CIBACRON-BLUE F3G-A ANCHORED MONOLAYERS WITH BIOSPECIFIC AFFINITY FORNAD(H)-DEPENDENT LACTATE-DEHYDROGENASE - CHARACTERIZATION BY FTIR-SPECTROSCOPY AND ATOMIC-FORCE MICROSCOPY

Fourier transform infrared reflection absorption spectroscopy (FT-IRAS ) and atomic force microscopy have been used to analyze several alkane thiol self-assembled monolayers derivatized with the triazine dye Ciba cron Blue F3G-A. This compound, when in solution, works as a competiti ve inhibitor for NAD(H)-dependent lactate dehydrogenase building a com plex by a site specific affinity binding at the enzyme's NAD(+)-bindin g pocket. Therefore, the chemisorption of alkanethiol self-assembled m onolayers bearing Cibacron Blue 3FG-A as affinity ligand should provid e a way to prepare gold surfaces with a high biospecific affinity for lactate dehydrogenase. The results presented in this work, together wi th previously reported data from electroenzymatic studies of lactate d ehydrogenase-modified gold electrode surfaces, show that the feasibili ty of such monolayers to bind enzyme strongly depends on the structure and composition of the alkanethiol underlayer used to anchor Cibacron Blue. Infrared spectra of the Cibacron Blue-anchored monolayers show that a sufficiently high amount of dye can be succesfully attached to functionalized alkanethiol self-assembled monolayers with a certain de gree of disorder in their structure. Atomic force images of lactate de hydrogenase-modified gold surfaces confirm that higher and more homoge neous surface coverage of the enzyme can be obtained from poorly order ed mixed short- and long-alkanethiol self-assembled monolayers. These results suggest that the higher electroenzymatic activities shown by l actate dehydrogenase-modified gold surfaces prepared from poorly order ed Cibacron Blue-anchored mixed alkanethiol self-assembled monolayers can be correlated with a higher loading of enzyme bound to the metal s urface as a result of the higher number of affinity sites (Cibacron Bl ue) available on the alkanethiol monolayer. (C) 1997 Elsevier Science Limited.