Electrochemical and quartz crystal microbalance detection of the cholera toxin employing horseradish peroxidase and GM1-functionalized liposomes

An ultrasensitive method for the detection of the cholera toxin (CT) using electrochemical or microgravimetric quartz crystal microbalance transductio n means is described. Horseradish peroxidase (HRP) and GM1-functionalized l iposomes act as catalytic recognition labels for the amplified detection of the cholera toxin based on highly specific recognition of CT by the gangli oside GM1. The sensing interface consists of monoclonal antibody against th e 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 liposo me. The enzyme-labeled liposome mediates the oxidation of 4-chloronaphthol (2) in the presence of H2O2 to form the insoluble product 3 on the electrod e support or the Au/quartz crystal. The biocatalytic precipitation of 3 pro vides the amplification route for the detection of the CT. Formation of the insulating film of 3 on the electrode increases the interfacial electron-t ransfer resistance, R-et, or enhances the electrode resistance, R', paramet ers that are quantitatively derived by Faradaic impedance measurements and chronopotentiometric analyses, respectively. Similarly, the precipitate 3 f ormed 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 CF with an unprecedented sensitivit y that corresponds to 1.0 x 10(-13) M.