Versatile nanozymes with fascinating catalytic properties provide inspiring and effective options for biosensing and pharmaceutical analysis. Herein, we report the first nanozyme-based ratiometric fluorescent platform for cysteine (Cys) and bleomycin (BLM) detection by harnessing the cost-effective and "mix & act" G-quadruplex/Cu(II) (G4/Cu) metal-nanozyme with satisfactory peroxidase-like activity, which was fully proven by circular dichroism (CD), electron paramagnetic resonance (EPR) spectra and reactive oxygen species (ROS) scavenging experiments. Based on the catalytic oxidation of G4/Cu metal-nanozyme toward two fluorescent substrates (Amplex Ultrared, AU; Scopoletin, Sc) with opposite responses in the presence of H2O2, and the specific interaction between Cu2+ and targets, we achieved the highly sensitive detection of Cys and BLM. Through recording the fluorescence changes of AU (emission at 590 nm, F590) and Sc (emission at 465 nm, F465), we obtained good linear relationships between ratiometric fluorescence values (F590/F465) and variable contents of targets, resulting in the competitive LODs of Cys (6.7 nM) and BLM (10 nM), respectively. Moreover, this platform presented high selectivity (without the need for masking agent) and acceptable performance in human serum samples. Furthermore, a library of DNA contrary logic pairs (CLPs) and multilevel concatenated circuits were fabricated based on the reverse dual-output of the above platform, enriching the building blocks of biocomputing. This work not only enlightened the design of affordable, "mix & act" type nanozyme-based ratiometric biosensors with high reliability, but also facilitated the pluralistic application of nucleic acid-templated nanozymes to innovative biocomputing.
Keywords: Cysteine and bleomycin; G-quadruplex; Mix & act” metal-nanozyme; Multilevel contrary logic computing; Ratiometric fluorescence.
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