Abstract

Plastic-based Nanofluidic Sensor for the Detection of Rare Nucleic Acids and Determining Their Sequence Variations from Liquid Biopsy Markers

Steve Soper, Foundation Distinguished Professor; Director, Center of BioModular Multi-scale System for Precision Medicine, Adjunct Professor, Ulsan National Institute of Science & Technology, The University of Kansas

Liquid biopsy markers (circulating tumor cells, CTCs; extracellular vesicles, EVs; and cell free DNA, cfDNA) are becoming extremely popular to manage a variety of cancer-related diseases due to the minimally invasive nature of their acquisition. However, the challenge with liquid biopsy markers is their rarity; for example, it is not uncommon to secure 1-100 CTCs per mL of whole blood supplying about 6-600 pg of genomic DNA. Because platforms like next generation sequencing require >30 ng of input DNA, significant amounts of amplification of the input are required that can generate a biased representation of the genome. To mitigate this issue, we have produced a mixed-scale nanofluidic sensor featuring a baffle area, high surface area pillar arrays, and nanometer flight tubes.  The pixel arrays can perform solid-phase ligase detection reactions (spLDRs) to score the presence of DNA mutations found in a diseased patient even when the mass of the marker is low (<1 ng), but does not require PCR amplification for the analysis. The spLDR can also expression profile mRNAs following reverse transcription. Successfully formed spLDR products are identified using a molecular-dependent time-of-flight (TOF) through a polymer nanofluidic channel flanked by two in-plane nanopores. Simulations (COMSOL) were used to guide the design and fabrication of the nanofluidic sensor to determine the loading efficiency and transport patterns of spLDR products from the pillar array into the flight tubes by evaluating operational parameters when using either hydrodynamic or electrokinetic flow. The nanofluidic sensor was fabricated from a Si master patterned using a combination of focused ion beam (FIB) milling and photolithography with inductively coupled plasma reactive ion etching. The Si master was used to produce resin stamps that were then used to transfer the relevant structures to a plastic via thermal nanoimprint lithography (NIL). The operational features of the device will be presented as well as detecting point mutations in KRAS genes from CTCs’ genomic DNA as well as mRNA expression profiling.