Laccase-MWCNT-chitosan biosensor-A new tool for total polyphenolic content evaluation from in vitro cultivated plants

Laccase from Trametes versicolor, an enzyme with broad substrate specificity for the phenolic substrates was employed as a biorecognition element in order to develop a biosensor for total content evaluation of phenolic secondary metabolites from two "in vitro" cultivated plants: Salvia officinalis and Mentha piperita. The enzyme immobilization was carried out by entrapment into the nanocomposite film during electrodeposition process from multiwall carbon nanotubes (MWCNT)-chitosan (CS) solution containing 25 U/mL laccase. Optimum conditions for MWCNT-CS film deposition (2 mg MWCNT/mL chitosan 1% solution prepared in 1% acetic acid) on gold support using a -1.5 V vs. Ag/AgCl controlled potential V for 5 min were established taking into consideration the layer capacity value. FTIR studies were performed to obtain information about the secondary structure of enzyme entrapped into the MWCNT-CS nanocomposite film. Calibration of the laccase biosensor was performed on four phenolic acids (caffeic acid, chlorogenic acid, gallic acids and rosmarinic acid) as substrates at -0.2 V vs. Ag/AgCl reference electrode. The developed biosensor was sensitive to micromolar concentration of the tested polyphenols. The performance characteristics of the biosensor for rosmarinic acid were: limit of detection 2.33 x 10(-7) mol L-1, response linear range 9.1 x 10(-7)-1.21 x 10(-5) mol L-1 and sensitivity 846 mu A/mmol. The obtained values of the Krev for all tested substrates proved that nanocomposite film provides a proper environment for enzyme immobilization, preserving enzyme catalytic specificity. The functionality of the developed biosensor was tested to evaluate the total polyphenolic content from real samples (S. officinalis and M. piperita extracts), results being expressed in equivalent rosmarinic acid. (C) 2010 Elsevier B.V. All rights reserved.