Probing Biological Systems Using Innovative Electrochemical Sensing and Imaging Platforms Inspired by Biology

Author/Creator ORCID

Date

2016-01-01

Department

Chemistry & Biochemistry

Program

Chemistry

Citation of Original Publication

Rights

This item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please see http://aok.lib.umbc.edu/specoll/repro.php or contact Special Collections at speccoll(at)umbc.edu
Distribution Rights granted to UMBC by the author.

Abstract

Electrochemical sensing and imaging platforms capable of detecting various analytes have attracted considerable attention in bio- and chemical analysis, as quantification and accurate detection of biologically relevant biomolecules may lead to early prognosis of certain diseases. Particularly, the utility of biological agents as biorecognition elements, such as nucleic acids and protein channels, have shown tremendous potential in the development of electrochemical-based biosensors owing to the high sensitivity and innate specificity that nature has already provided them with. In this thesis, the innovative use of nucleic acids and protein channels as biosensing and imaging devices to probe biological systems will be discussed in three parts: 1) in electrochemical DNA-based sensing that uses nucleic acids to monitor biochemical-binding mechanisms such as cooperativity; 2) in protein nanopore-based sensing that employs a ligand-gated, naturally occurring protein channel with specificity for adenosine triphosphate (ATP) to quantitatively detect physiological concentrations of ATP in solution; and 3) in protein channel-based imaging that demonstrates the potential of protein channels to be integrated into as imaging probes, which can be employed in scanning ion conductance microscopy to perform simultaneous topography imaging and specific molecule mapping across a synthetic membrane. In all parts, the development of the methodology is first described followed by its direct implementation in a number of biochemical and bioanalytical applications. The first part of this manuscript describes the use of an oligodeoxythymidylate [(dT)ₙ] electrochemical-DNA (E-DNA) based sensor for the direct monitoring of cooperative and non-cooperative binding of bacteriophage T4 gene 32 protein (g32p), a single-stranded DNA-binding protein that exhibits positive cooperativity when it binds to single-strand DNA (ssDNA). This demonstrates a new ability of E-DNA sensors, which may provide a rapid and simple methodology in understanding DNA-binding protein interactions, and, thus, adds to the growing toolbox enabled by this class of sensors. The second part of the manuscript describes the utility of the pore-forming nature of a ligand-gated heat shock cognate 70 (Hsc70) protein for the direct quantification of ATP. Hsc70 reconstitutes into phospholipid membranes and forms an ATP-regulated channel that exhibits multiple conductance states. The measurement of "charge flux" to characterize the ATP-regulated multi-conductance nature of Hsc70 is utilized as a new method of data analysis in order to achieve reproducible quantitation of ATP. This provides a universal method for quantifying ion-channel activity of proteins for the purpose of building specific and sensitive nanopore-based biosensors. The last part of the manuscript describes the development of a novel, bioinspired scanning ion conductance microscopy (bio-SICM) technique that couples the sensitivity and chemical selectivity of protein channels with the imaging ability of SICM to perform simultaneous pore imaging and specific molecule mapping. The framework of the bio-SICM platform is established using the single-molecule ability of the α-hemolysin (αHL) protein channel to map the spatial localization of β-cyclodextrin (βCD) target molecules crossing a single 25-µm-diameter substrate pore opening. This analytical approach not only demonstrates the potential of protein channels to act as sensing elements in imaging probes, but also extends the utility of SICM by enabling selective chemical imaging of specific target molecules, while simultaneously providing topographical information about the net ion flux through a pore under a concentration gradient. With further optimization, the bio-SICM platform will provide a powerful analytical methodology that is generalizable, and thus, offers significant utility in a myriad of bioanalytical applications.