In our laboratory we have been working to create a molecular scale analogy of a machine shop, using an atomic force microscope as our "milling"machine, an enzyme as our "tool bit", and various substrates for that enzyme as our "working stock". To realize our goal we have studied two enzyme-substrate systems: phospholipase C (PLC) hydrolyzing phosphorylcholine from a phospholipid modified surface and alpha- chymotrypsin cleaving a peptide from a peptide modified surface. A carboxylic acid derivative of dimyristoylphosphatidylcholine (DMPC) was coupled by amide bond formation to an amino-silane modified silica surface.
This lipid film was characterized using a variety of physical techniques including: x-ray photoelectron spectroscopy (XPS), secondary ion mass spectroscopy (SIMS), atomic force microscopy (AFM), ellipsometry and wettability. After treatment of the lipid film with PLC in solution, we observed a loss of the phosphate from the surface as measured with XPS and SIMS.
In parallel, we have also immobilized PLC on a silica surface and shown that it is enzymatically active on phospholipid in solution using an assay of our own design. Efforts to demonstrate enzymatic modification of the lipid surface using PLC immobilized on the tip of an AFM cantilever have yielded inconsistent results.
In light of the difficulties encountered with the PLC/lipid experiments we are investigating a second enzyme/substrate system based on a-chymotrypsin and the peptide N-t-BOC-phenylalanine. Once this peptide is immobilized on an amino-silane derivatized silica substrate via amide bond formation we should be able to completely cleave it from the surface using a-chymotrypsin, thus leaving the original amino acid surface. From a technical viewpoint, modification of this simple peptide could easily be translated to more complex peptides for possible development of biomaterial arrays.
Progress towards the
immobilization of the peptide and enzyme will be discussed, as well as
further experiments toward spatially controlled enzymatic modification
of the peptide surface.
Supported by the Naval Research Laboratory
Core Program.
Corresponding Author:
Dr. Bruce Paul Gaber
(202) 404-6003/FAX: (202) 767-9594
email: bgaber@cbmse.nrl.navy.mil