Perylene diimide and ferrocene functionalized MnO₂ nanorods for ratiometric detection of alkaline phosphatase

A positively charged perylene diimide (PDI) with strong π-π interaction was employed alongside aminoferrocene (Fc-NH₂) as potential-resolved probes to construct a ratiometric biosensor. MnO₂/Au nanorods first served as nanocarriers to load both probes. The proposed sensing mechanism relies on alkaline phosphatase (ALP)-triggered generation of ascorbic acid (AA), which reduces MnO₂ nanorods to Mn²⁺, leading to the release of Fc-NH₂ from the electrode surface while concurrently enhancing the exposure of trimethylaminoethyl perylene diimide (TMPDI) strongly absorbed on GCE through π-π conjugation interaction and electrostatic attractive interaction. With increasing AA concentration, the absolute value of reduction peak current at 0.56 V significantly decreased, while the absolute value of reduction peak current at -0.37 V gradually increased. The ratiometric signal (I_TMPDI/I_Fc-NH2) exhibits an excellent linear relationship with ALP activity over a wide linear range from 0.01 to 400 U/L with a lower detection limit of 8.5 × 10⁻³ U/L and satisfactory recoveries in the application to human serum samples.