Enzymatic determination of D-alanine using a cationic poly(fluorenylenephenylene) as the fluorescent probe and MnO2 nanosheets as quenchers.

A sensitive fluorometric assay is described here for sensitive determination of D-alanine (D-Ala). The method is based on the use of poly[9,9-bis(6-N,N,N-trimethylammonium)hexyl]fluorenylene phenylene (PFP) and manganese dioxide (MnO2) nanosheets. The blue fluorescence of PFP, peaking at 422 nm, is absorbed by the MnO2 nanosheets due to an inner filter effect (IFE). In the presence of D-Ala and D-α-amino acid oxidase (D-AAO), the enzymatic oxidation leads to the production of H2O2, triggering the decomposition of MnO2 nanosheets and the recovery of the fluorescence of PFP. Under the optimum conditions, the nanosensor has a wide linear range from 1.0 nM to 1.0 mM with a detection limit of 0.35 nM. This is about 10-100 times lower than most previously reported methods. The recovery experiment was performed with spiked serum and gave accuracy rates from 97.2 to 109%. The standard deviation is from 2.3% to 7.2%. Graphical abstract Schematic presentation of a sensitive "off-on" nanosensor for fluorimetric D-alanine detection.