Biological imaging with scanning electrochemical microscopy

Abstract

Scanning electrochemical microscopy (SECM) is a powerful and versatile technique for visualizing the local electrochemical activity of a surface as an ultramicroelectrode tip is moved towards or over a sample of interest using precise positioning systems. In comparison with other scanning probe techniques, SECM not only enables topographical surface mapping but also gathers chemical information with high spatial resolution. Considerable progress has been made in the analysis of biological samples, including living cells and immobilized biomacromolecules such as enzymes, antibodies and DNA fragments. Moreover, combinations of SECM with comple-mentary analytical tools broadened its applicability and facilitated multi-functional analysis with extended life science capabilities. The aim of this review is to present a brief topical overview on recent applications of biological SECM, with particular emphasis on important technical improvements of this surface imaging technique, recommended applications and future trends.

Keywords: bio-SECM; chemical imaging; microelectrochemistry; microelectrodes; nanoelectrodes; scanning electrochemical microscopy.

Figure 1.

Options for the scanning operation on single biological cells. Disc-shaped SECM tips of micrometre (a) or nanometre (b) dimensions can be used as electrochemical probes. (Online version in colour.)

Figure 2.

(a) Functional elements of the nervous and endocrine systems. (b) Parts of the human body most commonly affected by cancer appearance and progression. With a variety of analytical assays individual model nerve, endocrine and cancer cells are routine objects of fundamental pathological and (electro-) physiological research for mechanistic studies regarding chemical cell communication, tumour develop­ment and oncology. Accordingly, the cellular archetypes of common medical and life science research are also ideal candidates for diagnostic inspections via bio-SECM, targeting localized electrochemical detection of the structural and functional cell features with high spatial resolution. (Online version in colour.)

Figure 3.

Application of the voltage switching mode of SECM (VSM-SECM) with carbon nanoelectrodes for living A431 cell imaging. The cell topography from SECM scans in the VSM mode is shown in (a) while an electrochemical image of the presence of membranous epidermal growth factor (EGFR) on the outer cell surface is shown in (b). EGRF protein entities on A431 membranes were labelled with alkaline phosphatase prior to SECM experiments to facilitate electrochemical visualization by means of p-aminophenol phosphate (PAPP) substrate exposure and anodic p-aminophenol (PAP) detection. The carbon SECM tip with an active radius of 721.5 nm was held at −500 mV for negative feedback topography imaging and + 350 mV for PAP detection (versus Ag/AgCl), in HEPES buffer containing 10 mM [Ru(NH₃)₆]Cl₃ and 4.7 mM PAPP. Reprinted with permission from Takahashi et al. [51] (Copyright 2012 National Academy of Sciences). (Online version in colour.)

Figure 4.

Application of the 4D-SF/CD-RC mode of SECM for the visualization of the topography and respiration activity of HEK 293 model cells. The topography image (a) is the result of the acquisition of a shear force-based distance-dependent signal while high and low SECM tip currents for oxygen reduction at the point of closest approach (b) are representative of cellular respiration. Parameters for the pulsed electroche­mical oxygen monitoring were: E_tip,base = 500 mV, E_tip,pulse1 = 500 mV, t_pulse1 = 0.2 s, E_tip,pulse2 = −350 mV, t_pulse2 = 0.5 s; image taken at t = 15 ms (all potentials versus Ag/AgCl/3 M KCl). An optical image of the investigated cell area is shown in (c). Reprinted with permission from Nebel et al. [52] (Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA). (Online version in colour.)

Figure 5.

Schematic diagram comparing the feedback and generator/collector (G/C) modes of SECM used for enzyme imaging. (a) Enzyme-mediated positive feedback. The reaction only occurs when the tip is present. (b) G/C mode in the presence of a redox enzyme. A product of the enzymatic reaction is collected at the tip, either (i) the oxidized form of a redox mediator in solution or (ii) the enzymatically converted substrate. (Online version in colour.)

Figure 6.

(a) Illustration of the developed continuous nanoflow (CNF)-SECM system for imaging of enzyme spots. (b) Comparison of the resolution obtained with the conventional generator/collector mode (SG-TC) and the implemented CNF-SECM mode. Reprinted with permission from Kai et al. [124] (Copyright 2015 American Chemical Society). (Online version in colour.)

Figure 7.

SECM strategies for imaging of DNA modified surfaces and the detection of the hybridization event. (a) Use of labelled DNA strands for providing a redox signal measurable with the SECM tip. (b) Use of redox active intercalators capable of binding to virtually any dsDNA. (c) Repelling mode of SECM. While full recycling of the redox mediator in solution occurs at the electrode surface, the [Fe(CN)₆]⁴⁻ ions generated at the tip experience coulombic repulsions above a DNA modified spot, therefore leading to a decrease in tip current. (Online version in colour.)

Figure 8.

Schematics of ds-DNA (matched and mismatched) films evaluated and corresponding SECM images obtained. S1–S3: short complementary strands; L1–L3: long complementary strands; M: fully matched ds-DNA. The scale bar shows the intensity of the current. Adapted with permission from Shamsi & Kraatz [145] (Copyright 2013 Royal Society of Chemistry). (Online version in colour.)