Microneedle-based biosensor for minimally-invasive lactate detection.

Here we report the first mediated microneedles-based biosensor for minimally invasive continuous sensing of lactate in the dermal interstitial fluid (ISF). To further demonstrate the capability of microneedle arrays as second generation biosensors we have functionalized gold microneedles with nanocarbons at which mediated electron transfer of lactate oxidase takes place. In particular the gold surface of the microneedles electrode has been modified in 3 subsequent steps: i) electrodeposition of Au-multiwalled carbon nanotubes (MWCNTs); ii) electropolymerization of the mediator, methylene blue (MB); iii) immobilization of the enzyme lactate oxidase (LOX) by drop-casting procedure. The resulting microneedle-based LOX biosensor displays an interference-free lactate detection without compromising its sensitivity, stability, selectivity and response time. The performance of the microneedle array, second generation biosensor for lactate detection was assessed in artificial interstitial fluid and in human serum, both spiked with lactate. The results reveal that the new microneedles lactate sensor holds interesting promise for the development of a real-time monitoring device to be used in sport medicine and clinical care.

High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants.

A rapid method for identifying compounds that are potential substrates for the drug metabolising enzyme cytochrome P450 is described. The strategy is based on the detection of a degradation product of NAD(P)H oxidation during substrate turnover by the enzyme expressed in Escherichia coli cells spontaneously lysed under the experimental conditions. The performance of the method has been tested on two known substrates of the wild-type cytochrome P450 BM3, arachidonic (AA) and lauric (LA) acids, and two substrates with environmental significance, the anionic surfactant sodium dodecyl sulfate (SDS), and the solvent 1,1,2,2-tetrachloroethane (TCE). The minimal background signal given from cells expressing cytochrome P450 BM3 in the absence of added substrate is only 3% of the signal in the presence of saturating substrate. Control experiments have proven that this method is specifically detecting NADPH oxidation by catalytic turnover of P450 BM3. The assay has been adapted to a microtitre plate format and used to screen a series of furazan derivatives as potential substrates. Three derivatives were identified as substrates. The method gave a significant different signal for two isomeric furazan derivatives. All results found on the cell lysate were verified and confirmed with the purified enzyme. This strategy opens the way to automated high throughput screening of NAD(P)H-linked enzymatic activity of molecules of pharmacological and biotechnological interest and libraries of random mutants of NAD(P)H-dependent biocatalysts.