| Abstract
| - 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 ongold substrates via nanografting and characterize the in situ bioactivity of these proteins within the fabricatednanopatterns. This method exploits the high spatial and orientational control of the protein monolayerassembly 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 changethe AFM−tip−protein interaction, therefore causing the frictional signal to change. Our measurements showthat the protein ligand-binding function is maintained upon the immobilization process and is not affectedby (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 beregenerated, 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 maltoseconcentration, we determined the dissociation constant for the MBP-cys-cys/maltose system to be kd = (1± 0.04) μM. Our results show that the MBP-cys-cys system provides a very sensitive surface-based, proteinnanobiosensor for maltose detection at the attogram level (∼100 nM concentration). The implications ofour study for the fabrication of molecular-scale biological sensors are discussed at the end of the paper.
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