Hg. Hong et al., CYSTEINE-SPECIFIC SURFACE TETHERING OF GENETICALLY-ENGINEERED CYTOCHROMES FOR FABRICATION OF METALLOPROTEIN NANOSTRUCTURES, Langmuir, 10(1), 1994, pp. 153-158
The preparation of oriented metalloprotein nanostructures through intr
oduction of specific and complementary reactive groups on the solid an
d protein surfaces is critically dependent on the reaction conditions
used to prepare the solid surface. Key problems include the hydrolytic
stability of the Si-O bond, the low reactivity of simple nucleophilic
silane reagents, protein physisorption, and identification of conditi
ons for producing monolayer protein coverages. These problems are larg
ely circumvented by utilizing a two-step linker synthesis, in which th
e surface is first prepared with a monolayer of (3-aminopropyl)silane
(S-APS), and the resulting structure is derivatized with the heterobif
unctional reagent N-succinimidyl 6-maleimidocaproate (EMCS). The malei
mide functionality is then presented to the protein, into which a sing
le unique cysteine residue has been introduced by genetic engineering
techniques. Hydrolytic stability is dramatically enhanced by including
a postreaction curing step, in which the solid surface temperature is
elevated to drive the alkoxysilane condensation reaction to completio
n. Finally substituing a gas-phase chemical vapor deposition procedure
for the liquid-phase reaction of the 3-APS produces dramatically bett
er control over coverage and quality of the resulting films.