Label-Free Imaging of Flap Endonuclease 1 in Living Cells by Assembling Original and Multifunctional Nanoprobe.

Abstract

Flap endonuclease 1 (FEN1) becomes a potential tumor marker since it is closely related to cancer occurrence and development. Here, a poly dA20-mediated nanoprobe (AuNPs-poly dA20-poly dT20) was designed for FEN1 detection. Poly dA20 segment at the 3'- end of ssDNA adsorbed on AuNPs due to its strong affinity interaction with Au (stronger than Au-S bond), while the poly dT20 segment at the 5'- end overhangs. This nanoprobe not only worked as effective fluorescence quencher but also as the original nanosubstrate of FEN1. OliGreen adsorbed on poly dT20 emits strong green fluorescence because of its high sensitivity and selectivity toward thymine. However, it is quenched on the nanoprobe. In the presence of FEN1, it recognizes the overhanging poly dT20 segment and cleaves it efficiently, turning on the fluorescence of OliGreen. This indicates that the assembled nanoprobe is an effective artificial substrate to FEN1, although it is completely different from previously reported substrates that are all composed of dsDNA with a flap strand. This proposed nanoprobe was used to detect FEN1 not only in vitro but also in vivo. The method was simple, which avoided complex labeling procedures. It had a wide linear range from 0.05 U to 2 U, with the lowest detection limit of 0.007 U. Confocal imaging can distinguish cancer cells from normal cells, demonstrating its potential in clinical diagnostic and therapeutic monitoring.