Research Synopsis

Miniaturization in the field of analytical chemistry continues to advance measurement capabilities in a wide range of scientific disciplines. Our research group develops and implements bioanalytical techniques towards single-cell analysis for a better understanding of complex biological functions. Both electrochemical and electrogenerated chemiluminescence techniques are being developed to simultaneously detect relationships between classical neurotransmitter and free radical dynamics. Ultimately, we are interested in detecting these relationships within functional clusters (i.e. neurons and glia) to better understand intercellular free radical dynamics during neuronal communication involving different cell types as well as their contribution to disease states (i.e. neurodegeneration and cancer tumor formation). Our goals focus on contributing to the growing understanding that free-radicals have more of a biological role than just being toxic by-products of cellular function. Our over-arching goals are to exploit unique nanoscale phenomena for enhanced electrochemical-based sensing that could then be utilized for various analyses across disciplines. These goals are critically dependent on interdisciplinary efforts encompassing chemistry, biology, physics, and engineering.

Figure 1. SEM of nanopores in a polymethylmethacrylate (PMMA) layer milled with a 30-kV focused Ga+ ion beam. Pore diameters are ~ 170 ± 8 nm.

The transient nature of most of these free radical and reactive oxygen species makes the task of analysis a daunting challenge. The analytical challenges arise in providing temporal analysis of short-lived reactive species with sufficient sensitivity and spatial resolution to investigate cellular and subcellular processes. We are developing novel nanoelectrode fabrication technologies to perform faster measurements of small molecules associated with cellular function with higher spatial resolution. Specifically nanoelectrodes, with confined radial-diffusion reaction spheres, are coupled with electrogenerated chemiluminescence interrogations of transient species (i.e. reactive oxygen species) associated with cellular function. These nanostructures are fabricated by milling through a thin nonconductive polymer layer with a focused-ion beam (FIB - currently performed at the DOE sponsored Center for Materials Microanalysis at the University of Illinois at Urbana-Champaign) to produce a nanometer diameter through-holes that expose the underlying substrate (see Figure 1). We have successfully milled holes as small as 75 nm through 8-um thick poly(methylmethacrylate) layers. These unique structures serve either as nanoelectrode wells (both individual and arrays) or as templates for the creation of nanowire structures via electroless gold deposition followed by template removal via plasma etching. Incorporation of nanoelectrode arrays within microfluidic devices is also being pursued for efficient, high-throughput analysis of single cells. Combining nanoscale electrochemical architectures with ultra-sensitive optical microscopy for electrogenerated chemiluminescence will enhance the spatial and temporal interrogation of free radical dynamics in intercellular, single-cellular, and subcellular biological systems.

Fundamental studies associated with electrochemical miniaturization within our group include single molecule detection via the recycling amplification of electron transfer between a nanometer separated anode and cathode. The fabrication precision of FIB milling has easily achieved less than 100-nm separation of nanofabricated features. Chemical intermediates associated with fast electrochemical reactions are also investigated between two individually addressable nanometer scale electrodes, with electron tunneling measurements targeted for providing new analytical capabilities. Ideally, new technical areas will be enabled though the development of simpler methods for the reproducible preparation of individually addressable nanoelectrode structures of defined number and density.