WO2021190328A1 - Biosensor and preparation method therefor, and system and method for detecting viruses - Google Patents

Biosensor and preparation method therefor, and system and method for detecting viruses Download PDF

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WO2021190328A1
WO2021190328A1 PCT/CN2021/080531 CN2021080531W WO2021190328A1 WO 2021190328 A1 WO2021190328 A1 WO 2021190328A1 CN 2021080531 W CN2021080531 W CN 2021080531W WO 2021190328 A1 WO2021190328 A1 WO 2021190328A1
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biosensor
virus
electrode
output
nucleic acid
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PCT/CN2021/080531
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French (fr)
Chinese (zh)
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郭雪峰
李渝
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北京大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of biological detection, in particular to a biological sensor and a preparation method and a virus detection system and method.
  • a bioelectric sensor is a sensor that uses immobilized biological components or organisms as sensitive elements.
  • the basic principle is to detect the change in current or voltage caused by the biological action or chemical reaction between the sensitive element and the detected biological component or organism, and determine the detection result based on the change in current or voltage.
  • biosensor technology has achieved great development, and has been applied in environmental testing, genetic testing, bacteria and virus testing, etc., but the types of viruses that can actually be detected are still relatively small.
  • the purpose of the embodiments of the present invention is to provide a biosensor and a preparation method and a virus detection system and method to realize the detection of different viruses.
  • the embodiments of the present invention provide biosensors based on graphene and silicon nanowires, wherein the biosensors based on graphene include: a base layer and a graphene layer;
  • the graphene layer is located on the base layer
  • the graphene layer is plated with a metal electrode;
  • the metal electrode includes: an input electrode and an output electrode, and one input electrode corresponds to one output electrode; and,
  • the part of the non-metal electrode on the graphene layer is modified with bridging molecules for connecting biological macromolecules;
  • the bridging molecules include: pyrene or perylene or anthracene anchoring groups for connecting with graphene, and Active ester or disulfide bond or maleic acid glycoside connected to the biological macromolecule, and one or more linking groups that connect the anchor group to the active ester or disulfide bond or maleic acid glycoside;
  • the biological macromolecules connected to the bridging molecules are virus antigens or virus antibodies or nucleic acid probes.
  • a biosensor based on silicon nanowires including: a base layer;
  • the base layer is plated with metal electrodes; the metal electrodes include: input electrodes and output electrodes, and one input electrode corresponds to one output electrode; each input electrode and its corresponding output electrode are connected by silicon nanowires;
  • the silicon nanowire is modified with bridging molecules
  • the silicon nanowire is modified with bridging molecules for connecting biological macromolecules;
  • the bridging molecules are Ni 2+ probes or maleimides;
  • the biological macromolecules connected to the bridging molecules are virus antigens or virus antibodies or nucleic acid probes.
  • the embodiment of the present invention also provides a method for preparing the above-mentioned biosensor, wherein the method for preparing a graphene-based biosensor includes:
  • a metal electrode is vapor-deposited on the position of the metal electrode on the graphene layer to form a device to be modified; the metal electrode includes: an input electrode and an output electrode, and one input electrode corresponds to one output electrode;
  • the preparation method of a silicon nanowire-based biosensor includes:
  • the silicon nanowires generated on the substrate are functionalized on the surface
  • the shape of the metal electrode is etched by photolithography technology
  • a metal electrode is vapor-deposited on the position of the metal electrode on the substrate to form the device to be modified; the metal electrode includes: an input electrode and an output electrode, one input electrode corresponds to one output electrode, and each input electrode and its corresponding output electrode are different Connected by a silicon nanowire;
  • the embodiment of the present invention also provides a virus detection system, including: any of the above-mentioned biosensors, signal generators, signal collectors, signal analysis hosts, and displays;
  • the signal generator is connected to each input electrode of the biosensor, and sends a trigger electrical signal to each input electrode;
  • the signal collector is connected to each output electrode of the biosensor, collects the output current signal and sends it to the signal analysis host;
  • the output current signal is: virus antigen or virus antibody in the biosensor or virus detection Nucleic acid probes, the current signals obtained before and after the reaction with the sample to be tested;
  • the signal analysis host analyzes and detects the received output current signal, determines whether there is a virus in the sample to be tested, and sends the analysis and detection result to the display for display.
  • the embodiment of the present invention also provides a virus detection method, which is applied to the above-mentioned virus detection system, and includes the following steps:
  • the signal generator sends a trigger electrical signal to each input electrode of the biosensor
  • the signal collector is connected to each output electrode of the biosensor, collects the output current signal and sends it to the signal analysis host;
  • the output current signal is: virus antigen or virus antibody in the biosensor or virus detection Nucleic acid probes, the current signals obtained before and after the reaction with the sample to be tested;
  • the signal analysis host analyzes and detects the received output current signal, determines whether there is a virus in the sample to be tested, and sends the analysis and detection result to the display for display.
  • the biosensor and preparation method and virus detection system and method provided in the embodiments of the present invention through the bridging molecules that can connect a variety of biological macromolecules, modify virus antigens or virus antibodies on the biosensor, or are used to detect viruses.
  • the nucleic acid probe inputs the sample to be tested into the biosensor, and analyzes the received current signal, so as to be able to detect whether there is a virus in the sample to be tested; because the bridging molecule of the biosensor provided by the embodiment of the present invention can Connecting a variety of biological macromolecules, so it can realize the detection of different viruses.
  • Fig. 1a is a schematic diagram of a principle structure of a graphene-based biosensor provided by an embodiment of the present invention
  • Fig. 1b is a schematic diagram of a bridging molecule in the embodiment shown in Fig. 1a;
  • Fig. 1c is a structural example diagram of a biosensor prepared based on the principle structure of Fig. 1a;
  • FIG. 2 is a schematic diagram of a second principle structure of a graphene-based biosensor provided by an embodiment of the present invention
  • FIG. 3 is a schematic diagram of a third principle structure of a graphene-based biosensor provided by an embodiment of the present invention.
  • FIG. 4 is a schematic diagram of a principle structure of a silicon nanowire-based biosensor provided by an embodiment of the present invention.
  • FIG. 5 is a schematic diagram of a second principle structure of a silicon nanowire-based biosensor provided by an embodiment of the present invention.
  • FIG. 6 is a schematic diagram of a third principle structure of a silicon nanowire-based biosensor provided by an embodiment of the present invention.
  • Fig. 7a is a flow chart of a method for preparing a graphene-based biosensor
  • FIG. 7b is a diagram of a specific example of preparing a biosensor based on the method shown in FIG. 7a;
  • Fig. 7c is an exemplary diagram of a metal electrode of a biosensor prepared based on the method shown in Fig. 7a;
  • Figure 8a is a flow chart of a method for preparing a silicon nanowire-based biosensor
  • Fig. 8b is a diagram of a specific example of preparing a biosensor based on the method shown in Fig. 8a;
  • FIG. 8c is an exemplary diagram of metal electrodes and silicon nanowires of a biosensor prepared based on the method shown in FIG. 8a;
  • Fig. 8d is another specific example diagram of preparing a biosensor based on the method shown in Fig. 8a;
  • Fig. 9 is a schematic structural diagram of a virus detection system provided by an embodiment of the present invention.
  • embodiments of the present invention provide a biosensor and a preparation method and a virus detection system and method.
  • the biosensor provided by the embodiments of the present invention may be based on graphene materials or silicon nanowires, which will be described in detail below.
  • the graphene material-based biosensor provided by the embodiment of the present invention includes: a base layer and a graphene layer; the graphene layer is located on the base layer; a part of the non-metal electrode on the graphene layer is modified with Bridging molecules of biological macromolecules; the bridging molecules include: pyrene or perylene or anthracene anchoring groups used to connect to graphene, and active esters or disulfide bonds or cis-butylene used to connect to biological macromolecules Acrylic acid glycosides, and one or more linking groups that connect the anchor group to active esters or disulfide bonds or maleic acid glycosides; the biological macromolecules connected to the bridging molecule are: viral antigens Or virus antibodies or nucleic acid probes.
  • the bridging molecule may be one of the following:
  • the bridging molecule of the biosensor provided by the embodiment of the present invention can connect a variety of biological macromolecules (virus antigens or virus antibodies or nucleic acid probes), it can realize the detection of different viruses.
  • the graphene-based biosensor used for detecting new coronaviruses and influenza viruses is taken as an example below to describe in detail the graphene-based biosensor provided in the embodiments of the present invention.
  • the graphene-based biosensor for detecting the new coronavirus has three implementation methods, which are described in the following examples:
  • FIG 1a is a schematic structural diagram of a graphene-based biosensor provided by an embodiment of the present invention.
  • the biosensor includes: a Si/SiO 2 substrate 110, a graphene material 120, and a metal electrode (input/ Output) 130, bridging molecule 140, and COVID-19 antigen 150.
  • the Si/SiO 2 substrate 110 is composed of a silicon Si material layer located in the lower layer and a silicon dioxide SiO 2 material layer closely combined with the silicon Si material layer.
  • the function of SiO 2 is to prevent the interference of signal detection caused by bottom leakage.
  • the Si/SiO 2 substrate 110 may be Si/SiO 2 silicon wafers purchased in the market, and the Si/SiO 2 silicon wafers are integrated, and the silicon Si wafer has been covered with an SiO 2 layer.
  • the base layer in the embodiment of the present invention is not limited to the Si/SiO 2 base.
  • the Si/SiO 2 base 110 may be replaced with another insulating base.
  • the graphene material 120 is located on the upper layer of the Si/SiO 2 substrate 110 to form the graphene layer.
  • the graphene material 120 is plated with metal electrodes (input/output) 130.
  • the metal electrode 130 includes an input electrode and an output electrode. As shown in FIG. 1a, a metal electrode (input) 130 corresponds to a metal electrode (output) 130, and there is graphene between the metal electrode (input) 130 and the metal electrode (output) 130. Material 120.
  • bridging molecules 140 As shown in FIG. 1b, part of the non-metal electrode on the graphene material 120 is modified with bridging molecules 140.
  • the bridging molecule 140 is: 1-pyrene butyrate N-hydroxysuccinimide ester (ie, the bridging molecule 1 described above).
  • the anchor group can be replaced with a perylene or anthracene anchor group; the active ester (amino) can be replaced with a disulfide bond or maleic acid glycoside; the number of linking groups can also be adjusted.
  • the bridging molecule 140 can be replaced with any of the above-mentioned bridging molecules 2-12.
  • the bridging molecule 140 is connected to the new coronavirus COVID-19 antigen 150.
  • the COVID-19 antigen is: IgM and IgG antigen fragments;
  • the IgM and IgG antigen fragments are made of a 1:1 mixture of S protein and N protein;
  • S protein is: S1-RBD, and its amino acid sequence is:
  • amino acid sequence of N protein is:
  • the new coronavirus COVID-19 antigen is modified on the graphene material of the biosensor. If the sample to be tested contains COVID-19 antibody, the COVID-19 antigen and COVID-19 antibody Due to the specific effect, the COVID-19 antibody in the sample to be tested will bind to the COVID-19 antigen on the graphene material, so that the output electrode can output the pre-reaction and post-reaction current signals, thereby being able to detect the new coronavirus.
  • FIG. 1c is a structural example diagram of a biosensor prepared based on the principle structure of FIG. 1a;
  • the graphene material 120 is provided with a microchannel microreactor (PDMS ) 170;
  • the microchannel microreactor 170 is provided with a test sample reaction microcavity 171 that penetrates the upper and lower sides of the microchannel microreactor 170, so that the bridging molecules and COVID-19 antigen modified on the graphene material 120 are located
  • the sample is measured in the reaction microcavity 171.
  • the test sample is dripped into the test sample reaction microcavity 171 in liquid form for reaction, and the metal electrode (output) 130 outputs current signals before and after the reaction to detect the new coronavirus.
  • FIG. 2 is a schematic diagram of the second principle structure of a graphene-based biosensor provided by an embodiment of the present invention
  • the biosensor includes: a Si/SiO 2 substrate 210, a graphene material 220, and a metal electrode (input /Output) 230, bridging molecule 240, COVID-19 antibody 250.
  • the Si/SiO 2 substrate 210 is composed of a lower Si material layer and an SiO 2 material layer closely combined with the Si material layer.
  • the function of SiO 2 is to prevent the interference of signal detection caused by bottom leakage.
  • the Si/SiO 2 substrate 210 may be Si/SiO 2 silicon wafers purchased in the market, and the Si/SiO 2 silicon wafers are integrated, and the silicon Si wafer has been covered with an SiO 2 layer.
  • the graphene material 220 is located on the upper layer of the Si/SiO 2 substrate 210 to form the graphene layer.
  • the graphene material 220 is plated with metal electrodes (input/output) 230.
  • the metal electrode 230 includes an input electrode and an output electrode. As shown in FIG. 2, a metal electrode (input) 230 corresponds to a metal electrode (output) 230, and graphene is between the metal electrode (input) 230 and the metal electrode (output) 230. Material 220.
  • part of the non-metal electrode on the graphene material 220 is modified with bridging molecules 240.
  • the bridging molecule 240 is: the bridging molecule 8 described above.
  • the bridging molecule 240 can be replaced with any other of the 12 types of bridging molecules mentioned above.
  • a new coronavirus COVID-19 antibody 250 is connected to the bridging molecule 240.
  • the COVID-19 antibody in this example is: IgM and IgG antibody fragments.
  • the IgM and IgG antibodies are antibody fragments corresponding to IgM and IgG antigen fragments.
  • the new coronavirus COVID-19 antibody is modified on the graphene material of the biosensor.
  • the specific effect of the COVID-19 antigen in the sample to be tested will be combined with the COVID-19 antibody on the graphene material, so that the output electrode can output the current signal before and after the reaction, and then the new coronavirus can be detected.
  • FIG. 3 is a schematic diagram of a third principle structure of a graphene-based biosensor provided by an embodiment of the present invention.
  • the biosensor includes: a Si/SiO 2 substrate 310, a graphene material 320, and a metal electrode (input /Output) 330, bridging molecule 340, nucleic acid probe 350 and optional heating plate 360.
  • the Si/SiO 2 substrate 310 is composed of a lower Si material layer and an SiO 2 material layer closely combined with the Si material layer.
  • the function of SiO 2 is to prevent the interference of signal detection caused by bottom leakage.
  • the Si/SiO 2 substrate 310 may be Si/SiO 2 silicon wafers purchased in the market, and the Si/SiO 2 silicon wafers are integrated, and the silicon Si wafer has been covered with an SiO 2 layer.
  • the graphene material 320 is located on the upper layer of the Si/SiO 2 substrate 310 and forms the graphene layer.
  • the graphene material 320 is plated with metal electrodes (input/output) 330.
  • the metal electrode 330 includes an input electrode and an output electrode. As shown in FIG. 3, a metal electrode (input) 330 corresponds to a metal electrode (output) 330, and there is graphene between the metal electrode (input) 330 and the metal electrode (output) 330.
  • Material 320 is plated with metal electrodes (input/output) 330.
  • part of the non-metal electrode on the graphene material 320 is modified with bridging molecules 340.
  • the bridging molecule 340 is: the bridging molecule 12 described above.
  • the bridging molecule 340 can be replaced with any other of the 12 types of bridging molecules mentioned above.
  • a nucleic acid probe 350 is connected to the bridging molecule 340.
  • the nucleic acid probe 350 is used to detect the new coronavirus RNA, which can be specifically:
  • a heating plate 360 may also be provided under the Si/SiO 2 substrate 310.
  • the heating plate 360 may be an electric heating plate, which is a commonly used component of a biosensor.
  • the graphene material of the biosensor is modified with a nucleic acid probe for detecting the RNA of the new coronavirus. If the sample to be tested contains the COVID-19 virus, the COVID-19 virus will interact with the graphene.
  • the nucleic acid probes on the material are combined, so that the output electrode can output the current signal before and after the reaction, and then the new coronavirus can be detected.
  • the graphene-based biosensor for detecting influenza virus also has three implementation methods, which are described in the following embodiments:
  • the biosensor includes: Si/SiO 2 substrate, graphene material, metal electrodes (input/output), bridging molecules and influenza virus antigens.
  • the bridging molecule in this embodiment is the bridging molecule 5 among the 12 types of bridging molecules mentioned above.
  • the bridging molecule may be any of the other bridging molecules in 12 above.
  • the bridge molecule 140 is connected to influenza virus antigens instead of new coronavirus antigens.
  • influenza virus antigen sequence is:
  • the biosensor includes: Si/SiO 2 substrate, graphene material, metal electrodes (input/output), bridging molecules, and influenza virus antibodies.
  • the bridging molecule in this example is bridging molecule 3 among the 12 types of bridging molecules mentioned above.
  • the bridging molecule may be any of the other bridging molecules in 12 above.
  • the difference between this embodiment and the embodiment shown in FIG. 2 is that the bridge molecule 240 is connected to influenza virus antibodies instead of new coronavirus antibodies.
  • influenza virus antibodies in this example are antibodies corresponding to the aforementioned influenza virus antigens.
  • the biosensor includes: Si/SiO 2 substrate, graphene material, metal electrodes (input/output), bridging molecules, used to detect influenza Virus probe and heating plate.
  • the probe used to detect influenza virus is to detect the nucleotide sequence of the Hmi influenza virus (2009 mutant) against the HA gene, 5'-[FAM]-CAT TTC TTT CCA TT GCG-[ TAMRA or BHQ1]-3', 5'end modified amino group.
  • This sequence is a fluorescent short probe modified with LNA (Locked Nucleic Acid) to detect the HA gene of Hmi influenza virus (2009 mutant), that is, the bases at positions 4, 7, 10, 13, and 15 are modified with LNA.
  • the 5'end of the probe is labeled with the reporter fluorophore FAM (6-Carboxyfluorescein, 6-carboxyfluorescein), and the 3'end is labeled with the quenching group TAMRA (5-carboxytetramethylrhodamine) or BHQ1 (black hole quenching group) Group, or BQHI).
  • the bridging molecule in this example is bridging molecule 3 among the 12 types of bridging molecules mentioned above.
  • the bridging molecule may be any of the other bridging molecules in 12 above.
  • the difference between this embodiment and the embodiment shown in FIG. 3 is that the bridging molecule 340 is connected to a probe for detecting influenza virus instead of a probe for detecting new coronavirus.
  • biosensor provided in the embodiment of the present invention can also be used to detect influenza virus.
  • the silicon nanowire-based biosensor provided by the embodiment of the present invention includes: a base layer;
  • the base layer is plated with metal electrodes; the metal electrodes include: input electrodes and output electrodes, and one input electrode corresponds to one output electrode; each input electrode and its corresponding output electrode are connected by silicon nanowires;
  • the silicon nanowire is modified with bridging molecules for connecting biological macromolecules; the bridging molecules are Ni 2+ probes or maleimide; the biological macromolecules connected to the bridging molecules are virus antigens Or virus antibodies or nucleic acid probes.
  • the following uses a silicon nanowire-based biosensor for detecting new coronaviruses and influenza viruses as an example to describe in detail the silicon nanowire-based biosensor provided in the embodiments of the present invention.
  • the silicon nanowire-based biosensor for detecting the new coronavirus has three implementation methods, which are described in the following examples:
  • FIG. 4 is a schematic diagram of a principle structure of a silicon nanowire-based biosensor provided by an embodiment of the present invention; the biosensor includes: a Si/SiO 2 substrate 410, a silicon nanowire material 420, and a metal electrode ( Input/output) 430, bridging molecule 440, and COVID-19 antigen 450.
  • the biosensor includes: a Si/SiO 2 substrate 410, a silicon nanowire material 420, and a metal electrode ( Input/output) 430, bridging molecule 440, and COVID-19 antigen 450.
  • the Si/SiO 2 substrate 410 is composed of a silicon Si material layer located in the lower layer and a silicon dioxide SiO 2 material layer closely combined with the silicon Si material layer.
  • the function of SiO 2 is to prevent the interference of signal detection caused by bottom leakage.
  • the Si/SiO 2 substrate 110 may be Si/SiO 2 silicon wafers purchased in the market, and the Si/SiO 2 silicon wafers are integrated, and the silicon Si wafer has been covered with an SiO 2 layer.
  • the silicon nanowire material 420 is located on the upper layer of the Si/SiO 2 substrate 410.
  • the silicon nanowire material 420 is plated with metal electrodes (input/output) 430.
  • the metal electrode 430 includes an input electrode and an output electrode. As shown in FIG. 4, a metal electrode (input) 430 corresponds to a metal electrode (output) 430. There is silicon nanometer between the metal electrode (input) 430 and the metal electrode (output) 430. ⁇ 420 ⁇ Line material 420.
  • the silicon nanowire material 420 is modified with bridging molecules 440.
  • the bridging molecule 440 is a Ni 2+ probe:
  • the bridging molecule 440 may be maleimide:
  • the bridging molecule 440 is connected to the new coronavirus COVID-19 antigen 450.
  • the COVID-19 antigen is: IgM and IgG antigen fragments;
  • the IgM and IgG antigen fragments are made of a 1:1 mixture of S protein and N protein;
  • S protein is: S1-RBD, and its amino acid sequence is:
  • amino acid sequence of N protein is:
  • the new coronavirus COVID-19 antigen is modified on the silicon nanowire material of the biosensor. Due to the specific effect between the samples, the COVID-19 antibody in the sample to be tested will bind to the COVID-19 antigen on the silicon nanowire material 420, so that the output electrode can output the pre-reaction and post-reaction current signals, thereby being able to detect the new coronavirus.
  • FIG. 5 is a schematic diagram of the second principle structure of a silicon nanowire-based biosensor provided by an embodiment of the present invention
  • the biosensor includes: a Si/SiO 2 substrate 510, a silicon nanowire material 520, and a metal electrode (Input/output) 530, bridging molecule 550 and COVID-19 antibody 550.
  • the Si/SiO 2 substrate 510 is composed of a silicon Si material layer located in the lower layer and a silicon dioxide SiO 2 material layer closely combined with it.
  • the function of SiO 2 is to prevent the interference of signal detection caused by bottom leakage.
  • the Si/SiO 2 substrate 110 may be Si/SiO 2 silicon wafers purchased in the market, and the Si/SiO 2 silicon wafers are integrated, and the silicon Si wafer has been covered with an SiO 2 layer.
  • the silicon nanowire material 520 is located on the upper layer of the Si/SiO 2 substrate 510.
  • the silicon nanowire material 520 is plated with metal electrodes (input/output) 530.
  • the metal electrode 530 includes an input electrode and an output electrode. As shown in FIG. 5, a metal electrode (input) 530 corresponds to a metal electrode (output) 530. There is silicon nanometer between the metal electrode (input) 530 and the metal electrode (output) 530. ⁇ 520 ⁇ Line material 520.
  • the silicon nanowire material 520 is modified with bridging molecules 540.
  • the bridging molecule 540 is maleimide.
  • a new coronavirus COVID-19 antibody 550 is connected to the bridging molecule 540.
  • the COVID-19 antibody in this example is: IgM and IgG antibody fragments.
  • the IgM and IgG antibodies are antibody fragments corresponding to IgM and IgG antigen fragments.
  • the new coronavirus COVID-19 antibody is modified on the silicon nanowire material of the biosensor. Due to the specific effect between the samples, the COVID-19 antigen in the sample to be tested will bind to the COVID-19 antibody on the silicon nanowire material 520, so that the output electrode can output the pre-reaction and post-reaction current signals, thereby being able to detect the new coronavirus.
  • FIG. 6 is a schematic diagram of the third principle structure of a silicon nanowire-based biosensor provided by an embodiment of the present invention.
  • the biosensor includes: a Si/SiO 2 substrate 610, a silicon nanowire material 620, and a metal electrode (Input/Output) 630, bridging molecule 640, nucleic acid probe 650, and optional heating plate 660.
  • the Si/SiO 2 substrate 610 is composed of a lower Si material layer and an SiO 2 material layer closely combined with the Si material layer.
  • the function of SiO 2 is to prevent the interference of signal detection caused by bottom leakage.
  • the Si/SiO 2 substrate 610 may be Si/SiO 2 silicon wafers purchased in the market, and the Si/SiO 2 silicon wafers are integrated, and the silicon Si wafer has been covered with an SiO 2 layer.
  • the silicon nanowire material 620 is located on the upper layer of the Si/SiO 2 substrate 610.
  • the silicon nanowire material 620 is plated with metal electrodes (input/output) 630.
  • the metal electrode 630 includes an input electrode and an output electrode. As shown in FIG. 6, a metal electrode (input) 630 corresponds to a metal electrode (output) 630. There is silicon nanometer between the metal electrode (input) 630 and the metal electrode (output) 630. ⁇ 620 ⁇ Line material 620.
  • the silicon nanowire material 620 is modified with bridging molecules 640.
  • the bridging molecule 640 is:
  • a nucleic acid probe 650 is connected to the bridging molecule 640.
  • the nucleic acid probe 650 is used to detect the new coronavirus RNA, which can be specifically:
  • ORF1ab gene synthesis probe
  • a heating plate 660 may also be provided under the Si/SiO 2 substrate 610.
  • the heating plate 660 may be an electric heating plate, which is a commonly used component of a biosensor.
  • the silicon nanowire material of the biosensor is modified with a nucleic acid probe for detecting the RNA of the new coronavirus. If the sample to be tested contains the COVID-19 virus, the COVID-19 virus will interact with the silicon. The nucleic acid probe on the nanowire material is combined, so that the output electrode can output the current signal before and after the reaction, and then the new coronavirus can be detected.
  • Biosensors based on silicon nanowires for detecting influenza viruses 2. Biosensors based on silicon nanowires for detecting influenza viruses.
  • the silicon nanowire-based biosensor for detecting influenza virus also has three implementation methods, which are described in the following embodiments:
  • the biosensor includes: Si/SiO 2 substrate, silicon nanowire material, metal electrodes (input/output), bridging molecules, and influenza virus antigen.
  • the bridging molecule in this example is a Ni 2+ probe.
  • the bridging molecule may be maleimide.
  • the difference between this embodiment and the embodiment shown in FIG. 4 is that the bridging molecule 440 is connected to the influenza virus antigen instead of the new coronavirus antigen.
  • influenza virus antigen sequence is:
  • this biosensor includes: Si/SiO 2 substrate, graphene material, metal electrodes (input/output), bridging molecules, and influenza virus antibodies .
  • the bridging molecule in this example is maleimide.
  • the bridging molecule may be Ni 2+ .
  • the difference between this embodiment and the embodiment shown in FIG. 2 is that the bridge molecule 540 is connected to influenza virus antibodies instead of new coronavirus antibodies.
  • influenza virus antibodies in this example are antibodies corresponding to the aforementioned influenza virus antigens.
  • the biosensor includes: Si/SiO 2 substrate, graphene material, metal electrodes (input/output), bridging molecules, and detection Probe and heating plate for influenza virus.
  • the probe used to detect influenza virus is to detect the nucleotide sequence of the Hmi influenza virus (2009 mutant) against the HA gene, 5'-[FAM]-CAT TTC TTT CCA TT GCG-[ TAMRA or BHQ1]-3', 5'end modified amino group.
  • This sequence is a short fluorescent probe for detecting the LNA modification of the HA gene of the Hmi influenza virus (2009 mutant).
  • the end is labeled with the reporter fluorophore FAM (6-carboxyfluorescein), and the 3'end is labeled with the quencher TAMRA or BHQ1.
  • the bridging molecule in this example is a Ni 2+ probe.
  • the bridging molecule may be maleimide.
  • the difference between this embodiment and the embodiment shown in FIG. 6 is that a probe for detecting influenza virus is connected to the bridging molecule 640 instead of a probe for detecting new coronavirus.
  • biosensor provided in the embodiment of the present invention can also be used to detect influenza virus.
  • the embodiment of the present invention also provides a method for preparing a biosensor based on graphene and silicon nanowires, which will be described separately below.
  • the preparation method of graphene-based biosensor includes the following steps:
  • a metal electrode is vapor-deposited on the position of the metal electrode on the graphene layer to form a device to be modified; the metal electrode includes: an input electrode and an output electrode, and one input electrode corresponds to one output electrode;
  • the virus antigen may be a novel coronavirus COVID-19 antigen or an influenza virus antigen
  • the virus antibody may be a COVID-19 antibody or an influenza antibody
  • the nucleic acid probe may be a nucleic acid probe for detecting COVID-19 Or a nucleic acid probe used to detect influenza.
  • the prepared biosensor is a graphene field effect transistor.
  • a specific preparation process is shown in Fig. 7b, including the following steps:
  • PMMA polymethyl methacrylate
  • the shape of the metal electrode is etched by ultraviolet lithography technology, and then chromium (8nm) and gold (60nm) are deposited by thermal evaporation;
  • the graphene is etched into 25 ⁇ 2200 ⁇ m strips by ultraviolet lithography and oxygen plasma etching (RIE);
  • the graphene external electrodes (chromium (8nm) and gold (80nm)) are made, and finally the upper electrode protective layer silicon dioxide (40nm) is evaporated by electron beam.
  • FIG. 7c is an example diagram of the metal electrode of the biosensor prepared based on the method shown in FIG. 7a.
  • step 5 Immerse the device prepared in step 5 in 1 mM COVID-19 antigen or nucleic acid probe buffer, react for 12 hours, then rinse with the buffer and blow dry with nitrogen.
  • the bridging molecule is 1-pyrenebutyrate N-hydroxysuccinimide ester as an example.
  • the bridging molecule can be any of the above-mentioned 12 kinds of bridging molecules.
  • single-layer graphene can also be grown on the surface of other metals, and various sizes can also be adjusted according to actual conditions.
  • the graphene-based biosensor provided by the embodiments of the present invention is prepared through a “bottom-up” device manufacturing process, and the biosensor can detect multiple viruses, such as new coronavirus or influenza virus.
  • the preparation method of a silicon nanowire-based biosensor includes the following steps:
  • the silicon nanowires generated on the substrate are functionalized on the surface
  • the shape of the metal electrode is etched by photolithography technology
  • a metal electrode is vapor-deposited on the substrate to form the device to be modified; the metal electrode includes: an input electrode and an output electrode, one input electrode corresponds to one output electrode, and each input electrode and its corresponding output electrode are separated by a silicon Nanowire connection;
  • the virus antigen may be a novel coronavirus COVID-19 antigen or an influenza virus antigen
  • the virus antibody may be a COVID-19 antibody or an influenza antibody
  • the nucleic acid probe may be a nucleic acid probe for detecting COVID-19 Or a nucleic acid probe used to detect influenza.
  • the biosensor prepared in this embodiment is a silicon nanowire field-effect transistor.
  • a specific preparation process is shown in FIG. 8b and includes the following steps:
  • APTES Aminopropyltriethoxysilane
  • the shape of the metal electrode is etched by photolithography technology; and the metal electrode is vapor-deposited on the substrate to form the device to be modified; the metal electrode includes: an input electrode and Output electrodes, one input electrode corresponds to one output electrode, and each input electrode and its corresponding output electrode are connected by a silicon nanowire;
  • ABS-NTA N-(5-amino-1-carboxypentyl) iminoacetoacetic acid
  • FIG. 8c is an example diagram of the metal electrode and silicon nanowire of the biosensor prepared based on the method shown in FIG. 8a.
  • the bridging molecule is 1-pyrenebutyrate N-hydroxysuccinimide ester as an example.
  • the bridging molecule may also be maleimide.
  • maleimide is introduced to connect with the sulfhydryl group in the biomacromolecule, that is, the biomacromolecule is immobilized.
  • the biological macromolecules in Figure 8d can be viral antigens or viral antibodies or nucleic acid probes.
  • FIG. 9 is a schematic structural diagram of the virus detection system provided by an embodiment of the present invention.
  • the system includes: a biosensor 901, a signal generator 902, and a signal collector 903 , Signal analysis host 904 and display 905;
  • the biosensor 901 in this embodiment can use any of the biosensors shown in Figs. 1a to 6 above. In the reaction microcavity of the biosensor 901, there is a sample solution to be detected.
  • the signal generator 902 is connected to each input electrode of the biosensor 901, and sends a trigger electrical signal to each input electrode.
  • the signal collector 903 is connected to each output electrode of the biosensor, collects the output current signal and sends it to the signal analysis host 904; the output current signal is: the virus antigen or the virus antibody in the biosensor 901 or The nucleic acid probe that detects the virus is the current signal obtained before and after the reaction with the sample to be detected.
  • the signal analysis host 904 analyzes and detects the received output current signal, determines whether there is a virus in the sample to be tested, and sends the analysis and detection result to the display for display.
  • the current signals before and after the reaction can be collected separately and compared, so as to determine whether there is a virus in the sample to be tested according to the change of the current. Specifically, based on a preset concentration-current standard curve, the received current signals before and after the reaction can be analyzed to determine whether there is a virus in the sample to be tested, and the analysis and detection result can be sent to the display for display.
  • virus detection system using a biosensor modified with virus antigens or virus antibodies or nucleic acid probes for virus detection, a variety of viruses, such as new coronaviruses and influenza viruses, can be detected.
  • the embodiment of the present invention also provides a virus detection method, which is applied to the virus detection system shown in FIG. 9 and includes the following steps:
  • the signal generator sends a trigger electrical signal to each input electrode of the biosensor
  • the signal generator may send a trigger electrical signal to the biosensor after receiving the start instruction sent by the signal analysis host.
  • the signal collector is connected to each output electrode of the biosensor, collects the output current signal and sends it to the signal analysis host;
  • the output current signal is: virus antigen or virus antibody in the biosensor or virus detection Nucleic acid probe, the current signal obtained before and after the reaction with the sample to be tested;
  • the signal analysis host analyzes and detects the received output current signal, determines whether there is a virus in the sample to be tested, and sends the analysis and detection result to the display for display.
  • the embodiment of the present invention can detect a variety of viruses.
  • the graphene-based biosensor (graphene field effect transistor) shown in FIG. 1c was used to detect the sample to be tested.
  • the bridging molecule for generating the sensor is: 1-pyrene butyrate N-hydroxysuccinimide ester; the bridging molecule is connected to the new coronavirus antigen.
  • the antigen of the novel coronavirus is a specific antigen fragment selected against COVID-19.
  • the best IgM and IgG antigen fragments are selected.
  • the current antigen specificity exceeds 95.3%, and the specificity is 98%.
  • Detection speed For pre-processed serum samples, this method achieves rapid detection, and the detection time is less than 1 minute.
  • the pre-processing of the serum sample is to remove non-tested substances in the sample and improve the reliability of the results.
  • Positive group 8 positive patients detected by the colloidal gold nucleic acid test in clinical testing, including 7 positive cases of IgM or IgG;
  • Control group 8 cases of colloidal gold nucleic acid test were all negative, of which 4 cases were normal blood donors, and the other 4 were patients with negative clinical screening for the new coronavirus.
  • Test specificity Among the 16 on-site tests, there were 8 2019-nCoV positive patients and 8 negative controls. Compared with the colloidal gold test paper test method, the test results of this method showed that all 8 cases of 2019-nCoV positive patients were detected, and the positive accuracy rate was 100%; among the 8 negative controls, 4 cases were confirmed to be normal, and 4 cases were suspected or just. The sensitivity and specificity of the discharged patients are better than the colloidal gold method, which reflects the effectiveness of the method. The specific test results and comparison are as follows:
  • Detection sensitivity Dilute the samples of 2 patients with 2019-nCoV positive by 1000 times and use this method to detect the results, and the results are positive. This shows that the detection limit of this method is less than one-thousandth of the current current detection concentration, and the detection sensitivity is improved by 3 orders of magnitude. It is expected to develop new non-invasive detection methods and new technologies.
  • This experiment uses the graphene-based biosensor shown in Figure 3 to detect the sample to be tested.
  • the bridging molecule for generating the sensor is: 1-pyrene butyrate N-hydroxysuccinimide ester; connected to the bridging molecule is a nucleic acid probe for detecting the new coronavirus.
  • test results show that when the pre-processing conditions of the sample to be tested meet 65 degrees Celsius and the hybridization time is 30 minutes, the test results are very ideal. This will provide a new, effective and rapid solution for subsequent rapid and accurate detection of virus types.
  • This experiment uses a silicon nanowire-based biosensor (silicon nanowire field effect transistor) to detect the sample to be tested.
  • silicon nanowire-based biosensor silicon nanowire field effect transistor
  • the bridging molecule is a Ni 2+ probe:
  • nucleic acid probe for detecting influenza virus Connected to the linking molecule is a nucleic acid probe for detecting influenza virus:
  • This sequence is a short fluorescent probe for detecting the LNA modification of the HA gene of the Hmi influenza virus (2009 mutant).
  • the end is labeled with the reporter fluorophore FAM (6-carboxyfluorescein), and the 3'end is labeled with the quencher TAMRA or BHQ1.
  • Control group 2 cases of colloidal gold nucleic acid test were all negative, of which 1 case was a normal blood donor, and the other case was a positive patient in the follow-up clinical screening of influenza virus.

Abstract

Provided are a biosensor and a preparation method therefor, and a system and a method for detecting viruses. The method comprises the following steps: modifying a virus antigen or a virus antibody or a nucleic acid probe (350, 650) for detecting viruses on a biosensor by means of bridging molecules (140, 240, 340, 440, 540, 640) capable of connecting various biomacromolecules; inputting a sample to be detected into the biosensor; and analyzing current signals before and after reaction, so as to detect whether viruses exist in the sample to be detected or not. The bridging molecules (140, 240, 340, 440, 540, 640) of the biosensor provided by the present invention can be connected with various biomacromolecules, so that different viruses can be detected.

Description

一种生物传感器及制备方法和病毒检测***及方法Biosensor and preparation method and virus detection system and method
本申请要求于2020年3月27日提交中国专利局、申请号为202010233584.2、发明名称为“一种生物传感器及制备方法和病毒检测***及方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on March 27, 2020, the application number is 202010233584.2, and the invention title is "a biosensor and its preparation method, and virus detection system and method". The entire content is approved The reference is incorporated in this application.
技术领域Technical field
本发明涉及生物检测技术领域，特别是涉及一种生物传感器及制备方法和病毒检测***及方法。The invention relates to the technical field of biological detection, in particular to a biological sensor and a preparation method and a virus detection system and method.
背景技术Background technique
生物电学传感器是一种用固定化生物成分或生物体作为敏感元件的传感器。其基本原理是，对由敏感元件与被检测生物成分或生物体之间的生物作用或化学反应引起的电流或电压的变化进行检测，根据电流或电压的变化来确定检测结果。A bioelectric sensor is a sensor that uses immobilized biological components or organisms as sensitive elements. The basic principle is to detect the change in current or voltage caused by the biological action or chemical reaction between the sensitive element and the detected biological component or organism, and determine the detection result based on the change in current or voltage.
目前，生物传感器技术获得了很大的发展，在环境检测、基因检测、细菌和病毒检测等方面有所应用，但实际能够检测出的病毒类型还是比较少。At present, biosensor technology has achieved great development, and has been applied in environmental testing, genetic testing, bacteria and virus testing, etc., but the types of viruses that can actually be detected are still relatively small.
发明内容Summary of the invention
本发明实施例的目的在于提供一种生物传感器及制备方法和病毒检测***及方法，以实现对不同病毒的检测。The purpose of the embodiments of the present invention is to provide a biosensor and a preparation method and a virus detection system and method to realize the detection of different viruses.
为了实现本发明实施例的目的，本发明实施例提供了基于石墨烯和硅纳米线的生物传感器，其中，基于石墨烯的生物传感器，包括：基底层和石墨烯层；In order to achieve the purpose of the embodiments of the present invention, the embodiments of the present invention provide biosensors based on graphene and silicon nanowires, wherein the biosensors based on graphene include: a base layer and a graphene layer;
所述石墨烯层位于所述基底层上；The graphene layer is located on the base layer;
所述石墨烯层上镀有金属电极；所述金属电极包括：输入电极和输出电极，且一个输入电极对应一个输出电极；且，The graphene layer is plated with a metal electrode; the metal electrode includes: an input electrode and an output electrode, and one input electrode corresponds to one output electrode; and,
所述石墨烯层上非金属电极的部分修饰有用于连接生物大分子的桥连分子；所述桥连分子，包含：用于与石墨烯连接的芘或苝或蒽锚定基团、用于与生物大分子相连的活泼酯或二硫键或顺丁烯二酸苷、和将锚定基团与活泼酯或二硫键或顺丁烯二酸苷相连的一个或多个连接基团；The part of the non-metal electrode on the graphene layer is modified with bridging molecules for connecting biological macromolecules; the bridging molecules include: pyrene or perylene or anthracene anchoring groups for connecting with graphene, and Active ester or disulfide bond or maleic acid glycoside connected to the biological macromolecule, and one or more linking groups that connect the anchor group to the active ester or disulfide bond or maleic acid glycoside;
所述桥连分子上连接的生物大分子为：病毒抗原或病毒抗体或核酸探针。The biological macromolecules connected to the bridging molecules are virus antigens or virus antibodies or nucleic acid probes.
基于硅纳米线的生物传感器，包括：基底层；A biosensor based on silicon nanowires, including: a base layer;
所述基底层上镀有金属电极；所述金属电极包括：输入电极和输出电极，且一个输入电极对应一个输出电极；每个输入电极与其对应的输出电极之间由硅纳米线连接；The base layer is plated with metal electrodes; the metal electrodes include: input electrodes and output electrodes, and one input electrode corresponds to one output electrode; each input electrode and its corresponding output electrode are connected by silicon nanowires;
所述硅纳米线上修饰有桥连分子；The silicon nanowire is modified with bridging molecules;
所述硅纳米线上修饰有用于连接生物大分子的桥连分子；所述桥连分子为Ni 2+探针或马来酰亚胺； The silicon nanowire is modified with bridging molecules for connecting biological macromolecules; the bridging molecules are Ni 2+ probes or maleimides;
所述桥连分子上连接的生物大分子为：病毒抗原或病毒抗体或核酸探针。The biological macromolecules connected to the bridging molecules are virus antigens or virus antibodies or nucleic acid probes.
本发明实施例还提供了上述生物传感器的制备方法，其中基于石墨烯的生物传感器的制备方法，包括：The embodiment of the present invention also provides a method for preparing the above-mentioned biosensor, wherein the method for preparing a graphene-based biosensor includes:
A、将在金属表面生成的单层石墨烯转移到基底材料表面上，形成基底层和石墨烯层；A. Transfer the single-layer graphene generated on the metal surface to the surface of the base material to form the base layer and the graphene layer;
B、在所述石墨烯层上旋涂光刻胶后，通过光刻技术刻蚀出金属电极的形状；B. After spin-coating photoresist on the graphene layer, the shape of the metal electrode is etched by photolithography technology;
C、在石墨烯层上金属电极的位置蒸镀上金属电极，形成待修饰器件；所述金属电极包括：输入电极和输出电极，且一个输入电极对应一个输出电极；C. A metal electrode is vapor-deposited on the position of the metal electrode on the graphene layer to form a device to be modified; the metal electrode includes: an input electrode and an output electrode, and one input electrode corresponds to one output electrode;
D、在待修饰器件的石墨烯层上非金属电极的部分修饰上桥连分子；D. Modification of bridging molecules on part of the non-metal electrode on the graphene layer of the device to be modified;
E、在所述桥连分子上连接病毒抗原或病毒抗体或核酸探针。E. Connect viral antigens or viral antibodies or nucleic acid probes to the bridging molecule.
基于硅纳米线的生物传感器的制备方法，包括：The preparation method of a silicon nanowire-based biosensor includes:
a、在基底上生成的硅纳米线并表面功能化；a. The silicon nanowires generated on the substrate are functionalized on the surface;
b、在所述基底上旋涂光刻胶后，通过光刻技术刻蚀出金属电极的形状；b. After spin-coating photoresist on the substrate, the shape of the metal electrode is etched by photolithography technology;
c、在基底上金属电极的位置蒸镀上金属电极，形成待修饰器件；所述金属电极包括：输入电极和输出电极，一个输入电极对应一个输出电极，每个输入电极与其对应的输出电极之间由一硅纳米线连接；c. A metal electrode is vapor-deposited on the position of the metal electrode on the substrate to form the device to be modified; the metal electrode includes: an input electrode and an output electrode, one input electrode corresponds to one output electrode, and each input electrode and its corresponding output electrode are different Connected by a silicon nanowire;
d、在待修饰器件的硅纳米线上修饰上桥连分子；d. Modify bridging molecules on the silicon nanowire of the device to be modified;
e、在所述桥连分子上连接病毒抗原或病毒抗体或核酸探针。e. Connect viral antigens or viral antibodies or nucleic acid probes to the bridging molecule.
本发明实施例还提供了一种病毒检测***，包括：上述任一种生物传感器、信号发生器、信号采集器、信号分析主机和显示器；The embodiment of the present invention also provides a virus detection system, including: any of the above-mentioned biosensors, signal generators, signal collectors, signal analysis hosts, and displays;
所述信号发生器与所述生物传感器的各个输入电极相连，将触发电信号发送至各个输入电极；The signal generator is connected to each input electrode of the biosensor, and sends a trigger electrical signal to each input electrode;
所述信号采集器与所述生物传感器的各个输出电极相连，采集输出电流信号并发送至信号分析主机；该输出电流信号为：所述生物传感器中的病毒 抗原或病毒抗体或用于检测病毒的核酸探针，与待检测样品反应前和反应后分别获得的电流信号；The signal collector is connected to each output electrode of the biosensor, collects the output current signal and sends it to the signal analysis host; the output current signal is: virus antigen or virus antibody in the biosensor or virus detection Nucleic acid probes, the current signals obtained before and after the reaction with the sample to be tested;
所述信号分析主机，对接收到的输出电流信号进行分析检测，确定待测样品中是否存在病毒，将分析检测结果发送至显示器进行显示。The signal analysis host analyzes and detects the received output current signal, determines whether there is a virus in the sample to be tested, and sends the analysis and detection result to the display for display.
本发明实施例还提供了一种病毒检测方法，应用于上述的病毒检测***，包括如下步骤：The embodiment of the present invention also provides a virus detection method, which is applied to the above-mentioned virus detection system, and includes the following steps:
所述信号发生器将触发电信号发送至所述生物传感器的各个输入电极；The signal generator sends a trigger electrical signal to each input electrode of the biosensor;
将待测样品输入所述生物传感器；Input the sample to be tested into the biosensor;
所述信号采集器与所述生物传感器的各个输出电极相连，采集输出电流信号并发送至信号分析主机；该输出电流信号为：所述生物传感器中的病毒抗原或病毒抗体或用于检测病毒的核酸探针，与待检测样品反应前和反应后分别获得的电流信号；The signal collector is connected to each output electrode of the biosensor, collects the output current signal and sends it to the signal analysis host; the output current signal is: virus antigen or virus antibody in the biosensor or virus detection Nucleic acid probes, the current signals obtained before and after the reaction with the sample to be tested;
所述信号分析主机，对接收到的输出电流信号进行分析检测，确定待测样品中是否存在病毒，将分析检测结果发送至显示器进行显示。The signal analysis host analyzes and detects the received output current signal, determines whether there is a virus in the sample to be tested, and sends the analysis and detection result to the display for display.
本发明实施例有益效果：Beneficial effects of the embodiments of the present invention:
本发明实施例提供的一种生物传感器及制备方法和病毒检测***及方法，通过能够连接多种生物大分子的桥连分子，在生物传感器上修饰了病毒抗原或病毒抗体或用于检测病毒的核酸探针，将待测样品输入所述生物传感器，并对接收到的电流信号进行分析，从而能够检测出待测样品中是否有病毒；由于本发明实施例提供的生物传感器的桥连分子能够连接多种生物大分子，因此能够实现对不同病毒的检测。The biosensor and preparation method and virus detection system and method provided in the embodiments of the present invention, through the bridging molecules that can connect a variety of biological macromolecules, modify virus antigens or virus antibodies on the biosensor, or are used to detect viruses. The nucleic acid probe inputs the sample to be tested into the biosensor, and analyzes the received current signal, so as to be able to detect whether there is a virus in the sample to be tested; because the bridging molecule of the biosensor provided by the embodiment of the present invention can Connecting a variety of biological macromolecules, so it can realize the detection of different viruses.
附图说明Description of the drawings
为了更清楚地说明本发明实施例和现有技术的技术方案，下面对实施例和现有技术中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to explain the embodiments of the present invention and the technical solutions of the prior art more clearly, the following briefly introduces the drawings that need to be used in the embodiments and the prior art. Obviously, the drawings in the following description are merely the present invention. For some of the embodiments of the invention, those of ordinary skill in the art can obtain other drawings based on these drawings without creative work.
图1a为本发明实施例提供的基于石墨烯的生物传感器的一种原理结构示意图；Fig. 1a is a schematic diagram of a principle structure of a graphene-based biosensor provided by an embodiment of the present invention;
图1b为图1a所示实施例中的一种桥连分子的示意图；Fig. 1b is a schematic diagram of a bridging molecule in the embodiment shown in Fig. 1a;
图1c为基于图1a的原理结构制备的生物传感器的结构示例图；Fig. 1c is a structural example diagram of a biosensor prepared based on the principle structure of Fig. 1a;
图2为本发明实施例提供的基于石墨烯的生物传感器的第二种原理结构 示意图；2 is a schematic diagram of a second principle structure of a graphene-based biosensor provided by an embodiment of the present invention;
图3为本发明实施例提供的基于石墨烯的生物传感器的第三种原理结构示意图；3 is a schematic diagram of a third principle structure of a graphene-based biosensor provided by an embodiment of the present invention;
图4为本发明实施例提供的基于硅纳米线的生物传感器的一种原理结构示意图；4 is a schematic diagram of a principle structure of a silicon nanowire-based biosensor provided by an embodiment of the present invention;
图5为本发明实施例提供的基于硅纳米线的生物传感器的第二种原理结构示意图；5 is a schematic diagram of a second principle structure of a silicon nanowire-based biosensor provided by an embodiment of the present invention;
图6为本发明实施例提供的基于硅纳米线的生物传感器的第三种原理结构示意图；6 is a schematic diagram of a third principle structure of a silicon nanowire-based biosensor provided by an embodiment of the present invention;
图7a为基于石墨烯的生物传感器的制备方法的流程图；Fig. 7a is a flow chart of a method for preparing a graphene-based biosensor;
图7b为基于图7a所示方法制备生物传感器的具体示例图；FIG. 7b is a diagram of a specific example of preparing a biosensor based on the method shown in FIG. 7a;
图7c为基于图7a所示方法制备的生物传感器的金属电极的示例图；Fig. 7c is an exemplary diagram of a metal electrode of a biosensor prepared based on the method shown in Fig. 7a;
图8a为基于硅纳米线的生物传感器的制备方法的流程图；Figure 8a is a flow chart of a method for preparing a silicon nanowire-based biosensor;
图8b为基于图8a所示方法制备生物传感器的一种具体示例图；Fig. 8b is a diagram of a specific example of preparing a biosensor based on the method shown in Fig. 8a;
图8c为基于图8a所示方法制备的生物传感器的金属电极和硅纳米线的示例图；FIG. 8c is an exemplary diagram of metal electrodes and silicon nanowires of a biosensor prepared based on the method shown in FIG. 8a;
图8d为基于图8a所示方法制备生物传感器的另一种具体示例图；Fig. 8d is another specific example diagram of preparing a biosensor based on the method shown in Fig. 8a;
图9为本发明实施例提供的病毒检测***的结构示意图。Fig. 9 is a schematic structural diagram of a virus detection system provided by an embodiment of the present invention.
具体实施方式Detailed ways
为使本发明的目的、技术方案、及优点更加清楚明白，以下参照附图并举实施例，对本发明进一步详细说明。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。本领域普通技术人员基于本申请中的实施例所获得的所有其他实施例，都属于本申请保护的范围。In order to make the objectives, technical solutions, and advantages of the present invention clearer and more comprehensible, the following further describes the present invention in detail with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in this application fall within the protection scope of this application.
为了对多种不同病毒进行检测，本发明实施例提供了一种生物传感器及制备方法和病毒检测***及方法。In order to detect a variety of different viruses, embodiments of the present invention provide a biosensor and a preparation method and a virus detection system and method.
首先，对本发明实施例提供的生物传感器进行说明。First, the biosensor provided by the embodiment of the present invention will be described.
本发明实施例提供的生物传感器，可以基于石墨烯材料也可以基于硅纳米线，以下分别进行详细说明。The biosensor provided by the embodiments of the present invention may be based on graphene materials or silicon nanowires, which will be described in detail below.
本发明实施例提供的基于石墨烯材料的生物传感器，包括：基底层和石墨烯层；所述石墨烯层位于所述基底层上；所述石墨烯层上非金属电极的部分修饰有用于连接生物大分子的桥连分子；所述桥连分子，包含：用于与石 墨烯连接的芘或苝或蒽锚定基团、用于与生物大分子相连的活泼酯或二硫键或顺丁烯二酸苷、和将锚定基团与活泼酯或二硫键或顺丁烯二酸苷相连的一个或多个连接基团；所述桥连分子上连接的生物大分子为：病毒抗原或病毒抗体或核酸探针。具体的，所述的桥连分子可以为如下之一：The graphene material-based biosensor provided by the embodiment of the present invention includes: a base layer and a graphene layer; the graphene layer is located on the base layer; a part of the non-metal electrode on the graphene layer is modified with Bridging molecules of biological macromolecules; the bridging molecules include: pyrene or perylene or anthracene anchoring groups used to connect to graphene, and active esters or disulfide bonds or cis-butylene used to connect to biological macromolecules Acrylic acid glycosides, and one or more linking groups that connect the anchor group to active esters or disulfide bonds or maleic acid glycosides; the biological macromolecules connected to the bridging molecule are: viral antigens Or virus antibodies or nucleic acid probes. Specifically, the bridging molecule may be one of the following:
Figure PCTCN2021080531-appb-000001
Figure PCTCN2021080531-appb-000001
Figure PCTCN2021080531-appb-000002
Figure PCTCN2021080531-appb-000002
由于本发明实施例提供的生物传感器的桥连分子能够连接多种生物大分子(病毒抗原或病毒抗体或核酸探针)，因此能够实现对不同病毒的检测。Since the bridging molecule of the biosensor provided by the embodiment of the present invention can connect a variety of biological macromolecules (virus antigens or virus antibodies or nucleic acid probes), it can realize the detection of different viruses.
以下以用于检测新冠病毒和流感病毒的基于石墨烯的生物传感器为例，对本发明实施例提供的基于石墨烯的生物传感器进行详细说明。The graphene-based biosensor used for detecting new coronaviruses and influenza viruses is taken as an example below to describe in detail the graphene-based biosensor provided in the embodiments of the present invention.
一、用于检测新冠病毒的基于石墨烯的生物传感器。1. Graphene-based biosensor for detecting new coronavirus.
具体的，用于检测新冠病毒的基于石墨烯的生物传感器有三种实现方式，以下分别举实施例进行说明：Specifically, the graphene-based biosensor for detecting the new coronavirus has three implementation methods, which are described in the following examples:
用于检测新冠病毒的基于石墨烯的生物传感器实施例一Example 1 of graphene-based biosensor for detecting new coronavirus
如图1a所示，图1a为本发明实施例提供的基于石墨烯的生物传感器的一种原理结构示意图，该生物传感器包括：Si/SiO 2基底110、石墨烯材料120、金属电极(输入/输出)130、桥连分子140和COVID-19抗原150。 As shown in Figure 1a, Figure 1a is a schematic structural diagram of a graphene-based biosensor provided by an embodiment of the present invention. The biosensor includes: a Si/SiO 2 substrate 110, a graphene material 120, and a metal electrode (input/ Output) 130, bridging molecule 140, and COVID-19 antigen 150.
其中，Si/SiO 2基底110是由位于下层的硅Si材料层和与其紧密结合的二氧化硅SiO 2材料层构成。SiO 2的作用是防止由于底部漏电对信号检测造成的干扰。Si/SiO 2基底110可以是在市场中购买的Si/SiO 2硅片，Si/SiO 2硅片就是一体的，硅Si片上已经铺上了SiO 2层。 Wherein, the Si/SiO 2 substrate 110 is composed of a silicon Si material layer located in the lower layer and a silicon dioxide SiO 2 material layer closely combined with the silicon Si material layer. The function of SiO 2 is to prevent the interference of signal detection caused by bottom leakage. The Si/SiO 2 substrate 110 may be Si/SiO 2 silicon wafers purchased in the market, and the Si/SiO 2 silicon wafers are integrated, and the silicon Si wafer has been covered with an SiO 2 layer.
本发明实施例中的基底层不限于Si/SiO 2基底，在其他实施例中，可以用其他绝缘基底替换Si/SiO 2基底110。 The base layer in the embodiment of the present invention is not limited to the Si/SiO 2 base. In other embodiments, the Si/SiO 2 base 110 may be replaced with another insulating base.
石墨烯材料120位于Si/SiO 2基底110的上层，形成了所述的石墨烯层。 The graphene material 120 is located on the upper layer of the Si/SiO 2 substrate 110 to form the graphene layer.
石墨烯材料120上镀有金属电极(输入/输出)130。金属电极130包括：输入电极和输出电极，如图1a所示一个金属电极(输入)130对应一个金属电极(输出)130，金属电极(输入)130和金属电极(输出)130之间有石 墨烯材料120。The graphene material 120 is plated with metal electrodes (input/output) 130. The metal electrode 130 includes an input electrode and an output electrode. As shown in FIG. 1a, a metal electrode (input) 130 corresponds to a metal electrode (output) 130, and there is graphene between the metal electrode (input) 130 and the metal electrode (output) 130. Material 120.
如图1b所示，石墨烯材料120上非金属电极的部分修饰有桥连分子140。该桥连分子140为：1-芘丁酸N-羟基琥珀酰亚胺酯(即上述桥连分子1)。As shown in FIG. 1b, part of the non-metal electrode on the graphene material 120 is modified with bridging molecules 140. The bridging molecule 140 is: 1-pyrene butyrate N-hydroxysuccinimide ester (ie, the bridging molecule 1 described above).
如图1b所示，锚定基团可以替换为苝或蒽锚定基团；活泼酯(氨基)可以替换为二硫键或顺丁烯二酸苷；连接基团的数量也可以调整。As shown in Figure 1b, the anchor group can be replaced with a perylene or anthracene anchor group; the active ester (amino) can be replaced with a disulfide bond or maleic acid glycoside; the number of linking groups can also be adjusted.
在实际应用中，该桥连分子140可以用上述桥连分子2～12任一种替换。该桥连分子140上连接有新冠病毒COVID-19抗原150。In practical applications, the bridging molecule 140 can be replaced with any of the above-mentioned bridging molecules 2-12. The bridging molecule 140 is connected to the new coronavirus COVID-19 antigen 150.
所述COVID-19抗原为：IgM和IgG抗原片段；The COVID-19 antigen is: IgM and IgG antigen fragments;
所述IgM和IgG抗原片段是由S蛋白和N蛋白1：1混合成的；The IgM and IgG antigen fragments are made of a 1:1 mixture of S protein and N protein;
其中，S蛋白为：S1-RBD，其氨基酸排列顺序为：Among them, S protein is: S1-RBD, and its amino acid sequence is:
Figure PCTCN2021080531-appb-000003
Figure PCTCN2021080531-appb-000003
N蛋白的氨基酸排列顺序为：The amino acid sequence of N protein is:
Figure PCTCN2021080531-appb-000004
Figure PCTCN2021080531-appb-000004
由图1a～b所示实施例可见，在生物传感器的石墨烯材料上修饰了新冠病毒COVID-19抗原，如果待测样品中含COVID-19抗体，则由COVID-19抗原和COVID-19抗体之间的特异性作用，待测样品中的COVID-19抗体会与石墨烯材料上的COVID-19抗原结合，从而使输出电极输出反应前和反应后电流信号，进而能够检测出新冠病毒。It can be seen from the examples shown in Figures 1a to b that the new coronavirus COVID-19 antigen is modified on the graphene material of the biosensor. If the sample to be tested contains COVID-19 antibody, the COVID-19 antigen and COVID-19 antibody Due to the specific effect, the COVID-19 antibody in the sample to be tested will bind to the COVID-19 antigen on the graphene material, so that the output electrode can output the pre-reaction and post-reaction current signals, thereby being able to detect the new coronavirus.
为了能够对待测样品进行检测，实际应用中可以在生物传感器的输入电 极和输出电极之间，设置被测样品反应腔。具体的，参见图1c，图1c为基于图1a的原理结构制备的生物传感器的结构示例图；In order to be able to detect the sample to be tested, a reaction chamber for the sample to be tested can be set between the input electrode and the output electrode of the biosensor in practical applications. Specifically, refer to FIG. 1c, which is a structural example diagram of a biosensor prepared based on the principle structure of FIG. 1a;
如图1c所示，在图1a所示实施例的基础上，在金属电极(输入)130和金属电极(输出)130之间，石墨烯材料120上，设置有微流道微反应器(PDMS)170；在微流道微反应器170中设置有贯穿微流道微反应器170上下的被测样品反应微腔171，使得石墨烯材料120上修饰的桥连分子和COVID-19抗原位于被测样品反应微腔171中。被测样品以液态的形式滴入被测样品反应微腔171中进行反应，金属电极(输出)130输出反应前和反应后的电流信号，进而检测出新冠病毒。As shown in FIG. 1c, on the basis of the embodiment shown in FIG. 1a, between the metal electrode (input) 130 and the metal electrode (output) 130, the graphene material 120 is provided with a microchannel microreactor (PDMS ) 170; The microchannel microreactor 170 is provided with a test sample reaction microcavity 171 that penetrates the upper and lower sides of the microchannel microreactor 170, so that the bridging molecules and COVID-19 antigen modified on the graphene material 120 are located The sample is measured in the reaction microcavity 171. The test sample is dripped into the test sample reaction microcavity 171 in liquid form for reaction, and the metal electrode (output) 130 outputs current signals before and after the reaction to detect the new coronavirus.
用于检测新冠病毒的基于石墨烯的生物传感器实施例二Example 2 of graphene-based biosensor for detecting new coronavirus
如图2所示，图2为本发明实施例提供的基于石墨烯的生物传感器的第二种原理结构示意图；该生物传感器包括：Si/SiO 2基底210、石墨烯材料220、金属电极(输入/输出)230、桥连分子240、COVID-19抗体250。 As shown in Figure 2, Figure 2 is a schematic diagram of the second principle structure of a graphene-based biosensor provided by an embodiment of the present invention; the biosensor includes: a Si/SiO 2 substrate 210, a graphene material 220, and a metal electrode (input /Output) 230, bridging molecule 240, COVID-19 antibody 250.
其中，Si/SiO 2基底210是由位于下层的Si材料层和与其紧密结合的SiO 2材料层构成。SiO 2的作用是防止由于底部漏电对信号检测造成的干扰。Si/SiO 2基底210可以是在市场中购买的Si/SiO 2硅片，Si/SiO 2硅片就是一体的，硅Si片上已经铺上了SiO 2层。 Among them, the Si/SiO 2 substrate 210 is composed of a lower Si material layer and an SiO 2 material layer closely combined with the Si material layer. The function of SiO 2 is to prevent the interference of signal detection caused by bottom leakage. The Si/SiO 2 substrate 210 may be Si/SiO 2 silicon wafers purchased in the market, and the Si/SiO 2 silicon wafers are integrated, and the silicon Si wafer has been covered with an SiO 2 layer.
石墨烯材料220位于Si/SiO 2基底210的上层，形成了所述的石墨烯层。 The graphene material 220 is located on the upper layer of the Si/SiO 2 substrate 210 to form the graphene layer.
石墨烯材料220上镀有金属电极(输入/输出)230。金属电极230包括：输入电极和输出电极，如图2所示一个金属电极(输入)230对应一个金属电极(输出)230，金属电极(输入)230和金属电极(输出)230之间有石墨烯材料220。The graphene material 220 is plated with metal electrodes (input/output) 230. The metal electrode 230 includes an input electrode and an output electrode. As shown in FIG. 2, a metal electrode (input) 230 corresponds to a metal electrode (output) 230, and graphene is between the metal electrode (input) 230 and the metal electrode (output) 230. Material 220.
如图2所示，石墨烯材料220上非金属电极的部分修饰有桥连分子240。该桥连分子240为：上述桥连分子8。As shown in FIG. 2, part of the non-metal electrode on the graphene material 220 is modified with bridging molecules 240. The bridging molecule 240 is: the bridging molecule 8 described above.
同样的，在实际应用中该桥连分子240可以替换为上述12种桥连分子中的任何其他一种。Similarly, in practical applications, the bridging molecule 240 can be replaced with any other of the 12 types of bridging molecules mentioned above.
该桥连分子240上连接有新冠病毒COVID-19抗体250。A new coronavirus COVID-19 antibody 250 is connected to the bridging molecule 240.
本实施例中所述COVID-19抗体为：IgM和IgG抗体片段。该IgM和IgG抗体是与IgM和IgG抗原片段对应的抗体片段。The COVID-19 antibody in this example is: IgM and IgG antibody fragments. The IgM and IgG antibodies are antibody fragments corresponding to IgM and IgG antigen fragments.
由图2所示实施例可见，在生物传感器的石墨烯材料上修饰了新冠病毒COVID-19抗体，如果待测样品中含COVID-19抗原，则由COVID-19抗原和 COVID-19抗体之间的特异性作用，待测样品中的COVID-19抗原会与石墨烯材料上的COVID-19抗体结合，从而使输出电极输出反应前和反应后的电流信号，进而能够检测出新冠病毒。It can be seen from the embodiment shown in Figure 2 that the new coronavirus COVID-19 antibody is modified on the graphene material of the biosensor. The specific effect of the COVID-19 antigen in the sample to be tested will be combined with the COVID-19 antibody on the graphene material, so that the output electrode can output the current signal before and after the reaction, and then the new coronavirus can be detected.
用于检测新冠病毒的基于石墨烯的生物传感器实施例三Example 3 of graphene-based biosensor for detecting new coronavirus
如图3所示，图3为本发明实施例提供的基于石墨烯的生物传感器的第三种原理结构示意图；该生物传感器包括：Si/SiO 2基底310、石墨烯材料320、金属电极(输入/输出)330、桥连分子340、核酸探针350以及可选的加热板360。 As shown in FIG. 3, FIG. 3 is a schematic diagram of a third principle structure of a graphene-based biosensor provided by an embodiment of the present invention; the biosensor includes: a Si/SiO 2 substrate 310, a graphene material 320, and a metal electrode (input /Output) 330, bridging molecule 340, nucleic acid probe 350 and optional heating plate 360.
其中，Si/SiO 2基底310是由位于下层的Si材料层和与其紧密结合的SiO 2材料层构成。SiO 2的作用是防止由于底部漏电对信号检测造成的干扰。Si/SiO 2基底310可以是在市场中购买的Si/SiO 2硅片，Si/SiO 2硅片就是一体的，硅Si片上已经铺上了SiO 2层。 Among them, the Si/SiO 2 substrate 310 is composed of a lower Si material layer and an SiO 2 material layer closely combined with the Si material layer. The function of SiO 2 is to prevent the interference of signal detection caused by bottom leakage. The Si/SiO 2 substrate 310 may be Si/SiO 2 silicon wafers purchased in the market, and the Si/SiO 2 silicon wafers are integrated, and the silicon Si wafer has been covered with an SiO 2 layer.
石墨烯材料320位于Si/SiO 2基底310的上层，形成了所述的石墨烯层。 The graphene material 320 is located on the upper layer of the Si/SiO 2 substrate 310 and forms the graphene layer.
石墨烯材料320上镀有金属电极(输入/输出)330。金属电极330包括：输入电极和输出电极，如图3所示一个金属电极(输入)330对应一个金属电极(输出)330，金属电极(输入)330和金属电极(输出)330之间有石墨烯材料320。The graphene material 320 is plated with metal electrodes (input/output) 330. The metal electrode 330 includes an input electrode and an output electrode. As shown in FIG. 3, a metal electrode (input) 330 corresponds to a metal electrode (output) 330, and there is graphene between the metal electrode (input) 330 and the metal electrode (output) 330. Material 320.
如图3所示，石墨烯材料320上非金属电极的部分修饰有桥连分子340。该桥连分子340为：上述桥连分子12。As shown in FIG. 3, part of the non-metal electrode on the graphene material 320 is modified with bridging molecules 340. The bridging molecule 340 is: the bridging molecule 12 described above.
同样的，在实际应用中该桥连分子340可以替换为上述12种桥连分子中的任何其他一种。Similarly, in practical applications, the bridging molecule 340 can be replaced with any other of the 12 types of bridging molecules mentioned above.
该桥连分子340上连接有核酸探针350。核酸探针350用于检测新冠病毒RNA，具体可以是：A nucleic acid probe 350 is connected to the bridging molecule 340. The nucleic acid probe 350 is used to detect the new coronavirus RNA, which can be specifically:
5’-CCGTCTGCGGTATGTGGAAAGGTTATGG-3’，5’-CCGTCTGCGGTATGTGGAAAGGTTATGG-3’,
5’端修饰氨基5'modified amino group
为了保证测试时需要的温度，图3所示的实施例中，还可以在Si/SiO 2基底310的下方设置加热板360。该加热板360可以是电加热板，是生物传感器常用的部件。 In order to ensure the required temperature during the test, in the embodiment shown in FIG. 3, a heating plate 360 may also be provided under the Si/SiO 2 substrate 310. The heating plate 360 may be an electric heating plate, which is a commonly used component of a biosensor.
由图3所示实施例可见，在生物传感器的石墨烯材料上修饰了用于检测新冠病毒RNA的核酸探针，如果待测样品中含COVID-19病毒，则COVID-19病毒会与石墨烯材料上的核酸探针结合，从而使输出电极输出反应前和反应后的电流信号，进而能够检测出新冠病毒。It can be seen from the embodiment shown in Figure 3 that the graphene material of the biosensor is modified with a nucleic acid probe for detecting the RNA of the new coronavirus. If the sample to be tested contains the COVID-19 virus, the COVID-19 virus will interact with the graphene. The nucleic acid probes on the material are combined, so that the output electrode can output the current signal before and after the reaction, and then the new coronavirus can be detected.
二、用于检测流感病毒的基于石墨烯的生物传感器。2. Graphene-based biosensor for detecting influenza virus.
具体的，用于检测流感病毒的基于石墨烯的生物传感器也有三种实现方式，以下分别举实施例进行说明：Specifically, the graphene-based biosensor for detecting influenza virus also has three implementation methods, which are described in the following embodiments:
用于检测流感病毒的基于石墨烯的生物传感器实施例一Example 1 of graphene-based biosensor for detecting influenza virus
与图1a所示用于检测新冠病毒的基于石墨烯的生物传感器类似，该生物传感器包括：Si/SiO 2基底、石墨烯材料、金属电极(输入/输出)、桥连分子和流感病毒抗原。 Similar to the graphene-based biosensor used to detect the new coronavirus shown in Figure 1a, the biosensor includes: Si/SiO 2 substrate, graphene material, metal electrodes (input/output), bridging molecules and influenza virus antigens.
本实施例中的桥连分子是上述12种桥连分子中的桥连分子5。The bridging molecule in this embodiment is the bridging molecule 5 among the 12 types of bridging molecules mentioned above.
在其他实施例中，桥连分子可以是上述12中桥连分子中任一个其他的桥连分子。In other embodiments, the bridging molecule may be any of the other bridging molecules in 12 above.
可以参考图1a所示，本实施例与图1a所示实施例的区别在于：桥连分子140上连接的是流感病毒抗原，而不是新冠病毒抗原。As shown in Fig. 1a, the difference between this embodiment and the embodiment shown in Fig. 1a is that the bridge molecule 140 is connected to influenza virus antigens instead of new coronavirus antigens.
具体的，流感病毒抗原序列为：Specifically, the influenza virus antigen sequence is:
Figure PCTCN2021080531-appb-000005
Figure PCTCN2021080531-appb-000005
用于检测流感病毒的基于石墨烯的生物传感器实施例二Example 2 of graphene-based biosensor for detecting influenza virus
与图2所示用于检测新冠病毒的基于石墨烯的生物传感器类似，该生物传感器包括：Si/SiO 2基底、石墨烯材料、金属电极(输入/输出)、桥连分子和流感病毒抗体。 Similar to the graphene-based biosensor used to detect the new coronavirus shown in Figure 2, the biosensor includes: Si/SiO 2 substrate, graphene material, metal electrodes (input/output), bridging molecules, and influenza virus antibodies.
本实施例中的桥连分子是上述12种桥连分子中的桥连分子3。The bridging molecule in this example is bridging molecule 3 among the 12 types of bridging molecules mentioned above.
在其他实施例中，桥连分子可以是上述12中桥连分子中任一个其他的桥 连分子。In other embodiments, the bridging molecule may be any of the other bridging molecules in 12 above.
可以参考图2所示，本实施例与图2所示实施例的区别在于：桥连分子240上连接的是流感病毒抗体，而不是新冠病毒抗体。As shown in FIG. 2, the difference between this embodiment and the embodiment shown in FIG. 2 is that the bridge molecule 240 is connected to influenza virus antibodies instead of new coronavirus antibodies.
本实施例中流感病毒抗体，是与上述流感病毒抗原对应的抗体。The influenza virus antibodies in this example are antibodies corresponding to the aforementioned influenza virus antigens.
用于检测流感病毒的基于石墨烯的生物传感器实施例三Example 3 of graphene-based biosensor for detecting influenza virus
与图3所示用于检测新冠病毒的基于石墨烯的生物传感器类似，该生物传感器包括：Si/SiO 2基底、石墨烯材料、金属电极(输入/输出)、桥连分子、用于检测流感病毒的探针和加热板。 Similar to the graphene-based biosensor used to detect the new coronavirus shown in Figure 3, the biosensor includes: Si/SiO 2 substrate, graphene material, metal electrodes (input/output), bridging molecules, used to detect influenza Virus probe and heating plate.
本实施例中，用于检测流感病毒的探针，是针对HA基因检测甲型Hmi流感病毒(2009变异株)的核苷酸序列，5’-[FAM]-CAT TTC TTT CCA TT GCG-[TAMRA或BHQ1]-3’，5’端修饰氨基。In this example, the probe used to detect influenza virus is to detect the nucleotide sequence of the Hmi influenza virus (2009 mutant) against the HA gene, 5'-[FAM]-CAT TTC TTT CCA TT GCG-[ TAMRA or BHQ1]-3', 5'end modified amino group.
该序列为检测甲型Hmi流感病毒(2009变异株)HA基因的LNA(Locked Nucleic Acid)修饰的荧光短探针，即第4、7、10、13、15位的碱基用LNA进行修饰，探针的5’端标记报告荧光基团FAM(6-Carboxyfluorescein，6-羧基荧光素)，3’端标记淬灭基团TAMRA(5-羧基四甲基罗丹明)或BHQ1(黑洞淬灭基团，或称BQHI)。This sequence is a fluorescent short probe modified with LNA (Locked Nucleic Acid) to detect the HA gene of Hmi influenza virus (2009 mutant), that is, the bases at positions 4, 7, 10, 13, and 15 are modified with LNA. The 5'end of the probe is labeled with the reporter fluorophore FAM (6-Carboxyfluorescein, 6-carboxyfluorescein), and the 3'end is labeled with the quenching group TAMRA (5-carboxytetramethylrhodamine) or BHQ1 (black hole quenching group) Group, or BQHI).
本实施例中的桥连分子是上述12种桥连分子中的桥连分子3。The bridging molecule in this example is bridging molecule 3 among the 12 types of bridging molecules mentioned above.
在其他实施例中，桥连分子可以是上述12中桥连分子中任一个其他的桥连分子。In other embodiments, the bridging molecule may be any of the other bridging molecules in 12 above.
可以参考图3所示，本实施例与图3所示实施例的区别在于：桥连分子340上连接的是用于检测流感病毒的探针，而不是用于检测新冠病毒的探针。As shown in FIG. 3, the difference between this embodiment and the embodiment shown in FIG. 3 is that the bridging molecule 340 is connected to a probe for detecting influenza virus instead of a probe for detecting new coronavirus.
由上述的实施例可见，本发明实施例提供的生物传感器还可以用于检测流感病毒。It can be seen from the foregoing embodiment that the biosensor provided in the embodiment of the present invention can also be used to detect influenza virus.
其次，对本发明实施例提供的基于硅纳米线的生物传感器进行详细说明。Next, the silicon nanowire-based biosensor provided by the embodiment of the present invention will be described in detail.
本发明实施例提供的基于硅纳米线的生物传感器，包括：基底层；The silicon nanowire-based biosensor provided by the embodiment of the present invention includes: a base layer;
所述基底层上镀有金属电极；所述金属电极包括：输入电极和输出电极，且一个输入电极对应一个输出电极；每个输入电极与其对应的输出电极之间由硅纳米线连接；所述硅纳米线上修饰有用于连接生物大分子的桥连分子；所述桥连分子为Ni 2+探针或马来酰亚胺；所述桥连分子上上连接的生物大分子为：病毒抗原或病毒抗体或核酸探针。 The base layer is plated with metal electrodes; the metal electrodes include: input electrodes and output electrodes, and one input electrode corresponds to one output electrode; each input electrode and its corresponding output electrode are connected by silicon nanowires; The silicon nanowire is modified with bridging molecules for connecting biological macromolecules; the bridging molecules are Ni 2+ probes or maleimide; the biological macromolecules connected to the bridging molecules are virus antigens Or virus antibodies or nucleic acid probes.
以下以用于检测新冠病毒和流感病毒的基于硅纳米线的生物传感器为例， 对本发明实施例提供的基于硅纳米线的生物传感器进行详细说明。The following uses a silicon nanowire-based biosensor for detecting new coronaviruses and influenza viruses as an example to describe in detail the silicon nanowire-based biosensor provided in the embodiments of the present invention.
一、用于检测新冠病毒的基于硅纳米线的生物传感器。1. A silicon nanowire-based biosensor for the detection of new coronaviruses.
具体的，用于检测新冠病毒的基于硅纳米线的生物传感器有三种实现方式，以下分别举实施例进行说明：Specifically, the silicon nanowire-based biosensor for detecting the new coronavirus has three implementation methods, which are described in the following examples:
用于检测新冠病毒的基于硅纳米线的生物传感器实施例一Example 1 of a silicon nanowire-based biosensor for detecting new coronavirus
如图4所示，图4为本发明实施例提供的基于硅纳米线的生物传感器的一种原理结构示意图；该生物传感器包括：Si/SiO 2基底410、硅纳米线材料420、金属电极(输入/输出)430、桥连分子440和COVID-19抗原450。 As shown in FIG. 4, FIG. 4 is a schematic diagram of a principle structure of a silicon nanowire-based biosensor provided by an embodiment of the present invention; the biosensor includes: a Si/SiO 2 substrate 410, a silicon nanowire material 420, and a metal electrode ( Input/output) 430, bridging molecule 440, and COVID-19 antigen 450.
其中，Si/SiO 2基底410是由位于下层的硅Si材料层和与其紧密结合的二氧化硅SiO 2材料层构成。SiO 2的作用是防止由于底部漏电对信号检测造成的干扰。Si/SiO 2基底110可以是在市场中购买的Si/SiO 2硅片，Si/SiO 2硅片就是一体的，硅Si片上已经铺上了SiO 2层。 Among them, the Si/SiO 2 substrate 410 is composed of a silicon Si material layer located in the lower layer and a silicon dioxide SiO 2 material layer closely combined with the silicon Si material layer. The function of SiO 2 is to prevent the interference of signal detection caused by bottom leakage. The Si/SiO 2 substrate 110 may be Si/SiO 2 silicon wafers purchased in the market, and the Si/SiO 2 silicon wafers are integrated, and the silicon Si wafer has been covered with an SiO 2 layer.
硅纳米线材料420位于Si/SiO 2基底410的上层。 The silicon nanowire material 420 is located on the upper layer of the Si/SiO 2 substrate 410.
硅纳米线材料420上镀有金属电极(输入/输出)430。金属电极430包括：输入电极和输出电极，如图4所示一个金属电极(输入)430对应一个金属电极(输出)430，金属电极(输入)430和金属电极(输出)430之间有硅纳米线材料420。The silicon nanowire material 420 is plated with metal electrodes (input/output) 430. The metal electrode 430 includes an input electrode and an output electrode. As shown in FIG. 4, a metal electrode (input) 430 corresponds to a metal electrode (output) 430. There is silicon nanometer between the metal electrode (input) 430 and the metal electrode (output) 430.线材料420。 Line material 420.
如图4所示，硅纳米线材料420上修饰有桥连分子440。该桥连分子440为Ni 2+探针： As shown in FIG. 4, the silicon nanowire material 420 is modified with bridging molecules 440. The bridging molecule 440 is a Ni 2+ probe:
Figure PCTCN2021080531-appb-000006
Figure PCTCN2021080531-appb-000006
在其他实施例中，桥连分子440可以是马来酰亚胺：In other embodiments, the bridging molecule 440 may be maleimide:
Figure PCTCN2021080531-appb-000007
Figure PCTCN2021080531-appb-000007
该桥连分子440上连接有新冠病毒COVID-19抗原450。The bridging molecule 440 is connected to the new coronavirus COVID-19 antigen 450.
所述COVID-19抗原为：IgM和IgG抗原片段；The COVID-19 antigen is: IgM and IgG antigen fragments;
所述IgM和IgG抗原片段是由S蛋白和N蛋白1：1混合成的；The IgM and IgG antigen fragments are made of a 1:1 mixture of S protein and N protein;
其中，S蛋白为：S1-RBD，其氨基酸排列顺序为：Among them, S protein is: S1-RBD, and its amino acid sequence is:
Figure PCTCN2021080531-appb-000008
Figure PCTCN2021080531-appb-000008
N蛋白的氨基酸排列顺序为：The amino acid sequence of N protein is:
Figure PCTCN2021080531-appb-000009
Figure PCTCN2021080531-appb-000009
由图4所示实施例可见，在生物传感器的硅纳米线材料上修饰了新冠病毒COVID-19抗原，如果待测样品中含COVID-19抗体，则由COVID-19抗原和COVID-19抗体之间的特异性作用，待测样品中的COVID-19抗体会与硅纳米 线材料420上的COVID-19抗原结合，从而使输出电极输出反应前和反应后电流信号，进而能够检测出新冠病毒。It can be seen from the embodiment shown in Figure 4 that the new coronavirus COVID-19 antigen is modified on the silicon nanowire material of the biosensor. Due to the specific effect between the samples, the COVID-19 antibody in the sample to be tested will bind to the COVID-19 antigen on the silicon nanowire material 420, so that the output electrode can output the pre-reaction and post-reaction current signals, thereby being able to detect the new coronavirus.
用于检测新冠病毒的基于硅纳米线的生物传感器实施例二Embodiment 2 of a silicon nanowire-based biosensor for detecting new coronavirus
如图5所示，图5为本发明实施例提供的基于硅纳米线的生物传感器的第二种原理结构示意图；该生物传感器包括：Si/SiO 2基底510、硅纳米线材料520、金属电极(输入/输出)530、桥连分子550和COVID-19抗体550。 As shown in FIG. 5, FIG. 5 is a schematic diagram of the second principle structure of a silicon nanowire-based biosensor provided by an embodiment of the present invention; the biosensor includes: a Si/SiO 2 substrate 510, a silicon nanowire material 520, and a metal electrode (Input/output) 530, bridging molecule 550 and COVID-19 antibody 550.
其中，Si/SiO 2基底510是由位于下层的硅Si材料层和与其紧密结合的二氧化硅SiO 2材料层构成。SiO 2的作用是防止由于底部漏电对信号检测造成的干扰。Si/SiO 2基底110可以是在市场中购买的Si/SiO 2硅片，Si/SiO 2硅片就是一体的，硅Si片上已经铺上了SiO 2层。 Among them, the Si/SiO 2 substrate 510 is composed of a silicon Si material layer located in the lower layer and a silicon dioxide SiO 2 material layer closely combined with it. The function of SiO 2 is to prevent the interference of signal detection caused by bottom leakage. The Si/SiO 2 substrate 110 may be Si/SiO 2 silicon wafers purchased in the market, and the Si/SiO 2 silicon wafers are integrated, and the silicon Si wafer has been covered with an SiO 2 layer.
硅纳米线材料520位于Si/SiO 2基底510的上层。 The silicon nanowire material 520 is located on the upper layer of the Si/SiO 2 substrate 510.
硅纳米线材料520上镀有金属电极(输入/输出)530。金属电极530包括：输入电极和输出电极，如图5所示一个金属电极(输入)530对应一个金属电极(输出)530，金属电极(输入)530和金属电极(输出)530之间有硅纳米线材料520。The silicon nanowire material 520 is plated with metal electrodes (input/output) 530. The metal electrode 530 includes an input electrode and an output electrode. As shown in FIG. 5, a metal electrode (input) 530 corresponds to a metal electrode (output) 530. There is silicon nanometer between the metal electrode (input) 530 and the metal electrode (output) 530.线材料520。 Line material 520.
如图5所示，硅纳米线材料520上修饰有桥连分子540。该桥连分子540为：马来酰亚胺。As shown in FIG. 5, the silicon nanowire material 520 is modified with bridging molecules 540. The bridging molecule 540 is maleimide.
该桥连分子540上连接有新冠病毒COVID-19抗体550。A new coronavirus COVID-19 antibody 550 is connected to the bridging molecule 540.
本实施例中所述COVID-19抗体为：IgM和IgG抗体片段。该IgM和IgG抗体是与IgM和IgG抗原片段对应的抗体片段。The COVID-19 antibody in this example is: IgM and IgG antibody fragments. The IgM and IgG antibodies are antibody fragments corresponding to IgM and IgG antigen fragments.
由图5所示实施例可见，在生物传感器的硅纳米线材料上修饰了新冠病毒COVID-19抗体，如果待测样品中含COVID-19抗原，则由COVID-19抗原和COVID-19抗体之间的特异性作用，待测样品中的COVID-19抗原会与硅纳米线材料520上的COVID-19抗体结合，从而使输出电极输出反应前和反应后电流信号，进而能够检测出新冠病毒。It can be seen from the embodiment shown in Figure 5 that the new coronavirus COVID-19 antibody is modified on the silicon nanowire material of the biosensor. Due to the specific effect between the samples, the COVID-19 antigen in the sample to be tested will bind to the COVID-19 antibody on the silicon nanowire material 520, so that the output electrode can output the pre-reaction and post-reaction current signals, thereby being able to detect the new coronavirus.
用于检测新冠病毒的基于硅纳米线的生物传感器实施例三Embodiment 3 of a silicon nanowire-based biosensor for detecting new coronavirus
如图6所示，图6为本发明实施例提供的基于硅纳米线的生物传感器的第三种原理结构示意图；该生物传感器包括：Si/SiO 2基底610、硅纳米线材料620、金属电极(输入/输出)630、桥连分子640、核酸探针650以及可选的加热板660。 As shown in FIG. 6, FIG. 6 is a schematic diagram of the third principle structure of a silicon nanowire-based biosensor provided by an embodiment of the present invention; the biosensor includes: a Si/SiO 2 substrate 610, a silicon nanowire material 620, and a metal electrode (Input/Output) 630, bridging molecule 640, nucleic acid probe 650, and optional heating plate 660.
其中，Si/SiO 2基底610是由位于下层的Si材料层和与其紧密结合的SiO 2材料层构成。SiO 2的作用是防止由于底部漏电对信号检测造成的干扰。Si/SiO 2 基底610可以是在市场中购买的Si/SiO 2硅片，Si/SiO 2硅片就是一体的，硅Si片上已经铺上了SiO 2层。 Among them, the Si/SiO 2 substrate 610 is composed of a lower Si material layer and an SiO 2 material layer closely combined with the Si material layer. The function of SiO 2 is to prevent the interference of signal detection caused by bottom leakage. The Si/SiO 2 substrate 610 may be Si/SiO 2 silicon wafers purchased in the market, and the Si/SiO 2 silicon wafers are integrated, and the silicon Si wafer has been covered with an SiO 2 layer.
硅纳米线材料620位于Si/SiO 2基底610的上层。 The silicon nanowire material 620 is located on the upper layer of the Si/SiO 2 substrate 610.
硅纳米线材料620上镀有金属电极(输入/输出)630。金属电极630包括：输入电极和输出电极，如图6所示一个金属电极(输入)630对应一个金属电极(输出)630，金属电极(输入)630和金属电极(输出)630之间有硅纳米线材料620。The silicon nanowire material 620 is plated with metal electrodes (input/output) 630. The metal electrode 630 includes an input electrode and an output electrode. As shown in FIG. 6, a metal electrode (input) 630 corresponds to a metal electrode (output) 630. There is silicon nanometer between the metal electrode (input) 630 and the metal electrode (output) 630.线材料620。 Line material 620.
如图6所示，硅纳米线材料620上修饰有桥连分子640。该桥连分子640为：As shown in FIG. 6, the silicon nanowire material 620 is modified with bridging molecules 640. The bridging molecule 640 is:
Figure PCTCN2021080531-appb-000010
Figure PCTCN2021080531-appb-000010
该桥连分子640上连接有核酸探针650。核酸探针650用于检测新冠病毒RNA，具体可以是：A nucleic acid probe 650 is connected to the bridging molecule 640. The nucleic acid probe 650 is used to detect the new coronavirus RNA, which can be specifically:
ORF1ab基因合成探针：ORF1ab gene synthesis probe:
5’-CCGTCTGCGGTATGTGGAAAGGTTATGG-3’，5’端修饰氨基。5'-CCGTCTGCGGTATGTGGAAAGGTTATGG-3', 5'end is modified with amino group.
为了保证测试时需要的温度，图6所示的实施例中，还可以在Si/SiO 2基底610的下方设置加热板660。该加热板660可以是电加热板，是生物传感器常用的部件。 In order to ensure the required temperature during the test, in the embodiment shown in FIG. 6, a heating plate 660 may also be provided under the Si/SiO 2 substrate 610. The heating plate 660 may be an electric heating plate, which is a commonly used component of a biosensor.
由图6所示实施例可见，在生物传感器的硅纳米线材料上修饰了用于检测新冠病毒RNA的核酸探针，如果待测样品中含COVID-19病毒，则COVID-19病毒会与硅纳米线材料上的核酸探针结合，从而使输出电极输出反应前和反应后的电流信号，进而能够检测出新冠病毒。It can be seen from the embodiment shown in Fig. 6 that the silicon nanowire material of the biosensor is modified with a nucleic acid probe for detecting the RNA of the new coronavirus. If the sample to be tested contains the COVID-19 virus, the COVID-19 virus will interact with the silicon. The nucleic acid probe on the nanowire material is combined, so that the output electrode can output the current signal before and after the reaction, and then the new coronavirus can be detected.
二、用于检测流感病毒的基于硅纳米线的生物传感器。2. Biosensors based on silicon nanowires for detecting influenza viruses.
具体的，用于检测流感病毒的基于硅纳米线的生物传感器也有三种实现方式，以下分别举实施例进行说明：Specifically, the silicon nanowire-based biosensor for detecting influenza virus also has three implementation methods, which are described in the following embodiments:
用于检测流感病毒的基于硅纳米线的生物传感器实施例一The first embodiment of a silicon nanowire-based biosensor for detecting influenza virus
与图4所示用于检测新冠病毒的基于硅纳米线的生物传感器类似，该生物 传感器包括：Si/SiO 2基底、硅纳米线材料、金属电极(输入/输出)、桥连分子和流感病毒抗原。 Similar to the silicon nanowire-based biosensor used to detect the new coronavirus shown in Figure 4, the biosensor includes: Si/SiO 2 substrate, silicon nanowire material, metal electrodes (input/output), bridging molecules, and influenza virus antigen.
本实施例中的桥连分子是Ni 2+探针。在其他实施例中，桥连分子可以是马来酰亚胺。 The bridging molecule in this example is a Ni 2+ probe. In other embodiments, the bridging molecule may be maleimide.
可以参考图4所示，本实施例与图4所示实施例的区别在于：桥连分子440上连接的是流感病毒抗原，而不是新冠病毒抗原。As shown in FIG. 4, the difference between this embodiment and the embodiment shown in FIG. 4 is that the bridging molecule 440 is connected to the influenza virus antigen instead of the new coronavirus antigen.
具体的，流感病毒抗原序列为：Specifically, the influenza virus antigen sequence is:
Figure PCTCN2021080531-appb-000011
Figure PCTCN2021080531-appb-000011
用于检测流感病毒的基于硅纳米线的生物传感器实施例二Second embodiment of silicon nanowire-based biosensor for detecting influenza virus
与图5所示用于检测新冠病毒的基于硅纳米线的生物传感器类似，该生物传感器包括：Si/SiO 2基底、石墨烯材料、金属电极(输入/输出)、桥连分子和流感病毒抗体。 Similar to the silicon nanowire-based biosensor used to detect the new coronavirus shown in Figure 5, this biosensor includes: Si/SiO 2 substrate, graphene material, metal electrodes (input/output), bridging molecules, and influenza virus antibodies .
本实施例中的桥连分子是马来酰亚胺。The bridging molecule in this example is maleimide.
在其他实施例中，桥连分子可以是Ni 2+。 In other embodiments, the bridging molecule may be Ni 2+ .
可以参考图5所示，本实施例与图2所示实施例的区别在于：桥连分子540上连接的是流感病毒抗体，而不是新冠病毒抗体。As shown in FIG. 5, the difference between this embodiment and the embodiment shown in FIG. 2 is that the bridge molecule 540 is connected to influenza virus antibodies instead of new coronavirus antibodies.
本实施例中流感病毒抗体，是与上述流感病毒抗原对应的抗体。The influenza virus antibodies in this example are antibodies corresponding to the aforementioned influenza virus antigens.
用于检测流感病毒的基于硅纳米线的生物传感器实施例三Embodiment 3 of a silicon nanowire-based biosensor for detecting influenza virus
与图6所示用于检测新冠病毒的基于硅纳米线的生物传感器类似，该生物传感器包括：Si/SiO 2基底、石墨烯材料、金属电极(输入/输出)、桥连分子、 用于检测流感病毒的探针和加热板。 Similar to the silicon nanowire-based biosensor used to detect the new coronavirus shown in Figure 6, the biosensor includes: Si/SiO 2 substrate, graphene material, metal electrodes (input/output), bridging molecules, and detection Probe and heating plate for influenza virus.
本实施例中，用于检测流感病毒的探针，是针对HA基因检测甲型Hmi流感病毒(2009变异株)的核苷酸序列，5’-[FAM]-CAT TTC TTT CCA TT GCG-[TAMRA或BHQ1]-3’，5’端修饰氨基。In this example, the probe used to detect influenza virus is to detect the nucleotide sequence of the Hmi influenza virus (2009 mutant) against the HA gene, 5'-[FAM]-CAT TTC TTT CCA TT GCG-[ TAMRA or BHQ1]-3', 5'end modified amino group.
该序列为检测甲型Hmi流感病毒(2009变异株)HA基因的LNA修饰的荧光短探针，即第4、7、10、13、15位的碱基用LNA进行修饰，探针的5’端标记报告荧光基团FAM(6-羧基荧光素)，3’端标记淬灭基团TAMRA或BHQ1。This sequence is a short fluorescent probe for detecting the LNA modification of the HA gene of the Hmi influenza virus (2009 mutant). The end is labeled with the reporter fluorophore FAM (6-carboxyfluorescein), and the 3'end is labeled with the quencher TAMRA or BHQ1.
本实施例中的桥连分子是Ni 2+探针。在其他实施例中，桥连分子可以是马来酰亚胺。 The bridging molecule in this example is a Ni 2+ probe. In other embodiments, the bridging molecule may be maleimide.
可以参考图6所示，本实施例与图6所示实施例的区别在于：桥连分子640上连接的是用于检测流感病毒的探针，而不是用于检测新冠病毒的探针。As shown in FIG. 6, the difference between this embodiment and the embodiment shown in FIG. 6 is that a probe for detecting influenza virus is connected to the bridging molecule 640 instead of a probe for detecting new coronavirus.
由上述的实施例可见，本发明实施例提供的生物传感器还可以用于检测流感病毒。It can be seen from the foregoing embodiment that the biosensor provided in the embodiment of the present invention can also be used to detect influenza virus.
本发明实施例还提供了基于石墨烯和硅纳米线的生物传感器的制备方法，以下分别进行说明。The embodiment of the present invention also provides a method for preparing a biosensor based on graphene and silicon nanowires, which will be described separately below.
参见图7a，基于石墨烯的生物传感器的制备方法，包括如下步骤：Referring to Fig. 7a, the preparation method of graphene-based biosensor includes the following steps:
A、将在金属表面生成的单层石墨烯转移到基底材料表面上，形成基底层和石墨烯层；A. Transfer the single-layer graphene generated on the metal surface to the surface of the base material to form the base layer and the graphene layer;
B、在所述石墨烯层上旋涂光刻胶后，通过光刻技术刻蚀出金属电极的形状；B. After spin-coating photoresist on the graphene layer, the shape of the metal electrode is etched by photolithography technology;
C、在石墨烯层上金属电极的位置蒸镀上金属电极，形成待修饰器件；所述金属电极包括：输入电极和输出电极，且一个输入电极对应一个输出电极；C. A metal electrode is vapor-deposited on the position of the metal electrode on the graphene layer to form a device to be modified; the metal electrode includes: an input electrode and an output electrode, and one input electrode corresponds to one output electrode;
D、在待修饰器件的石墨烯层上非金属电极的部分修饰上桥连分子；D. Modification of bridging molecules on part of the non-metal electrode on the graphene layer of the device to be modified;
E、在所述桥连分子上连接病毒抗原或病毒抗体或核酸探针。E. Connect viral antigens or viral antibodies or nucleic acid probes to the bridging molecule.
其中，所述病毒抗原可以为新冠病毒COVID-19抗原或流感病毒抗原，所述病毒抗体可以为COVID-19抗体或流感抗体，所述核酸探针可以为用于检测COVID-19的核酸探针或用于检测流感的核酸探针。Wherein, the virus antigen may be a novel coronavirus COVID-19 antigen or an influenza virus antigen, the virus antibody may be a COVID-19 antibody or an influenza antibody, and the nucleic acid probe may be a nucleic acid probe for detecting COVID-19 Or a nucleic acid probe used to detect influenza.
基于石墨烯的生物传感器的制备方法实施例：Examples of preparation methods of graphene-based biosensors:
本实施例中，制备的生物传感器是石墨烯场效应晶体管，一种具体的制备过程如图7b所示，包括如下步骤：In this embodiment, the prepared biosensor is a graphene field effect transistor. A specific preparation process is shown in Fig. 7b, including the following steps:
1、通过化学气相沉积法(CVD)在铜箔表面生长单层石墨烯；1. Grow a single layer of graphene on the surface of copper foil by chemical vapor deposition (CVD);
2、利用聚甲基丙烯酸甲酯(PMMA)将铜箔上的石墨烯通过湿法转移到硅片(尺寸1.5×1.5cm，300nm二氧化硅)表面；2. Use polymethyl methacrylate (PMMA) to transfer the graphene on the copper foil to the surface of the silicon wafer (size 1.5×1.5cm, 300nm silicon dioxide) by wet method;
3、在所述石墨烯层上旋涂光刻胶后，通过紫外光刻技术刻蚀出金属电极的形状，再通过热蒸镀沉积上铬(8nm)和金(60nm)；3. After spin-coating photoresist on the graphene layer, the shape of the metal electrode is etched by ultraviolet lithography technology, and then chromium (8nm) and gold (60nm) are deposited by thermal evaporation;
4、通过紫外光刻技术和氧等离子体刻蚀(RIE)将石墨烯刻蚀成25×2200μm的条带；4. The graphene is etched into 25×2200μm strips by ultraviolet lithography and oxygen plasma etching (RIE);
5、利用紫外光刻和热蒸镀，制作石墨烯外接电极(铬(8nm)和金(80nm))，最后通过电子束蒸镀上电极保护层二氧化硅(40nm)。5. Using ultraviolet lithography and thermal evaporation, the graphene external electrodes (chromium (8nm) and gold (80nm)) are made, and finally the upper electrode protective layer silicon dioxide (40nm) is evaporated by electron beam.
具体的金属电极的结构，如图7c所示，图7c为基于图7a所示方法制备的生物传感器的金属电极的示例图。The specific structure of the metal electrode is shown in FIG. 7c, which is an example diagram of the metal electrode of the biosensor prepared based on the method shown in FIG. 7a.
6、对石墨烯表面进行修饰，具体的，包括：6. Modify the graphene surface, specifically, including:
61、将石墨烯晶体管浸泡在1mM的1-芘丁酸N-羟基琥珀酰亚胺酯的乙腈溶液12小时，通过π-πstacking相互作用将桥连分子修饰到石墨烯上，然后乙腈润洗，氮气小心吹干备用；61. Soak the graphene transistor in 1 mM 1-pyrene butyrate N-hydroxysuccinimide ester in acetonitrile solution for 12 hours, modify the bridging molecules to graphene through π-πstacking interaction, and then rinse with acetonitrile. Dry the nitrogen carefully for later use;
62、把步骤5制备的器件浸润在1mM的COVID-19抗原或核酸探针缓冲液中，反应12小时，然后缓冲液润洗，氮气吹干。62. Immerse the device prepared in step 5 in 1 mM COVID-19 antigen or nucleic acid probe buffer, react for 12 hours, then rinse with the buffer and blow dry with nitrogen.
另外需要说明的是，图7b中仅是以桥连分子是1-芘丁酸N-羟基琥珀酰亚胺酯为例，实际应用中桥连分子可以是上述12种桥连分子的任一种。同时，也可以在其他金属的表面生长单层石墨烯，各种尺寸也可以根据实际情况进行调整。In addition, it should be noted that in Fig. 7b, the bridging molecule is 1-pyrenebutyrate N-hydroxysuccinimide ester as an example. In practical applications, the bridging molecule can be any of the above-mentioned 12 kinds of bridging molecules. . At the same time, single-layer graphene can also be grown on the surface of other metals, and various sizes can also be adjusted according to actual conditions.
可见，本发明实施例提供的基于石墨烯的生物传感器，是通过“自下而上”的器件制备工艺制备的，该生物传感器能够检测多种病毒，如：新冠病毒或流感病毒。It can be seen that the graphene-based biosensor provided by the embodiments of the present invention is prepared through a “bottom-up” device manufacturing process, and the biosensor can detect multiple viruses, such as new coronavirus or influenza virus.
参见图8a，基于硅纳米线的生物传感器的制备方法，包括如下步骤：Referring to Fig. 8a, the preparation method of a silicon nanowire-based biosensor includes the following steps:
a、在基底上生成的硅纳米线并表面功能化；a. The silicon nanowires generated on the substrate are functionalized on the surface;
b、在所述基底上旋涂光刻胶后，通过光刻技术刻蚀出金属电极的形状；b. After spin-coating photoresist on the substrate, the shape of the metal electrode is etched by photolithography technology;
c、在基底上蒸镀上金属电极，形成待修饰器件；所述金属电极包括：输入电极和输出电极，一个输入电极对应一个输出电极，每个输入电极与其对应的输出电极之间由一硅纳米线连接；c. A metal electrode is vapor-deposited on the substrate to form the device to be modified; the metal electrode includes: an input electrode and an output electrode, one input electrode corresponds to one output electrode, and each input electrode and its corresponding output electrode are separated by a silicon Nanowire connection;
d、在待修饰器件的硅纳米线上修饰上桥连分子；d. Modify bridging molecules on the silicon nanowire of the device to be modified;
e、在所述桥连分子上连接病毒抗原或病毒抗体或核酸探针。e. Connect viral antigens or viral antibodies or nucleic acid probes to the bridging molecule.
其中，所述病毒抗原可以为新冠病毒COVID-19抗原或流感病毒抗原，所述病毒抗体可以为COVID-19抗体或流感抗体，所述核酸探针可以为用于检测COVID-19的核酸探针或用于检测流感的核酸探针。Wherein, the virus antigen may be a novel coronavirus COVID-19 antigen or an influenza virus antigen, the virus antibody may be a COVID-19 antibody or an influenza antibody, and the nucleic acid probe may be a nucleic acid probe for detecting COVID-19 Or a nucleic acid probe used to detect influenza.
基于硅纳米线的生物传感器的制备方法实施例：Examples of methods for preparing biosensors based on silicon nanowires:
本实施例制备的生物传感器是硅纳米线场效应晶体管，一种具体的制备过程如图8b所示，包括如下步骤：The biosensor prepared in this embodiment is a silicon nanowire field-effect transistor. A specific preparation process is shown in FIG. 8b and includes the following steps:
1、在硅片基底上，用化学气相沉积法(CVD)生长硅纳米线并表面功能化。1. On the silicon wafer substrate, use chemical vapor deposition (CVD) to grow silicon nanowires and functionalize the surface.
将组装上金纳米粒子(20nm，Ted Pella)催化剂的硅片基底置于钨舟上，放入CVD生长***的石英管内，管内抽空气至0.5Pa以下，通入7.5sccm的H 2气作为载气，加热至465摄氏度，稳定40分钟，生长出硅纳米线。 Place the silicon wafer substrate assembled with gold nanoparticle (20nm, Ted Pella) catalyst on a tungsten boat, put it into the quartz tube of the CVD growth system, evacuate the inside of the tube to below 0.5Pa, and introduce 7.5sccm of H 2 gas as the carrier Heat it to 465 degrees Celsius and stabilize for 40 minutes to grow silicon nanowires.
2、将氨丙基三乙氧基硅烷(APTES)通过气相表面功能化将硅纳米线表面氨基化；2. Aminopropyltriethoxysilane (APTES) was used to aminate the surface of silicon nanowires through gas phase surface functionalization;
3、将硅纳米线通过机械滑移到Si/SiO 2基底上； 3. Sliding the silicon nanowire onto the Si/SiO 2 substrate mechanically;
4、在所述基底上旋涂光刻胶后，通过光刻技术刻蚀出金属电极的形状；并在基底上蒸镀上金属电极，形成待修饰器件；所述金属电极包括：输入电极和输出电极，一个输入电极对应一个输出电极，每个输入电极与其对应的输出电极之间由一硅纳米线连接；4. After spin-coating photoresist on the substrate, the shape of the metal electrode is etched by photolithography technology; and the metal electrode is vapor-deposited on the substrate to form the device to be modified; the metal electrode includes: an input electrode and Output electrodes, one input electrode corresponds to one output electrode, and each input electrode and its corresponding output electrode are connected by a silicon nanowire;
5、将桥连分子双功能基团对苯基异硫氰酸酯(PDITC)接续到表面氨基化的硅纳米线上；5. Connect the bifunctional group p-phenyl isothiocyanate (PDITC) of the bridging molecule to the surface aminated silicon nanowire;
6、将N-(5-氨基-1-羧基戊基)亚胺基乙酰乙酸(AB-NTA)通过PDITC桥连基团键合到硅纳米线表面；6. Bond the N-(5-amino-1-carboxypentyl) iminoacetoacetic acid (AB-NTA) to the surface of the silicon nanowire through the PDITC bridging group;
7、将Ni 2+螯合到NTA末端形成Ni-NTA分子探针； 7. Chelate Ni 2+ to the end of NTA to form a Ni-NTA molecular probe;
8、在上述表面功能化硅纳米线上将抗原/抗体/核酸探针固定在硅纳米线表面。8. Fix the antigen/antibody/nucleic acid probe on the surface of the silicon nanowire on the surface-functionalized silicon nanowire.
本实施例具体的金属电极和硅纳米的结构，如图8c所示，图8c为基于图8a所示方法制备的生物传感器的金属电极和硅纳米线的一种示例图。The specific metal electrode and silicon nanostructures of this embodiment are shown in FIG. 8c, which is an example diagram of the metal electrode and silicon nanowire of the biosensor prepared based on the method shown in FIG. 8a.
另外，需要说明的是，图8b中仅是以桥连分子是1-芘丁酸N-羟基琥珀酰亚胺酯为例，实际应用中桥连分子还可以是马来酰亚胺。具体的，如图8d所示，在对硅纳米线表面进行氢键算处理后，引入马来酰亚胺，与生物大分子中巯 基进行连接，即进行生物大分子固定。图8d中的生物大分子，可以是病毒抗原或病毒抗体或核酸探针。In addition, it should be noted that in FIG. 8b, the bridging molecule is 1-pyrenebutyrate N-hydroxysuccinimide ester as an example. In practical applications, the bridging molecule may also be maleimide. Specifically, as shown in Figure 8d, after hydrogen bonding is performed on the surface of the silicon nanowire, maleimide is introduced to connect with the sulfhydryl group in the biomacromolecule, that is, the biomacromolecule is immobilized. The biological macromolecules in Figure 8d can be viral antigens or viral antibodies or nucleic acid probes.
本发明实施例还提供了一种病毒检测***，参见图9，图9为本发明实施例提供的病毒检测***的结构示意图，该***包括：生物传感器901、信号发生器902、信号采集器903、信号分析主机904和显示器905；The embodiment of the present invention also provides a virus detection system. Refer to FIG. 9. FIG. 9 is a schematic structural diagram of the virus detection system provided by an embodiment of the present invention. The system includes: a biosensor 901, a signal generator 902, and a signal collector 903 , Signal analysis host 904 and display 905;
本实施例中的生物传感器901可以采用上述图1a～图6所示的任一种生物传感器。该生物传感器901的反应微腔中，有待检测样品溶液。The biosensor 901 in this embodiment can use any of the biosensors shown in Figs. 1a to 6 above. In the reaction microcavity of the biosensor 901, there is a sample solution to be detected.
所述信号发生器902与所述生物传感器901的各个输入电极相连，将触发电信号发送至各个输入电极。The signal generator 902 is connected to each input electrode of the biosensor 901, and sends a trigger electrical signal to each input electrode.
所述信号采集器903与所述生物传感器的各个输出电极相连，采集输出电流信号并发送至信号分析主机904；该输出电流信号为：所述生物传感器901中的病毒抗原或病毒抗体或用于检测病毒的核酸探针，与待检测样品反应前和反应后分别获得的电流信号。The signal collector 903 is connected to each output electrode of the biosensor, collects the output current signal and sends it to the signal analysis host 904; the output current signal is: the virus antigen or the virus antibody in the biosensor 901 or The nucleic acid probe that detects the virus is the current signal obtained before and after the reaction with the sample to be detected.
所述信号分析主机904，对接收到的输出电流信号进行分析检测，确定待测样品中是否存在病毒，将分析检测结果发送至显示器进行显示。The signal analysis host 904 analyzes and detects the received output current signal, determines whether there is a virus in the sample to be tested, and sends the analysis and detection result to the display for display.
由于反应前后电阻发生变化，因此可以分别采集反应前和反应后的电流信号，进行比较，从而根据电流的变化来判断待测样品中是否存在病毒。具体的，可以基于预设的浓度-电流标准曲线，对接收到的反应前和反应后的电流信号进行分析，来判断待测样品中是否存在病毒，将分析检测结果发送至显示器进行显示。Because the resistance changes before and after the reaction, the current signals before and after the reaction can be collected separately and compared, so as to determine whether there is a virus in the sample to be tested according to the change of the current. Specifically, based on a preset concentration-current standard curve, the received current signals before and after the reaction can be analyzed to determine whether there is a virus in the sample to be tested, and the analysis and detection result can be sent to the display for display.
采用本发明实施例提供的病毒的检测***，采用修饰病毒抗原或病毒抗体或用于检测病毒的核酸探针的生物传感器，能够检测出多种病毒，如新冠病毒和流感病毒等。Using the virus detection system provided by the embodiments of the present invention, using a biosensor modified with virus antigens or virus antibodies or nucleic acid probes for virus detection, a variety of viruses, such as new coronaviruses and influenza viruses, can be detected.
本发明实施例还提供了一种病毒的检测方法，应用于图9所示的病毒检测***，包括如下步骤：The embodiment of the present invention also provides a virus detection method, which is applied to the virus detection system shown in FIG. 9 and includes the following steps:
所述信号发生器将触发电信号发送至所述生物传感器的各个输入电极；The signal generator sends a trigger electrical signal to each input electrode of the biosensor;
在有的实施例中，信号发生器可以在接收到信号分析主机发送的开启指令后，向生物传感器发送触发电信号。In some embodiments, the signal generator may send a trigger electrical signal to the biosensor after receiving the start instruction sent by the signal analysis host.
将待测样品输入所述生物传感器；Input the sample to be tested into the biosensor;
所述信号采集器与所述生物传感器的各个输出电极相连，采集输出电流信号并发送至信号分析主机；该输出电流信号为：所述生物传感器中的病毒 抗原或病毒抗体或用于检测病毒的核酸探针，与待检测样品反应前和反应后获得的电流信号；The signal collector is connected to each output electrode of the biosensor, collects the output current signal and sends it to the signal analysis host; the output current signal is: virus antigen or virus antibody in the biosensor or virus detection Nucleic acid probe, the current signal obtained before and after the reaction with the sample to be tested;
所述信号分析主机，对接收到的输出电流信号进行分析检测，确定待测样品中是否存在病毒，将分析检测结果发送至显示器进行显示。The signal analysis host analyzes and detects the received output current signal, determines whether there is a virus in the sample to be tested, and sends the analysis and detection result to the display for display.
下面对本发明实施例实际应用的效果进行说明。The actual application effects of the embodiments of the present invention will be described below.
本发明实施例可以检测多种病毒，以下以对新冠病毒和对流感病毒的检测结果进行说明：The embodiment of the present invention can detect a variety of viruses. The following describes the detection results of the new coronavirus and the influenza virus:
第一、对新冠病毒的检测实验一First, the first test for the detection of the new coronavirus
该实验中，采用的是图1c所示的基于石墨烯的生物传感器(石墨烯场效应晶体管)对待检测样品进行了检测。该生成传感器的桥连分子为：1-芘丁酸N-羟基琥珀酰亚胺酯；桥连分子上连接的是新冠病毒抗原。In this experiment, the graphene-based biosensor (graphene field effect transistor) shown in FIG. 1c was used to detect the sample to be tested. The bridging molecule for generating the sensor is: 1-pyrene butyrate N-hydroxysuccinimide ester; the bridging molecule is connected to the new coronavirus antigen.
具体的，本发明实施例中，新型冠状病毒(COVID-19)的抗原是筛选出的针对COVID-19的特异性抗原片段。是经过数名核酸检测阳性(患者)和阴性(非患者或者密切反应人群)对照的拎出临床测试，优选出最佳IgM和IgG抗原片段。目前的抗原特异性超过95.3％，特异性98％。对于通过蛋白表达***扩增，已经具备产业化的生产能力，可满足疫区临床的检测大量需求。此类免疫方法检测的结果通过纳米技术元件，可大大提高检测的敏感性，缩短检测时长。Specifically, in the embodiments of the present invention, the antigen of the novel coronavirus (COVID-19) is a specific antigen fragment selected against COVID-19. After a number of nucleic acid tests positive (patients) and negative (non-patients or closely responding population) controlled clinical tests, the best IgM and IgG antigen fragments are selected. The current antigen specificity exceeds 95.3%, and the specificity is 98%. For amplification through the protein expression system, it already has industrialized production capacity, which can meet a large number of clinical testing needs in epidemic areas. The results of this type of immune method detection through nanotechnology components can greatly improve the sensitivity of the detection and shorten the detection time.
本发明实施例实际应用的结果如下：The practical application results of the embodiment of the present invention are as follows:
一、检测速度：对于前处理的血清样品，本方法实现了快速检测，检测时间不到1分钟。本实施例中，对血清样品进行前处理，是为了去除样品中非被测物质，提高结果可信度。1. Detection speed: For pre-processed serum samples, this method achieves rapid detection, and the detection time is less than 1 minute. In this embodiment, the pre-processing of the serum sample is to remove non-tested substances in the sample and improve the reliability of the results.
二、实验情况：2. Experimental situation:
实验分组：Experiment grouping:
1.阳性组：采用临床检测中8例胶体金核酸检法测得的阳性患者，其中有IgM或IgG阳性的病例7例；1. Positive group: 8 positive patients detected by the colloidal gold nucleic acid test in clinical testing, including 7 positive cases of IgM or IgG;
2.对照组：8例胶体金核酸检测法均呈阴性，其中4例为正常献血人员，另外4人为新冠病毒临床筛查阴性患者。2. Control group: 8 cases of colloidal gold nucleic acid test were all negative, of which 4 cases were normal blood donors, and the other 4 were patients with negative clinical screening for the new coronavirus.
检测特异性：在16例现场检测中，有8例2019-nCoV阳性患者，8例阴性对照者。与胶体金试纸检测方法比较，该方法检测结果显示：对8例2019-nCoV阳性患者全部检出，阳性准确率100％；在8例阴性对照中有4例确定正常，4 例疑似患者或刚愈出院患者，灵敏性和特异性优于胶体金法，体现了该方法的有效性。具体测试结果和对照如下表：Test specificity: Among the 16 on-site tests, there were 8 2019-nCoV positive patients and 8 negative controls. Compared with the colloidal gold test paper test method, the test results of this method showed that all 8 cases of 2019-nCoV positive patients were detected, and the positive accuracy rate was 100%; among the 8 negative controls, 4 cases were confirmed to be normal, and 4 cases were suspected or just. The sensitivity and specificity of the discharged patients are better than the colloidal gold method, which reflects the effectiveness of the method. The specific test results and comparison are as follows:
编号serial number 胶体金检测结果Colloidal gold test results 石墨烯场效应晶体管结果Graphene field effect transistor results 一致性 consistency
11 阴性feminine 阴性feminine 是Yes
22 阳性positive 阳性positive 是Yes
33 阳性positive 阳性positive 是Yes
44 阳性positive 阳性positive 是Yes
55 阴性feminine 偏高High 优于胶体金法Better than colloidal gold method
66 阳性positive 阳性positive 是Yes
77 阳性positive 阳性positive 是Yes
88 阳性positive 阳性positive 是Yes
99 阳性positive 阳性positive 是Yes
1010 阳性positive 阳性positive 是Yes
1111 阴性feminine 阴性feminine 是Yes
1212 阴性feminine 偏高High 优于胶体金法Better than colloidal gold method
1313 阴性feminine 阴性feminine 是Yes
1414 阴性feminine 阴性feminine 是Yes
1515 阴性feminine 偏高High 优于胶体金法Better than colloidal gold method
1616 阴性feminine 偏高High 优于胶体金法Better than colloidal gold method
三、检测灵敏度：将2例2019-nCoV阳性患者样品稀释1000倍，用该方法检测，结果为阳性。这表明该方法的检测限在现有通行检测浓度的千分之一以下，检测灵敏度提升3个数量级，有望发展无创检测新方法和新技术。3. Detection sensitivity: Dilute the samples of 2 patients with 2019-nCoV positive by 1000 times and use this method to detect the results, and the results are positive. This shows that the detection limit of this method is less than one-thousandth of the current current detection concentration, and the detection sensitivity is improved by 3 orders of magnitude. It is expected to develop new non-invasive detection methods and new technologies.
第二、对新冠病毒的检测实验二Second, the new coronavirus detection experiment two
该实验采用的是图3所示的基于石墨烯的生物传感器对待检测样品进行了检测。该生成传感器的桥连分子为：1-芘丁酸N-羟基琥珀酰亚胺酯；桥连分子上连接的是用于检测新冠病毒的核酸探针。This experiment uses the graphene-based biosensor shown in Figure 3 to detect the sample to be tested. The bridging molecule for generating the sensor is: 1-pyrene butyrate N-hydroxysuccinimide ester; connected to the bridging molecule is a nucleic acid probe for detecting the new coronavirus.
实验结果表明，当待测样品的前处理条件满足65摄氏度，杂交时间30分钟的情况下，检测的结果非常理想。这对于后续开展病毒种类快速精准区分的检测，将会提供一种全新、有效的快速解决方案。The experimental results show that when the pre-processing conditions of the sample to be tested meet 65 degrees Celsius and the hybridization time is 30 minutes, the test results are very ideal. This will provide a new, effective and rapid solution for subsequent rapid and accurate detection of virus types.
第三、对流感病毒的检测实验一Third, the first test of influenza virus detection
该实验采用的是基于硅纳米线的生物传感器(硅纳米线场效应晶体管)对待检测样品进行了检测。This experiment uses a silicon nanowire-based biosensor (silicon nanowire field effect transistor) to detect the sample to be tested.
所述桥连分子为Ni 2+探针： The bridging molecule is a Ni 2+ probe:
Figure PCTCN2021080531-appb-000012
Figure PCTCN2021080531-appb-000012
该连接分子上连接的是用于检测流感病毒的核酸探针：Connected to the linking molecule is a nucleic acid probe for detecting influenza virus:
具体的，是针对HA基因检测甲型Hmi流感病毒(2009变异株)的核苷酸序列，5’-[FAM]-CAT TTC TTT CCA TT GCG-[TAMRA或BHQ1]-3’，5’端修饰氨基。Specifically, it is to detect the nucleotide sequence of the Hmi influenza virus (2009 mutant) against the HA gene, 5'-[FAM]-CAT TTC TTC TTT CCA TT GCG-[TAMRA or BHQ1]-3', 5'end Modified amino.
该序列为检测甲型Hmi流感病毒(2009变异株)HA基因的LNA修饰的荧光短探针，即第4、7、10、13、15位的碱基用LNA进行修饰，探针的5’端标记报告荧光基团FAM(6-羧基荧光素)，3’端标记淬灭基团TAMRA或BHQ1。This sequence is a short fluorescent probe for detecting the LNA modification of the HA gene of the Hmi influenza virus (2009 mutant). The end is labeled with the reporter fluorophore FAM (6-carboxyfluorescein), and the 3'end is labeled with the quencher TAMRA or BHQ1.
实验分组：Experiment grouping:
1.阳性组：采用临床检测中6例胶体金核酸检测阳性的患者；1. Positive group: 6 cases of colloidal gold nucleic acid test positive patients in clinical testing;
2.对照组：2例胶体金核酸检测法测得均为阴性，其中1例为正常献血人员，另外1例在后续的流感病毒临床筛查为阳性患者。2. Control group: 2 cases of colloidal gold nucleic acid test were all negative, of which 1 case was a normal blood donor, and the other case was a positive patient in the follow-up clinical screening of influenza virus.
检测结果如下：The test results are as follows:
编号serial number 胶体金检测结果Colloidal gold test results 硅纳米线场效应晶体管结果Silicon nanowire field effect transistor results 一致性 consistency
11 阳性positive 阳性positive 是Yes
22 阳性positive 阳性positive 是Yes
33 阳性positive 阳性positive 是Yes
44 阴性feminine 阴性feminine 是Yes
55 阳性positive 阳性positive 是Yes
66 阳性positive 阳性positive 是Yes
77 阴性feminine 阳性positive 优于胶体金法Better than colloidal gold method
88 阳性positive 阳性positive 是Yes
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明保护的范围之内。The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the present invention. Within the scope of protection.

Claims (10)

  1. 一种生物传感器，其特征在于，包括：基底层和石墨烯层；A biosensor, characterized by comprising: a base layer and a graphene layer;
    所述石墨烯层位于所述基底层上；The graphene layer is located on the base layer;
    所述石墨烯层上镀有金属电极；所述金属电极包括：输入电极和输出电极，且一个输入电极对应一个输出电极；且，The graphene layer is plated with a metal electrode; the metal electrode includes: an input electrode and an output electrode, and one input electrode corresponds to one output electrode; and,
    所述石墨烯层上非金属电极的部分修饰有用于连接生物大分子的桥连分子；所述桥连分子，包含：用于与石墨烯连接的芘或苝或蒽锚定基团、用于与生物大分子相连的活泼酯或二硫键或顺丁烯二酸苷、和将锚定基团与活泼酯或二硫键或顺丁烯二酸苷相连的一个或多个连接基团；The part of the non-metal electrode on the graphene layer is modified with bridging molecules for connecting biological macromolecules; the bridging molecules include: pyrene or perylene or anthracene anchoring groups for connecting with graphene, and Active ester or disulfide bond or maleic acid glycoside connected to the biological macromolecule, and one or more linking groups that connect the anchor group to the active ester or disulfide bond or maleic acid glycoside;
    所述桥连分子上连接的生物大分子为：病毒抗原或病毒抗体或核酸探针。The biological macromolecules connected to the bridging molecules are virus antigens or virus antibodies or nucleic acid probes.
  2. 根据权利要求1所述的生物传感器，其特征在于，所述的桥连分子为如下之一：The biosensor according to claim 1, wherein the bridging molecule is one of the following:
    Figure PCTCN2021080531-appb-100001
    Figure PCTCN2021080531-appb-100001
    Figure PCTCN2021080531-appb-100002
    Figure PCTCN2021080531-appb-100002
  3. 一种生物传感器，其特征在于，包括：基底层；A biosensor, characterized by comprising: a base layer;
    所述基底层上镀有金属电极；所述金属电极包括：输入电极和输出电极，且一个输入电极对应一个输出电极；每个输入电极与其对应的输出电极之间由硅纳米线连接；The base layer is plated with metal electrodes; the metal electrodes include: input electrodes and output electrodes, and one input electrode corresponds to one output electrode; each input electrode and its corresponding output electrode are connected by silicon nanowires;
    所述硅纳米线上修饰有用于连接生物大分子的桥连分子；所述桥连分子为Ni 2+探针或马来酰亚胺； The silicon nanowire is modified with bridging molecules for connecting biological macromolecules; the bridging molecules are Ni 2+ probes or maleimides;
    所述桥连分子上连接的生物大分子为：病毒抗原或病毒抗体或核酸探针。The biological macromolecules connected to the bridging molecules are virus antigens or virus antibodies or nucleic acid probes.
  4. 根据权利要求3所述的生物传感器，其特征在于，The biosensor according to claim 3, wherein:
    所述的桥连分子为如下之一：The bridging molecule is one of the following:
    Figure PCTCN2021080531-appb-100003
    Figure PCTCN2021080531-appb-100003
  5. 根据权利要求1或3所述的生物传感器，其特征在于，，The biosensor according to claim 1 or 3, wherein,
    所述病毒抗原为新冠病毒COVID-19抗原或流感病毒抗原，所述病毒抗体为COVID-19抗体或流感抗体，所述核酸探针为用于检测COVID-19病毒的核酸探针或用于检测流感病毒的核酸探针。The virus antigen is a novel coronavirus COVID-19 antigen or an influenza virus antigen, the virus antibody is a COVID-19 antibody or an influenza antibody, and the nucleic acid probe is a nucleic acid probe for detecting COVID-19 virus or for detecting Nucleic acid probe for influenza virus.
  6. 根据权利要求5所述的生物传感器，其特征在于，The biosensor according to claim 5, wherein:
    所述COVID-19抗原为：IgM和IgG抗原片段；The COVID-19 antigen is: IgM and IgG antigen fragments;
    所述IgM和IgG抗原片段是由S蛋白和N蛋白1：1混合成的；The IgM and IgG antigen fragments are made of a 1:1 mixture of S protein and N protein;
    其中，S蛋白为：S1-RBD，其氨基酸排列顺序为：Among them, S protein is: S1-RBD, and its amino acid sequence is:
    RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF(Seq ID No.1)；RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQNGVCGVNGVCGVNGVCGVCGVCGVCGVNGVG
    N蛋白的氨基酸排列顺序为：The amino acid sequence of N protein is:
    MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADL DDFSKQLQQSMSSADSTQA(Seq ID No.2)；MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADL DDFSKQLQQSMSSADSTQA (Seq ID No.2);
    所述COVID-19抗体为：IgM和IgG抗体片段；The COVID-19 antibody is: IgM and IgG antibody fragments;
    所述用于检测新冠病毒核酸探针，为：The nucleic acid probe used to detect the new coronavirus is:
    ORF1ab基因合成探针：ORF1ab gene synthesis probe:
    5’-CCGTCTGCGGTATGTGGAAAGGTTATGG-3’，5’-CCGTCTGCGGTATGTGGAAAGGTTATGG-3’,
    5’端修饰氨基；Modified amino group at the 5'end;
    所述流感病毒抗原为：The influenza virus antigen is:
    MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKIDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI(Seq ID No.3)；MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKIDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI (Seq ID No.3);
    所述流感病毒抗体为：上述流感病毒对应的抗体；The influenza virus antibody is: the antibody corresponding to the above influenza virus;
    所述用于检测流感的核酸探针，为：The nucleic acid probe for detecting influenza is:
    5’-[FAM]-CAT TTC TTT CCA TT GCG-[TAMRA或BHQ1]-3’，5’端修饰氨基。5’-[FAM]-CAT TTC TTT CCA TT GCG-[TAMRA or BHQ1]-3’, 5’ end modified amino group.
  7. 一种权利要求1所述生物传感器的制备方法，其特征在于，包括：A method for preparing the biosensor according to claim 1, characterized in that it comprises:
    A、将在金属表面生成的单层石墨烯转移到基底材料表面上，形成基底层和石墨烯层；A. Transfer the single-layer graphene generated on the metal surface to the surface of the base material to form the base layer and the graphene layer;
    B、在所述石墨烯层上旋涂光刻胶后，通过光刻技术刻蚀出金属电极的形状；B. After spin-coating photoresist on the graphene layer, the shape of the metal electrode is etched by photolithography technology;
    C、在石墨烯层上金属电极的位置蒸镀上金属电极，形成待修饰器件；所述金属电极包括：输入电极和输出电极，且一个输入电极对应一个输出电极；C. A metal electrode is vapor-deposited on the position of the metal electrode on the graphene layer to form a device to be modified; the metal electrode includes: an input electrode and an output electrode, and one input electrode corresponds to one output electrode;
    D、在待修饰器件的石墨烯层上非金属电极的部分修饰上桥连分子；D. Modification of bridging molecules on part of the non-metal electrode on the graphene layer of the device to be modified;
    E、在所述桥连分子上连接病毒抗原或病毒抗体或核酸探针。E. Connect viral antigens or viral antibodies or nucleic acid probes to the bridging molecule.
  8. 一种权利要求3所述生物传感器的制备方法，其特征在于，包括：A method for preparing the biosensor according to claim 3, characterized in that it comprises:
    a、在基底上生成的硅纳米线并表面功能化；a. The silicon nanowires generated on the substrate are functionalized on the surface;
    b、在所述基底上旋涂光刻胶后，通过光刻技术刻蚀出金属电极的形状；b. After spin-coating photoresist on the substrate, the shape of the metal electrode is etched by photolithography technology;
    c、在基底上蒸镀上金属电极，形成待修饰器件；所述金属电极包括：输入电极和输出电极，一个输入电极对应一个输出电极，每个输入电极与其对应的输出电极之间由硅纳米线连接；c. A metal electrode is vapor-deposited on the substrate to form the device to be modified; the metal electrode includes: an input electrode and an output electrode, one input electrode corresponds to one output electrode, each input electrode and its corresponding output electrode Wire connection
    d、在待修饰器件的硅纳米线上修饰上桥连分子；d. Modify bridging molecules on the silicon nanowire of the device to be modified;
    e、在所述桥连分子上连接病毒抗原或病毒抗体或核酸探针。e. Connect viral antigens or viral antibodies or nucleic acid probes to the bridging molecule.
  9. 一种病毒检测***，其特征在于，包括：权利要求1～6任一项所述的生物传感器、信号发生器、信号采集器、信号分析主机和显示器；A virus detection system, characterized by comprising: the biosensor, signal generator, signal collector, signal analysis host, and display according to any one of claims 1 to 6;
    所述信号发生器与所述生物传感器的各个输入电极相连，将触发电信号发送至各个输入电极；The signal generator is connected to each input electrode of the biosensor, and sends a trigger electrical signal to each input electrode;
    所述信号采集器与所述生物传感器的各个输出电极相连，采集输出电流信号并发送至信号分析主机；该输出电流信号为：所述生物传感器中的病毒抗原或病毒抗体或用于检测病毒的核酸探针，与待检测样品反应前和反应后分别获得的电流信号；The signal collector is connected to each output electrode of the biosensor, collects the output current signal and sends it to the signal analysis host; the output current signal is: virus antigen or virus antibody in the biosensor or virus detection Nucleic acid probes, the current signals obtained before and after the reaction with the sample to be tested;
    所述信号分析主机，对接收到的输出电流信号进行分析检测，确定待测样品中是否存在病毒，将分析检测结果发送至显示器进行显示。The signal analysis host analyzes and detects the received output current signal, determines whether there is a virus in the sample to be tested, and sends the analysis and detection result to the display for display.
  10. 一种病毒检测方法，其特征在于，应用于权利要求9所述的病毒检测***，包括如下步骤：A virus detection method, characterized in that it is applied to the virus detection system of claim 9, and comprises the following steps:
    所述信号发生器将触发电信号发送至所述生物传感器的各个输入电极；The signal generator sends a trigger electrical signal to each input electrode of the biosensor;
    将待测样品输入所述生物传感器；Input the sample to be tested into the biosensor;
    所述信号采集器与所述生物传感器的各个输出电极相连，采集输出电流信号并发送至信号分析主机；该输出电流信号为：所述生物传感器中的病毒抗原或病毒抗体或用于检测病毒的核酸探针，与待检测样品反应前和反应后分别获得的电流信号；The signal collector is connected to each output electrode of the biosensor, collects the output current signal and sends it to the signal analysis host; the output current signal is: virus antigen or virus antibody in the biosensor or virus detection Nucleic acid probes, the current signals obtained before and after the reaction with the sample to be tested;
    所述信号分析主机，对接收到的输出电流信号进行分析检测，确定待测样品中是否存在病毒，将分析检测结果发送至显示器进行显示。The signal analysis host analyzes and detects the received output current signal, determines whether there is a virus in the sample to be tested, and sends the analysis and detection result to the display for display.
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