Imaging of biological macromolecules on mica in humid air by scanning electrochemical microscopy

Abstract

Imaging of DNA, keyhole limpet hemocyanin, mouse monoclonal IgG, and glucose oxidase on a mica substrate has been accomplished by scanning electrochemical microscopy with a tungsten tip. The technique requires the use of a high relative humidity to form a thin film of water on the mica surface that allows electrochemical reactions to take place at the tip and produce a faradaic current (approximately 1 pA) that can be used to control tip position. The effect of relative humidity and surface pretreatment with buffer solutions on the ionic conductivity of a mica surface was investigated to find appropriate conditions for imaging. Resolution of the order of 1 nm was obtained.

Figure 1

Schematic diagram for the SECM chamber with controlled humidity, and the EC processes that control the current. The tip was located laterally ca. 1–2 mm away from the Au counter electrode. For illustration purposes, the thickness of the liquid layer is exaggerated to accommodate equations for the various EC processes. V, voltage bias between the tip and Au contact. i, current flow through the tip. R and Ox represent the reduced and oxidized forms of an electroactive species. ⊕ and ⊝ represent cations and anions in the liquid layer and in the mica sheet.

Figure 2

A series of semilogarithmic plots of current–voltage curves for a bare mica substrate at different RH: 33% (a); 58% (b); 65% (c); 74% (d); 81% (e); and 93% (f).

Figure 3

Conductance values at various RH for the same mica sheet before (□) and after (✠) the treatment with TE or phosphate (+) buffer. The conductance was measured as the slope of the current–voltage curve in the bias range of 1.5–2.0 V.

Figure 4

Typical voltammetric curves at 100% RH and 25°C over a bare mica substrate (A) or a mica substrate treated with TE buffer solution (B). Radius of curvature of the W tip is ≈10 μm. In all cases, the voltage was scanned from 0 V (the starting point) in either direction and then returned to 0 V; scan rate, 0.2 V/s.

Figure 5

Cyclic voltammograms of a polyurethane-coated W tip in deaerated 1 mM Ru(NH₃)₆³⁺/10 mM NaClO₄ solution (a) and 10 mM NaClO₄ only (b). Potential scan rate is 5 mV/s.

Figure 6

Typical parallel conductance (A) and parallel capacitance (B) vs. tip displacement curves for a blunt W tip at 100% RH at 25°C for a mica substrate treated with TE buffer solution. Tip bias was 3 V with respect to the Au counter electrode. The tip approached the substrate surface at 3 nm/s.

Figure 7

(A) Constant-current image of fragments of DNA specimen in humid air (80% RH at 25°C). The DNA specimen was deposited on a TE buffer-treated mica substrate. Image was taken with a sharp W tip at a reference current of 0.3 pA and a tip bias of 3 V. The tip raster rate was 0.25 Hz. (B) A high-resolution image of a DNA molecule obtained by scanning over a smaller area.

Figure 8

(A and B) Constant-current images showing several mouse monoclonal IgG molecules on mica substrates treated with phosphate buffer. The images were recorded with a W tip in humid air (80% RH at 25°C). The reference current was 1 pA at a tip bias of 1.5 V (vs. the Au contact). (C) Schematic of an IgG molecule showing two Fab arms and the Fc portion (16).

Figure 9

(A) Constant-current image showing several GOD molecules on mica substrates treated with phosphate buffer. The images were recorded with a W tip in humid air (80% RH at 25°C). The reference current was 1 pA at a tip bias of 1.5 V. (B) C^α tracing of the dimer structure of a GOD molecule (20). Contacts between molecules forming the dimer are confined to a long, narrow stretch.

Figure 10

(A) Constant-current image of a KLH molecule on a mica surface treated with phosphate buffer. The image was taken at 65% RH (25°C) with a W tip biased at 1.7 V with a reference current of 1 pA. (B) Analogous Mellema and Klug model (22) for a nine parallel-row Gastropod hemocyanin molecule.