Discriminative intracellular and extracellular ATP detection based on magnetically controlled antimicrobial peptide

摘要

Extracellular free adenosine triphosphate (ATP) is often encountered as an interferent in the detection of intracellular ATP, especially in biofilm systems. To resolve this problem, in this study, a novel impedimetric aptasensor was designed for the discriminative detection of intracellular and extracellular ATP using antimicrobial peptide-functionalized magnetic particles (M-AMPs). Successive fabrication of reduced graphene sheets (rGSs) and gold nano-clusters (Au NCs) on a glassy carbon electrode was conducted by simple electrodeposition methods to achieve improved detection sensitivity. Single-stranded aptamers were attached to the surface of Au NCs for selective ATP binding, and electrochemical impedance spectroscopy was conducted to analyze the change of surface state induced by ATP binding. For discriminative intracellular and extracellular ATP detection, magnetically controlled M-AMPs were introduced into the detection system for mild and rapid bacterial disintegration. In bacterial samples, the M-AMPs were firstly constrained by a magnetic field, and extracellular ATP was selectively bound to the prepared aptasensor, leading to increased impedance response. Thereafter, the magnetic field was shut down to liberate the M-AMPs for cell disruption, and the released intracellular ATP was determined by the further impedance increasement. The results demonstrated that the proposed aptasensor exhibited excellent sensitivity and selectivity, with a wide linear range from 1 nM to 100 nM and a detection limit of 0.65 nM. Remarkably, this electrochemical aptasensor provided a convenient and label-free approach for discriminative intracellular and extracellular ATP detection, and the detection reliability was verified in various bacterial samples and E. coli biofilms.