Cibacron Blue F3G-A anchored monolayers with biospecific affinity for NAD(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 alkanethiol self-assembled monolayers derivatized with the triazine dye Cibacron Blue F3G-A. This compound, when in solution, works as a competitive inhibitor for NAD(H)-dependent lactate dehydrogenase building a complex by a site specific affinity binding at the enzyme's NAD(+)-binding pocket. Therefore, the chemisorption of alkanethiol self-assembled monolayers bearing Cibacron Blue 3FG-A as affinity ligand should provide a way to prepare gold surfaces with a high biospecific affinity for lactate dehydrogenase. The results presented in this work, together with previously reported data from electroenzymatic studies of lactate dehydrogenase-modified gold electrode surfaces, show that the feasibility 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 degree of disorder in their structure. Atomic force images of lactate dehydrogenase-modified gold surfaces confirm that higher and more homogeneous surface coverage of the enzyme can be obtained from poorly ordered mixed short- and long-alkanethiol self-assembled monolayers. These results suggest that the higher electroenzymatic activities shown by lactate dehydrogenase-modified gold surfaces prepared from poorly ordered Cibacron Blue-anchored mixed alkanethiol self-assembled monolayers can be correlated with a higher loading of enzyme bound to the metal surface as a result of the higher number of affinity sites (Cibacron Blue) available on the alkanethiol monolayer. (C) 1997 Elsevier Science Limited.