Immobilization of Alcohol Dehydrogenase in Phospholipid Langmuir−Blodgett Films To Detect Ethanol

Abstract

Enzyme immobilization in nanostructured films may be useful for a number of biomimetic systems, particularly if suitable matrixes are identified. Here we show that alcohol dehydrogenase (ADH) has high affinity toward a negatively charged phospholipid, dimyristoylphosphatidic acid (DMPA), which forms a Langmuir monolayer at an air-water interface. Incorporation of ADH into the DMPA monolayer was monitored with surface pressure measurements and polarization-modulation infrared reflection absorption spectroscopy, with the alpha-helices from ADH being mainly oriented parallel to the water surface. ADH remained at the interface even at high surface pressures, thus allowing deposition of Langmuir-Blodgett (LB) films from the DMPA-ADH film. Indeed, interaction with DMPA enhances the transfer of ADH, where the mass transferred onto a solid support increased from 134 ng for ADH on a Gibbs monolayer to 178 ng for an LB film with DMPA. With fluorescence spectroscopy it was possible to confirm that the ADH structure was preserved even after one month of the LB deposition. ADH-containing films deposited onto gold-interdigitated electrodes were employed in a sensor array capable of detecting ethanol at concentrations down to 10 ppb (in volume), using impedance spectroscopy as the method of detection.

Recent advances in electronic tongues

“…[32][33][34][35][36][37][38] Furthermore, using label-free detection processes is advantageous since uncertainties are removed which are associated with the effects of labels on molecular conformation, blocking of active binding sites, steric hindrance, and the inability to encounter available labels for specific molecules. [32][33][34][35][36][37][38] Furthermore, using label-free detection processes is advantageous since uncertainties are removed which are associated with the effects of labels on molecular conformation, blocking of active binding sites, steric hindrance, and the inability to encounter available labels for specific molecules.…”

Biomimetic biosensor based on lipidic layers containing tyrosinase and lutetium bisphthalocyanine for the detection of antioxidants

“…Tyrosinase biosensors have been immobilised using a range of techniques that include immobilisation onto carbonaceous electrodes via the cross-linking step by glutaraldehyde (Sanz et al, 2005;Zhao et al, 2009) or immobilisation in polymeric matrixes (Yildiz et al, 2006). The layer-by-layer (LbL) and the Langmuir-Blodgett (LB) techniques are of special interest for enzyme immobilisation because they allow the preparation of biomimetic systems where the enzyme is adsorbed in a lipidic layer via COOH group interaction (Cajab et al, 2009;Caseli et al, 2009). Electrodes incorporating lipids can enhance the enzymatic activity by preserving the conformation of the enzyme (Goto et al, 2010;Schmidt et al, 2008).…”

Interactions of bioactive molecules & nanomaterials with Langmuir monolayers as cell membrane models

“…Immobilizing enzymes with some affinity with substrates can be used to identify analytes by using principal component analyses comparing the impedance in several samples, as used for alcohol dehydrogenase for detecting ethanol [67]. The selectivity of enzymes for binding in certain lipids has also been demonstrated.…”