We propose the development of a DNA-based electrochemical device using a new two-electrode strategy for DNA array patterning and detection. This renewal proposal is based on DNA-mediated electrochemistry and should allow the detection of nucleic acid and DNA-binding protein biomarkers with high sensitivity, suitable for quantitative diagnostics and research. Our 2-electrode platform provides a means to fabricate a DNA array on a single electrode, along with quantitative, multiplexed electrocatalytic sensing. We propose first to optimize the platform, including the incorporation of pin- based patterning. Click chemistry with copper activation will be the primary means for potential- dependent array formation. With respect to detection, we will optimize electrocatalysis partners and we will assess limits of detection (attomoles) through analysis of TATA-binding protein binding to DNA on the patterned platform. Microfluidics will be incorporated into the device. Once optimized, we propose developing the platform first for nucleic acid detection, specifically for two target microRNA sequences, miR-200c and let-7a. MicroRNAs are differentially expressed in healthy and cancerous tissues, which make them ideal targets for early cancer detection and profiling. We will monitor differences in expression levels using cultured colorectal cell lines with and without cancerous transformation. We also propose to test this sensor in detecting the human methylase DNMT1. DNA methylation modulates gene regulation and transcription, and both hyper and hypomethylation are associated with disease. We will take advantage of our "turn-on" methylase/restriction assay. We will quantify DNMT1 from cell lysates differing in expression of DNMT1, followed by measurements of tissue samples. Correlations will be drawn between different cancers and levels of methylase activity in order to establish a new early diagnostic based upon aberrant methylation. We will develop the platform also to screen potential therapeutics that inhibit methylation. Next we will move to simultaneous detection of disease-related miRNA expression and DNMT1 levels. Given the high sensitivity and reproducibility in detection with this device, we will also explore single cell detection of ou biomarkers. We will explore our miRNA and methylase targets to compare results between the bulk average and distribution among single cells. Combining assays for protein binding, RNA and DNA analysis already developed in our laboratory with new array fabrication methods and a two-electrode detection scheme, we propose an innovative approach to multiple biomarker detection through a robust sensor suitable for both basic research in systems biology as well as multiplexed applications for diagnosis and screening.

Public Health Relevance

This project involves the construction of new electrical sensors for RNA and proteins that bind to DNA. These sensors will serve as completely new diagnostic tools to detect the proteins and RNAs from cells and tissues that are associated with different disease states. These sensors could represent a new technology for the early diagnosis of cancer and other diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
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Edmonds, Charles G
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California Institute of Technology
Schools of Engineering
United States
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Furst, Ariel L; Muren, Natalie B; Hill, Michael G et al. (2014) Label-free electrochemical detection of human methyltransferase from tumors. Proc Natl Acad Sci U S A 111:14985-9
Furst, Ariel; Hill, Michael G; Barton, Jacqueline K (2014) Electrocatalysis in DNA Sensors. Polyhedron 84:150-159
Pheeney, Catrina G; Barton, Jacqueline K (2013) Intraduplex DNA-mediated electrochemistry of covalently tethered redox-active reporters. J Am Chem Soc 135:14944-7
Muren, Natalie B; Barton, Jacqueline K (2013) Electrochemical assay for the signal-on detection of human DNA methyltransferase activity. J Am Chem Soc 135:16632-40
Pheeney, Catrina G; Barton, Jacqueline K (2012) DNA electrochemistry with tethered methylene blue. Langmuir 28:7063-70
Slinker, Jason D; Muren, Natalie B; Renfrew, Sara E et al. (2011) DNA charge transport over 34 nm. Nat Chem 3:228-33
Genereux, Joseph C; Boal, Amie K; Barton, Jacqueline K (2010) DNA-mediated charge transport in redox sensing and signaling. J Am Chem Soc 132:891-905
Slinker, Jason D; Muren, Natalie B; Gorodetsky, Alon A et al. (2010) Multiplexed DNA-modified electrodes. J Am Chem Soc 132:2769-74
Gorodetsky, Alon A; Hammond, William J; Hill, Michael G et al. (2008) Scanning electrochemical microscopy of DNA monolayers modified with Nile Blue. Langmuir 24:14282-8
Guo, Xuefeng; Gorodetsky, Alon A; Hone, James et al. (2008) Conductivity of a single DNA duplex bridging a carbon nanotube gap. Nat Nanotechnol 3:163-7

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