Electrochemical and Quartz Crystal Microbalance Detection of the Cholera Toxin Employing Horseradish Peroxidase And GM1-Functionalized Liposomes

Abstract

An ultrasensitive method for the detection of the cholera toxin (CT) using electrochemical or microgravimetric quartz crystal microbalance transduction means is described. Horseradish peroxidase (HRP) and GM1-functionalized liposomes act as catalytic recognition labels for the amplified detection of the cholera toxin based on highly specific recognition of CT by the ganglioside GM1. The sensing interface consists of monoclonal antibody against the B subunit of CT that is linked to protein G, assembled as a monolayer on an Au electrode or an Au/quartz crystal. The CT is detected by a “sandwich-type” assay on the electronic transducers, where the toxin is first bound to the anti-CT-Ab and then to the HRP-GM1-ganglioside-functionalized liposome. The enzyme-labeled liposome mediates the oxidation of 4-chloronaphthol (2) in the presence of H₂O₂ to form the insoluble product 3 on the electrode support or the Au/quartz crystal. The biocatalytic precipitation of 3 provides the amplification route for the detection of the CT. Formation of the insulating film of 3 on the electrode increases the interfacial electron-transfer resistance, R_et, or enhances the electrode resistance, R‘, parameters that are quantitatively derived by Faradaic impedance measurements and chronopotentiometric analyses, respectively. Similarly, the precipitate 3 formed on the Au/quartz crystal results in a mass increase on the transducer that is reflected by a decrease in the resonance frequency of the crystal. The methods allow the detection of the CT with an unprecedented sensitivity that corresponds to 1.0 × 10^-13 M.