WO2007140497A1 - Nanoréseau de virus - Google Patents

Nanoréseau de virus Download PDF

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Publication number
WO2007140497A1
WO2007140497A1 PCT/AT2007/000264 AT2007000264W WO2007140497A1 WO 2007140497 A1 WO2007140497 A1 WO 2007140497A1 AT 2007000264 W AT2007000264 W AT 2007000264W WO 2007140497 A1 WO2007140497 A1 WO 2007140497A1
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Prior art keywords
virus
viral
nanoarray
proteins
spots
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PCT/AT2007/000264
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English (en)
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WO2007140497A8 (fr
Inventor
Ferry Kienberger
Helga Artelsmaier
Christian Rankl
Ali Tinazli
Robert Tampe
Dieter Blaas
Peter Hinterdorfer
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Universität Linz
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Publication of WO2007140497A1 publication Critical patent/WO2007140497A1/fr
Publication of WO2007140497A8 publication Critical patent/WO2007140497A8/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
    • G01Q60/24—AFM [Atomic Force Microscopy] or apparatus therefor, e.g. AFM probes
    • G01Q60/38—Probes, their manufacture, or their related instrumentation, e.g. holders
    • G01Q60/42—Functionalisation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00436—Maskless processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00617—Delimitation of the attachment areas by chemical means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/0063—Other, e.g. van der Waals forces, hydrogen bonding
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632—Introduction of reactive groups to the surface
    • B01J2219/00637—Introduction of reactive groups to the surface by coating it with another layer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00677—Ex-situ synthesis followed by deposition on the substrate
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/0074—Biological products

Definitions

  • the present invention provides means for the label-free detection and characterization of viral particles, in particular a nano-structured surface which can be provided in form of a nanoarray.
  • PCR polymerase chain reaction
  • the atomic force microscope (AFM) (Binnig et al., 1986) allows for imaging individual molecules under physiological conditions and for monitoring and visualizing dynamic processes at the single molecule level (Kienberger et al, 2004). Furthermore, the capability of AFM to measure forces in the pico-Newton range has opened the possibility to investigate inter- and intramolecular forces. Using this methodology, the interaction between tip-bound ligands and surface-bound receptor molecules can be analyzed in terms of affinity and rate constants (Hinter- dorfer et al, 1996) . In its application to viruses and virus related processes, AFM has successfully complemented EM and X-ray diffraction studies (Kuznetsov et al., 2001).
  • AFM virus particles can be visualized in appropriate buffers at room temperature (Drygin et al., 1998). Most importantly, AFM yields three-dimensional images with a very good resolution in the vertical direction (less than a nanometer) .
  • HRV human rhinovirus
  • AFM was used to study the architecture of the human rhinovirus (HRV) (Kien- berger et al., 2005). For the first time the structure of the rhinovirus capsid, the protein shell covering the viral genome, was imaged under physiological conditions. The resolution obtained was 2 nanometers and visually revealed the individual protein capsomeres of the capsid. Based on this resolution, viruses from different families can be differentiated by topographical imaging on the basis of their capsid structure alone.
  • the US 2005/239193 Al describes a chip of immobilized antibodies for the detection of Coxsackie viruses.
  • the antibody spots on the chip are formed by a microjet device and have a spot diameter of 30 ⁇ m to 80 ⁇ m with a spot distance of 20 ⁇ m.
  • the bound viruses can be analysed by a variety of methods including fluorescence and atomic force microscopy (AFM) .
  • AFM atomic force microscopy
  • the scan range is 120 ⁇ m which limits the chip to an amount of 2x2 spots.
  • Using larger areas with larger amount of detection spots on a single chip other detection means like fluorescence labeled antibodies are routinely used (Nettikadan et al., 2003) .
  • the tip of an AFM probe can be adapted with deposition devices to form smaller spots onto a carrier material.
  • spot sizes of below 0,2 ⁇ m of deposited material were reached.
  • microarray for viral detection via AFM was demonstrated.
  • the microarray pattern was created using a microjet device creating spots with 30 to 80 ⁇ m diameter.
  • the immobilized antibodies could then be used to bind viral particles which can be visualized by AFM below the size requirements of fluorescence detection methods.
  • a method for creating a nanoarray wherein a layer of oligo (ethylene glycol) (OEG) - covalently bound to a silicon surface via Si-C bonds - which is oxidized in localized spots with an AFM probe under voltage and biomolecules are immobilized on said spots.
  • oligo ethylene glycol
  • biomolecules are immobilized on said spots.
  • biomolecule is avidin a multitude of other possible biomolecules can be attached through a biotin-tag. The ensuing avidin-biotin linkage is one of the strongest biochemical non-covalent interactions.
  • spots of l ⁇ -mercaptohexadecanoic acid were created on a gold surface using an AFM probe tip as deposition device "dip-pen nanolithography tm ".
  • AFM probe tip as deposition device "dip-pen nanolithography tm ".
  • PEG polyethyleneglycol
  • the US 2005/0009206 describes a "dip-pen nanolithography tra " patterning method wherein the AFM probe tip is treated with a solution of certain biomolecules to be deposited onto a chip surface and brought into contact (using a force of 1.5 nN) with a negatively charged surface thus transferring the biomolecules. It was also shown that it is possible to deposit different proteins onto different spots, although this serial procedure is time consuming as it requires the steps of unmounting the AFM tip, immersing the tip in a solution of the desired protein, blow-drying, mounting the AFM tip and the contacting procedure for each protein.
  • the dip pen method is used under controlled conditions with a certain air humidity with the goal to prevent denaturation of the proteins by dehydration.
  • Wadu-Mesthrige et al. (Biophys. J. 80(4), (2001): 1891-9) describe the manufacture of immobilized protein patterns with AFM. Alkanethioles which form a monolayer on a gold surface are removed with an AFM probe and the resulting gap in the monolayer is filled with proteins.
  • Patterning of gaps with AFM is further described in the US 2004/0131843 Al, in particular wherein such patterns in an al- kanethiole layer is filled with biomolecules such as oligonucleotides .
  • the DE 10 2004 008 241 Al concerns enzymatic modification of a surface with an enzyme which is immobilized on the tip of an AFM probe.
  • the present invention provides a nanoarray of viral ligands immobilized in a pattern of spots each of a diameter size of less than 1000 nm, characterized in that the spots are provided embedded in a layer of antiadsorptive proteins which encompasses the area between the spots.
  • the antiadsorptive proteins are for example first provided as a layer, especially a single molecular layer on a surface of a chip.
  • An AFM tip can be used to pattern the protein layer and the viral ligands are subsequently immobilized (via attachment to the surface beneath) . This process is carried out in physiological solution maintaining the native fold of the proteins (including the viral ligands) .
  • the pattern e.g. created by the AFM tip in form of e.g. 2x2 or 3x3 squares, is a regular pattern distinctly different from arbitrary immobilized molecules. How many viral ligands are immobilized per spot depends on the size of the ligand and the size of the spot. It is possible to create spots with diameters of 5 run where even (only) single proteins can be immobilized.
  • the spot form is not restricted to squares or circular shapes. In this context diameter can be understood as the largest dimension of the spot on the plane of the surface.
  • the shape of the spots is rectangular or substantially rectangular (in contrast to less preferred spots which are substantially circular (which can in principle be created with the dip pen technology) but are more complex to form with AFM methods according to the present invention) .
  • Preferred spot diameters are less than 500 nm, preferably less than 300 nm, more preferred less than 100 nm, even more preferred less than 60 nm, most preferred less than 10 nm.
  • the antiadsorptive protein is in special embodiments bovine serum albumin, maltose-binding protein or a mixture thereof - especially binding marker labelled (e.g. His-tagged) variants of these proteins are immobilized specifically to the underlying layer.
  • BSA is regularly used as blocking agent in immunoassays and is readily available as byproduct of the beef industry.
  • other protein blocking agents e.g. other blood proteins of other sources can be used.
  • a characteristics of the antiadsorptive protein is the prevention of unspecific binding and maintaining a native environment which helps to stabilize proteins, in particular viral ligands and viruses.
  • the immobilized viral ligands are preferably proteins selected form viral receptors, i.e. proteins that interact with viral proteins such as ICAM-I or LDLR, viral proteins, e.g. in particular viral antigens or viral receptor binding proteins, and anti-viral antibodies or a determinant portion thereof.
  • the determinant portion is the portion of the ligand which is responsible for the interaction, e.g. the Fab portion of an antibody (which can also be produced recombinantly as single chain molecule) .
  • These can be either provided bound via linker or directly to the layer be- neath. Linkers normally increase the flexibility of a bound molecule.
  • topographical imaging flexible molecules can not be detected (therefore direct binding or using small molecular linkers is preferred for these applications) , however specific detection through measurement of the detachment force or oscillating AFM tips is possible using recognition imaging AFM (Stroh et al., 2004) .
  • the nanoarray comprises at least two, preferably four, more preferred at least 10, even more preferred at least 100, most preferred at least 1000, spots with different viral ligands.
  • at least 10000, 100000, 1 million or even 10 million spots per chip are possible due to the enormous miniaturisation possible with the nanoarray of the present invention. It is also feasible that such a number of spots (2, 4, 10, 100, 1000, 10000, 100000, 1 million or 10 million) have different viral ligands immobilized.
  • the spots can be immobilized in distinct patterns since the spatial control of the AFM tip during the removal of the antiad- sorptive protein is in the nm range.
  • the pattern of spots is a rectangular pattern of regular rows and columns of spots with at least 2, preferably at least 3, more preferred at least 5, even more preferred at least 8, rows or columns, including any combination thereof.
  • the number of rows and columns can be selected independently of each other and can also be at least 10, 20, 50, 100, 200, 500, 1000, 2000 or 10000.
  • the distance between individual spots is less than 1000 nm, preferably less than 500 nm, preferably less than 300 nm, more preferred less than 100 nm, even more preferred less than 60 nm, most preferred less than 10 nm, e.g. between 1 nm and 5 nm.
  • the nanoarray is provided on a chip surface, preferably a glass or silicon chip surface, especially a flat surface, in particular flat in the 0.1 nm to 10 nm range.
  • a chip surface preferably a glass or silicon chip surface, especially a flat surface, in particular flat in the 0.1 nm to 10 nm range.
  • nanoarray can be either used to refer to the nanometric arrangement or to the chip with the nanometric arrangement, similar to the usage of the term “microarray”, which is used for arrangements in the ⁇ m scale or chips comprising these arrangements .
  • viral ligands and the antiadsorptive proteins are provided in a single molecular layer, i.e. side by side on the surface area.
  • the nanoarray also comprises a gold layer in the most preferred embodiments.
  • This gold layer can be directly located on the chip support and is preferably 0.1 nm to 100 nm in thickness.
  • the nanoarray comprises a monolayer of self assembling monolayer molecules, preferably alkanethioles, preferably located on a gold layer.
  • Ultraflat gold substrates can be functionalized with a metal-chelating self-assembled monolayer, e.g. of alkane thioles for high-affinity capturing of His-tagged proteins and covered by a dense monolayer of immobilized his-tagged proteins.
  • a metal-chelating self-assembled monolayer e.g. of alkane thioles for high-affinity capturing of His-tagged proteins and covered by a dense monolayer of immobilized his-tagged proteins.
  • the antiadsorptive proteins and the viral ligands are located in a well-defined manner.
  • This layer may comprise binding elements for protein tags, e.g. metal chelating organic acid groups as disclosed by Tinazli et al. (2005), e.g.
  • thioalkanes modified with 2- r 3-, 4-, 5-, 6-, 7-, 8-, 9-carboxylic acid groups (multi valent chelator thiols) , or mixtures thereof (together with monovalent chelator thiols) .
  • One such modification is via grafting of N-nitrilotriacetic acid (NTA) groups, which are good chelators of His 6 -tags on proteins.
  • NTA N-nitrilotriacetic acid
  • the layer of viral ligands and antiadsorptive proteins is preferably detachably attached to the self assembled monolayer, preferably via biotin-avidin interaction or interaction of a His-tag with mono- or multivalent metal-chelating self assembling monolayer molecules.
  • the His-tag is preferably a His 6 -tag, but can also be a His 4 -i 6 -tag or His 6 -io-tag, or contain even more His residues. Through a strong AFM force (e.g. 15-30 nN) this attachment can be disengaged leaving the chelating monolayer free to bind other molecules (e.g. the viral ligands) .
  • a method for the manufacture of the nanoarray characterized in that a layer of antiadsorptive proteins is provided on a support, antiadsorptive proteins are removed in a pattern of discrete spots with the tip of an atomic force microscope (AFM) probe and viral ligands are immobilized on the discrete spots.
  • AFM atomic force microscope
  • the antiadsorptive proteins are preferably removed with a force of between 10 nN and 40 nN, preferably 15 nN and 30 nN, between the tip of the AFM probe and the support.
  • the viral ligands which fill the empty spots left by the removed antiadsorptive proteins preferably are viral receptors or anti-viral antibodies.
  • the viral receptors can be used in the buffer solution which is used during the removal or afterwards.
  • the removal of antiadsorptive proteins is performed in an aqueous solution, preferably a buffered water solution or buffered aqueous solution with physiological ionic strength.
  • the viral ligands are provided in the aqueous solution for binding to the layer beneath (e.g. the multivalent metal chelating NTA monolayer) .
  • the nanoarray may comprise a multitude of spots as indicated above, e.g. at least two, preferably four, more preferred at least 10, even more preferred at least 100, most preferred at least 1000, spots with different immobilized viral ligands.
  • the ligands are preferably provided in different solutions, which are sequentially used to contact the support after in one (or the desired amount of spots with identical viral ligands) spot the antiadsorptive proteins have been removed. This cycle of protein removal and attachment of viral ligands can be sequentially repeated for all spots.
  • a monolayer of self assembling monolayer molecules preferably alkanethioles, preferably located on a gold layer on the support, is provided between the layer of antiadsorptive proteins and the support, preferably by self assembly in a liquid, fluid or gaseous medium.
  • the thioles readily attach to a gold surface, but other monolayers can be used as well to provide a molecular or even atomic flat surface suitable to immobilize further proteins.
  • the monolayer of self assembling monolayer molecules remains intact. This can be achieved by a suitable "scratching" or displacement force e.g. 15-30 nN. This force of course may vary depending on the interaction forces between the proteins and molecules and the shape and constitution of the AFM tip .
  • the present invention provides a method for the detection and identification of viruses, characterized in that a sample potentially comprising a virus is contacted with the nanoarray of the present invention, wherein the viral ligands located in a spot are specific for one virus, binding of the potentially present virus on a spot and detecting the virus with AFM measurement.
  • This AFM measurement is preferably a topographical AFM measurement, whereby the virus is identified by assignment of the detected virus on a spot with viral ligands specific for the virus, since it is known which ligands are located on which spot.
  • Virions virus particles
  • Virions can be further characterized and detailed information of their biophysical properties obtained. It is also possible to assign a virus group or family to the detected virus based on measured shape and appearance.
  • the AFM measurement is performed preferably in a liquid medium such as a physiological water/buffer solution.
  • a liquid medium such as a physiological water/buffer solution.
  • This medium can of course also be fluid and comprises a supercritical medium as well.
  • the AFM probe is preferably modified with a viral ligand, preferably an antibody and the AFM measurement method is recognition imaging.
  • recognition imaging further characteristics can be evaluated, e.g. by using serotype specific antibodies it is possible to further characterize an immobilized virus species by measuring individual force profiles in tapping mode or oscillation mode.
  • the viral ligands are specific for a virus group, virus family, virus subfamily, a virus genus, a virus species, a virus subspecies or a virus serotype for viral classification.
  • Another application of the nano-array of different receptors is the parallel investigation of virus-receptor interactions, realized by the immobilization of single viral particles to the AFM-tip.
  • a method for the characterization of viral ligands or a virus is provided, wherein a virus is immobilized on an AFM probe tip and is used for recognition imaging AFM and force measurements on a nanoar- ray of viral ligands.
  • potential drug candidates can be quickly screened by measuring the interaction profiles of the virus bound AFM-tip and the viral receptors on the chip surface, in presence and absence of the antiviral drugs within the liquid cell of the AFM.
  • the viral ligand is provided on a flexible linker to enable various spatial conformations for the interaction with the virus, which is preferably a complete virus particle or a viral antigen or functional determinant, e.g. responsible for natural interaction with a host cell.
  • the AFM measurement is performed in a liquid medium, preferably aqueous medium.
  • the medium can be for example a physiological buffer or salt solution.
  • antiviral drugs are present in the liquid medium. This facilitates a competitive assay with viral ligands on the chip surface, the virus on the AFM or scanning probe and antiviral drugs in the liquid medium. Thereby especially strong virus-binding viral ligands can be detected and assayed, at least in the range of the used antiviral drugs. Alternatively, potential antiviral drugs with unknown binding power to the virus can be assayed in comparison to specific viral ligands by the competitive AFM . measurement .
  • Figure 1 Patterns with various numbers of spots fabricated within the anti-adsorptive layer and imaged with the AFM.
  • A Topographical image of the anti-adsorptive protein-layer with a surface roughness of about 1 nm.
  • B A single rectangular element (800 nm x 800 nm) was fabricated into the anti-adsorptive layer.
  • C Two rectangular elements (each element with 200 nm x 200 nm in size) fabricated and imaged.
  • D 2 x 2 array of rectangular elements (each element 300 nm x 300 nm) .
  • E 2 x 2 array of rectangular elements (each element 200 nm x 200 nm) .
  • F 3 x 3 array of rectangular elements (each element 300 nm x 300 nm) .
  • FIG. 1 Refilling the spots with virus receptors.
  • A Sketch showing the Au surface with the self assembled monolayer and the anti-adsorptive protein layer. The AFM tip is used to fabricate the holes (left panel) . The holes are filled up with viral receptors (right panel) .
  • B A 2 x 2 array of holes before receptor filling (left panel) and after injection of virus receptors into the liquid cell of the AFM (right panel) . The holes are filled up by the receptors.
  • C The final prototype will be 5 x 5 array, i.e. 25 elements carry a different type of viral re ⁇ ceptor.
  • FIG. 3 Detection of viral particles using topographical imaging.
  • A Topographical image of a 2 x 2 array of 200 nm wide holes scratched into a protein (BSA) layer (left panel) .
  • B The holes are refilled with LDLR receptors (middle panel) .
  • C After injection of a solution containing human rhinovirus particles (HRV) , specific binding on the preformed pattern is obtained. Roughly 60 HRV particles can be counted on each element.
  • HRV human rhinovirus particles
  • D Topographical image of a single rotavirus particle and cross-section profile.
  • E Topographical image of a single rotavirus particle where the genomic DNA protrudes from the viral capsid.
  • F Scheme of the detection of single viral particles.
  • Figure 4 Recognition imaging of HRV particles with antibodies attached to the AFM-tip.
  • A Topographical imaging of a dense layer of HRV particles using an antibody modified tip.
  • B Simultaneously acquired recognition image showing the black spots on virus particles that were recognized by the antibody on the tip.
  • C Black recognition spots disappear when the virus-antibody interaction is blocked, proofing the specificity of the recognition signal.
  • FIG. 1 Parallel screening of virus-receptor interactions.
  • A Single viral particles are specifically attached to the AFM-tip, using a three-step chemical protocol. The virus-modified tip can be used for the parallel screening of virus-receptor interactions, in presence and absence of antiviral drugs.
  • B Interaction force measurements of virus particles attached to the AFM- tip and receptor molecules immobilized to the surface.
  • Examples The following demonstrates: 1) the fabrication of nano-ar- rays of different viral receptors under physiological conditions, 2) the application of the nano-array for the detection of single viral particles using topographical imaging, 3) a further characterization of virus particles on the virus-chip using recognition imaging, and 4) the use of the nano-array for the parallel investigation of virus-receptor interactions.
  • Example 1 Fabrication of a nano-array of different viral receptors
  • a nano-array of different receptors was fabricated using AFM-based native protein nanolithography combined with a flow- through liguid cell.
  • ultraflat gold substrates are covered with a self-assembled monolayer (SAM) of multivalent metal-chelating (multi-NTA) alkane-thioles that are suitable for high affinity immobilization of hexahistidine- tagged (His 6 ) proteins.
  • SAM self-assembled monolayer
  • multi-NTA multivalent metal-chelating alkane-thioles
  • His 6 hexahistidine- tagged
  • his-tagged proteins form a densely packed self-assembled protein monolayer on the chelating SAM and are used as a biocompatible and ductile patterning material, termed "structuring matrix".
  • His 6 - tagged proteins either maltose-binding protein (MBP) or bovine serum albumin (BSA) , were specifically immobilized on the multivalent metal-chelating SAM.
  • Figure IA shows a topographical image of the resulting protein layer.
  • the second step is the production of a nano-pattern with precise control over the pattern size and geometry.
  • the patterns were produced within the structuring matrix using AFM-based native protein nanolithography.
  • a high force typically 15 - 30 nN
  • a strongly reduced imaging forces typically ⁇ 100 pN
  • the generated nanostructures in the protein layer can be characterized in situ by repeated imaging of the same surface area.
  • Figure IB shows a single rectangular element (800 x 800 nm 2 ) structured into the self-assembled protein layer with a depth of ⁇ 5 nm.
  • FIG. 1C shows two rectangular elements fabricated into the structuring matrix, and it is straightforward to fabricate 2 x 2 and 3 x 3 arrays ( Figure ID, E, and F) .
  • the lateral dimensions of the islands can be easily adjusted from 10 nm to 1 ⁇ m, and arrays with up to 5 x 5 elements can be fabricated within several minutes .
  • the patterns can be refilled specifically with virus receptors ( Figure 2) . Therefore, His 6 -tagged receptor molecules are injected into the liquid cell which results in specific immobilization to the multivalent chelator groups of the SAM ( Figure 2A) .
  • Figure 2B, left panel shows a 2 x 2 array of rectangular nanostructures in the self-assembled protein monolayer using native protein nanolithography.
  • His 6 -tagged viral receptors low-density lipoprotein receptors, LDLR
  • the structures are filled up with receptors as observed in the cross-section profile ( Figure 2B, right panel) .
  • native protein nanolithography was combined with a flow-through liquid cell.
  • Example 2 Detection of single virus particles using topographical imaging
  • the nano-array of viral receptor molecules can be used for the detection of single virus particles (Figure 3).
  • Figure 3A After the nanopatterns had been arrayed (Figure 3A) and refilled with LDL receptors (Figure 3B), single human rhinovirus particles (HRV) were detected on the preformed pattern ( Figure 3C) .
  • HRV single human rhinovirus particles
  • Figure 3C single human rhinovirus particles
  • a virus sample was injected and the time-resolved binding of single HRV particles observed on topographical images. Specific binding of viruses was obtained only on areas where receptor molecules had been immobilized.
  • On each element of the 2 x 2 array roughly 60 virus particles were counted with diameters of 30 nm (see cross-section profile, lower panel in Figure 3C) .
  • FIG. 3D shows a topographical image of a single rotavirus particle (causing human diarrhea) with a signal-to-noise ratio of ⁇ 100 (see corresponding cross- section profile) .
  • the diameter of the rotavirus is 70 nm, which is significantly larger than the diameter of the rhinovirus particle (30 nm) , showing that it is possible to discriminate viruses from different families solely by their size, geometry and capsid structure.
  • Figure 3E shows a single rotavirus particle where the genomic DNA protrudes from the capsid, indicating that this virus particle is not infectious anymore. Hence, relevant information for medical diagnosis can be obtained on the single particle level.
  • Example 3 Multiplexing the read-out of the virus-chip using recognition imaging
  • the virus-chip can be read out using the recently developed technique of recognition imaging (Stroh et al., 2004).
  • ligand molecules e.g. antibodies, drugs
  • a multiplexed read-out of the virus chip is realized based on the molecular interactions between tip-bound ligands and immobilized virions.
  • various parameters like cross-reactivities, binding probabilities, as well as kinetic and energetic parameters of virus-ligand interactions can be measured in a single experiment (Kienberger et al., 2006). For instance, this information can be used to discriminate different serotypes within a single virus family where the geometry and capsid structure is the same for all members.
  • FIG 4 shows an example where recognition imaging has been applied to rhinovirus particles. Neutralizing antibodies had been bound to the AFM-tip and simultaneous topography (Figure 4A) and recognition images (Figure 4B) were acquired. In the recognition image, viral particles that have been recognized by the tip-bound antibody appear black ( Figure 4B, arrows) . In order to proof that the black spots correspond indeed with specific molecular recognition, the interaction was blocked resulting in abolishment of the reconition spots ( Figure 4C) . In this way, recognition imaging can be used to distinguish different virus particles on the basis of their binding properties to the ligand molecules on the AFM-tip.
  • Example 4 The nano-array of different receptors used for parallel screening of virus-receptor interactions
  • the AFM-tip was modified with ligands in order to get further information of the virus particles that had been detected with the virus chip.
  • a modification of the AFM-tip with single viral particles allows for the parallel investigation of single virus-receptor interactions.
  • a nano-array of different receptors is constructed and read out via a virus-modified tip.
  • 25 different (in case of a 5 x 5 array) virus-ligand interactions can be studied using force-volume, recognition imaging or any other force-measuring mode. The measurements can be carried out in the presence and absence of antiviral drugs.
  • Figure 5 shows force measurements of a virus-modified AFM- tip and receptor molecules that are immobilized to the surface.
  • Figure 5A shows a sketch of the chemistry used for specifically binding single viral particles to the AFM-tip. Single-molecular interaction forces between the virus and immobilized receptor molecules can then be measured in force-distance cycles (Figure 5B) .
  • the parabolic shaped signal in the retrace experiment shows the unbinding force of a single rhinovirus-receptor interaction. The specificity is shown by blocking the interaction resulting in a loss of the binding signal (Figure 5B, inset) .
  • Virus identification is thereby based on type-specific im- munocapture and the morphological properties of the captured viruses obtained by the AFM. Virus particles can thereby be detected either from clarified cell lysates, body fluids, or environmental samples. Detection and identification will be at the level of single virus particles. Using recognition imaging, further characterization of the detected virus particles can be obtained.
  • the nano-array of different host-cell receptors will be used to measure and characterize various virus-receptor binding interactions in a single scan.
  • the parallel screening of virus-receptor interactions at the single molecule level in the absence and presence of antiviral drug is optimally suited to improve current entry inhibitors and antiviral drugs.
  • the nano-array viewed as the melding of a nanotechnological tool (AFM) with biotechnology (solid phase immuno-capture of virus particles) is suggested as a clinically relevant platform for the detection and characterization of single viral particles in a variety of samples without the need of labelling and pre-de- rivatization of viruses.
  • AFM nanotechnological tool
  • biotechnology solid phase immuno-capture of virus particles

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Abstract

L'invention concerne un nanoréseau de ligands viraux immobilisés dans un motif de points, le diamètre desquels est inférieur à 1000 nm, et dans lequel les points sont encastrés dans une couche de protéines anti-adsorbantes qui englobe la zone située entre les points. L'invention concerne également un procédé de fabrication de ce nanoréseau et son utilisation analytique spécifique dans une mesure AFM.
PCT/AT2007/000264 2006-06-02 2007-06-01 Nanoréseau de virus WO2007140497A1 (fr)

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CN113265379A (zh) * 2021-05-14 2021-08-17 厦门依加成科技有限公司 一种表面具有功能化斑点的微球及其制备方法和应用
CN113265379B (zh) * 2021-05-14 2022-04-29 厦门依加成科技有限公司 一种表面具有功能化斑点的微球及其制备方法和应用
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