This Small Business Innovation Research (SBIR) Phase I project aims to develop a commercial nanofluidic chip for the rapid identification of epigenetic marks on individual molecules. The instrument will dramatically improve existing methods of epigenomic analysis by removing two major limitations of the current technology. The proposed device will enable automated quantification of multiple epigenetic marks simultaneously using minute inputs of chromatin and high throughput single molecule observations. The approach involves measuring distinct fluorescent signals from antibodies bound specifically to epigenetic marks on individual chromatin fragments that are electrophoretically transported through lasers focused within a nanoscale fluidic channel. The objectives of this proposal are to transform this laboratory setup into a prototype commercial product by increasing the throughput using parallel fluidic channels and transforming to a free space optical system. It is anticipated that this Phase I proposal will result in the fabrication of devices containing 96 parallel nanofluidic channels and the design of the free space optical system that will facilitate this transformation.
The broader impact/commercial potential of this project is that it will result in commercially available products that overcome two key limitations of current epigenomic technology. Current technology requires an abundant amount of input material and can query only one epigenetic mark at a time. The proposed single molecule analytical methods can overcome both of these limitations. It is anticipated that this effort will yield a new disruptive epigenomics technology to serve commercial, academic, and clinical needs. By developing an automated instrument that can interrogate multiple epigenetic marks simultaneously on single chromatin molecules extracted from very small inputs of cells, this technology will enable epigenomic analyses that are far more information rich and lower in cost than is currently possible.
It has become clear over the last 20 years that epigenetic variations are of profound importance to human health and understanding cell development. Among the most important of these variations are chemical modifications to the DNA and histone proteins associated with the DNA. These modifications include methylation of cytosines in chromosomal DNA and at least six modifications affecting more than 50 amino acid residues in the four core histones. The epigenetic state does not alter the primary DNA sequence but it does expert a potent influence on gene expression. Environmental variables also influence health, but evidence indicates that their influence is often mediated by epigenetic mechanisms. Current methods for obtaining epigenetic information are limited by the requirement for analyzing large amounts of chromosomal material, detecting only one epigenetic mark per sample, and by the time required to obtain quantitative information. The most refined current methods for epigenomic analysis include bisulfite-based methods or methyl DNA immunoprecipitation to assess DNA methylation, and chromatin immunoprecipitation (ChIP) to monitor histone modifications. Each of these can be used for genome wide analyses. Odyssey Scientific's Phase I SBIR research project has addressed these limitations by advancing the development of a single molecule detection (SMD) analytical device and approach. By interrogating individual molecules with the same reagents currently used for ChIP, one can detect several distinct epigenetic marks simultaneously, and determine which combinations are actually present on an individual DNA molecule. Because very small inputs of material are needed for single molecule analysis, it is possible to do studies with very few cells. This solves the first problem of ambiguity created by combining chromosomal material from a heterogeneous cell population. Our solution also addresses the second problem that limits ChIP, the ability to interrogate one epigenetic mark at most when materials are limiting. Our approach is directed toward developing an integrated system that can rapidly detect and quantify the presence of selected epigenetic signatures for diagnostic or research use. The SMD methods afforded by our platform have the potential to revolutionize basic research, environmental testing, therapeutics development and disease diagnosis by providing exquisitely sensitive, information rich, scalable assays for important biomarkers.
