Technical description MicroRNA are small 20-26 nucleotide (nt) long noncoding RNA that are effective biomarkers for a variety of diseases, including cancer, heart, obesity, and mental illness. During the last two decades, it has become evident that profiling 100s of microRNA (miRNA) molecules found in blood, urine, and tissue can lead to early diagnoses before clinical symptoms appear. Today, there is no inexpensive, reliable method for profiling microRNA that can be translated to a clinic setting. Microarray is an inexpensive technology but not reliable due to large background signal from nonspecific binding. The objective of this proposal is to explore a novel electrochemical method for profiling miRNA by measuring local redox reactions on a monolith electrode by simply scanning a laser beam over various microarray binding sites. The electrochemical (redox current) signal can potentially distinguish a perfect match between an immobilized probe and a target binding, and no redox occurs on nonspecific binding. The applied electric potential for the redox signal shifts when the binding has a single mismatch relative to a perfect binding case. The "active" biosensing based on the redox current has the potential to measure binding of target analyte molecules to immobilized probes at a sensitivity of 1 atto-moles. The electrochemical method to map local redox current density over an electrode by laser scanning is called "Scanning Electrometer for Electrical Double-layer" (SEED).
Non-technical description This proposal will develop a new way to sense small pieces of nucleic acid chains that serve as biomarkers of multiple diseases including cancer. The proposal will enable detection of several different nucleic acid molecules simultaneously using electrical measurements. Importantly, instead of having to create individual electrodes for each biomarker, the authors will develop a way to detect all of the biomarkers for one electrode. This project will result in simpler, more powerful and informative way of analyzing biomarkers for human diseases.
MicroRNA profiles have the potential to be highly effective in the early diagnosis of range of diseases including, neurological disorder, obesity and various type of cancers before clinical signs emerge. Owing to its small size in the range of 20-30 nucleotides, the current technology for miRNA profiling are difficult to multiplex and too expensive to be used for screening. A disruptive technology is needed to read microarrays of miRNA with minimal background and high sensitivity. In this one year EAGER program, a proof-of-concept study to evaluate a technology called Scanning Electrometer for Electrical Double-layer (SEED) as a screening tool for miRNA profiling is explored. Electrochemical detection using (redox) dye (such as methylene blue) is blind to nonspecific binding and can distinguish single base mismatch. SEED is proposed as a potential method to multiplex electrochemical detection by measuring local redox current on a monolith electrode by simple laser scanning. The key outcome of the preliminary study are two. (1) Successful measurement on 12 spot array was possible to perform spot-to-spot redox current measurement where the probe sequence was perfectly complimentary to the target (i.e., perfect match (PM)), had a single base mismatch (MM1), and was non-complimentary (non-specific) to the target sequence (Fig. 1). (2) Using Electric Field Influenced Binding (EFIB) where a cyclic potential was applied between the electrode (with the immobilized probe molecules) and the solution, the immunospecific binding time was reduced from 18 hours to 30 mins. (Fig. 2). EFIB will significantly reduce the time to result from over 20 hours to below 90 mins. The lowest target concentration measured was 0.1 nM. All the studies were performed on synthetic nucleotides corresponding to miRNA, hsa-MiR-155, a biomarker for early detection of Pancreatic Cancer. The data suggests, that SEED is a promising method to profile miRNA on 50 to 100 spot array with high specificity. The study was conducted by one graduate student. Intellectual Merit: SEED is potentially a disruptive technology that will translate the single analyte detection per electrode to multiple analyte on single electrode. The study will also allow quantitative measurement of immunospecific binding kinetics using EFIB. The SEED application to miRNA will serve as a motivation to develop other combinatorial analytics based on electrochemical detection on monolith electrode. Broad impact: MiRNA profiling is a highly desirable platform application for SEED which may lead to a reliable diagnostic tool for early detection of diseases before clinical signatures emerge. As miRNA is in the blood stream, potentially, a screening based on genomics can be developed. The project exposes graduate students to physical-biology, instrumentation and micro-fabrication methods.
