Evaluating the Use of Edge Detection in Extracting Feature Size from Scanning Electrochemical Microscopy Images.

The edge of a reactive or topographical feature is hard to estimate from feedback-based scanning electrochemical microscopy due to diffusional blurring, but is crucial to determining the accurate size and shape of these features. In this work, numerical simulations are used to demonstrate that the inflection point in a 1D line scan corresponds well to the true feature edge. This approach is then applied in 2D using the Canny algorithm to experimental images of two model substrates and a biological sample. This approach circumvents the need for aligning the imaged region between separate microscopy techniques, reveals hidden details embedded in SECM images, and allows individual features to be separated from their background more effectively.

Determination of the Relationship between Expression and Functional Activity of Multidrug Resistance-Associated Protein 1 using Scanning Electrochemical Microscopy.

Cancer cells can develop multidrug resistance (MDR) after prolonged exposure to chemotherapeutic drugs, which is a severe impediment to successful treatment. MDR is typically associated with transmembrane proteins mediating efflux of administered drugs, thereby keeping their intracellular concentration below the threshold required to kill cells. Although expression assays based on flow cytometry and immunostaining have shown that multidrug resistance-associated protein 1 (MRP1) is prevalent in many cancer types, the functional activity of this efflux pump is more difficult to elucidate, especially at the single-cell level. Herein, we report the measurement of MRP1 functional activity in individual cancer cells using scanning electrochemical microscopy (SECM). Cells were cultured onto plastic substrates containing selective adhesion sites. Optical microscopy and SECM revealed that cells adapt to the underlying surface, while MRP1 functional activity increases once the dimensions of the adhesive islands become smaller than those of the cell itself. Time-lapse SECM imaging revealed a suitable window of 30 min to complete each measurement before the cell undergoes blebbing, which is associated with a considerable increase in functional activity. Distinct cell populations were produced by performing a doxorubicin drug challenge on two parental cell lines (e.g., wild-type HeLa cells and MRP1-overexpressing HeLa-R cells). Expression and functional activity of MRP1 were determined using flow cytometry and SECM, and our findings show that these parameters do not directly correlate. This suggests that functional activity may represent a powerful indicator of a cancer cell's response to chemotherapeutic treatment and should improve our understanding of efflux mechanisms based on MRP1.

Scanning Electrochemical Microscopy: A Comprehensive Review of Experimental Parameters from 1989 to 2015.

Scanning electrochemical microscopy (SECM) is an electroanalytical scanning probe technique capable of imaging substrate topography and local reactivity with high resolution. Since its inception in 1989, it has expanded into a wide variety of research areas including biology, corrosion, energy, kinetics, instrumental development, and surface modification. In the past 25 years, over 1800 peer-reviewed publications have focused on SECM, including several topical reviews. However, these reviews often omit key details, forcing readers to search the literature. In this review, we provide a comprehensive summary of the experimental parameters (e.g., solvents, probes, and mediators) used in all SECM publications since 1989, irrespective of the application. It can be used to rapidly assess experimental possibilities and make an informed decision about experimental design. In other words, it is a practical guide to SECM.

Fabrication of carbon, gold, platinum, silver, and mercury ultramicroelectrodes with controlled geometry.

A simple, fast, and reproducible method for the fabrication of disk ultramicroelectrodes (UMEs) with controlled geometry is reported. The use of prepulled soda-lime glass capillaries allows one to bypass the irreproducible torch-sealing and experimentally challenging tip-sharpening steps used in conventional fabrication protocols. A micron-sized electroactive wire is sealed inside this capillary producing UMEs with a highly reproducible geometry. Total fabrication time (1 h) and experimental difficulty are significantly reduced. Disk UMEs with various diameters and cores were fabricated, including carbon fiber (7 and 11 µm), gold (10 and 25 µm), platinum (10 and 25 µm), silver (25 µm), and mercury (25 µm). The ratio of the insulating sheath to the electroactive core of the UMEs was 2.5-3.6. Silver UMEs were also used to produce a Ag/AgCl microreference electrode. This general fabrication method can readily be applied to other electroactive cores and could allow any research group to produce high quality disk UMEs, which are a prerequisite for quantitative scanning electrochemical microscopy.

Disk-shaped amperometric enzymatic biosensor for in vivo detection of D-serine.

At the synapse, D-serine is an endogenous co-agonist for the N-methyl-D-aspartate receptor (NMDAR). It plays an important role in synaptic transmission and plasticity and has also been linked to several pathological diseases such as schizophrenia and Huntington's. The quantification of local changes in D-serine concentration is essential to further understanding these processes. We report herein the development of a disk-shaped amperometric enzymatic biosensor for detection of D-serine based on a 25 µm diameter platinum disk microelectrode with an electrodeposited poly-m-phenylenediamine (PPD) layer and an R. gracilis D-amino acid oxidase (RgDAAO) layer. The disk-shaped D-serine biosensor is 1-5 orders of magnitude smaller than previously reported probes and exhibits a sensitivity of 276 µA cm(-2) mM(-1) with an in vitro detection limit of 0.6 µM. We demonstrate its usefulness for in vivo applications by measuring the release of endogenous D-serine in the brain of Xenopus laevis tadpoles.

Assessment of multidrug resistance on cell coculture patterns using scanning electrochemical microscopy.

The emergence of resistance to multiple unrelated chemotherapeutic drugs impedes the treatment of several cancers. Although the involvement of ATP-binding cassette transporters has long been known, there is no in situ method capable of tracking this transporter-related resistance at the single-cell level without interfering with the cell's environment or metabolism. Here, we demonstrate that scanning electrochemical microscopy (SECM) can quantitatively and noninvasively track multidrug resistance-related protein 1-dependent multidrug resistance in patterned adenocarcinoma cervical cancer cells. Nonresistant human cancer cells and their multidrug resistant variants are arranged in a side-by-side format using a stencil-based patterning scheme, allowing for precise positioning of target cells underneath the SECM sensor. SECM measurements of the patterned cells, performed with ferrocenemethanol and [Ru(NH3)6](3+) serving as electrochemical indicators, are used to establish a kinetic "map" of constant-height SECM scans, free of topography contributions. The concept underlying the work described herein may help evaluate the effectiveness of treatment administration strategies targeting reduced drug efflux.