Single-molecule observation of protein adsorption onto an inorganic surface

Abstract

Understanding the interactions between silicon-based materials and proteins from the bloodstream is of key importance in a myriad of realms, such as the design of nanofluidic devices and functional biomaterials, biosensors, and biomedical molecular diagnosis. By using nanopores fabricated in 20 nm-thin silicon nitride membranes and highly sensitive electrical recordings, we show single-molecule observation of nonspecific protein adsorption onto an inorganic surface. A transmembrane potential was applied across a single nanopore-containing membrane immersed into an electrolyte-filled chamber. Through the current fluctuations measured across the nanopore, we detected long-lived captures of bovine serum albumin (BSA), a major multifunctional protein present in the circulatory system. Based upon single-molecule electrical signatures observed in this work, we judge that the bindings of BSA to the nitride surface occurred in two distinct orientations. With some adaptation and further experimentation, this approach, applied on a parallel array of synthetic nanopores, holds potential for use in methodical quantitative studies of protein adsorption onto inorganic surfaces.

Figure 1

Representative Si_xN_y nanopores imaged by a Technia F-20 S/TEM in TEM mode. The diameters of the nanopores were 5 nm (A), 10 nm (B), and 20 nm (C).

Figure 2

Single-channel electrical recordings with a 12 nm-diameter Si_xN_y nanopore, revealing long-lived BSA captures. (A) A uniform, stable and fluctuation-free single-channel current was observed in the absence of the BSA protein. (B) Short-lived and long-lived gating current blockades were detected when 180 nM BSA was added to the cis side of the chamber. (C) The dwell-time histogram of the long-lived current blockades. The transmembrane potential was +150 mV. A two-exponential fit was made, giving time constants of τ₁=110 ± 11 ms and τ₂=440 ± 62 ms with the associated probabilities of P₁=0.58 ± 0.05 and P₂=0.42 ± 0.05, respectively. The fit was based upon a log likelihood ratio (LLR) test,, with a given confidence level of 0.95. The buffer solution contained 1 M KCl, 10 mM potassium phosphate, pH 7.4. For the sake of the clarity, the single-channel electrical traces were low-pass Bessel filtered at 400 Hz.

Figure 3

Representative single-channel electrical recording with a 9 nm-diameter Si_xN_y nanopore. The electrical trace was low-pass Bessel filtered at 2 kHz. 10 nM BSA was added to the cis side of the chamber. The other experimental conditions were similar to those presented in Fig. 2.

Figure 4

The voltage dependence of the long-lived current fluctuations. The single-channel electrical traces from the top panels are recorded at +100 mV (A) and +300 mV (B). These experiments were carried out with a 12 nm-diameter nanopore. The BSA concentration in the cis chamber was 20 nM. The middle panels represent a schematic model of the voltage-dependent partitioning of the negatively charged, unattached part of the BSA protein into the nanopore interior at a transmembrane potential V=0 mV (A) and V >> 0 mV (B). These panels show the attached BSA protein in the open (A) and closed (partitioned) (B) states, respectively. The bottom panels illustrate free energy landscapes of the BSA-nanopore complex at zero (A) and much greater than zero (B) voltages, respectively. The other experimental conditions were similar to those presented in Fig. 2.

Figure 5

Diagrams show the proposed mechanism for the long-lived protein captures. The upper panels indicate the position of adsorbed BSA (red) within the nanopore interior (grey), in cross-section. (A) BSA is attached within the interior of the nanopore causing a long-lived current blockade (middle panel) without additional long-lived current fluctuations of the resulting current state. The short-lived current spikes were in a sub-millisecond range. 20 nM BSA was added to the cis chamber; (B) BSA is attached to the nanopore interior, but in a different orientation than in (A). Additional current fluctuations occur (middle panel) in which a movable “unattached” part of the BSA protein wiggles between the nanopore interior and the aqueous phase, while the other end remains attached to the Si_xN_y surface of the nanopore interior. This results in a gating of the current between the open and the partially occluded (closed) state (Fig. 4). The left-hand bottom panel presents an all-points amplitude histogram of the trace in (A). The right-hand bottom panel is a dwell time histogram of the trace in (B), with τ_off-1=240 ± 6.9 ms (P₁=0.70 ± 0.02) and τ_off-2=3020 ± 730 ms (P₂=0.31 ± 0.04). 60 nM BSA was added to the cis chamber. The fit was based upon a log likelihood ratio (LLR) test,, with a given confidence level of 0.95. The diameter of the nanopore was 15 nm, as judged by the least square linear fit to an I–V curve (Supporting Information, Fig. S2). The other experimental conditions were similar as those presented in Fig. 2.