Adenosine deaminase biosensor combining cationic conjugated polymer-based FRET with deoxyguanosine-based photoinduced electron transfer.

Abstract

We demonstrated a sensitive and selective adenosine deaminase (ADA) detection by modulating the fluorescence resonance energy transfer (FRET) between cationic conjugated poly(9,9-bis(6'-N,N,N-trimethylammonium) hexyl)fluorine phenylene) (PFP) and the deoxyguanosine-tailored hairpin aptamer. The hairpin aptamer was labeled with a fluorophore FAM at one end and three deoxyguanosines (Gs) at the other end as a quencher. In the absence of ADA, aptamer forms hairpin-like conformation with adenosines making close affinity of Gs and FAM, which results in the weak FRET from PFP to FAM because of FAM fluorescence being quenched by Gs via photoinduced electron transfer (PET). After addition of ADA, adenosine was hydrolyzed by ADA, followed by the release of free aptamer. In this case, FAM being far away from Gs, the strong FRET thus was obtained due to the quenching process being blocked. Therefore, the new strategy based on the FRET ratio enhancement is reasonably used to detect the ADA sensitively, combining the fluorescence signal amplification of conjugated polymers with the initiative signal decreasing by Gs. The detection limit of the ADA assay is 0.3 U/L in both buffer solution and human serum, which is more sensitive than most of those previously documented methods. Importantly, the assay is rapid, homogeneous, and simple without a complicated treating process. The ADA inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA), was also studied based on this assay, and the detection limit of EHNA is 10 pM. This strategy provides a new platform for the detection of other biomolecules and enzymes.