The sample is processed to extract virus nucleic acid, the extraction method is that 200 microliter of sample is extracted from a sample collecting tube of the sample and put into a nucleic acid extraction kit, then the kit is put into an automatic or semi-automatic nucleic acid extractor, and the extracted novel coronavirus SARS-CoV-2 nucleic acid is stored at 4 ℃.
Noise was reduced by keeping the detection strand on the DNA probe and the electrochemical tag away from the graphene by applying-0.7 volts to the working electrode before testing. Electrochemical test parameters were set for the reaction of methylene blue at-0.6 volts initial, 0.2 volts final, 0.005 volts step and 0.05 volts pulse. Adding 80 microliters of the solution to be detected, and enabling the DNA probe modified on the electrochemical microelectrode to be complementarily paired and combined with the coronavirus nucleic acid sequence in the solution to be detected. After the detection sample is added and incubation is finished for 30 minutes, the strength of the applied repulsive electric field is adjusted by controlling the magnitude of the negative voltage, the detection chain combined with the coronavirus nucleic acid is distinguished from the detection chain not combined with the coronavirus nucleic acid, the top end of the detection chain not combined with the coronavirus nucleic acid is far away from graphene, meanwhile, the top end of the detection chain combined with the coronavirus nucleic acid is close to the graphene, the detection sensitivity and precision are improved, and the lowest detection concentration is 5 copies/microliter.
FIG. 1 is a schematic view of the surface of an electrochemical microelectrode and a schematic view of connection to an electrochemical test device in example 1.
In fig. 1, an electrochemical testing device 1 is connected with a reference electrode 3, a working electrode 4 and a counter electrode 5 through a lead 2, wherein the lead 2, the reference electrode 3, the working electrode 4 and the counter electrode 5 are all prepared on an insulating substrate 6, graphene is completely covered on the surface of the working electrode 4, and a graphene modification immobilized DNA probe 7 on the surface of the working electrode 4; the DNA probe 7 is formed by adding a plurality of detection strands 8 for complementary pairing with a coronavirus nucleic acid sequence and an electrochemical tag 9 positioned at the top end of the detection strands 8 to a three-dimensional nucleic acid nanostructure, wherein the three-dimensional nucleic acid nanostructure is formed by self-assembly through oligonucleotide strand complementary pairing. The micro-liquid container 10 is placed on the graphene working electrode 4, and the capacity of the micro-liquid container is about 80-100 microliters.
FIG. 2 is a differential pulse voltammetry current response curve for the novel coronavirus SARS-CoV-2 nucleic acid extracted from a sample in example 1.
Example 2
This example provides a specific microelectrode for detecting coronavirus nucleic acid, its preparation method and application for non-disease diagnosis and treatment
Firstly, preparing a chromium/gold (5/40 nanometer) lead, a working electrode (a rounded square, the length and the width of which are both 300 micrometers), a counter electrode (surrounding the working electrode) and a silver (40 nanometer) electrode (positioned on the left side of the working electrode) by a thermal evaporation technology, and then obtaining a silver/silver chloride reference electrode on the silver electrode by a dropping coating method to finally obtain the electrochemical microelectrode.
Preparing single-layer graphene on a copper foil with the thickness of 25 microns by using a chemical vapor deposition method, and transferring the prepared graphene to the surface of an electrochemical microelectrode by using a chemical etching stripping method.
Preparing a patterned electrode by adopting an ultraviolet lithography method and an oxygen plasma etching technology, then soaking the graphene electrochemical microelectrode in a benzoic acid N-hydroxysuccinimide ester solution for 0.5-2 hours, washing the graphene electrochemical microelectrode with ultrapure water, and adsorbing a pyrenyl group at one end of the molecule to the surface of the electrochemical microelectrode through pi-pi stacking.
And manufacturing a micro liquid container, and placing the micro liquid container on the graphene working electrode, wherein the capacity of the micro liquid container is about 100-200 microliters.
The nano structure based on the DNA prism is designed, three top ends of the DNA prism are respectively provided with a detection chain combined with reverse transcription DNA of a coding non-structural protein gene (ORF 1 ab), a coding thorn protein gene (S) and a coding nucleocapsid protein gene (N) for SARS virus nucleic acid detection, the top end of the detection chain is modified with methylene blue, a section of nucleic acid single chain which is not matched with the DNA subjected to reverse transcription of a coronavirus nucleic acid sequence is added between the detection chain and the three-dimensional nucleic acid nano structure, and the detection chain can rotate in multiple degrees of freedom. The probe is formed by self-assembling nine DNA single-strands through base complementary pairing, and the method for synthesizing the DNA probe is to heat a DNA mixed solution to 95 ℃ for 5 minutes, then uniformly cool the mixed solution to 15 ℃ within 1 minute, and finally store the mixed solution at 4 ℃. Then, the DNA probe solution is placed in a micro-liquid container, amino groups connected to a base of the DNA probe can be combined with N-hydroxysuccinimide groups to fix the DNA probe on an interface on the surface of the graphene electrochemical microelectrode, the required time is about 8-10 hours, and then the graphene electrochemical microelectrode is washed clean by using ultra-pure water.
Treating a sample to extract virus nucleic acid, and carrying out reverse transcription treatment on the extracted SARS virus nucleic acid by using a reverse transcription kit, wherein the treatment method comprises the steps of treating by using the reverse transcription kit, heating and annealing, keeping the heating temperature at 20-30 ℃ for 5-20 minutes, keeping the heating temperature at 35-45 ℃ for 1-2.5 hours, keeping the heating temperature at 80-90 ℃ for 2-10 minutes, and storing a DNA sequence obtained after reverse transcription of the SARS virus nucleic acid at 4 ℃.
Noise was reduced by keeping the detection strand on the DNA probe and the electrochemical tag away from the graphene by applying-0.8 volts to the working electrode before testing. Electrochemical test parameters were set to react methylene blue using square wave voltammetry with an initial voltage of-0.7 volts, an end voltage of-0.1 volts, a step voltage of 0.005 volts, and a pulse voltage of 0.05 volts. Adding 150 microliters of solution to be detected, and enabling the DNA probe modified on the electrochemical microelectrode to be combined with the DNA complementary pair obtained after the reverse transcription of the coronavirus nucleic acid sequence in the solution to be detected. After the detection sample is added and incubation is finished for 30 minutes, the strength of the applied repulsive electric field is adjusted by controlling the magnitude of the negative voltage, the detection chain which is combined with the DNA obtained after reverse transcription is distinguished from the detection chain which is not combined with the DNA obtained after reverse transcription, the top end of the detection chain which is not combined with the DNA obtained after reverse transcription is far away from graphene, and meanwhile, the top end of the detection chain which is combined with the DNA obtained after reverse transcription is close to graphene, so that the detection sensitivity and precision are improved. Finally, the microelectrode has high sensitivity response to SARS virus nucleic acid extracted from a specimen, and the detection limit is 0.5 copy number/microliter.
Example 3
This example provides a specific microelectrode for detecting coronavirus nucleic acid, its preparation method and application for non-disease diagnosis and treatment
Firstly, preparing a chromium/gold (5/40 nanometer) lead, a working electrode (a fillet octagon, the length and the width of which are both 400 micrometers), a counter electrode (surrounding the working electrode) and a silver (40 nanometer) electrode (positioned on the left side of the working electrode) by a thermal evaporation technology, and then obtaining a silver/silver chloride reference electrode on the silver electrode by a dropping coating method, thereby finally obtaining the electrochemical microelectrode.
Preparing single-layer graphene on a copper foil with the thickness of 25 microns by using a chemical vapor deposition method, and transferring the prepared graphene to the surface of an electrochemical microelectrode by using an electrochemical stripping method.
Preparing a patterned electrode by adopting an ultraviolet lithography method and an oxygen plasma etching technology, then soaking the graphene electrochemical microelectrode into a benzoic acid N-hydroxysuccinimide ester solution for 0.5-2 hours, washing the graphene electrochemical microelectrode with ultrapure water, and adsorbing a pyrenyl group at one end of the molecule to the surface of the electrochemical microelectrode through pi-pi stacking effect.
And manufacturing a micro liquid container, and placing the micro liquid container on the graphene working electrode, wherein the capacity of the micro liquid container is about 500-1000 microliters.
A DNA cube structure is designed, four vertexes of the DNA cube are respectively provided with a detection chain combined with a coding non-structural protein gene (ORF 1 ab), a coding envelope protein gene (E), a coding spike protein gene (S) and a coding nucleocapsid protein gene (N) for detecting middle east respiratory syndrome virus, the top end of the detection chain is modified with ferrocene, a section of nucleic acid single chain which is not paired with DNA subjected to reverse transcription of a coronavirus nucleic acid sequence is added between the detection chain and a three-dimensional nucleic acid nanostructure, and the detection chain can rotate in multiple degrees of freedom. The probe is formed by self-assembling twelve DNA single strands through base complementary pairing, and the method for synthesizing the DNA probe is to heat DNA mixed solution to 95 ℃ for 5 minutes, then uniformly cool to 15 ℃ within 1 minute, and finally store at 4 ℃. Then, the DNA probe solution is placed in a micro-liquid container, amino groups connected to a base of the DNA probe can be combined with N-hydroxysuccinimide groups to fix the DNA probe on an interface on the surface of the graphene electrochemical microelectrode, the required time is about 8-10 hours, and then the graphene electrochemical microelectrode is washed clean by using ultra-pure water.
The method comprises the steps of using the reverse transcription kit to process, then heating and annealing, keeping the heating temperature at 20-30 ℃ for 5-20 minutes, then keeping the heating temperature at 35-45 ℃ for 1-2.5 hours, keeping the heating temperature at 80-90 ℃ for 2-10 minutes, and storing the extracted DNA sequence obtained after reverse transcription of the middle east respiratory syndrome virus nucleic acid at 4 ℃.
Before testing, noise was reduced by applying a-0.4 volt voltage across the working electrode to keep the detection strand on the DNA probe and the electrochemical tag away from the graphene. Electrochemical test parameters were set to react ferrocene using differential pulse voltammetry with an initial voltage of 0 volts, an end voltage of 0.6 volts, a step voltage of 0.005 volts, and a pulse voltage of 0.05 volts. Adding 800 microliters of solution to be detected, and enabling the DNA probe modified on the electrochemical microelectrode to be combined with the DNA complementary pair obtained after the coronavirus nucleic acid in the solution to be detected is subjected to reverse transcription. After the detection sample is added and incubation is finished for 30 minutes, the strength of the applied repulsive electric field is adjusted by controlling the magnitude of the negative voltage, the detection chain combined with the DNA obtained after reverse transcription is distinguished from the detection chain not combined with the DNA obtained after reverse transcription, the top end of the detection chain not combined with the DNA obtained after reverse transcription is far away from graphene, and the top end of the detection chain combined with the DNA obtained after reverse transcription is close to graphene, so that the detection sensitivity and precision are improved. Finally, the microelectrode has high sensitivity response to the nucleic acid of the respiratory syndrome virus of the middle east of China extracted from the specimen, and the detection limit is 0.1 copy number/microliter.
Example 4
This example provides a specific microelectrode for detecting coronavirus nucleic acid, its preparation method and application for non-disease diagnosis and treatment
Firstly, preparing a gold (1000 nm) lead, a working electrode (circular with the radius of 400 microns), a counter electrode (surrounding the working electrode) and a silver/silver chloride reference electrode by an ink-jet printing technology, and finally obtaining the electrochemical microelectrode.
Preparing single-layer graphene on a copper foil with the thickness of 25 microns by using a chemical vapor deposition method, and transferring the prepared graphene to the surface of an electrochemical microelectrode by using a chemical etching method.
Preparing a patterned electrode by adopting an ultraviolet lithography method and an oxygen plasma etching technology, then soaking the graphene electrochemical microelectrode in a benzoic acid N-hydroxysuccinimide ester solution for 0.5-2 hours, washing the graphene electrochemical microelectrode with ultrapure water, and adsorbing a pyrenyl group at one end of the molecule to the surface of the electrochemical microelectrode through pi-pi stacking.
And (3) preparing a micro liquid container, and placing the micro liquid container on the graphene working electrode, wherein the capacity of the micro liquid container is about 10-50 microliters.
A DNA-based dodecahedron structure is designed, a detection chain combined with a coding non-structural protein gene (ORF 1 ab), a coding envelope protein gene (E), a coding thorn protein gene (S) and a coding nucleocapsid protein gene (N) for SARS virus nucleic acid detection is arranged at four vertexes of a DNA cube respectively, prussian blue is modified at the top end of the detection chain, a section of nucleic acid single chain which is not matched with a coronavirus nucleic acid sequence is added between the detection chain and a three-dimensional nucleic acid nanostructure, and the detection chain can rotate in multiple degrees of freedom. The probe is formed by self-assembling twelve DNA single strands through base complementary pairing, and the method for synthesizing the DNA probe is to heat DNA mixed solution to 95 ℃ for 5 minutes, then uniformly cool to 15 ℃ within 1 minute, and finally store at 4 ℃. Then, the DNA probe solution is placed in a micro-liquid container, amino groups connected to a base of the DNA probe can be combined with N-hydroxysuccinimide groups to fix the DNA probe on an interface on the surface of the graphene electrochemical microelectrode, the required time is about 8-10 hours, and then the graphene electrochemical microelectrode is washed clean by using ultra-pure water.
The sample is treated to extract virus nucleic acid, and the extraction process includes drawing 200 microliter sample from the sample collecting tube, setting the sample into nucleic acid extracting kit, setting the kit into automatic or semi-automatic nucleic acid extractor, and storing SARS virus nucleic acid at 4 deg.c. Noise was reduced by keeping the detection strand on the DNA probe and the electrochemical tag away from the graphene by applying-0.5 volts to the working electrode before testing. Electrochemical test parameters for reacting prussian blue were set using square wave voltammetry with an initial voltage of 0 volts, a final voltage of 0.6 volts, a step voltage of 0.005 volts, and a pulse voltage of 0.05 volts. Adding 40 microliters of the solution to be detected, so that the DNA probe modified on the electrochemical microelectrode is complementarily paired and combined with the coronavirus nucleic acid sequence in the solution to be detected. After the detection sample is added and incubation is finished for 30 minutes, the strength of the applied repulsive electric field is adjusted by controlling the magnitude of the negative voltage, the detection chain combined with the coronavirus nucleic acid is distinguished from the detection chain not combined with the coronavirus nucleic acid, the top end of the detection chain not combined with the coronavirus nucleic acid is far away from graphene, and meanwhile, the top end of the detection chain combined with the coronavirus nucleic acid is close to the graphene, so that the detection sensitivity and the detection precision are improved. Finally, the microelectrode has high sensitivity response to SARS virus nucleic acid extracted from a specimen, and the detection limit is 0.1 copy number/microliter.
Example 5
This example provides a specific microelectrode for detecting coronavirus nucleic acid, its preparation method and application for non-disease diagnosis and treatment
Firstly, preparing a chromium/gold (5/40 nanometer) lead, an interdigital electrode (the length is 1000 micrometers and the width is 20 micrometers) and a silver/silver chloride reference electrode (positioned below the interdigital electrode) by an ink-jet printing technology, and finally obtaining the electrochemical microelectrode.
Preparing single-layer graphene on a copper foil with the thickness of 25 microns by using a chemical vapor deposition method, and transferring the prepared graphene to the surface of an electrochemical microelectrode by using a chemical etching method.
Preparing a patterned electrode by adopting an ultraviolet lithography method and an oxygen plasma etching technology, then soaking the graphene electrochemical microelectrode in a benzoic acid N-hydroxysuccinimide ester solution for 0.5-2 hours, washing the graphene electrochemical microelectrode with ultrapure water, and adsorbing a pyrenyl group at one end of the molecule to the surface of the electrochemical microelectrode through pi-pi stacking.
And (3) preparing a micro liquid container, and placing the micro liquid container on the graphene working electrode, wherein the capacity of the micro liquid container is about 80-100 microliters.
Designing a probe based on a DNA tetrahedral nano structure, wherein the top end of the DNA tetrahedral nano structure is provided with a detection chain for combining with novel coronavirus SARS-CoV-2 nucleic acid, the top end of the detection chain is methylene blue, and a section of nucleic acid single chain which is not matched with a coronavirus nucleic acid sequence is added between the detection chain and the three-dimensional nucleic acid nano structure, so that the detection chain can rotate in multiple degrees of freedom. The probe is formed by self-assembling four DNA single strands through base complementary pairing, and the method for synthesizing the DNA probe is to heat DNA mixed liquor to 95 ℃ for 5 minutes, then uniformly cool the mixed liquor to 15 ℃ within 1 minute, and finally store the mixed liquor at 4 ℃. Then, the DNA probe solution is placed in a micro-liquid container, amino groups connected to a base of the DNA probe can be combined with N-hydroxysuccinimide groups to fix the DNA probe on an interface on the surface of the graphene electrochemical microelectrode, the required time is about 8-10 hours, and then the graphene electrochemical microelectrode is washed clean by using ultra-pure water.
The sample is treated to extract virus nucleic acid, the extraction method is that 200 microliter of sample is extracted from the sample collecting tube and put into a nucleic acid extraction kit, then the kit is put into an automatic or semi-automatic nucleic acid extractor, and the extracted novel coronavirus SARS-CoV-2 nucleic acid is stored at 4 ℃.
Noise was reduced by keeping the detection strand on the DNA probe and the electrochemical tag away from the graphene by applying-0.7 volts to the working electrode before testing. Electrochemical test parameters were set to react methylene blue using square wave voltammetry with an initial voltage of-0.7 volts, an end voltage of-0.1 volts, a step voltage of 0.005 volts, and a pulse voltage of 0.05 volts. Adding 80 microliters of the solution to be detected, and enabling the DNA probe modified on the electrochemical microelectrode to be complementarily paired and combined with the coronavirus nucleic acid sequence in the solution to be detected. After the detection sample is added and incubation is finished for 30 minutes, the strength of the applied repulsive electric field is adjusted by controlling the magnitude of the negative voltage, the detection chain combined with the coronavirus nucleic acid is distinguished from the detection chain not combined with the coronavirus nucleic acid, the top end of the detection chain not combined with the coronavirus nucleic acid is far away from graphene, and meanwhile, the top end of the detection chain combined with the coronavirus nucleic acid is close to the graphene, so that the detection sensitivity and the detection precision are improved. Finally, the microelectrode has high sensitivity response to the novel coronavirus SARS-CoV-2 nucleic acid extracted from the specimen, and the detection limit is 0.05 copy number/microliter.
FIG. 3 is a square wave voltammetry current response curve of the novel coronavirus SARS-CoV-2 nucleic acid extracted from the sample in example 5.
Example 6
This example provides a specific microelectrode for detecting coronavirus nucleic acid, its preparation method and application for non-disease diagnosis and treatment
Firstly, preparing a chromium/gold (5/40 nm) wire, an interdigital electrode (with the length of 1000 microns and the width of 20 microns) and a silver/silver chloride reference electrode (positioned below the interdigital electrode) by an ink-jet printing technology to finally obtain the electrochemical microelectrode.
Preparing single-layer graphene on a copper foil with the thickness of 25 microns by using a chemical vapor deposition method, and transferring the prepared graphene to the surface of an electrochemical microelectrode by using a chemical etching method.
Preparing a patterned electrode by adopting an ultraviolet lithography method and an oxygen plasma etching technology, then soaking the graphene electrochemical microelectrode in a benzoic acid N-hydroxysuccinimide ester solution for 0.5-2 hours, washing the graphene electrochemical microelectrode with ultrapure water, and adsorbing a pyrenyl group at one end of the molecule to the surface of the electrochemical microelectrode through pi-pi stacking.
And (3) preparing a micro liquid container, and placing the micro liquid container on the graphene working electrode, wherein the capacity of the micro liquid container is about 200-300 microliters.
A probe based on a DNA tetrahedral nano structure is designed, wherein a detection chain used for being combined with nucleic acid of the middle east respiratory syndrome virus is arranged at the top end of the DNA tetrahedral nano structure, methylene blue is arranged at the top end of the detection chain, a section of nucleic acid single chain which is not matched with a nucleic acid sequence of the coronavirus is added between the detection chain and the three-dimensional nucleic acid nano structure, and the detection chain can rotate in multiple degrees of freedom. The probe is formed by self-assembling four DNA single strands through base complementary pairing, and the method for synthesizing the DNA probe is to heat DNA mixed liquor to 95 ℃ for 5 minutes, then uniformly cool the mixed liquor to 15 ℃ within 1 minute, and finally store the mixed liquor at 4 ℃. Then, the DNA probe solution is placed in a micro-liquid container, amino groups connected to a base of the DNA probe can be combined with N-hydroxysuccinimide groups to fix the DNA probe on an interface on the surface of the graphene electrochemical microelectrode, the required time is about 8-10 hours, and then the graphene electrochemical microelectrode is washed clean by using ultra-pure water.
The sample is processed to extract virus nucleic acid, the extraction method comprises the steps of extracting 200 microliters of sample in a sample collection tube, placing the sample into a nucleic acid extraction kit, then placing the kit into an automatic or semi-automatic nucleic acid extraction instrument, and storing the extracted middle east respiratory syndrome virus nucleic acid at 4 ℃.
Noise was reduced by keeping the detection strand on the DNA probe and the electrochemical tag away from the graphene by applying-0.7 volts to the working electrode before testing. Electrochemical test parameters were set to react methylene blue using square wave voltammetry, initial voltage-0.7 volts, final voltage-0.1 volts, step voltage 0.005 volts, and pulse voltage 0.05 volts. Adding 200 microliters of the solution to be detected, so that the DNA probe modified on the electrochemical microelectrode is complementarily paired and combined with the coronavirus nucleic acid sequence in the solution to be detected. After the detection sample is added and incubation is finished for 30 minutes, the strength of the applied repulsive electric field is adjusted by controlling the magnitude of the negative voltage, the detection chain combined with the coronavirus nucleic acid is distinguished from the detection chain not combined with the coronavirus nucleic acid, the top end of the detection chain not combined with the coronavirus nucleic acid is far away from graphene, and meanwhile, the top end of the detection chain combined with the coronavirus nucleic acid is close to the graphene, so that the detection sensitivity and the detection precision are improved. Finally, the microelectrode has high sensitivity response to the nucleic acid of the respiratory syndrome virus in the middle east, which is extracted from the specimen, and the detection limit is 0.05 copy number/microliter.
The virus detection based on the protocols of examples 1-6 above and the qualitative results of the detection are shown in table 1.
TABLE 1 Virus detected and qualitative results of detection according to the protocols of examples 1-6
Through the above embodiments, it is confirmed that the present invention can accurately and quantitatively detect coronavirus nucleic acid, and compared with the common polymerase chain reaction and fluorescent quantitative polymerase chain reaction methods used for detecting virus nucleic acid at present, the technical scheme provided by the present invention has the advantages of short detection time, high sensitivity, good specificity and good application prospect.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. A microelectrode for detecting coronavirus nucleic acid is characterized by comprising an insulating substrate, a microelectrode and a lead, wherein the microelectrode is prepared on the insulating substrate and comprises a working electrode, a counter electrode and a reference electrode, the surface of the working electrode is completely covered with graphene, and a DNA probe is modified and fixed by the graphene on the surface of the working electrode; the DNA probe can be directly combined with a nucleic acid sequence of the coronavirus to be detected or DNA subjected to reverse transcription with a nucleic acid characteristic sequence of the coronavirus to be detected through base complementary binding, and an electrochemical label on the DNA probe is contacted with graphene on the surface of the microelectrode to generate an oxidation-reduction reaction due to a repulsion effect caused by the combination, so that an electrochemical signal is generated.
2. The microelectrode for nucleic acid detection of coronavirus of claim 1, wherein the DNA probe is prepared by adding one or more detection strands for complementary pairing with a coronavirus nucleic acid sequence and an electrochemical tag at the top of the detection strand on a single DNA strand or a three-dimensional nucleic acid nanostructure, wherein the three-dimensional nucleic acid nanostructure is self-assembled by oligonucleotide strand complementary pairing.
3. The microelectrode for nucleic acid detection of coronavirus of claim 1, wherein the coronavirus nucleic acid signature sequence is one or more of a gene encoding a non-structural protein, a gene encoding a spike protein, a gene encoding a nucleocapsid protein, or a gene encoding an envelope protein.
4. The microelectrode for nucleic acid detection of coronavirus of claim 1, wherein a single nucleic acid strand not paired with coronavirus nucleic acid sequence or DNA reverse transcribed from 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 with multiple degrees of freedom.
5. The microelectrode for nucleic acid detection of coronavirus of claim 1, wherein the working electrode pattern is a centrosymmetric pattern; the insulating substrate is made of silicon dioxide; the graphene is single-layer graphene.
6. The method for preparing a microelectrode for nucleic acid detection of coronaviruses according to any one of claims 1 to 5, comprising the steps of:
step 1: preparing a working electrode, a reference electrode, a counter electrode and a lead on the surface of an insulating substrate;
and 2, step: transferring graphene to the surface of a microelectrode, patterning the graphene by utilizing a photoetching technology to enable the graphene to completely cover a working electrode, and removing the graphene covering the surfaces of a counter electrode and a reference electrode to obtain a device to be modified;
and step 3: adding a device to be modified into a connecting molecule solution for 0.5-2 hours to enable the connecting molecules to be adsorbed to the surface of the graphene, and simultaneously synthesizing a DNA probe;
and 4, step 4: manufacturing a micro-liquid container on the micro-electrode;
and 5: adding a DNA probe solution into a liquid container, connecting the DNA probe with a connecting molecule through a covalent bond, and standing for reacting for 2-10 hours to modify the DNA probe on the surface of the graphene;
step 6: and after finishing modification, taking out the DNA probe solution, adding a buffer solution containing magnesium ions, and storing the device at normal temperature to obtain the microelectrode for detecting the coronavirus nucleic acid.
7. The method for using the microelectrode for the detection of coronavirus nucleic acids according to any of claims 1 to 5 for non-disease diagnostic and therapeutic purposes, comprising the following steps:
before testing, the microelectrode is respectively electrically connected with an interface of an electrochemical testing device, negative voltage is applied to the working electrode, during detection, a sample to be tested is added into the micro liquid container, so that the sample can contact and cover graphene on the surface of the working electrode, and high-sensitivity detection of coronavirus nucleic acid is realized by detecting electrochemical current change.
8. The application method of claim 7, wherein the corresponding electrochemical detection parameters are set according to the redox potential of the electrochemical label of the DNA probe, and comprise a voltage scanning starting voltage, a termination voltage, a stepping voltage and a pulse voltage; the initial voltage and the final voltage are respectively increased or decreased by 0.3 volt to 0.5 volt according to the oxidation-reduction potential of the electrochemical tag, the stepping voltage is 0.005 volt to 0.01 volt, and the pulse voltage is 0.01 volt to 0.1 volt; before testing, the detection chain on the DNA probe and the electrochemical label are far away from the graphene by applying a negative voltage of-0.9 volt to-0.1 volt on the working electrode, so that the noise is reduced.
9. The application method of claim 7, wherein in the detection, a solution to be detected is added into a micro liquid container of the electrochemical microelectrode, so that the DNA probe modified on the electrochemical microelectrode is combined with the coronavirus nucleic acid sequence in the solution to be detected or the DNA obtained after the retrovirus nucleic acid is reversely transcribed;
after adding a detection sample and incubating for 5-30 minutes, regulating the intensity of the applied repulsive electric field by controlling the magnitude of negative voltage, and distinguishing the detection chain combined with the coronavirus nucleic acid from the detection chain not combined with the coronavirus nucleic acid, so that the top end of the detection chain not combined with the coronavirus nucleic acid is far away from graphene, and the top end of the detection chain combined with the coronavirus nucleic acid is close to the graphene, thereby improving the detection sensitivity and precision;
the electrochemical response in the detection process is used as a signal for nucleic acid detection, namely, the nucleic acid can be judged to be positive if an oxidation-reduction current peak appears near the oxidation-reduction potential of the electrochemical tag, and the nucleic acid can be judged to be negative if the oxidation-reduction current peak does not appear near the oxidation-reduction potential of the electrochemical tag;
and finally, calculating the concentration of the coronavirus nucleic acid by detecting the change of the redox current in real time and a concentration formula.
10. The method of use of claim 9, wherein the occurrence of a redox current peak is indicated by more than 120%, including 120%, of the average current value corresponding to the electrochemical tag redox potential ± 0.2 volts;
the absence of a redox current peak is indicated by less than 120%, excluding 120%, of the mean value of the current corresponding to the electrochemical tag redox potential ± 0.2 volts.
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