US20120325669A1 - Nanohair structure and an application therefor - Google Patents

Nanohair structure and an application therefor Download PDF

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US20120325669A1
US20120325669A1 US13/225,167 US201113225167A US2012325669A1 US 20120325669 A1 US20120325669 A1 US 20120325669A1 US 201113225167 A US201113225167 A US 201113225167A US 2012325669 A1 US2012325669 A1 US 2012325669A1
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nanotemplate
nanohair
nanohair structure
structure according
nickel
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Young Keun Kim
Jee Won Lee
Jin Seung Park
Moon Kyu Cho
Eun Jung Lee
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Korea University Research and Business Foundation
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
    • B82B1/00—Nanostructures formed by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
    • C25D—PROCESSES FOR THE ELECTROLYTIC OR ELECTROPHORETIC PRODUCTION OF COATINGS; ELECTROFORMING; APPARATUS THEREFOR
    • C25D5/00—Electroplating characterised by the process; Pretreatment or after-treatment of workpieces
    • C25D5/02—Electroplating of selected surface areas
    • C25D5/022—Electroplating of selected surface areas using masking means
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a nanohair structure and a use thereof.
  • a producing method can largely be classified into an infiltration method (after immersing a support into a solution dissolving an active material, the active material is supported at the support by evaporating or adding a precipitate), an ion exchange method (an active material is exchanged to a support by contacting the support with the solution dissolving the active material), a precipitation method (passing through a activating process by precipitating the active material in a solution state), and the like.
  • the present invention which is a specific method out of the infiltration method of metal catalyst, uses a metal nickel as a catalyst.
  • the nickel is easily made in desired shapes and desired sizes by using a nanotemplate as compared with a metal line, a metal thin film, a metal crystal, and the like by using the nickel are often used to study a catalytic action.
  • the surface of the nickel nanohair structure can be biofunctionalized to achieve applicability in the field of biotechnology (BT).
  • the exposed part of nickel nanowire is possible to use in a biosensor using an affinity of antibody-antigen and biotin-avidin through a surface modification.
  • the applicability of nickel is greatly improved because the nickel can be possible to selectively bind with a variety of ligands such as amine and histidine.
  • the nickel nanohair structure according to the present invention is a very useful nanomaterial for chemically detecting because it is uniform in height; the agglomeration phenomenon is prevented by being inside the nanotemplate; and it has a high density. Accordingly, a method for synthesizing a nanostructure for studying the nickel nanohair structure is required.
  • An object of the present invention which is created by a necessity as mentioned above to solve the above problems, provides a nanohair structure which is necessary to prepare nanosensor.
  • Another object of the present invention provides a three-dimensional nanostructure-based ultra-sensitive biosensor based on the nanohair structure.
  • the present invention provides a nanohair structure comprising:
  • nanotemplate comprising a plurality of pores; and (b) a plurality of nanowires grown through the pores of the nanotemplate, wherein one portion of the nanowires is embedded inside the nanotemplate, while the other portion of the nanowires is exposed vertically on top of the surface of the nanotemplate.
  • nanohair structure which is used as its widest mean, in the present invention means the structure of exposing the nanowire array equalizing the length of nanowire by using semiconductor process such as the chemical mechanical planarization (CMP) process and the reactive ion etching (RIE) process, after filling metal (for example, nickel) in nanotemplate (for example, AAO nanotemplate).
  • CMP chemical mechanical planarization
  • RIE reactive ion etching
  • the exposed nanowire is derived from electrically conductive material such as transition metal species and alloy thereof comprising Ni, Fe, Co, Ni, Cu, Ag, Au, Pd and Pt or electrically conductive polymer.
  • electrically conductive material such as transition metal species and alloy thereof comprising Ni, Fe, Co, Ni, Cu, Ag, Au, Pd and Pt or electrically conductive polymer.
  • the ‘electrically conductive polymer’ in the present invention means a light electrically conductive polymer with easy processing.
  • the polymer is a long chain molecule compared with prior low molecular material and become solid state (crystal) by aggregation of the polymer chains.
  • the electrically conductive polymer is preferably polyacetylene, polyaniline, polypyrrole, polythiophene, poly sulfur nitride etc, but is not limited thereto.
  • the nanotemplate is preferably hard material like anodized aluminum oxide (AAO) or soft material like poly-carbonate, but is not limited thereto.
  • AAO anodized aluminum oxide
  • the present invention provides the method of the nanohair structure comprising, a) preparing a nanotemplate with a plurality of pores; b) generating a conductive electrode layer on one side of the nanotemplate; c) putting the nanotemplate into the solution containing the metal ion and growing the metal nanowire through pores of the nanotemplate by electrodeposition method employing it as a cathode; d) planarization of the metal nanowires through chemical mechanical planarization (CMP); and e) selective reactive ion etching (RIE) of the nanotemplate.
  • CMP chemical mechanical planarization
  • RIE selective reactive ion etching
  • the thickness of deposition of step b) preferably is 250-350 nm, but is not limited thereto.
  • the metal ion is preferably selected from the group consisting of Ni, Fe, Co, Ni, Cu, Ag, Au, Pd and Pt, but is not limited thereto.
  • the solution containing the metal ion of step c) preferably is the mixture solution of metal sulfate, nickel chloride and boric acid, but is not limited thereto.
  • the process of reactive ion etching of step e) preferably is to etch the nanotemplate for 10 min in an etching rate of 0.25 ⁇ m/min using BCl 3 gas, but is not limited thereto.
  • precious metal such as Cu, Ag, Au, or Pt is mainly used in the electrode layer, but all kind of conductive thin film can be used in the electrode layer
  • Pt is mainly used in the anode layer, but Pd or Ir can be used in the anode layer, but is not limited thereto.
  • the present invention provides a three-dimensional nanostructure-based biosensor produced by adding chimeric nanoparticles on the surface of nanohair structure and immobilizing the chimeric nanoparticle to the structure.
  • the chimeric nanoparticle is preferably HBV derived-chimeric protein, but is not limited thereto.
  • the nanosensor preferably further comprises antibody which recognizes specific disease marker, but is not limited thereto.
  • the disease marker is preferably Troponin I, but is not limited thereto.
  • the producing method of the present invention preferably includes as follows: a) obtaining two gene clones that are derived from Hepatitis B virus core protein (HBVcAg) gene encoding synthesizes of N-NdeI-hexahistidine-HBVcAg(1-78)-G4SG4T-XhoI-C and N-BamH1-G4SG4-HBVcAg (81-149)-HindIII-C; b) producing other two clones, i.e., N-XhoI-SPA B -EcoRI-C and N-EcoRI-SPA B -BamHI-C in order to substituting P79A80 of HBVcAg with tandem repeat of 209-270 residues of B domain of Staphylococcal protein A (SPA B ); c) producing a plasmid expressing vector encoding the synthesis of N-His 6 -HBVcAg (1-78)-SPA B
  • the ‘chimeric protein’ or ‘chimeric nanoparticle,’ which is used as its widest mean, in the present invention means the protein or protein nanoparticles with various functionalities by combining a foreign bio material to the surface of the protein nanoparticles based on a genetic engineering and a protein engineering technique.
  • HBV capsid of the present invention is used as a model virus scaffold for a surface display of SPA B
  • other viruses or virus-like particles can be used for the production of chimeric protein or chimeric nanoparticles displaying a surface SPA B .
  • the ‘HBV-derived chimeric protein means the protein or protein nanoparticles with various functionailities by combining a foreign protein to the HBV-derived protein.
  • the nanosensor in the present invention means device which detects specific compound, molecule or biomaterial like DNA or protein in the gas or liquid, or measures partial pressure or concentration of specific molecule or measures degree of vacuum of vacuum device or vacuum chamber or searching a site of gas leak.
  • the present invention provides nanohair-based electrodes which offer increased surface area compared to the conventional flat surface electrodes, and thereby increase the electrochemical or biological reactions of materials (for example, ions or enzymes).
  • FIG. 1 shows a process mimetic diagram of a fabrication method of nanohair wire structure:
  • FIG. 1( a ) shows the synthesize of anodized aluminum oxide nanotemplate;
  • FIG. 1( b ) shows the deposition of conductive layer (Ag, 300 ⁇ 400 nm) by E-beam evaporation;
  • FIG. 1( c ) shows the synthesize of nanowires inside the nanotemplate;
  • FIG. 1( d ) shows the planarization process of nanowires and nanotemplate;
  • FIG. 1( e ) shows a selective etching process of nanotemplate for nanohair wire structure.
  • FIG. 2 shows Field Emission-Scanning Electron Microscope (FE-SEM) images of nickel nanohair wire structure according to Example of the present invention:
  • FIGS. 2( a ) and ( b ) show floor plans that are planarized by chemical mechanical planarization (CMP) process;
  • FIGS. 2( c ) and ( d ) show cross-sectional diagrams that are planarized by the CMP process;
  • FIGS. 2( e ) and ( f ) show cross-sectional diagrams of nickel nanohair wire structure by selective reactive ion etching (RIE) process of nanotemplate.
  • CMP chemical mechanical planarization
  • FIGS. 2( e ) and ( f ) show cross-sectional diagrams of nickel nanohair wire structure by selective reactive ion etching (RIE) process of nanotemplate.
  • RIE reactive ion etching
  • FIGS. 3 and 4 show three-dimensional diagnostic assay based on virus nanoparticles.
  • FIG. 3 shows schematic and TEM images of native hepatitis B virus (HBV) capsid particles and chimeric nanoparticles synthesized in E. coli and
  • FIG. 4 shows schematic, of the diagnostic system performed in a 96-well microplate and the assay principle in detail. Briefly, antibodies that recognize the disease marker (Troponin I in this case) bind to the chimeric nanoparticle and are oriented in a specific way. Troponin I binds to the antibodies and detection is achieved with secondary antibodies conjugated with quantum dots.
  • HBV hepatitis B virus
  • FIGS. 5 and 6 show the detection of Troponin I.
  • FIG. 5 shows that the conventional ELISA assay could not detect Troponin I at concentrations lower than 0.1 nM
  • FIG. 6 shows that the assay using chimeric nanoparticles and nickel nanohairs shows 10 ⁇ 18 (atto-molar, aM) sensitivities in both PBS and human sera.
  • “Control” refers to the experiment in which only quantum-dot-secondary antibodies were added to the nickel nanohair surface, which was covered with a sufficient amount of chimeric nanoparticles (30 nM in PBS buffer 50
  • FIGS. 7 and 8 show a washable and reuseable assay system.
  • FIG. 7 shows Four-step protocol for washing and reusing the nickel nanohairs for detection.
  • FIG. 8 shows the consecutive assays of eight different Troponin I (Tn) samples using three separate systems, showing good reproducibility.
  • A, B, C, D, dotted and solid arrows, and PL1/PL2 correspond to those in FIG. 7 .
  • Black areas of the rectangle represent net PL increase.
  • FIGS. 9 and 10 show Troponin I assay on PVDF membranes.
  • FIG. 9 shows that chimeric nanoparticles immobilized on PVDF membranes show similar detection sensitivities in both PBS and human sera.
  • FIG. 10 shows that antibodies immobilized directly on the PVDF membranes show significantly lower sensitivities than those immobilized on chimeric nanoparticles.
  • FIGS. 11 and 12 show clinical specificity and sensitivity of the viral chimeric nanoparticle-based assay.
  • assays of sera derived from 16 healthy individuals and 26 AMI patients using the chimeric nanoparticles and PVDF membrane system [see FIG. 11( a )] unambiguously detected Troponin I in all patients, but using an ELISA-based diagnosis [see FIG. 11( b )] failed to detect three patients (Nos. 4, 9, and 18) and revealed nine ambiguous signals close to the clinical cutoff signal (horizontal dotted line).
  • FIG. 12 shows assays with antibodies attached to chimeric nanoparticles, but not those immobilized directly on PVDF membranes, could detect Troponin I in a sample diluted 1,000 times. 74/M and 75/F represent the age and gender of the AMI patients.
  • FIG. 13 shows a graph for determining an amount of HVB capsid-derived chimeric nanoparticles added to bare nickel surface, preventing the binding of non-specific quantum-dot-secondary antibodies to the nickel surface.
  • Example 1 of the present invention relates to a method for forming Nanohair structure having an exposed nanowire structure on the anodized aluminum oxide (AAO) nanotemplate after synthesizing the nickel nanowires in the AAO nanotemplate.
  • An object of the present invention is to provide a development of bio-nano catalyst material that can be possible to apply in vitro by using the nanowire as a catalyst for the application in the biochemical field.
  • the technique according to Example 1 of the present invention is based on an electrochemistry, and relates to a producing method that would allow the mass production of a catalyst at low cost, in which the catalyst can increase the reaction rate, that is, can make the reaction of low activation energy by contacting with a reactant.
  • the technique can be implemented by exposing the nickel nanowire through a selective isotropy reactive ion etching (RIE), after synthesizing the nickel nanowire in AAO nanotemplate produced by an electrodeposition method.
  • RIE selective isotropy reactive ion etching
  • a producing process of the nickel nanostructure according to the present invention includes regularly planarizing the heights of nanowire and AAO through a chemical mechanical planarization (CMP), after synthesizing the nanowire in the AAO nanotemplate by using an electrochemical method.
  • AAO nanotemplate of the sample resulted from the above steps is selectively etched through an etching process by using a reactive ion etching (RIE) apparatus.
  • RIE reactive ion etching
  • the nanohair structure (Ni nanohair structure) exposed on the final AAO nanotemplate like hairs can be synthesized through the processes as mentioned above.
  • the nanohair structure implemented as mentioned above does not have an agglomeration phenomenon of nanowires, and has very high density (10 8 wires/cm 2 ) and a regular height (max. 60 ⁇ m) so that its applicability is largely increased as a catalyst in the biochemical and environmental fields.
  • the ‘chemical mechanical planarization (CMP)’ is one of the methods generally used in the process of planarizing, and includes pressing the action surface to the rotation polishing pad and then introducing a polishing and/or chemical reaction solution that is known as slurry on the polishing pad.
  • the mechanical effect of pressure is applied through the polishing pad, and the chemical reaction caused by the input of slurry allows the materials to be selectively removed from the action surface thereby a little more equalizing the layer.
  • deionized water having a high purity is applied to the polishing solution as base, and a particle and/or chemical additive having the effect of polishing is added therein.
  • the more information about the chemical mechanical polishing, the slurry, and the like is disclosed in U.S. Pat. Nos. 6,914,001 and 6,887,137.
  • Troponin I disclosed in the specification of the present invention is a type of proteins found in the blood of patients suffered from a cardiac infarction, and when detecting in the existence of Troponin I, it can be judged to have a disorder of heart.
  • the ‘nickel nanohair structure’ disclosed in the specification of the present invention is the structure of exposing the nanowire by selectively etching AAO nanotemplate using the reactive ion etching (RIE) process, and equalizing the length of nanowire by using the chemical mechanical planarization (CMP) process, after synthesizing nickel nanowire in AAO nanotemplate.
  • RIE reactive ion etching
  • CMP chemical mechanical planarization
  • PVDF membrane poly(vinyl difluoride) membrane
  • the ‘PVDF membrane (poly(vinyl difluoride) membrane)’ disclosed in the specification of the present invention is the polymer membrane having small pores and hydrophobic property (no compatibility with water), and the present invention used the membrane having the pore of 450 nm size, in which the membrane has the property that allows the surface thereof to easily well take the nanoparticles.
  • bio-nano-probe disclosed in the specification of the present invention is used as the material of sensor that is accumulated with the probe for detecting a target to the protein nanoparticles.
  • the present inventors showed that combining the three-dimensional nanostructure including the nickel nanohair and virus nanoparticles that are designed to have a dual affinity for antibodies and nickel can detect at low level of Troponin, i.e., 10 6 ⁇ 10 7 in human serum as compared with using the typical ELISA assay [Hirsch, L. R., Jackson, J. B., Lee, A., Halas, N. J. & West, J. Anal. Chem. 75, 2377-2381 (2003); Nam, J. M., Park, S. J. &Mirkin, C. A. J. Am. Chem. Soc. 124, 3820-3821 (2002); Niemeyer, C. M. & Ceyhan, B. Angew.
  • the virus nanoparticles help the orientation of antibodies for the maximum capture of Troponin marker.
  • Troponin marker is largely bound to antibody in the high density combined to the nanostructure, the sensitivity of detecting is largely increased.
  • the nickel nanohair is able to reproduce and also to regeneratively distinguish a healthy serum from unhealthy serum.
  • the present inventors anticipate other virus nanoparticles forming a diagnosis assay with high similar sensitivity to other various protein markers.
  • HBV core protein consisting of four long alpha-helix bundles ( FIG. 3 ), when expressed in bacteria, assembles into core-shell particles that closely resemble the native capsid structure of virus [Bottcher, B., Wynne, S. A. & Crowther, R. A. Nature 386, 88-91 (1997); Crowther, R. A. et al. Cell 77, 943-950 (1994)].
  • Bottcher and colleagues demonstrated that a single HBV core protein truncated after residue 149 forms the core-shell particles that contain 240 subunits and have an overall diameter of 36 nm [Bottcher, B., Wynne, S. A.
  • the dimer clustering of the subunits produces spikes on the surface of the shell particle, and the immunogenic epitope is located at the tips of prominent surface spikes ( FIG. 3 ).
  • the surface-exposed spike tip corresponds to the loop segment consisting of the residues from D78 to D83 of the single core protein.
  • the present inventors made that the P79A80 in the loop segment was replaced with the tandem repeated SPA B sequences (NCBI nucleotide accession No. M18264 nucleotide sequence 625-813, Sequence No. 11), which were subsequently exposed on the surface of the synthesized chimeric nanoparticles with high density ( FIG. 3 ).
  • the present inventors also added the hexahistidine sequence to the N-terminus of the truncated HBV core protein so that the chimeric nanoparticles would have a strong affinity for nickel ( FIG. 3 ).
  • Transmission electron microscopy (TEM) image analysis FIG. 3 ) revealed that the HBV capsid-derived chimeric nanoparticles that were expressed in E. coli assembled into spherical nanoparticles with a nearly native diameter. Consequently, the chimeric nanoparticles have a dual affinity for the Fc domain of antibodies (IgG) and nickel.
  • IgG antibodies
  • These viral particles can display on their surface various peptides and proteins that are used for detecting and/or quantifying bimolecular of interest.
  • nanohair refers to an array of nanowires in which some wires are exposed to the air, the rest being embedded in the body of a supporting organic or inorganic template ( FIG. 4 ). In these structures, the air-exposed portion of the nanowires has a greatly increased surface-to-volume ratio.
  • the efficient three-dimensional assay system was developed with the following significant advantages: (i) maximum accessibility of protein markers to antibodies, enabled both by the controlled orientation of the antibodies and the three-dimensional manner of protein capture, and (ii) a dramatically increased density and ratio of antibodies to protein markers on the three-dimensional nanohair surface.
  • the captured markers were detected by sensing photoluminescence emitted by quantum dots conjugated to the secondary antibodies ( FIG. 4 ).
  • one nickel nanohair structure can be repeatedly used for multiple samples. Through washing and rinsing (i.e., in the step A of FIG. 7 ), the used nickel nanohair was refreshed and reused for another sample assay. All of the three separate nickel nanohair structures were successfully used for the consecutive assay of eight different samples. Each consecutive assay showed reproducible and consistent signals for all the samples tested.
  • PVDF polyvinylidene fluoride
  • the present inventors also tested the PVDF-based assay system in the clinical diagnosis of 26 AMI patients (Table 1) who were confirmed to have experienced an AMI, and the assay results were compared with the ELISA-based assay (a and b of FIG. 11 ).
  • Table 1 the clinical diagnosis of 26 AMI patients
  • the assay results were compared with the ELISA-based assay (a and b of FIG. 11 ).
  • three (Nos. 4, 9, and 18) AMI patient sera were not positively detected; that is, the absorbance signals were below the clinical cutoff value (indicated by the horizontal dotted line), and the signals from nine (Nos. 1, 2, 3, 5, 8, 11, 16, 17, and 23) AMI patient sera were positive but very close to the clinical cutoff.
  • the chimeric nanoparticles and PVDF-based assay gave clear positive signals for all 26 AMI patient sera, therefore indicating 100% clinical specificity ( FIG. 11 ).
  • the ELISA assay results were not surprising because the clinical specificity of the ELISA kit is reported by the supplier to be 87.5%.
  • antibodies directly immobilized on the PVDF surface failed to diagnose the 1,000-times diluted AMI patient sera, whereas the chimeric nanoparticles and PVDF-based assay could detect Troponin I in the diluted patient sera ( FIG. 12 ), indicating that this three-dimensional assay can discriminate the onset of AMI even with an extremely small quantity of patient sera.
  • HBV capsid-derived chimeric nanoparticles and three-dimensional nanostructures we were able to develop a highly sensitive and specific assay system for the specific AMI marker, Troponin I.
  • HBV capsid according to the present invention was used in this study as a model viral scaffold for the surface display of SPA B , other viruses or virus-like particles could also be used for the production of chimeric nanoparticles, displaying the surface SPA B . Owing to the controlled orientation of densely immobilized antibodies and the three-dimensional manner of protein capture, the assay sensitivity and clinical specificity were significantly enhanced as compared to the conventional ELISA assay.
  • the nanohair wire structure according to the present invention which is a method for chemically detecting that has a yield of a high efficiency due to the exclusion of agglomeration phenomenon among the nanowires, has a high applicability in the flied of Biotechnology (BT) as well as Nanotechnology (NT) by synthesizing the nanowire material having a biological functionality inside the nanotemplate.
  • BT Biotechnology
  • NT Nanotechnology
  • the present invention showed that the assay system according to the present invention using chimeric nanoparticles and three-dimensional nanostructure (nickel nanohair and PVDF membrane) has a very higher sensitivity and specificity as the use of detecting the disease diagnosis marker and has very high sensitivity and specificity to the protein marker, such as Troponin I or specific AMI marker.
  • the anodized aluminum oxide (AAO) nanotemplate with uniform pore diameter (tens to hundreds nm) as shown in FIG. 1( a ) was synthesized. And then, Ag as a working electrode was deposited one side of AAO in a thickness of 250-350 nm using E-beam evaporator as shown in FIG. 1( b ). Then, after adding AAO to the solution of nickel sulfate (NiSO 4 6H 2 O, 0.5 M)+nickel chloride (NiCl 2 .6H 2 O, 0.1M)+boric acid (H 3 BO 3 , 0.1M) as shown in FIG. 1( c ), Pt as a counter electrode was deposited to the nickel nanowires.
  • nickel sulfate is a main component of plating; nickel chloride was used for increasing an electrical conductivity; and boric acid was used as a buffer solution for pH homeostasis.
  • polishing was performed about 10 ⁇ m through a chemical mechanical planarization (CMP) process for identifying AAO nanotemplate with the height of nanowires that were overflow or underflow-gtown inside the AAO.
  • CMP chemical mechanical planarization
  • a selective reactive ion etching (RIE) process of AAO nanotemplate was performed in order to expose the nickel nanowires.
  • the process was performed by etching AAO for 10 min at an etching rate of 0.25 ⁇ m/min using BCl 3 (100%) gas.
  • a cleaning process DI water: ultrapure water, ethanol was completed, finishing the process of the nickel nanohairs structure with a clean surface.
  • HBV core protein HBV core protein
  • N-XhoI-SPA a -EcoRI-C and N-EcoRI-SPA a -BamHI-C were prepared.
  • plasmid expression vector pT7-Chimera-HBV encoding the synthesis of N-His 6 -HBVcAg(1-78)-SPA B -SPA B -HBVcAg(81-149)-C.
  • Table 1 shows primer sequences, in which bold types represent restriction enzymes sequences; underlined parts represent linker sequences; and italic types represent 6 histidine sequences.
  • HBV capsid-derived chimeric nanoparticles can be largely divided into 1-78 amino acid sequence regions of capsid protein, the region including continuously two Staphylococcal protein A, and 81-149 amino acid sequences regions of capsid protein (1-78 sequences of capsid protein is NCBI Nucleotide accession number: AF286594 sequences: 1901-2134 (Sequence No. 12) and amino acid sequence (Sequence No. 13); 81-149 sequences of capsid protein is 2141-2347 sequences (Sequence No. 14) of AF286594; and the amino acid sequence is Sequence No. 15, and Protein A sequence is NCBI nucleotide accession No. M18264, nucleotide sequence 625-813 (Sequence No. 11); and the amino acid is Sequence No. 16).
  • the first region was subjected to extension PCR using the primer sequence region 1 including 6 histidines using the gene sequence of HBV capsid protein (1901-2452 sequences of NCBI Nucleotide accession number: AF286594 sequences) as a template, and the primer sequences 2 and 3 including a linker sequence (amino sequences GGGGSGGGGT).
  • PCR was performed by using the primer sequences 1 and 3 using the synthesized PCR products as the templates.
  • PCR product consisting of 5′-NdeI-HBV capsid protein (1-78 amino acid sequences)-linker sequence (GGGGSGGGT)-XhoI-3′ was obtained.
  • the third region was subjected to extension PCR using the primer sequence 6, and the primer sequences 4 and 5 including the linker sequence (Amino acid sequence GGGGSGGGG) through using HBV capsid protein gene sequence as a template.
  • PCR was performed by using the primer sequences 4 and 6 through using the synthesized PCR products as a template.
  • PCR product consisting of 5′-BamHI-linker sequence (GGGGSGGGG)-HBV capsid protein (81-149 amino acid sequence)-HindIII-3′ was obtained.
  • the nickel nanohair structure prepared by the above Example 1 was placed in the Costar 96-well plate (Cat. No. 3599, Corning, N.Y., USA). Before immobilizing the chimeric nanoparticles, the nickel nanohair in each well was washed four times for 15 min using 0.3 M sulphuric acid and then six times for 10 min using distilled water, then completely dried. Next, the background photoluminescence from the nickel nanohair structure was measured using a microplate reader (GENios, Tecan, Austria) with excitation and emission at 420 and 650 nm, respectively.
  • PBS buffer 50 ⁇ l containing the 38-nM chimeric nanoparticles prepared from Example 2 was added to the nickel nanohair structure, followed by slow agitation for 30 min, after which it was washed with 50 mM Tris buffer (pH 7.4).
  • Rabbit anti-troponin polyclonal antibody (5 ⁇ g/ml, Cat. No. ab470003, Abcam, Cambridge, UK) in 200 ⁇ l PBS buffer was added to the chimeric nanoparticles that were already immobilized on the nickel nanohair, by slowly stirring the nickel nanohair in the antibody-containing PBS buffer for 2 h.
  • PVDF membrane (Immobilion-FL, IPFL 10100, Millipore, Mass., U.S.A.) in a Costar 96-well plate was pre-wetted with methanol for 1 min and washed with a PBS buffer (137 mM, NaCl; 2.7 mM, KCl; 10 mM, Na 2 HPO 4 ; 2 mM, KH 2 PO 4 ; pH, 7.4) for 5-10 min. Before the PVDF membrane was completely dried, 10 a of PBS buffer containing the chimeric nanoparticles purified in Example 2 was dropped onto a designated spot on the membrane.
  • PBS buffer 137 mM, NaCl; 2.7 mM, KCl; 10 mM, Na 2 HPO 4 ; 2 mM, KH 2 PO 4 ; pH, 7.4
  • the membrane was then slowly stirred for 1 h in the blocking solution (1% skimmed milk) and washed twice with the PBS buffer for 30 min.
  • Goat anti-Troponin I polyclonal antibodies (20 ⁇ g/ml in PBS buffer; Cat. No. 70-XG82, Fitzgerald, Mass., USA) was added to the chimeric protein nanoparticles that were already immobilized onto the PVDF membrane, by slowly stirring the membrane in the antibody-containing 200 ⁇ l PBS buffer for 2 h.
  • ELISA assay experiments in the present invention were conducted using the commercial ELISA Troponin assay kit (Troponin I EIA, Cat. No 25-TR1HU-E01, 96 wells, ALPCO Diagnostics, NH, USA) that was developed for in vitro diagnostic use. In short, it is as follows: 1) 100 ⁇ l of human serum (AMI patient or healthy serum) or Troponin (human cardiac Troponin I-T-C complex, Cat. No.
  • Troponin I EIA provides a reliable assay for the quantitative measurement of human cardiac-specific Troponin I with a clinical specificity of 87.5
  • the procedure disclosed in Troponin I EIA protocol was strictly followed for the Troponin I assay, and the assay procedure is as follows. The entire list of AMI patients and healthy sera are disclosed in Table 2.

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KR102309495B1 (ko) 2021-01-11 2021-10-05 한국화학연구원 사스 코로나바이러스 2 항체 검출용 나노선 기반 면역형광 키트 및 이의 용도
KR20230102652A (ko) 2021-12-30 2023-07-07 한국화학연구원 결핵균 항원 또는 항체 검출용 산화아연 나노선 어레이의 제조방법
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