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Detecting sequential bond formation using three-dimensional thermal fluctuation analysis

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    0333759 - ÚPT 2010 RIV DE eng J - Journal Article
    Bartsch, T. - Fisinger, S. - Kochanczyk, M.D. - Huang, R. - Jonáš, Alexandr - Florin, E. L.
    Detecting sequential bond formation using three-dimensional thermal fluctuation analysis.
    ChemPhysChem. Roč. 10, 9-10 (2009), s. 1541-1547. ISSN 1439-4235
    Institutional research plan: CEZ:AV0Z20650511
    Keywords : biophysics * mechanical properties * optical tweezers * single-molecule studies * thermal fluctuations
    Subject RIV: BO - Biophysics
    Impact factor: 3.453, year: 2009

    We present a novel experimental method that solves two key problems in nondestructive mechanical studies of small biomolecules at the single-molecule level, namely the confirmation of single-molecule conditions and the discrimination against nonspecific binding. A biotin avidin ligand-receptor couple is spanned between a glass slide and a 1 mu m latex particles using short linker molecules. Optical tweezers are used to initiate bond formation and to follow the particles thermal position fluctuations with nanometer spatial and microsecond temporal resolution. Here we show that each step in the specific binding process leads to an abrupt change in the magnitude of the particle's thermal position fluctuations, allowing us to count the number of bonds formed one by one. Moreover, three-dimensional position histograms calculated from the particle's fluctuations can be separated into well-defined categories reflecting different binding conditions.
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