A reusable ratiometric fluorescent biosensor with simple operation for cysteine detection in biological sample

Highly sensitive detection of cysteine is critical for early warning and diagnosis of cysteine-associated diseases. For the first time, herein, a reusable and ratiometric fluorescent biosensor with simple operation was developed for sensitive and rapid detection of Cys on the basis of configuration transformation of guanine-rich DNA (G-DNA). The sensing system is quite simple and consisted of G-DNA and Ag+ with NMM (N-methylmesoporphyrin IX) and terbium ion (Tb3+) as the two fluorescence probes. The Ag+ assists the G-DNA to keep its random single-strand structure (ssG-DNA), generating a distinct fluorescence of Tb3+ and a faint fluorescence of NMM. The analyte of cysteine (Cys) can strongly coordinate with Ag+, thus, the ssG-DNA is transferred into G-quadruplex (G4) configuration. The fluorescence of NMM is then outstandingly augmented, while the fluorescence of Tb3+ is significantly depressed. With the fluorescent intensity ratio of FNMM/FTb as readout signal, the detection limit of the developed ratiometric fluorescent biosensor reaches as low as 4.1 nM, confirming the excellent sensitivity. After completing the Cys detection, Ag+ is further added to disrupt the G-quadruplex into random single-strand G-DNA, which restores the sensing system into the initial state. The reversible configuration change between G-quadruplex and single-stand G-DNA endows the as-developed sensing system with charming reusability for specific detection of Cys. The developed sensing assay is cost-effective and operated in an enzyme-free and label-free condition, providing a straightforward and effective “mix-then-test” strategy to meet the requirement for practical application in disease monitoring and diagnosis.