Raster image correlation spectroscopy as a novel tool for the quantitative assessment of protein diffusional behaviour in solution

Zahra Hamrang, Alain Pluen, Egor Zindy, David Clarke

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    Abstract

    The application of raster image correlation spectroscopy (RICS) as a tool for the characterisation of protein diffusion was assessed using a model protein, bovine serum albumin (BSA), as a function of formulation and denaturing conditions. RICS results were also validated against dynamic light scattering and fluorescence correlation spectroscopy. Results from this study demonstrate correlation between outputs obtained from the three experimental techniques. Ionic strength independency was observed at pH 7, and a reduction in the corresponding diffusion coefficients was noted at pH 4.5 for 1 μM BSA-Alexa Fluor 488. Conversely, at pH 5.2, higher-concentration samples exhibited ionic strength dependency. Buffer composition, sample pretreatment, thermal denaturation and freeze-thaw cycling were also found to influence RICS output, with a reduction in the diffusion coefficient and the number of particles observed for both pH values. In conclusion, RICS analysis of images acquired using a commercial confocal microscope offers a potential scope for application to both quantitative and qualitative characterisation of macromolecular behaviour in solution. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association.
    Original languageEnglish
    Article numberjps.23105
    Pages (from-to)2082-2093
    Number of pages11
    JournalJournal of Pharmaceutical Sciences
    Volume101
    Issue number6
    DOIs
    Publication statusPublished - Jun 2012

    Keywords

    • Albumin
    • Diffusion
    • Light scattering (dynamic)
    • Microscopy
    • PH
    • Protein aggregation
    • Raster image correlation spectroscopy

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