A real time S1 assay at neutral pH based on graphene oxide quenched fluorescence probe

• A sensitive and low-cost fluorescence sensor for S1 assay at neutral pH was constructed.
• This is the first systematic study about the activity assay and kinetics study of S1 at neutral pH.
• The strategy has been successfully used to search partial effectors of S1 nuclease activity in vitro.

As the extracellular nuclease of Aspergillus, S1 nuclease can split single and double-stranded DNA into oligo- or mononucleotides, while preferentially digests single-stranded nucleic acids. Furthermore, the existence of S1 can be the standard to identify Aspergillus and used to evaluate the severity of Aspergillosis. Herein, a simple and sensitive fluorescent sensing platform for S1 assay was developed based on the S1-induced DNA strand scission and the difference in affinity of graphene oxide (GO) for single-stranded DNA containing different bases. This platform was applied to monitor S1 activity and study the kinetics in real time. Results indicated that the detection limit is 0.5 U/mL. The Km and kcat at 45 °C, are 1.4 ± 0.12 μM and 0.6 min− 1, respectively. Moreover, by monitoring the effect of chemical drugs on S1 activity, we found that 2 mM of erythromycin, sodium penicillin, carbenicillin disodium and ampicillin can inhibit S1 activity about 8%, 60%, 61% and 66%, respectively, while gentamycin sulfate is a stimulator. Overall, the assay platform based on graphene oxide quenched fluorescence probe is successfully constructed to study the enzymatic activity of S1 and used for screening antibiotics.