Effect of Polystyrene Microsphere Surface to Fluorescence Lifetime Under Two-Photon Excitation

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.

Quantitative Analysis of In Situ Locked Nucleic Acid and DNA Competitive Displacement Events on Microspheres

“…Importantly, all dsprobes studied here, including the flipped system, were designed to keep the toehold segment closest to the 5′ end of the immobilized sequence. To assuage concerns regarding the labeled primary target in the flipped dsprobe system that the proximity of the FAM fluorophore to the penultimate 3′ guanine residue as well as to the microsphere surface could lead to quenching of the FAM signal, 68 trial runs employed soluble L 3 B9F primary target sequences with a 3′ FAM fluorophore both with and without alkane spacers of 3−6 carbon atoms (data not shown). Surprisingly, the primary duplex densities obtained with these modifications that shift the FAM fluorophore further away from the microsphere surface resulted in even lower duplex densities than those observed with the original 5′ FAM-labeled L 3 B9F target for the flipped immobilized strand.…”

Conjugation of (E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine to Hydrophilic Microspheres: Development of a Mobile Microscale UV Light Actinometer

“…In all cases, the conjugation reaction was either inefficient or there was considerable quenching of the photoreaction and/or fluorescence. Problems associated with quenching when fluorophores are closely spaced within a matrix or on a surface are well-recognized – .…”

Sensitivity of fluorophore-quencher labeled microbubbles to externally applied static pressure

“…The theory in using F-Q labeled microbubbles as a pressure sensor is that variation in the externally applied pressure induces a bubble's radius change, [18][19][20][21][22][23][24][25][26][27][28][29] and the bubble's radius change results in a change in distance between the fluorophore and its quencher; this change in the distance between these molecules causes a shift in quenching efficiency and an alteration of the fluorescence lifetime and quantum yield. [30][31][32][33][34] By imaging the fluorophore lifetime ͑or emission intensity͒, the pressure distribution may be potentially quantified. The feasibility of this technique is highly dependent on the sensitivity of the bubble system to hydrostatic pressure.…”