CN111474365B - Biosensor and preparation method thereof, and virus detection system and method - Google Patents

Biosensor and preparation method thereof, and virus detection system and method Download PDF

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CN111474365B
CN111474365B CN202010233584.2A CN202010233584A CN111474365B CN 111474365 B CN111474365 B CN 111474365B CN 202010233584 A CN202010233584 A CN 202010233584A CN 111474365 B CN111474365 B CN 111474365B
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biosensor
virus
antibody
antigen
nucleic acid
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CN111474365A (en
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郭雪峰
李渝
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Peking University
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    • 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
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    • 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
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    • G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
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    • 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
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    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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    • 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
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    • 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

Abstract

The embodiment of the invention provides a biosensor, a preparation method thereof, a virus detection system and a virus detection method, wherein a bridging molecule capable of connecting various biological macromolecules is used for modifying a virus antigen or a virus antibody or a nucleic acid probe for detecting viruses on the biosensor, a sample to be detected is input into the biosensor, and current signals before and after reaction are analyzed, so that whether the virus exists in the sample to be detected can be detected; the bridging molecules of the biosensor provided by the embodiment of the invention can be connected with various biological macromolecules, so that detection of different viruses can be realized.

Description

Biosensor and preparation method thereof, and virus detection system and method
Technical Field
The invention relates to the technical field of biological detection, in particular to a biosensor, a preparation method thereof, a virus detection system and a virus detection method.
Background
A bioelectrical sensor is a sensor using an immobilized biological component or organism as a sensitive element. The basic principle is to detect a change in current or voltage caused by a biological action or chemical reaction between the sensing element and the biological component or organism to be detected, and to determine the detection result from the change in current or voltage.
At present, the biosensor technology has been greatly developed and applied to environmental detection, genetic detection, detection of bacteria and viruses, etc., but there are still few types of viruses that can be detected actually.
Disclosure of Invention
The embodiment of the invention aims to provide a biosensor, a preparation method thereof, a virus detection system and a virus detection method, so as to realize detection of different viruses.
In order to achieve the object of the embodiments of the present invention, the embodiments of the present invention provide a graphene and silicon nanowire based biosensor, wherein the graphene based biosensor includes: a substrate layer and a graphene layer;
the graphene layer is positioned on the substrate layer;
the graphene layer is plated with a metal electrode; the metal electrode includes: the device comprises input electrodes and output electrodes, wherein one input electrode corresponds to one output electrode; and the number of the first and second electrodes,
the part of the non-metal electrode on the graphene layer is modified with bridging molecules for connecting biomacromolecules; the bridging molecule comprising: the graphene material comprises a pyrene or perylene or anthracene anchoring group used for being connected with graphene, an active ester or disulfide bond or maleic acid glycoside used for being connected with biological macromolecules, and one or more connecting groups used for connecting the anchoring group with the active ester or disulfide bond or maleic acid glycoside;
the biomacromolecule connected on the bridging molecule is as follows: a viral antigen or a viral antibody or a nucleic acid probe.
A silicon nanowire-based biosensor comprising: a base layer;
the base layer is plated with a metal electrode; the metal electrode includes: the device comprises input electrodes and output electrodes, wherein one input electrode corresponds to one output electrode; each input electrode is connected with the corresponding output electrode through a silicon nanowire;
the silicon nanowire is modified with bridging molecules;
bridging molecules for connecting biomacromolecules are modified on the silicon nano-wire; the bridging molecule is Ni2+A probe or maleimide;
the biomacromolecule connected on the bridging molecule is as follows: a viral antigen or a viral antibody or a nucleic acid probe.
The embodiment of the invention also provides a preparation method of the biosensor, wherein the preparation method of the biosensor based on graphene comprises the following steps:
A. transferring the single-layer graphene generated on the metal surface to the surface of a substrate material to form a substrate layer and a graphene layer;
B. after photoresist is spin-coated on the graphene layer, the shape of the metal electrode is etched through the photoetching technology;
C. evaporating a metal electrode at the position of the metal electrode on the graphene layer to form a device to be modified; the metal electrode includes: the device comprises input electrodes and output electrodes, wherein one input electrode corresponds to one output electrode;
D. bridging molecules on partial modification of the non-metal electrode on the graphene layer of the device to be modified;
E. a viral antigen or a viral antibody or a nucleic acid probe is attached to the bridging molecule.
The preparation method of the biosensor based on the silicon nanowire comprises the following steps:
a. generating silicon nanowires on a substrate and functionalizing the surfaces of the silicon nanowires;
b. after photoresist is spin-coated on the substrate, the shape of the metal electrode is etched through the photoetching technology;
c. evaporating a metal electrode at the position of the metal electrode on the substrate to form a device to be modified; the metal electrode includes: the silicon nanowire array comprises input electrodes and output electrodes, wherein one input electrode corresponds to one output electrode, and each input electrode is connected with the corresponding output electrode through a silicon nanowire;
d. modifying a bridging molecule on a silicon nanowire of a device to be modified;
e. a viral antigen or a viral antibody or a nucleic acid probe is attached to the bridging molecule.
The embodiment of the present invention further provides a virus detection system, including: any one of the biosensors, the signal generator, the signal collector, the signal analysis host and the display;
the signal generator is connected with each input electrode of the biosensor and sends a trigger electric signal to each input electrode;
the signal collector is connected with each output electrode of the biosensor, collects output current signals and sends the output current signals to the signal analysis host; the output current signal is: the virus antigen or virus antibody or nucleic acid probe for detecting virus in the biosensor respectively obtains current signals before and after reaction with a sample to be detected;
and the signal analysis host computer is used for analyzing and detecting the received output current signal, determining whether viruses exist in the sample to be detected or not, and sending an analysis and detection result to the display for displaying.
The embodiment of the invention also provides a virus detection method, which is applied to the virus detection system and comprises the following steps:
the signal generator sends a trigger electric signal to each input electrode of the biosensor;
inputting a sample to be detected into the biosensor;
the signal collector is connected with each output electrode of the biosensor, collects output current signals and sends the output current signals to the signal analysis host; the output current signal is: the virus antigen or virus antibody or nucleic acid probe for detecting virus in the biosensor respectively obtains current signals before and after reaction with a sample to be detected;
and the signal analysis host computer is used for analyzing and detecting the received output current signal, determining whether viruses exist in the sample to be detected or not, and sending an analysis and detection result to the display for displaying.
The embodiment of the invention has the following beneficial effects:
according to the biosensor, the preparation method and the virus detection system and method provided by the embodiment of the invention, the bridging molecules capable of being connected with various biological macromolecules are used for modifying virus antigens or virus antibodies or nucleic acid probes for detecting viruses on the biosensor, a sample to be detected is input into the biosensor, and a received current signal is analyzed, so that whether the virus exists in the sample to be detected can be detected; the bridging molecules of the biosensor provided by the embodiment of the invention can be connected with various biological macromolecules, so that detection of different viruses can be realized.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Fig. 1a is a schematic structural diagram of a principle of a graphene-based biosensor provided in an embodiment of the present invention;
FIG. 1b is a schematic representation of a bridging molecule in the embodiment of FIG. 1 a;
FIG. 1c is a view showing an exemplary structure of a biosensor manufactured based on the principle structure of FIG. 1 a;
fig. 2 is a schematic structural diagram of a second principle of a graphene-based biosensor provided in an embodiment of the present invention;
fig. 3 is a schematic structural diagram of a third principle of a graphene-based biosensor provided in an embodiment of the present invention;
FIG. 4 is a schematic structural diagram of a biosensor based on silicon nanowires according to an embodiment of the present invention;
FIG. 5 is a schematic structural diagram of a second principle of a biosensor based on silicon nanowires according to an embodiment of the present invention;
FIG. 6 is a schematic structural diagram of a third principle of a biosensor based on silicon nanowires according to an embodiment of the present invention;
FIG. 7a is a flow chart of a method of making a graphene-based biosensor;
FIG. 7b is a diagram illustrating an exemplary embodiment of a biosensor manufactured according to the method shown in FIG. 7 a;
FIG. 7c is an exemplary view of a metal electrode of the biosensor manufactured based on the method shown in FIG. 7 a;
FIG. 8a is a flow chart of a method of fabricating a silicon nanowire-based biosensor;
FIG. 8b is a diagram showing an example of a biosensor manufactured according to the method shown in FIG. 8 a;
FIG. 8c is an exemplary view of a metal electrode and a silicon nanowire of a biosensor manufactured based on the method shown in FIG. 8 a;
FIG. 8d is a diagram showing another embodiment of the biosensor manufactured according to the method shown in FIG. 8 a;
fig. 9 is a schematic structural diagram of a virus detection system according to an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to detect a plurality of different viruses, the embodiment of the invention provides a biosensor, a preparation method thereof, a virus detection system and a virus detection method.
First, a biosensor provided in an embodiment of the present invention will be described.
The biosensor provided by the embodiment of the invention can be based on graphene materials and silicon nanowires, and the details are respectively described below.
The biosensor based on the graphene material provided by the embodiment of the invention comprises: a substrate layer and a graphene layer; the graphene layer is positioned on the substrate layer; the part of the non-metal electrode on the graphene layer is modified with bridging molecules for connecting biomacromolecules; the bridging molecule comprising: the graphene material comprises a pyrene or perylene or anthracene anchoring group used for being connected with graphene, an active ester or disulfide bond or maleic acid glycoside used for being connected with biological macromolecules, and one or more connecting groups used for connecting the anchoring group with the active ester or disulfide bond or maleic acid glycoside; the biomacromolecule connected on the bridging molecule is as follows: a viral antigen or a viral antibody or a nucleic acid probe. Specifically, the bridging molecule may be one of the following:
Figure BDA0002428357150000051
Figure BDA0002428357150000061
the bridging molecule of the biosensor provided by the embodiment of the invention can be connected with various biological macromolecules (virus antigens or virus antibodies or nucleic acid probes), so that the detection of different viruses can be realized.
The graphene-based biosensor provided in the embodiments of the present invention is described in detail below, taking a graphene-based biosensor for detecting a new coronavirus and an influenza virus as an example.
A graphene-based biosensor for detecting new coronavirus.
Specifically, there are three implementation ways of the graphene-based biosensor for detecting the new coronavirus, which are described below by way of examples respectively:
embodiment one of graphene-based biosensor for detecting novel coronavirus
As shown in fig. 1a, fig. 1a is a schematic structural diagram of a graphene-based biosensor according to an embodiment of the present invention, where the biosensor includes: Si/SiO2Substrate 110, graphene material 120, metal electrodes (input/output) 130, bridging molecules 140, and COVID-19 antigen 150.
Wherein, Si/SiO2The substrate 110 is composed of an underlying silicon Si material layer and silicon dioxide SiO tightly bonded thereto2A layer of material. SiO 22The function of (a) is to prevent interference with signal detection due to bottom leakage. Si/SiO2The substrate 110 may be Si/SiO commercially available in the market2Silicon wafer, Si/SiO2The silicon chip is integrated, and SiO is paved on the silicon Si chip2And (3) a layer.
The base layer in embodiments of the invention is not limited to Si/SiO2Substrate, in other embodiments, Si/SiO may be replaced with other insulating substrates2A substrate 110.
The graphene material 120 is located on Si/SiO2The upper layer of the substrate 110 forms the graphene layer.
The graphene material 120 is plated with metal electrodes (input/output) 130. The metal electrode 130 includes: input electrodes and output electrodes, as shown in fig. 1a, one metal electrode (input) 130 corresponds to one metal electrode (output) 130, and a graphene material 120 is disposed between the metal electrode (input) 130 and the metal electrode (output) 130.
As shown in fig. 1b, a part of the non-metal electrode on the graphene material 120 is modified with a bridging molecule 140. The bridging molecule 140 is: 1-pyrenebutanoic acid N-hydroxysuccinimide ester (i.e., bridging molecule 1 above).
As shown in fig. 1b, the anchoring group may be replaced with a perylene or anthracene anchoring group; the active ester (amino) can be replaced by disulfide bond or maleic acid glucoside; the number of linking groups can also be adjusted.
In practical applications, the bridging molecule 140 can be replaced with any one of the bridging molecules 2-12 described above. The bridging molecule 140 is linked to a new coronavirus COVID-19 antigen 150.
The COVID-19 antigen is: IgM and IgG antigen fragments;
the IgM and IgG antigen fragments are composed of S protein and N protein 1: 1, mixing;
wherein, the S protein is: S1-RBD, the amino acid sequence of which is:
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVL YNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEI YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPAT VCGPKKSTNLVKNKCVNF；
the amino acid sequence of the N protein is as follows:
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNN TASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGK MKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNP ANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSR GTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAA EASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWP QIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLN KHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSK QLQQSMSSADSTQA。
as can be seen from the embodiments shown in FIGS. 1 a-b, the graphene material of the biosensor is modified with the CoVID-19 antigen of the new coronavirus, and if the sample to be detected contains the COVID-19 antibody, the COVID-19 antibody in the sample to be detected is combined with the COVID-19 antigen on the graphene material due to the specific action between the COVID-19 antigen and the COVID-19 antibody, so that the output electrode outputs pre-reaction and post-reaction current signals, and the new coronavirus can be detected.
In order to detect a sample to be detected, a sample reaction chamber to be detected may be disposed between an input electrode and an output electrode of the biosensor in practical applications. Specifically, referring to fig. 1c, fig. 1c is a view illustrating a structure of a biosensor prepared based on the principle structure of fig. 1 a;
as shown in fig. 1c, in addition to the embodiment shown in fig. 1a, a micro-channel microreactor (PDMS) 170 is disposed between the metal electrode (input) 130 and the metal electrode (output) 130 and on the graphene material 120; the micro-channel microreactor 170 is provided with a sample reaction microcavity 171 to be measured which penetrates through the micro-channel microreactor 170 from top to bottom, so that the modified bridging molecule and the COVID-19 antigen on the graphene material 120 are located in the sample reaction microcavity 171 to be measured. The sample to be detected is dropped into the sample reaction microcavity 171 in a liquid state for reaction, and the metal electrode (output) 130 outputs current signals before and after the reaction, so as to detect the new coronavirus.
Graphene-based biosensor embodiment two for the detection of novel coronavirus
As shown in fig. 2, fig. 2 is a schematic structural diagram of a second principle of a graphene-based biosensor provided in an embodiment of the present invention; the biosensor includes: Si/SiO2Substrate 210, graphene material 220, metal electrode (input/output) 230, bridging molecule 240, COVID-19 antibody 250.
Wherein, Si/SiO2The substrate 210 is composed of an underlying Si material layer and SiO tightly bonded thereto2A layer of material. SiO 22The function of (a) is to prevent interference with signal detection due to bottom leakage. Si/SiO2The substrate 210 may be Si/SiO commercially available in the marketplace2Silicon wafer, Si/SiO2The silicon wafer is integral, the silicon Si waferOn which SiO has been laid2And (3) a layer.
The graphene material 220 is located on Si/SiO2The graphene layer is formed on the upper layer of the substrate 210.
The graphene material 220 is plated with metal electrodes (input/output) 230. The metal electrode 230 includes: input electrodes and output electrodes, as shown in fig. 2, one metal electrode (input) 230 corresponds to one metal electrode (output) 230, and a graphene material 220 is disposed between the metal electrode (input) 230 and the metal electrode (output) 230.
As shown in fig. 2, a part of the non-metal electrode on the graphene material 220 is modified with a bridging molecule 240. The bridging molecule 240 is: the bridging molecule 8 described above.
Likewise, in practice, the bridging molecule 240 may be replaced with any other of the 12 bridging molecules described above.
The bridging molecule 240 is linked to a novel coronavirus COVID-19 antibody 250.
The COVID-19 antibody described in this example is: IgM and IgG antibody fragments. The IgM and IgG antibodies are antibody fragments corresponding to IgM and IgG antigen fragments.
As can be seen from the embodiment shown in FIG. 2, the graphene material of the biosensor is modified with the antibody of the new coronavirus COVID-19, and if the sample to be detected contains the COVID-19 antigen, the COVID-19 antigen in the sample to be detected is combined with the antibody of the COVID-19 on the graphene material due to the specific action between the COVID-19 antigen and the antibody of the COVID-19, so that the output electrode outputs current signals before and after reaction, and the new coronavirus can be detected.
Graphene-based biosensor embodiment three for the detection of new coronavirus
As shown in fig. 3, fig. 3 is a schematic structural diagram of a third principle of a graphene-based biosensor provided in an embodiment of the present invention; the biosensor includes: Si/SiO2Substrate 310, graphene material 320, metal electrodes (input/output) 330, bridging molecules 340, nucleic acid probes 350, and optional heating plate 360.
Wherein, Si/SiO2The substrate 310 is formed of an underlying layer of Si material andSiO tightly bound thereto2A layer of material. SiO 22The function of (a) is to prevent interference with signal detection due to bottom leakage. Si/SiO2The substrate 310 may be Si/SiO commercially available in the market2Silicon wafer, Si/SiO2The silicon chip is integrated, and SiO is paved on the silicon Si chip2And (3) a layer.
The graphene material 320 is located on Si/SiO2The upper layer of the substrate 310 forms the graphene layer.
The graphene material 320 is plated with a metal electrode (input/output) 330. The metal electrode 330 includes: as shown in fig. 3, one metal electrode (input) 330 corresponds to one metal electrode (output) 330, and a graphene material 320 is disposed between the metal electrode (input) 330 and the metal electrode (output) 330.
As shown in fig. 3, a part of the non-metal electrode on the graphene material 320 is modified with a bridging molecule 340. The bridging molecule 340 is: the bridging molecule 12 described above.
Likewise, the bridging molecule 340 may be replaced with any other of the 12 bridging molecules described above in practice.
The bridging molecule 340 has attached thereto a nucleic acid probe 350. The nucleic acid probe 350 is used for detecting the new coronavirus RNA, and specifically can be:
5’-CCGTCTGCGGTATGTGGAAAGGTTATGG-3’，
5' end modified amino
In order to ensure the required temperature during the test, the embodiment shown in FIG. 3 can also be implemented in Si/SiO2A heating plate 360 is disposed under the substrate 310. The heating plate 360 may be an electrical heating plate, which is a common component of biosensors.
As can be seen from the embodiment shown in FIG. 3, the nucleic acid probe for detecting RNA of the new coronavirus is modified on the graphene material of the biosensor, and if the sample to be detected contains the COVID-19 virus, the COVID-19 virus will bind to the nucleic acid probe on the graphene material, so that the output electrode outputs current signals before and after the reaction, and the new coronavirus can be detected.
And secondly, a graphene-based biosensor for detecting influenza virus.
Specifically, there are three implementation manners of the graphene-based biosensor for detecting influenza virus, which are described below by referring to the following examples:
embodiment one of biosensor based on graphene for detecting influenza virus
Similar to the graphene-based biosensor for detecting the new coronavirus shown in fig. 1a, the biosensor includes: Si/SiO2A substrate, graphene material, metal electrodes (input/output), bridging molecules, and influenza virus antigens.
The bridging molecule in this example is bridging molecule 5 of the 12 bridging molecules described above.
In other embodiments, the bridging molecule can be any of the other bridging molecules described above for bridging molecule 12.
Referring to fig. 1a, the present embodiment is different from the embodiment shown in fig. 1a in that: attached to the bridging molecule 140 is an influenza antigen, not a neocoronavirus antigen.
Specifically, the influenza virus antigen sequence is:
MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVN LLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETS SSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAAC PHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSAD QQSLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGD KITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPF QNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMV DGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGK EFNHLEKRIENLNKKIDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYE KVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNR EEIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI。
graphene-based biosensor for detecting influenza virus embodiment two
Similar to the graphene-based biosensor for detecting a new coronavirus shown in fig. 2, the biosensor includes: Si/SiO2Substrate, graphene material, metal electrodes (input/output), bridging molecules and flowAn influenza virus antibody.
The bridging molecule in this example is bridging molecule 3 of the 12 bridging molecules described above.
In other embodiments, the bridging molecule can be any of the other bridging molecules described above for bridging molecule 12.
Referring to fig. 2, the present embodiment is different from the embodiment shown in fig. 2 in that: attached to bridging molecule 240 is an influenza antibody, not a new coronavirus antibody.
In the present example, the influenza virus antibody is an antibody corresponding to the influenza virus antigen.
Graphene-based biosensor for detecting influenza virus
Similar to the graphene-based biosensor for detecting a new coronavirus shown in fig. 3, the biosensor includes: Si/SiO2A substrate, graphene material, metal electrodes (input/output), bridging molecules, probes for detecting influenza virus, and a heating plate.
In this example, the probe for detecting influenza virus is a nucleotide sequence for detecting Hmi influenza a virus (2009 variant) against the HA gene, and amino groups are modified at the 5 ' - [ FAM ] -CAT TTC TTT CCA TT GCG- [ TAMRA or BHQI ] -3 ', 5 ' -end.
The sequence is an LNA-modified short fluorescent probe for detecting HA genes of the influenza A Hmi virus (2009 variants), namely bases at positions 4, 7, 10, 13 and 15 are modified by LNA, the 5 'end of the probe is marked with a reporter fluorophore FAM (6-carb0 Xy-fluorescein), and the 3' end of the probe is marked with a quencher TAMRA or BHQI.
The bridging molecule in this example is bridging molecule 3 of the 12 bridging molecules described above.
In other embodiments, the bridging molecule can be any of the other bridging molecules described above for bridging molecule 12.
Referring to fig. 3, the present embodiment is different from the embodiment shown in fig. 3 in that: attached to bridging molecule 340 is a probe for detecting influenza virus, not a probe for detecting new coronavirus.
As can be seen from the above embodiments, the biosensor provided in the embodiments of the present invention can also be used for detecting influenza virus.
Next, a biosensor based on silicon nanowires provided by an embodiment of the present invention will be described in detail.
The embodiment of the invention provides a biosensor based on silicon nanowires, which comprises: a base layer;
the base layer is plated with a metal electrode; the metal electrode includes: the device comprises input electrodes and output electrodes, wherein one input electrode corresponds to one output electrode; each input electrode is connected with the corresponding output electrode through a silicon nanowire; bridging molecules for connecting biomacromolecules are modified on the silicon nano-wire; the bridging molecule is Ni2+A probe or maleimide; the biomacromolecule connected to the bridging molecule is as follows: a viral antigen or a viral antibody or a nucleic acid probe.
The silicon nanowire-based biosensor provided by the embodiment of the present invention is described in detail below by taking a silicon nanowire-based biosensor for detecting new coronavirus and influenza virus as an example.
A biosensor based on silicon nanowires for detecting new coronavirus.
Specifically, there are three implementation ways for the silicon nanowire-based biosensor for detecting the new coronavirus, which are described below by referring to the examples respectively:
embodiment one of biosensor based on silicon nanowire for detecting new coronavirus
Fig. 4 is a schematic structural diagram of a biosensor based on silicon nanowires according to an embodiment of the present invention, as shown in fig. 4; the biosensor includes: Si/SiO2A substrate 410, a silicon nanowire material 420, metal electrodes (input/output) 430, a bridging molecule 440, and a COVID-19 antigen 450.
Wherein, Si/SiO2The substrate 410 is composed of an underlying silicon Si material layer and silicon dioxide SiO tightly bonded thereto2A layer of material. SiO 22The function of (a) is to prevent interference with signal detection due to bottom leakage. Si/SiO2Substrate 110 may be a commercially available Si/SiO2Silicon wafer, Si/SiO2The silicon chip is integrated, and SiO is paved on the silicon Si chip2And (3) a layer.
The silicon nanowire material 420 is located in Si/SiO2The upper layer of the substrate 410.
The silicon nanowire material 420 is plated with metal electrodes (input/output) 430. The metal electrode 430 includes: input electrodes and output electrodes, as shown in fig. 4, one metal electrode (input) 430 corresponds to one metal electrode (output) 430, and a silicon nanowire material 420 is arranged between the metal electrode (input) 430 and the metal electrode (output) 430.
As shown in fig. 4, the silicon nanowire material 420 is modified with a bridging molecule 440. The bridging molecule 440 is Ni2+And (3) probe:
Figure BDA0002428357150000141
in other embodiments, the bridging molecule 440 can be a maleimide:
Figure BDA0002428357150000142
the bridging molecule 440 is connected with a new coronavirus COVID-19 antigen 450.
The COVID-19 antigen is: IgM and IgG antigen fragments;
the IgM and IgG antigen fragments are composed of S protein and N protein 1: 1, mixing;
wherein, the S protein is: S1-RBD, the amino acid sequence of which is:
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVL YNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEI YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPAT VCGPKKSTNLVKNKCVNF；
the amino acid sequence of the N protein is as follows:
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNN TASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGK MKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNP ANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSR GTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAA EASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWP QIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLN KHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSK QLQQSMSSADSTQA。
as can be seen from the embodiment shown in fig. 4, the silicon nanowire material of the biosensor is modified with the COVID-19 antigen of the new coronavirus, and if the sample to be detected contains the COVID-19 antibody, the COVID-19 antibody in the sample to be detected is combined with the COVID-19 antigen on the silicon nanowire material 420 due to the specific action between the COVID-19 antigen and the COVID-19 antibody, so that the output electrode outputs pre-reaction and post-reaction current signals, and the new coronavirus can be detected.
Silicon nanowire-based biosensor embodiment two for the detection of novel coronavirus
As shown in fig. 5, fig. 5 is a schematic structural diagram of a second principle of a silicon nanowire-based biosensor provided in an embodiment of the present invention; the biosensor includes: Si/SiO2Substrate 510, silicon nanowire material 520, metal electrodes (input/output) 530, bridging molecules 550, and COVID-19 antibodies 550.
Wherein, Si/SiO2The substrate 510 is composed of an underlying silicon Si material layer and silicon dioxide SiO tightly bonded thereto2A layer of material. SiO 22The function of (a) is to prevent interference with signal detection due to bottom leakage. Si/SiO2The substrate 110 may be Si/SiO commercially available in the market2Silicon wafer, Si/SiO2The silicon chip is integrated, and SiO is paved on the silicon Si chip2And (3) a layer.
The silicon nanowire material 520 is located on Si/SiO2The upper layer of the substrate 510.
The silicon nanowire material 520 is plated with metal electrodes (input/output) 530. The metal electrode 530 includes: input electrodes and output electrodes, as shown in fig. 5, one metal electrode (input) 530 corresponds to one metal electrode (output) 530, and a silicon nanowire material 520 is disposed between the metal electrode (input) 530 and the metal electrode (output) 530.
As shown in fig. 5, the silicon nanowire material 520 is modified with a bridging molecule 540. The bridging molecule 540 is: a maleimide.
The bridging molecule 540 is linked to a novel coronavirus COVID-19 antibody 550.
The COVID-19 antibody described in this example is: IgM and IgG antibody fragments. The IgM and IgG antibodies are antibody fragments corresponding to IgM and IgG antigen fragments.
As can be seen from the embodiment shown in FIG. 5, the silicon nanowire material of the biosensor is modified with the antibody of the new coronavirus COVID-19, and if the sample to be detected contains the COVID-19 antigen, the COVID-19 antigen in the sample to be detected is combined with the antibody of COVID-19 on the silicon nanowire material 520 due to the specific action between the COVID-19 antigen and the COVID-19 antibody, so that the output electrode outputs the pre-reaction and post-reaction current signals, and the new coronavirus can be detected.
Biosensor embodiment three based on silicon nanowires for detecting new coronavirus
As shown in fig. 6, fig. 6 is a schematic structural diagram of a third principle of a silicon nanowire-based biosensor provided in an embodiment of the present invention; the biosensor includes: Si/SiO2A substrate 610, a silicon nanowire material 620, metal electrodes (input/output) 630, bridging molecules 640, nucleic acid probes 650, and an optional heating plate 660.
Wherein, Si/SiO2The substrate 610 is composed of an underlying Si material layer and SiO tightly bonded thereto2A layer of material. SiO 22The function of (a) is to prevent interference with signal detection due to bottom leakage. Si/SiO2The substrate 610 may be Si/SiO commercially available in the marketplace2Silicon wafer, Si/SiO2The silicon chip is integrated, and SiO is paved on the silicon Si chip2And (3) a layer.
The silicon nanowire material 620 is located on Si/SiO2The upper layer of the substrate 610.
The silicon nanowire material 620 is plated with metal electrodes (input/output) 630. The metal electrode 630 includes: input electrodes and output electrodes, one metal electrode (input) 630 corresponds to one metal electrode (output) 630 as shown in fig. 6, with the silicon nanowire material 620 between the metal electrode (input) 630 and the metal electrode (output) 630.
As shown in fig. 6, the silicon nanowire material 620 is modified with a bridging molecule 640. The bridging molecule 640 is:
Figure BDA0002428357150000161
the bridging molecule 640 has attached thereto a nucleic acid probe 650. The nucleic acid probe 650 is used for detecting the new coronavirus RNA, and specifically can be:
ORF1ab gene synthesis probe:
5'-CCGTCTGCGGTATGTGGAAAGGTTATGG-3', amino group is modified at 5 ' end.
In order to ensure the required temperature during the test, the embodiment shown in FIG. 6 can also be implemented in Si/SiO2A heating plate 660 is disposed under the substrate 610. The heating plate 660 may be an electrical heating plate, which is a common component of biosensors.
As can be seen from the example shown in FIG. 6, the nucleic acid probe for detecting RNA of the new coronavirus is modified on the silicon nanowire material of the biosensor, and if the sample to be detected contains the COVID-19 virus, the COVID-19 virus will bind to the nucleic acid probe on the silicon nanowire material, so that the output electrode outputs current signals before and after the reaction, and the new coronavirus can be detected.
And secondly, a silicon nanowire-based biosensor for detecting influenza virus.
Specifically, there are three implementation ways for the silicon nanowire-based biosensor for detecting influenza virus, which are described below by way of examples:
embodiment one of biosensor based on silicon nanowire for detecting influenza virus
Similar to the silicon nanowire-based biosensor for detecting a new coronavirus shown in fig. 4, the biosensor includes: Si/SiO2A substrate, a silicon nanowire material, metal electrodes (input/output), a bridging molecule, and an influenza virus antigen.
This exampleThe bridging molecule in (1) is Ni2+And (3) a probe. In other embodiments, the bridging molecule may be a maleimide.
Referring to fig. 4, the present embodiment is different from the embodiment shown in fig. 4 in that: linked to the bridging molecule 440 is an influenza virus antigen, not a neocoronavirus antigen.
Specifically, the influenza virus antigen sequence is:
MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVN LLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETS SSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAAC PHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSAD QQSLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGD KITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPF QNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMV DGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGK EFNHLEKRIENLNKKIDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYE KVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNR EEIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI。
biosensor embodiment two based on silicon nanowires for detecting influenza virus
Similar to the silicon nanowire-based biosensor for detecting a new coronavirus shown in fig. 5, the biosensor includes: Si/SiO2A substrate, graphene material, metal electrodes (input/output), bridging molecules, and influenza virus antibodies.
The bridging molecule in this example is maleimide.
In other embodiments, the bridging molecule may be Ni2+。
Referring to fig. 5, the present embodiment is different from the embodiment shown in fig. 2 in that: attached to bridging molecule 540 are influenza antibodies, not new coronavirus antibodies.
In the present example, the influenza virus antibody is an antibody corresponding to the influenza virus antigen.
Biosensor embodiment three based on silicon nanowires for detecting influenza virus
FIG. 6 shows the detection of the novel coronavirusLike the silicon nanowire-based biosensor, the biosensor includes: Si/SiO2A substrate, graphene material, metal electrodes (input/output), bridging molecules, probes for detecting influenza virus, and a heating plate.
In this example, the probe for detecting influenza virus is a nucleotide sequence for detecting Hmi influenza a virus (2009 variant) against the HA gene, and amino groups are modified at the 5 ' - [ FAM ] -CAT TTC TTT CCA TT GCG- [ TAMRA or BHQI ] -3 ', 5 ' -end.
The sequence is an LNA-modified short fluorescent probe for detecting HA genes of the influenza A Hmi virus (2009 variants), namely bases at positions 4, 7, 10, 13 and 15 are modified by LNA, the 5 'end of the probe is marked with a reporter fluorophore FAM (6-carb0 Xy-fluorescein), and the 3' end of the probe is marked with a quencher TAMRA or BHQI.
The bridging molecule in this example is Ni2+And (3) a probe. In other embodiments, the bridging molecule may be a maleimide.
Referring to fig. 6, the present embodiment is different from the embodiment shown in fig. 6 in that: attached to bridging molecule 640 is a probe for detecting influenza virus, not a probe for detecting new corona virus.
As can be seen from the above embodiments, the biosensor provided in the embodiments of the present invention can also be used for detecting influenza virus.
The embodiment of the invention also provides a preparation method of the biosensor based on the graphene and the silicon nanowire, which is respectively explained below.
Referring to fig. 7a, a method for preparing a graphene-based biosensor includes the steps of:
A. transferring the single-layer graphene generated on the metal surface to the surface of a substrate material to form a substrate layer and a graphene layer;
B. after photoresist is spin-coated on the graphene layer, the shape of the metal electrode is etched through the photoetching technology;
C. evaporating a metal electrode at the position of the metal electrode on the graphene layer to form a device to be modified; the metal electrode includes: the device comprises input electrodes and output electrodes, wherein one input electrode corresponds to one output electrode;
D. bridging molecules on partial modification of the non-metal electrode on the graphene layer of the device to be modified;
E. a viral antigen or a viral antibody or a nucleic acid probe is attached to the bridging molecule.
The virus antigen can be a new coronavirus COVID-19 antigen or an influenza virus antigen, the virus antibody can be a COVID-19 antibody or an influenza antibody, and the nucleic acid probe can be a nucleic acid probe for detecting COVID-19 or a nucleic acid probe for detecting influenza.
Embodiment of the preparation method of the biosensor based on graphene:
in this embodiment, the prepared biosensor is a graphene field effect transistor, and a specific preparation process is shown in fig. 7b, and includes the following steps:
1. growing single-layer graphene on the surface of the copper foil by a Chemical Vapor Deposition (CVD) method;
2. transferring graphene on a copper foil to the surface of a silicon wafer (silicon dioxide with the size of 1.5 multiplied by 1.5cm and the size of 300 nm) by utilizing polymethyl methacrylate (PMMA) through a wet method;
3. after photoresist is spin-coated on the graphene layer, the shape of the metal electrode is etched by an ultraviolet lithography technology, and then chromium (8nm) and gold (60nm) are deposited by thermal evaporation;
4. etching graphene into strips of 25 × 2200 μm by ultraviolet lithography and oxygen plasma etching (RIE);
5. and (3) manufacturing a graphene external electrode (chromium (8nm) and gold (80nm)) by utilizing ultraviolet lithography and thermal evaporation, and finally evaporating and plating an electrode protection layer silicon dioxide (40nm) by utilizing an electron beam.
Specific structure of metal electrode, as shown in fig. 7c, fig. 7c is an exemplary view of a metal electrode of a biosensor prepared based on the method shown in fig. 7 a.
6. The method for modifying the surface of the graphene specifically comprises the following steps:
61. soaking a graphene transistor in 1mM acetonitrile solution of 1-pyrenebutanoic acid N-hydroxysuccinimide ester for 12 hours, modifying bridging molecules onto graphene through pi-pi stacking interaction, then rinsing with acetonitrile, and carefully drying with nitrogen for later use;
62. the device prepared in step 5 was immersed in 1mM COVID-19 antigen or nucleic acid probe buffer for 12 hours, followed by rinsing with buffer and drying with nitrogen.
It should be noted that fig. 7b only shows an example that the bridging molecule is 1-pyrenebutanoic acid N-hydroxysuccinimide ester, and in practical applications, the bridging molecule may be any of the above 12 bridging molecules. Meanwhile, single-layer graphene can also be grown on the surfaces of other metals, and various sizes can be adjusted according to actual conditions.
It can be seen that the biosensor based on graphene provided by the embodiment of the present invention is prepared by a "bottom-up" device preparation process, and the biosensor can detect various viruses, such as: a new coronavirus or influenza virus.
Referring to fig. 8a, a method for preparing a silicon nanowire-based biosensor includes the steps of:
a. generating silicon nanowires on a substrate and functionalizing the surfaces of the silicon nanowires;
b. after photoresist is spin-coated on the substrate, the shape of the metal electrode is etched through the photoetching technology;
c. evaporating a metal electrode on the substrate to form a device to be modified; the metal electrode includes: the silicon nanowire array comprises input electrodes and output electrodes, wherein one input electrode corresponds to one output electrode, and each input electrode is connected with the corresponding output electrode through a silicon nanowire;
d. modifying a bridging molecule on a silicon nanowire of a device to be modified;
e. a viral antigen or a viral antibody or a nucleic acid probe is attached to the bridging molecule.
The virus antigen can be a new coronavirus COVID-19 antigen or an influenza virus antigen, the virus antibody can be a COVID-19 antibody or an influenza antibody, and the nucleic acid probe can be a nucleic acid probe for detecting COVID-19 or a nucleic acid probe for detecting influenza.
The preparation method of the biosensor based on the silicon nanowire comprises the following steps:
the biosensor prepared in this embodiment is a silicon nanowire field effect transistor, and a specific preparation process is shown in fig. 8b, and includes the following steps:
1. growing silicon nanowires on a silicon wafer substrate by using a Chemical Vapor Deposition (CVD) method and functionalizing the surface of the silicon nanowires.
Placing the silicon wafer substrate assembled with gold nanoparticle (20nm, Ted Pella) catalyst on a tungsten boat, placing into a quartz tube of a CVD growth system, pumping air in the tube to below 0.5Pa, and introducing H of 7.5sccm2And taking the gas as a carrier gas, heating to 465 ℃, stabilizing for 40 minutes, and growing the silicon nanowire.
2. Performing gas phase surface functionalization on Aminopropyltriethoxysilane (APTES) to aminate the surface of the silicon nanowire;
3. mechanically sliding silicon nanowires to Si/SiO2A substrate;
4. after photoresist is spin-coated on the substrate, the shape of the metal electrode is etched through the photoetching technology; and a metal electrode is evaporated on the substrate to form a device to be modified; the metal electrode includes: the silicon nanowire array comprises input electrodes and output electrodes, wherein one input electrode corresponds to one output electrode, and each input electrode is connected with the corresponding output electrode through a silicon nanowire;
5. connecting a bridging molecule bifunctional group to p-phenylene isothiocyanate (PDITC) to the surface aminated silicon nanowire;
6. bonding N- (5-amino-1-carboxyl amyl) imido acetoacetic acid (AB-NTA) to the surface of the silicon nanowire through a PDITC bridging group;
7. mixing Ni2+Chelating to NTA terminal to form Ni-NTA molecular probe;
8. and (3) immobilizing an antigen/antibody/nucleic acid probe on the surface of the silicon nanowire on the surface functionalized silicon nanowire.
In the specific structure of the metal electrode and the silicon nano-wire of this embodiment, as shown in fig. 8c, fig. 8c is an exemplary diagram of the metal electrode and the silicon nano-wire of the biosensor prepared based on the method shown in fig. 8 a.
In addition, it should be noted that, in FIG. 8b, the bridging molecule is 1-pyrenebutanoic acid N-hydroxysuccinimide ester, but in practical application, the bridging molecule may also be maleimide. Specifically, as shown in fig. 8d, after the hydrogen bonding process is performed on the surface of the silicon nanowire, maleimide is introduced to connect with thiol in the biomacromolecule, i.e., the biomacromolecule is immobilized. The biomacromolecule in FIG. 8d can be a viral antigen or a viral antibody or a nucleic acid probe.
An embodiment of the present invention further provides a virus detection system, referring to fig. 9, where fig. 9 is a schematic structural diagram of the virus detection system provided in the embodiment of the present invention, and the system includes: a biosensor 901, a signal generator 902, a signal collector 903, a signal analysis host 904 and a display 905;
the biosensor 901 in this embodiment may be any one of the biosensors shown in fig. 1a to 6. In the reaction microcavity of the biosensor 901, a sample solution is 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 with each output electrode of the biosensor, collects output current signals and sends the output current signals to the signal analysis host 904; the output current signal is: the virus antigen or virus antibody in the biosensor 901 or the nucleic acid probe for detecting virus respectively obtain current signals before and after reaction with the sample to be detected.
The signal analysis host 904 analyzes and detects the received output current signal, determines whether a virus exists in the sample to be detected, and sends an analysis and detection result to a display for displaying.
Because the resistance changes before and after the reaction, current signals before and after the reaction can be respectively collected and compared, and whether viruses exist in the sample to be detected or not can be judged according to the change of the current. Specifically, the received current signals before and after the reaction can be analyzed based on a preset concentration-current standard curve to judge whether viruses exist in the sample to be detected, and the analysis and detection result is sent to a display for displaying.
By adopting the virus detection system provided by the embodiment of the invention, various viruses such as new coronavirus, influenza virus and the like can be detected by adopting the biosensor for modifying virus antigens or virus antibodies or nucleic acid probes for detecting the viruses.
The embodiment of the invention also provides a virus detection method, which is applied to the virus detection system shown in fig. 9 and comprises the following steps:
the signal generator sends a trigger electric signal to each input electrode of the biosensor;
in some embodiments, the signal generator may send a trigger electrical signal to the biosensor after receiving a start command sent by the signal analysis host.
Inputting a sample to be detected into the biosensor;
the signal collector is connected with each output electrode of the biosensor, collects output current signals and sends the output current signals to the signal analysis host; the output current signal is: the virus antigen or virus antibody or nucleic acid probe for detecting virus in the biosensor and current signals obtained before and after reaction of a sample to be detected;
and the signal analysis host computer is used for analyzing and detecting the received output current signal, determining whether viruses exist in the sample to be detected or not, and sending an analysis and detection result to the display for displaying.
The following explains the effects of practical application of the embodiments of the present invention.
The embodiment of the invention can detect various viruses, and the detection results of the new coronavirus and the influenza virus are explained as follows:
first, assay for New coronavirus
In this experiment, a graphene-based biosensor (graphene field effect transistor) shown in fig. 1c was used to detect a sample to be detected. The bridging molecules of the resulting sensor are: 1-pyrenebutanoic acid N-hydroxysuccinimide ester; the bridging molecule is linked to a new coronavirus antigen.
Specifically, in the embodiment of the invention, the antigen of the novel coronavirus (COVID-19) is a specific antigen fragment which is screened and aims at the COVID-19. The optimal IgM and IgG antigen fragments are preferably selected through carrying out clinical tests of a plurality of nucleic acid detection positive (patients) and negative (non-patients or close reaction population) controls. The specificity of the current antigen is over 95.3 percent, and the specificity is 98 percent. The protein expression system has industrialized production capacity for amplification, and can meet a great amount of requirements for clinical detection of epidemic areas. The detection result of the immunological method can greatly improve the detection sensitivity and shorten the detection time through a nanotechnology element.
The practical application results of the embodiment of the invention are as follows:
firstly, detecting speed: for the pre-treated serum sample, the method realizes the rapid detection, and the detection time is less than 1 minute. In this example, the serum sample was pretreated to remove non-analyte substances in the sample, thereby improving the reliability of the result.
II, experimental conditions:
grouping experiments:
1. positive group: positive patients detected by 8 colloidal gold nucleic acid detection methods in clinical detection comprise 7 IgM or IgG positive cases;
2. control group: the 8 colloidal gold nucleic acid detection methods were all negative, 4 of them were normal blood donors, and 4 were negative patients for clinical screening of new coronavirus.
Detection specificity: of the 16 field tests, there were 8 2019-nCoV positive patients and 8 negative controls. Compared with a colloidal gold test paper detection method, the detection result of the method shows that: all 8 2019-nCoV positive patients are detected, and the positive accuracy rate is 100%; 4 negative controls of 8 cases are determined to be normal, 4 suspected patients or patients just cured are discharged from hospital, the sensitivity and the specificity are superior to those of a colloidal gold method, and the effectiveness of the method is reflected. The specific test results and controls are as follows:
Figure BDA0002428357150000241
Figure BDA0002428357150000251
thirdly, detection sensitivity: the sample of 2 cases of 2019-nCoV positive patients is diluted 1000 times and detected by the method, and the result is positive. The detection limit of the method is less than one thousandth of the existing passing detection concentration, the detection sensitivity is improved by 3 orders of magnitude, and a novel noninvasive detection method and a novel technology are expected to be developed.
Second, assay for New coronavirus
In the experiment, the biosensor based on graphene shown in fig. 3 is used for detecting a sample to be detected. The bridging molecules of the resulting sensor are: 1-pyrenebutanoic acid N-hydroxysuccinimide ester; connected to the bridging molecule is a nucleic acid probe for detecting the novel coronavirus.
The experimental result shows that the detection result is very ideal when the pretreatment condition of the sample to be detected meets 65 ℃ and the hybridization time is 30 minutes. The method can provide a brand-new and effective rapid solution for the subsequent detection of rapid and accurate virus species distinction.
Third, detection experiment for influenza Virus I
The experiment adopts a biosensor (silicon nanowire field effect transistor) based on a silicon nanowire to detect a sample to be detected.
The bridging molecule is Ni2+And (3) probe:
Figure BDA0002428357150000252
the nucleic acid probe for detecting the influenza virus is connected to the connecting molecule:
specifically, the nucleotide sequence of influenza A Hmi virus (2009 variant) is detected aiming at HA gene, and amino is modified at 5 ' - [ FAM ] -CAT TTC TTT CCA TT GCG- [ TAMRA or BHQI ] -3 ', 5 '.
The sequence is an LNA-modified short fluorescent probe for detecting HA genes of the influenza A Hmi virus (2009 variants), namely bases at positions 4, 7, 10, 13 and 15 are modified by LNA, the 5 'end of the probe is marked with a reporter fluorophore FAM (6-carb0 Xy-fluorescein), and the 3' end of the probe is marked with a quencher TAMRA or BHQI.
Grouping experiments:
1. positive group: 6 cases of colloidal gold nucleic acid in clinical detection are adopted to detect positive patients;
2. control group: the 2 cases of colloidal gold nucleic acid detection methods are all negative, wherein 1 case is a normal blood donor, and the other 1 case is a positive patient after the subsequent influenza virus clinical screening.
The detection results are as follows:
numbering Colloidal gold test results Silicon nanowire field effect transistor results Consistency
1 Positive for Positive for Is that
2 Positive for Positive for Is that
3 Positive for Positive for Is that
4 Negative of Negative of Is that
5 Positive for Positive for Is that
6 Positive for Positive for Is that
7 Negative of Positive for Superior to colloidal gold method
8 Positive for Positive for Is that
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims (8)

1. A biosensor, comprising: a substrate layer and a graphene layer;
the graphene layer is positioned on the substrate layer;
the graphene layer is plated with a metal electrode; the metal electrode includes: the device comprises input electrodes and output electrodes, wherein one input electrode corresponds to one output electrode; and the number of the first and second electrodes,
the part of the non-metal electrode on the graphene layer is modified with bridging molecules for connecting biomacromolecules; the bridging molecule comprising: the graphene material comprises a pyrene or perylene or anthracene anchoring group used for being connected with graphene, an active ester or disulfide bond or maleic acid glycoside used for being connected with biological macromolecules, and one or more connecting groups used for connecting the anchoring group with the active ester or disulfide bond or maleic acid glycoside;
the biomacromolecule connected on the bridging molecule is as follows: a viral antigen or a viral antibody or a nucleic acid probe;
the virus antigen is a new 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 the COVID-19 virus or a nucleic acid probe for detecting the influenza virus;
the COVID-19 antigen is: IgM and IgG antigen fragments;
the IgM and IgG antigen fragments are composed of S protein and N protein 1: 1, mixing;
wherein, the S protein is: S1-RBD, the amino acid sequence of which is:
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF；
the amino acid sequence of the N protein is as follows:
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA；
the antibody COVID-19 is: IgM and IgG antibody fragments;
the nucleic acid probe for detecting the novel coronavirus is as follows:
ORF1ab gene synthesis probe:
5’-CCGTCTGCGGTATGTGGAAAGGTTATGG-3’，
modifying amino at the 5' end;
the influenza virus antigens are:
MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKIDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI；
the influenza virus antibody is: an antibody against the influenza virus;
the nucleic acid probe for detecting influenza comprises the following components:
5 ' - [ FAM ] -CAT TTC TTT CCA TT GCG- [ TAMRA or BHQI ] -3 ', 5 ' end modified amino.
2. The biosensor of claim 1,
the bridging molecule is one of the following:
Figure FDA0003126123860000031
Figure FDA0003126123860000041
3. a biosensor, comprising: a base layer;
the base layer is plated with a metal electrode; the metal electrode includes: the device comprises input electrodes and output electrodes, wherein one input electrode corresponds to one output electrode; each input electrode is connected with the corresponding output electrode through a silicon nanowire;
bridging molecules for connecting biomacromolecules are modified on the silicon nano-wire; the bridging molecule is Ni2+A probe or maleimide;
the biomacromolecule connected on the bridging molecule is as follows: a viral antigen or a viral antibody or a nucleic acid probe;
the virus antigen is a new 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 the COVID-19 virus or a nucleic acid probe for detecting the influenza virus;
the COVID-19 antigen is: IgM and IgG antigen fragments;
the IgM and IgG antigen fragments are composed of S protein and N protein 1: 1, mixing;
wherein, the S protein is: S1-RBD, the amino acid sequence of which is:
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF；
the amino acid sequence of the N protein is as follows:
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA；
the antibody COVID-19 is: IgM and IgG antibody fragments;
the nucleic acid probe for detecting the novel coronavirus is as follows:
ORF1ab gene synthesis probe:
5’-CCGTCTGCGGTATGTGGAAAGGTTATGG-3’，
modifying amino at the 5' end;
the influenza virus antigens are:
MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGTSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKIDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI；
the influenza virus antibody is: an antibody against the influenza virus;
the nucleic acid probe for detecting influenza comprises the following components:
5 ' - [ FAM ] -CAT TTC TTT CCA TT GCG- [ TAMRA or BHQI ] -3 ', 5 ' end modified amino.
4. The biosensor of claim 3,
the bridging molecule is one of the following:
Figure FDA0003126123860000061
5. a method for preparing the biosensor in accordance with claim 1, comprising:
A. transferring the single-layer graphene generated on the metal surface to the surface of a substrate material to form a substrate layer and a graphene layer;
B. after photoresist is spin-coated on the graphene layer, the shape of the metal electrode is etched through the photoetching technology;
C. evaporating a metal electrode at the position of the metal electrode on the graphene layer to form a device to be modified; the metal electrode includes: the device comprises input electrodes and output electrodes, wherein one input electrode corresponds to one output electrode;
D. bridging molecules on partial modification of the non-metal electrode on the graphene layer of the device to be modified;
E. a viral antigen or a viral antibody or a nucleic acid probe is attached to the bridging molecule.
6. A method for preparing the biosensor in accordance with claim 3, comprising:
a. generating silicon nanowires on a substrate and functionalizing the surfaces of the silicon nanowires;
b. after photoresist is spin-coated on the substrate, the shape of the metal electrode is etched through the photoetching technology;
c. evaporating a metal electrode on the substrate to form a device to be modified; the metal electrode includes: the silicon nanowire array comprises input electrodes and output electrodes, wherein one input electrode corresponds to one output electrode, and each input electrode is connected with the corresponding output electrode through a silicon nanowire;
d. modifying a bridging molecule on a silicon nanowire of a device to be modified;
e. a viral antigen or a viral antibody or a nucleic acid probe is attached to the bridging molecule.
7. A virus detection system, comprising: the biosensor, the signal generator, the signal collector, the signal analysis host and the display of any one of claims 1 to 4;
the signal generator is connected with each input electrode of the biosensor and sends a trigger electric signal to each input electrode;
the signal collector is connected with each output electrode of the biosensor, collects output current signals and sends the output current signals to the signal analysis host; the output current signal is: the virus antigen or virus antibody or nucleic acid probe for detecting virus in the biosensor respectively obtains current signals before and after reaction with a sample to be detected;
and the signal analysis host computer is used for analyzing and detecting the received output current signal, determining whether viruses exist in the sample to be detected or not, and sending an analysis and detection result to the display for displaying.
8. A virus detection method applied to the virus detection system according to claim 7, comprising the steps of:
the signal generator sends a trigger electric signal to each input electrode of the biosensor;
inputting a sample to be detected into the biosensor;
the signal collector is connected with each output electrode of the biosensor, collects output current signals and sends the output current signals to the signal analysis host; the output current signal is: the virus antigen or virus antibody or nucleic acid probe for detecting virus in the biosensor respectively obtains current signals before and after reaction with a sample to be detected;
and the signal analysis host computer is used for analyzing and detecting the received output current signal, determining whether viruses exist in the sample to be detected or not, and sending an analysis and detection result to the display for displaying.
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