Cytochrome c self-assembly on alkanethiol monolayer electrodes as characterized by AFM, IR, QCM, and direct electrochemistry.
With the advantage of carbodiimide coupling chemistry, horse heart cytochrome c (cyt c) has been covalently immobilized onto self-assembled monolayers (SAMs) from 11-mercaptoundecanoic acid (MUDA) developed on single-crystal or polycrystalline gold substrate surfaces. The cyt c immobilized substrates thus prepared have been characterized by atomic force microscopy (AFM); we have succeeded in obtaining surface topographical images down to single-protein resolution. AFM imaging has also shown densely packed, uniform protein monolayer formation that is highly suggestive of self-assembly of cyt c molecules on MUDA SAMs. Covalent attachment of cyt c has been further evidenced by reflection-absorption FT-IR as well as microgravimetric analysis using a quartz crystal microbalance (QCM). In the latter, the specific MUDA and cyt c surface concentrations were determined to be 0.86 +/- 0.11 nmol cm-2 (n = 5) and 28 +/- 12 pmol cm-2 (n = 5), both of which agree fairly well with their theoretical counterparts. The obtained QCM chips having the cyt c/MUDA/Au interfacial structure were found to be capable of the direct electrochemistry of the surface-attached cyt c molecules. Cyclic voltammetric measurements on the chips gave particular redox waves showing characteristics of surface process. The electroactive protein surface concentration was determined to be 7.2 +/- 4.8 pmol cm-2 (n = 6); it was almost consistent with values found in literature, while it was limited to 26% in magnitude for the QCM data. This was deemed to have arisen from the orientation variation of the surface-confined cyt c molecules and is discussed briefly.
Nakano
Koji
K
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. kojiitcm@mbox.nc.kyushu-u.ac.jp
Yoshitake
Tadateru
T
Yamashita
Yasunori
Y
Bowden
Edmond F
EF
D000473:Alkanes / Q000737:chemistry; D000818:Animals; D015374:Biosensing Techniques; D045304:Cytochromes c / Q000737:chemistry*; D004563:Electrochemistry; D004566:Electrodes; D006736:Horses; D066298:In Vitro Techniques; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared; D013438:Sulfhydryl Compounds / Q000737:chemistry
D000473:Alkanes; D046912:Multiprotein Complexes; D013438:Sulfhydryl Compounds; D045304:Cytochromes c