Silver ion-regulated ratiometric fluorescence assay for alkaline phosphatase detection based on carbon dots and o-phenylenediamine

In this work, a novel silver ion (Ag+)-regulated ratiometric fluorescence method for the effective and sensitive determination of alkaline phosphatase (ALP) was established based on carbon dots (CDs) and o-phenylenediamine (OPD). OPD can be oxidized by Ag+ to generate fluorescent 2, 3-diaminophenazine (DAP). Thus, based on inner-filter effect (IFE) or/and fluorescence resonance energy transfer (FRET) between CDs and DAP, the CDs-Ag+-OPD system can generate dual-emission at 454 nm and 570 nm respectively when excited at 360 nm. The introduction of ascorbic acid (AA) can react with Ag+ to produce dehydroascorbic acid (DHAA), which inhibits the generation of DAP, resulting in the fluorescence decrease at 570 nm and fluorescence recovery of CDs at 454 nm. Meanwhile, DHAA can react with OPD to generate quoxaline (QX), which emits strong blue fluorescence at 440 nm, further inhibiting the IFE or/and FRET between CDs and DAP. An obvious ratiometric fluorescence response was observed with the increase of the concentration of AA introduced. Due to the fact that AA can be generated by the enzyme catalysis reaction between ALP and 2-phospho-l-ascorbic acid (AAP), the CDs-Ag+-OPD ratiometric system was applied to the determination of ALP successfully. The ratiometric fluorescence value of F454/F570 increases with increasing ALP concentration, with a linear range of 0.2 to 40 U/L and detection limit of 0.1 U/L. In addition, the CDs-Ag+-OPD ratiometric system was successfully applied to the detection of ALP in human serum samples.