Resistive Pulse Sensing of Protein and Protein/Antibody Complexes Using Single Conical Nanopores

Current work in the Martin group has focused on utilizing single conical nanopores prepared by the track-etch method [1] to detect proteins via the resistive pulse sensing technique by recording current-time (i-t) traces through a single pore. When an analyte passes through the pore, translocation events are observed in the i-t trace. In this study, we recorded translocation events for an individual protein and a protein/antibody complex. We then analyzed the difference between the protein and the complex events. Bovine serum albumin (BSA) and its antibody were used as a model system. Membranes containing single nanopores were prepared by irradiating 12-μm thick polyethylene terephthalate (PET) foils with individual heavy ions (e.g. Au, Pb, U of 11.4 MeV/u) from the UNILAC. Through a chemical track etching process, using 9M NaOH and 1M formic acid, the single track was developed into a conical pore [1]. The base diameter of the resulting conical pore was ~520 nm, and the small diameter opening after etching was ~45 nm. The pores were then electrolessly gold plated and subsequently functionalized with a poly(ethylene glycol) (PEG) monolayer [2]. This was done to minimize non-specific protein absorption [3] and accomplished with a thiolated PEG chain (MW 5000). After these steps the base diameter remained approximately the same diameter because the Au layer and PEG monolayer deposited are very thin, however, the tip diameter is decreased to ~17 nm. The PEG functionalized single conical pore was mounted in a conductivity cell, and each side was filled with 10 mM PBS buffer (pH = 7.4) that was also 100 mM in KCl. A Ag/AgCl electrode was placed on each side of the cell and connected to an Axopatch 200B (Molecular Devices Corporation, Union City, CA) patch clamp amplifier which was used to apply the desired potential, and measure the resulting ionic current flowing through the nanopore. Current time recordings were taken in the presence of (A) buffer only, (B) increasing concentrations of BSA, and (C) a solution of BSA with excess anti-BSA. Control experiments with streptavidin (SA) in the presence of anti-BSA were also done. The translocation events for BSA showed a voltage dependence between 400-1000 mV, as well as a concentration dependence with the four concentrations used between 25-100 nM at an applied potential of 1000 mV. After addition of excess anti-BSA (270 nM) to a solution that was 100nM in BSA facing the tip side of the conical nanopore, the duration and amplitude of events changed dramatically. Histograms of event duration for the BSA/anti-BSA complex were compared with those associated with BSA only. The event durations observed for the complex were much larger (~2213 ± 187 ms) than those for the protein only (~520 ± 11 ms), which indicates that the BSA/anti-BSA complex was formed and translocated the nanopore. Scatter plots of event amplitude vs. event duration for BSA and the BSA/anti-BSA complex were also compared (Fig. 1). One unexpected observation is the decrease in amplitude for the events associated with the protein/antibody complex. One would expect an increase in event amplitude with a larger analyte [4]. A possible explanation for this occurrence could be due to conformational changes that must take place in order for the complex to translocate the nanopore. Control experiments with SA and anti-BSA showed that events associated with SA do not change in amplitude or duration after addition of anti-BSA.