Nöll Junior Research Group, Organic Chemistry, Chem. Biol. Dept., Faculty IV, Siegen University, Adolf-Reichwein-Str. 2, 57068 Siegen, Germany.
Analyst. 2016 Oct 21;141(20):5784-5791. doi: 10.1039/c6an01129b. Epub 2016 Aug 3.
For the detection of oligonucleotides a sandwich-like detection strategy has been developed by which the background fluorescence is significantly lowered in comparison with surface-bound molecular beacons. Surface bound optical molecular beacons are DNA hairpin structures comprising a stem and a loop. The end of the stem is modified with a fluorophore and a thiol anchor for chemisorption on gold surfaces. In the closed state the fluorophore is in close proximity to the gold surface, and most of the fluorescence is quenched. After hybridization with a target the hairpin opens, the fluorophore and surface become separated, and the fluorescence drastically increases. Using this detection method the sensitivity is limited by the difference in the fluorescence intensity in the closed and open state. As the background fluorescence is mainly caused by non-quenched fluorophores, a strategy to reduce the background fluorescence is to cut the beacon in two halves. First a thiolated ssDNA capture probe strand (first half) is chemisorbed to a gold surface together with relatively short thiol spacers. Next the target is hybridized by one end to the surface-anchored capture probe and by the other to a fluorophore-labeled reporter probe DNA (second half). The signal readout is done by surface plasmon fluorescence spectroscopy (SPFS). Using this detection strategy the background fluorescence can be significantly lowered, and the detection limit is lowered by more than one order of magnitude. The detection of a target takes only a few minutes and the sensor chips can be used for multiple detection steps without a significant decrease in performance.
为了检测寡核苷酸，开发了一种夹心式检测策略，与表面结合的分子信标相比，这种策略大大降低了背景荧光。表面结合的光学分子信标是包含茎和环的 DNA 发夹结构。茎的末端用荧光团和硫醇锚修饰，用于化学吸附在金表面上。在闭合状态下，荧光团与金表面接近，大部分荧光被猝灭。与靶标杂交后，发夹打开，荧光团和表面分离，荧光急剧增加。使用这种检测方法，灵敏度受到闭合和打开状态荧光强度差异的限制。由于背景荧光主要是由未猝灭的荧光团引起的，因此降低背景荧光的策略是将信标切成两半。首先，将巯基化的 ssDNA 捕获探针链（上半部分）与相对较短的硫醇间隔物一起化学吸附到金表面上。接下来，目标通过一端与表面锚定的捕获探针杂交，通过另一端与荧光标记的报告探针 DNA（下半部分）杂交。信号读出是通过表面等离子体荧光光谱法（SPFS）完成的。使用这种检测策略，可以显著降低背景荧光，并且检测限降低了一个数量级以上。目标的检测只需几分钟，并且传感器芯片可以在不显著降低性能的情况下用于多个检测步骤。
