Abstract
Molecular assays such as immunoassays are often performed using solid carriers and fluorescent labels. In such an assay format a question can be raised on how much the fluorescence of the label is influenced by the bio-affinity binding events and the solid carrier surface. Since changes in fluorescence intensity as labels bind to surfaces are notoriously difficult to quantify other approaches are preferred. A good indicator, independent of the fluorescence intensity of the label, is the fluorescence lifetime of the marker fluorophore. Changes in fluorescence lifetime reliably indicate the presence of dynamic quenching, energy transfer or other de-excitation processes. A microsphere based assay system is studied under two-photon excitation. Changes in fluorescence lifetime are studied as labeled protein conjugates bind on microsphere surfaces – both direct on the surface and with a few nanometer distance from the surface. Fluorescence signal is measured from individual polystyrene microspheres and the fluorescence lifetime histogram is simultaneously recorded. The results indicate that self-quenching and quenching by the polystyrene surface are both present in such a system. However, the effect of the surface can be avoided by increasing the distance between the surface and the label. Typical distances achieved by a standard sandwich type of assay, are already sufficient to overcome the surface induced quenching in fluorescence detection.
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Acknowledgement
This work was supported by Finnish Graduate School of Chemical Sensors and Micro Analytical Systems, and Turku University Foundation (M.T.), Academy of Finland (R.W. and P.H.), Arctic Diagnostics Inc. and European Commission (N.M. and J.T.).
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Tirri, M.E., Wahlroos, R., Meltola, N.J. et al. Effect of Polystyrene Microsphere Surface to Fluorescence Lifetime Under Two-Photon Excitation. J Fluoresc 16, 809–816 (2006). https://doi.org/10.1007/s10895-006-0124-6
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DOI: https://doi.org/10.1007/s10895-006-0124-6