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

•A reusable ratiometric fluorescent biosensor for Cys has been developed.•Cys in serum sample could be detected sensitively and selectively.•The sensing system could be reused time after time with excellent repeatability.•The ratiometric strategy could eliminate false positive and false negative results.•The sensing assay offers an effective “mix-then-test” way for Cys detection. 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.