Resistive-pulse studies of proteins and protein/antibody complexes using a conical nanotube sensor.
There is increasing interest in using nanopores in synthetic membranes as resistive-pulse sensors for molecular and macromolecule analytes. In general, this method entails measuring current pulses associated with translocation of the analyte through the nanopore sensor element. A key challenge for this sensing paradigm is building selectivity into the protocol so that the current pulses for the target analyte can be distinguished from current pulses for other species that might be present in the sample. We show here that this can be accomplished with a protein analyte by adding to the solution an antibody that selectively binds the protein. We demonstrate this concept using bovine serum albumin (BSA) and a Fab fragment from a BSA-binding polyclonal antibody. Because the complex formed upon binding of the Fab to BSA is larger than the free BSA molecule, the current-pulse signature for the BSA/Fab complex can be easily distinguished from the free BSA. Furthermore, the BSA/Fab pulses can be easily distinguished from the pulses obtained for the free Fab and from pulses obtained for a control protein that does not bind to the Fab. Finally, we also show that the current-pulse signature for the BSA/Fab complex can provide information about the size and stoichiometry of the complex.
Sexton
Lindsay T
LT
Department of Chemistry and Center for Research at the Bio/Nano Interface, University of Florida, Gainesville, Florida 32611-7200, USA.
Horne
Lloyd P
LP
Sherrill
Stefanie A
SA
Bishop
Gregory W
GW
Baker
Lane A
LA
Martin
Charles R
CR
D000818:Animals; D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000276:immunology*; D015374:Biosensing Techniques / Q000379:methods*; D002417:Cattle; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008854:Microscopy, Electron; D043942:Nanotubes / Q000737:chemistry* / Q000648:ultrastructure; D012710:Serum Albumin, Bovine / Q000276:immunology
D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D012710:Serum Albumin, Bovine