Reagentless amperometric formaldehyde-selective biosensors based on the recombinant yeast formaldehyde dehydrogenase

摘要

Novel formaldehyde-selective amperometric biosensors were developed based on NAD(+)- and glutathione-dependent formaldehyde dehydrogenase isolated from a gene-engineered strain of the methylotrophic yeast Hansenula polymorpha. Electron transfer between the immobilized enzyme and a platinized graphite electrode was established using a number of different low-molecular free-diffusing redox mediators or positively charged cathodic electrodeposition paints modified with Os-bis-N,N-(2,2'-bipyridil)-chloride ([Os(bpy)(2)Cl]) complexes. Among five tested Os-containing redox polymers of different chemical structure and properties, complexes of osmium-modified poly(4-vinylpyridine)with molecular mass of about 60 kDa containing diaminopropyl groups were selected. The positively charged cathodic paint exhibited the best electron-transfer characteristics. Moreover, the polymer layers Simultaneously served as a matrix for keeping the negatively charged low-molecular cofactors, glutathione and NAD(+), in the bioactive layer. Additionally, covering the enzyme/polymer layer with a negatively charged Nafion membrane significantly decreased cofactors leakage and simultaneously enhanced the sensor' stability. The developed sensors revealed a high selectivity to formaldehyde (FA) and a low cross-sensitivity to other substances (such as, e.g. butyraldehyde, propionaldehyde, acetaldehyde, methylglyoxal). The maximum current value was 34.2 +/- 0.72 mu A/mm(2) (3.05 mm diameter electrode) and the apparent Michaelis-Menten constant (K-M(app)) derived from the FA calibration curves was 120 +/- 5 mM with a linear detection range for FA up to 20 mM. The best observed sensitivity for reagentless sensor was 1.8 nA mu M-1 (358 A m(-2) M-1). The developed sensors had a good operational and storage stability. The laboratory prototype of the sensor was applied for FA testing in some real samples of pharmaceutical (formidron), disinfectant (descoton forte) and industrial product (formalin). A good correlation was revealed between the concentration values measured using the developed FdDH-based sensor, an enzymatic method and standard chemical methods of FA determination. (C) 2008 Elsevier B.V. All rights reserved.