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-GM1ganglioside-functionalized liposome. The enzyme-labeled liposome mediates the oxidation of 4-chloronaphthol (2) in the presence of H 2 O 2 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.

Dopamine Transporter Activity Is Modulated by α-Synuclein

“…Recently, Cremona et al (55) have shown that localization of the transporter in the cholesterol-rich membrane raft microdomains is essential for the AMPH-induced reversed transport of dopamine in dopaminergic neurons. GM1 (monosialotetrahexosylganglioside) is a well characterized membrane ganglioside that is distributed in the cholesterol-enriched membrane microdomains (56) and has been show to interact with and recruit ␣-synuclein to membrane rafts (57). We used confocal microscopy to examine the hypothesis that elevated ␣-synuclein affects the membrane microdomain distribution of DAT to regulate its activity.…”

An electrochemical amplification immunoassay using biocatalytic metal deposition coupled with anodic stripping voltammetric detection

“…Thus, the flow system arrangement coupled with ac adsorptive stripping voltammetry was used to demonstrate detection of pneumolysin and interkeulin at picogram per milliliter levels by employing a conventional ELISA approach with alkaline phosphatase label [16]. Willner's group reported on the use of chronopotentiometry and Faradic impedance spectroscopy for the transduction of an amplified immunosensing scheme that consisted of the biocatalytic precipitation of an insoluble product onto a gold electrode surface [17][18][19]. Hwang et al developed a DNA sensor that used stripping voltammetry to detect the accumulated silver metal reduced by enzyme-generated product [20].…”

Quantitative Detection of Vibrio cholera Toxin by Real-Time and Dynamic Cytotoxicity Monitoring

“…In addition, classic CT determinations require the use of either animal methods (5,6) or tissue culture methods (7,8), which are also time-consuming and are subjective in the interpretation of results. Efforts have been made recently to develop more-sensitive methods, including enzyme-linked immunosorbent assays (9), latex agglutination assays (10), coagglutination assays (5), liposome-based assays (9,11), radioimmunoassays (12), hydrogel-based immunoassays (13), monosaccharide-and antibody array-based assays (14)(15)(16)(17), PCR-based molecular assays (10,(18)(19)(20)(21), and biosensor-based assays (22)(23)(24)(25)(26)(27)(28)(29)(30)(31). A reliable laboratory tool is desirable for clinical services and epidemiological investigation, to characterize CT activities rapidly and quantitatively.…”