title: Molecular Occupancy of Nanodot Arrays.
abstract: Single-molecule nanodot arrays, in which a biomolecule of choice (protein, nucleic acid, etc.) is bound to a metallic nanoparticle on a solid substrate, are becoming an increasingly important tool in the study of biomolecular and cellular interactions. We have developed an on-chip measurement protocol to monitor and control the molecular occupancy of nanodots. Arrays of widely spaced nanodots and nanodot clusters were fabricated on glass surfaces by nanolithography and functionalized with fluorescently labeled proteins. The molecular occupancy was determined by monitoring individual fluorophore bleaching events, while accounting for fluorescence quenching effects. We found that the occupancy can be interpreted as a packing problem, and depends on nanodot size and binding ligand concentration, where the latter is easily adjusted to compensate the flexibility of dimension control in nanofabrication. The results are scalable with nanodot cluster size, extending to large area close packed arrays. As an example, the nanoarray platform was used to probe the geometric requirement of T-cell activation at the single-molecule level.
authors.forename: Haogang
authors.initials: H
authors.lastname: Cai
authors.forename: Haguy
authors.initials: H
authors.lastname: Wolfenson
authors.affiliation: The Kennedy Institute of Rheumatology, University of Oxford , Oxford, OX3 7FY, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Depoil
authors.affiliation: The Kennedy Institute of Rheumatology, University of Oxford , Oxford, OX3 7FY, United Kingdom.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Dustin
authors.forename: Michael P
authors.initials: MP
authors.lastname: Sheetz
authors.forename: Shalom J
authors.initials: SJ
authors.lastname: Wind
mesh_terms: D001710:Biotin / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006046:Gold / Q000737:chemistry; D006801:Humans; D008213:Lymphocyte Activation; D053768:Metal Nanoparticles / Q000737:chemistry*; D046228:Microarray Analysis / Q000379:methods; D010165:Palladium / Q000737:chemistry; D010316:Particle Size; D011485:Protein Binding; D000072760:Single Molecule Imaging / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013499:Surface Properties; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D005456:Fluorescent Dyes; D013438:Sulfhydryl Compounds; D010165:Palladium; D001710:Biotin; D006046:Gold; D019809:Streptavidin
keywords: electron beam lithography; fluorescence quenching; molecular occupancy; photobleaching; single-molecule assays; stoichiometry