Verification of biochemical activity for proteins nanografted on gold surfaces.
We demonstrate that the Atomic Force Microscope (AFM) can be used to immobilize a dicysteine-terminated protein (Maltose Binding Protein, MBP-cys-cys for short) at well-defined locations directly on gold substrates via nanografting and characterize the in situ bioactivity of these proteins within the fabricated nanopatterns. This method exploits the high spatial and orientational control of the protein monolayer assembly allowed by nanografting, combined with the high sensitivity of the AFM for detecting ligand-binding events. The maltose-mediated conformational changes within the MBP have been found to change the AFM-tip-protein interaction, therefore causing the frictional signal to change. Our measurements show that the protein ligand-binding function is maintained upon the immobilization process and is not affected by (a) the addition of the cysteine dipeptide, (b) the spatial confinement associated with nanografting, and (c) the interaction between the protein and the Au substrate. These surface-confined proteins can also be regenerated, and their frictional response is reproducible through several maltose exposure/washing cycles. By measuring the change in the frictional force above the protein nanopatterns as a function of maltose concentration, we determined the dissociation constant for the MBP-cys-cys/maltose system to be kd = (1 +/- 0.04) microM. Our results show that the MBP-cys-cys system provides a very sensitive surface-based, protein nanobiosensor for maltose detection at the attogram level (approximately 100 nM concentration). The implications of our study for the fabrication of molecular-scale biological sensors are discussed at the end of the paper.
Staii
Cristian
C
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA. cstaii@princeton.edu
Wood
David W
DW
Scoles
Giacinto
G
D015374:Biosensing Techniques; D002352:Carrier Proteins / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D058619:Maltose-Binding Proteins; D018625:Microscopy, Atomic Force; D036103:Nanotechnology / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011506:Proteins; D006046:Gold