Electrochemical coupled-enzyme assays at carbon nanotubes.

The recently developed internally calibrated electrochemical continuous enzyme assay (ICECEA) has proved to work well for single-enzyme systems. In the present work, its relevance to more challenging coupled-enzyme measurements was investigated by using a model enzyme pair comprising aspartate transaminase (AST) and malic dehydrogenase. The ICECEA was performed at an electrode modified with carbon nanotubes (CNTs), which were dispersed in a polysaccharide chitosan that acted as an adhesive. The 7 min assay required a 100 μL sample and relied on an AST-free calibration. It had a limit of detection equal to 5.0 pM AST (0.10 U L(-1)) with no need for the incubation period. Its linear range extended up to 3500 pM (70 U L(-1)). Perhaps the most promising was the fact that the assay and its calibration could be performed in the same solution even though the composition of the assay solution for the coupled-enzyme assays is typically more complex than that for the single-enzyme assays. This and the fast electrode kinetics of the signal transducing reaction of nicotinamide adenine dinucleotide at CNTs accounted for the low limit of detection. The unique shape of the ICECEA amperogram allowed for the selective determination of AST in the complex matrix of serum samples containing redox active potentially interfering species. Given these advantages, the prospects for the ICECEA in the development of other coupled-enzyme assays were also discussed.