Biosensors & Bioelectronics Vol. 12. No. 8, pp. 839–852, 1997  1997 Elsevier Science Limited All rights reserved. Printed in Great Britain 0956–5663/97/$17.00 PII: S0956–5663(97)00049-3 Cibacron Blue F3G-A anchored monolayers with biospecific affinity for NAD(H)-dependent lactate dehydrogenase: characterization by FTIR-spectroscopy and atomic force microscopy Lars Bertilsson,* Hans-Ju ¨ rgen Butt,† Gabriele Nelles† & Daniela D. Schlereth‡§ *Institut fu ¨r Physik. Fakulta ¨t fu ¨r Elektrotechnik. Universita ¨t der Bundeswehr Mu ¨nchen, Werner-Heißenberg-weg 39, Neubiberg D-85577, Germany †Max-Planck Institut fu ¨r Biophysik. Kennedyallee 70, Frankfurt am Main D-60596, Germany ‡Lehrstuhl fu ¨r Allgemeine Chemie und Biochemie. Technische Universita ¨t Mu ¨nchen, Vo ¨ ttingerstraße 40, Freising D-85354, Germany Abstract: Fourier transform infrared reflection absorption spectroscopy (FT- IRAS) and atomic force microscopy have been used to analyze several alka- nethiol 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 feasi- bility 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 mono- layers 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. 1997 Elsevier Science Limited Keywords: Cibacron Blue F3G-A, self-assembled monolayers, alkanethiolates, lactate dehydrogenase, gold surfaces, Fourier transform infrared reflection absorption spectroscopy, atomic force microscopy §To whom correspondence should be addressed. 839