A sandwich-like strategy for the label-free detection of oligonucleotides by surface plasmon fluorescence spectroscopy (SPFS)

Abstract

Cutting surface-bound optical molecular beacons results in a sandwich-like detection strategy with lower background fluorescence.

Influence of the Thiol Anchor on the Orientation of Surface‐Grafted dsDNA Assemblies

“…To compare fluorophore‐labeled dsDNA assemblies of different length at the same capture probe modified surface, the previously mentioned sandwich‐like surface modification procedure was adopted . For sensor chip preparation, the thiol‐anchored ssDNA capture probes comprising 20 bases were chemisorbed in buffer solution for 60 min, followed by incubation with a mixture of MCB and MPA (0.5 m m each) in pure water for 30 min, before the surface was rinsed with buffer (2 m m Tris‐buffer at pH 7.5 containing 200 m m NaCl and 6 m m MgCl 2 was used for all measurements).…”

“…[9] For sensor chip preparation, the thiol-anchored ssDNA capturep robes comprising 20 bases were chemisorbed in buffer solution for 60 min, followed by incubation with am ixture of MCB and MPA( 0.5mm each) in pure water for 30 min, before the surface was rinsed with buffer (2 mm Trisbuffera tp H7.5 containing 200 mm NaCl and 6mm MgCl 2 was used for all measurements).A ss hown previously by using the capturep robe moleculese quipped with three dithiane rings, this procedure resultsi narather high capture probe surface coverage of 1.6 10 À11 mol cm À2 ,l eading to ac enter-to-center distance of 3.2 nm between two adjacent capturep robe strands, when surface roughness is neglected. [9] By following the same procedure, as urface coverage of 2.5 10 À11 mol cm À2 and ac enter-to-center distance of 2.6 nm between two adjacent ssDNA capture probe strands was estimated, when as ingle mercaptohexyl anchor was appliedi nstead( neglecting surfaceroughness). [1a] For detection, the captureprobe was hybridized with the complementary end of at arget T n comprising 40, 60, or 80 bases, and the other ends of these targets were hybridized with ac omplementary Cy5-labeled reporter probe of 20 bases.…”

“…For the first SPFS measurements, shown in Figure 1, an ordinary sensor chip was prepared by chemisorptiono ft he capture probe strandse quipped with an (1,2-dithiane) 3 anchora s used previously. [9] After incubation with the target T 40 (prehybridized with an excess of reporter probe),t he increase in fluorescencew as already quite large, which can be explained by the assumption that the resulting dsDNA hybrid comprising 40 base pairs (bp) with al ength of about 14 nm stands on the surfacea tacertain angle. As mentioned before, the capture probe molecules are grafted to the surface at high density,r e-sulting in electrostatic repulsion between individual capture probe strands and the target-containing dsDNA structure.…”

“…By following this sandwich‐like detection procedure the detection limit could be lowered by more than one order of magnitude in comparison to conventional surface‐grafted MBs . As shown in this previous work, the background fluorescence before target binding had been minimized; thus, it was the goal in the current contribution to maximize the fluorescence intensity after target binding. For this purpose, the distance between fluorophore and gold surface was optimized; that is, a distance needed to be established at which fluorescence quenching is minimized and, at the same time, efficient fluorescence excitation is possible .…”

“…A strategy to lower the detection limit even further is to minimize the background fluorescence of the MBs in the closed state and/or to maximize the fluorescence in the open state. As the background fluorescence is mainly caused by MBs that are not entirely quenched, a successful strategy that has been demonstrated already is to cut the MBs in two halves, resulting in a sandwich‐like detection procedure as outlined in the first line of Scheme ( A → B ). For sensor chip preparation, a thiolated ssDNA capture probe (first half of the MB) was chemisorbed on to a gold surface.…”