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. Author manuscript; available in PMC: 2013 Nov 6.
Published in final edited form as: Anal Chem. 2012 Oct 29;84(21):9379–9387. doi: 10.1021/ac302165h

Figure 4.

Figure 4

Comparison of FRET analysis in bulk (solution) and printed array spots on solid substrate for two different orientation of dye (as described in Figure S-1). A) Oligo DNA target Cy5 emission shift indicating FRET in bulk solution duplexes for 5’-Cy3-oligo1-probe:Oligo2–3’-Cy5-target (Case 2 dye on the same side of duplexes indicated here by dotted line) and not for 5’-Cy3-oligo1-probe:5’-Cy5-oligo2-target (Case 1 with anti-parallel dye duplexes indicated by solid line). B) Single spot analysis of fluorescence signals for Cy3 probe and Cy5 target emissions from surface capture assays monitored in real-time by confocal microscopy for 5’-Cy3 oligo1-probe:5’-Cy5-Oligo2-target parallel duplex (dotted lines) and for 5’-Cy3 oligo1-probe:Oligo2–3’-Cy5-target antiparallel duplex (solid lines) indicating FRET with increasing Cy5 emission intensity and simultaneously decrease in Cy3 probe intensity. Solution-phase FRET occurs in (A) for two fluorophores in close proximity (i.e., on same duplex ends) whereas single spot array assays (B) indicated FRET for both duplex dye orientations upon hybridization in printed spots.