Reagentless amperometric formaldehyde-selective biosensors based on the recombinant yeast formaldehyde dehydrogenase.
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 microA/mm(2) (3.05 mm diameter electrode) and the apparent Michaelis-Menten constant (K(M)(app)) derived from the FA calibration curves was 120+/-5mM with a linear detection range for FA up to 20mM. The best observed sensitivity for reagentless sensor was 1.8 nA microM(-1) (358 Am(-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.
Demkiv
Olha
O
Department of Analytical Biotechnology, Institute of Cell Biology, Drahomanov Street 14/16, 79005 Lviv, Ukraine.
Smutok
Oleh
O
Paryzhak
Solomiya
S
Gayda
Galyna
G
Sultanov
Yusif
Y
Guschin
Dmitrii
D
Shkil
Halyna
H
Schuhmann
Wolfgang
W
Gonchar
Mykhailo
M
D000445:Aldehyde Oxidoreductases / Q000032:analysis*; D015374:Biosensing Techniques; D004202:Disinfectants / Q000032:analysis; D004563:Electrochemistry / Q000379:methods*; D004867:Equipment Design; D005557:Formaldehyde / Q000032:analysis / Q000494:pharmacology*; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010843:Pichia / Q000201:enzymology; D011108:Polymers / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D004202:Disinfectants; D011108:Polymers; D011994:Recombinant Proteins; D005557:Formaldehyde; D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent