A dual-signal sensing platform based on nanosheet materials for ratiometric fluorescence and colorimetric detection of enzyme activities in human blood

Abstract A dual-signal sensing platform comprising g-C3N4/MnO2 nanosheet composite and a routine chromogenic reagent o-phenylenediamine (OPD) was constructed for activity evaluation of several enzymes including Alkaline phosphatase (ALP), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) in human blood. MnO2 nanosheets was employed as a catalyst for oxidizing OPD to fluorescent 2,3-diaminophenazine (oxOPD), while g-C3N4 nanosheets served as a fluorescent indicator. MnO2 nanosheets also exhibited inner filter effect (IFE) on the fluorescence of g-C3N4 nanosheets due to the overlap of their respective absorption band and fluorescence emission band. Similarly, oxOPD would quench the fluorescence of g-C3N4 nanosheets via photoinduced electron transfer (PET) process. By using L-ascorbic acid-2-phosphate (AAP) and acetylthiocholine iodide (ATCh) as substrates, products of target enzyme-catalyzed hydrolysis reactions would reduce MnO2 nanosheets to Mn2+ cations, resulting in the inhibition of OPD oxidation and the fluorescence recovery of g-C3N4 nanosheets. Based on these reactions and effects, activities of target enzymes were indicated by the intensity ratio of fluorescence emitted from g-C3N4 nanosheets and oxOPD (F440/F560), as well as by the absorbance decrease of oxOPD at 420 nm (ΔA420). Limits of detection (LODs) obtained by the sensing platform for ALP, AChE, and BChE were respectively 0.28 U L-1, 0.19 U L-1, and 0.15 U L-1 for ratiometric fluorescence mode, and 1.3 U L-1, 1.0 U L-1 and 0.9 U L-1 for colorimetric mode. This dual-signal sensing platform can be extended for testing other enzymes after applying appropriate substrates for target enzyme-catalyzed hydrolysis reactions and demonstrates good potential for clinical diagnosis.