Abstract

A Solid-State Nanopore microRNA Quantification Platform

Hsueh-Chia Chang, Bayer Professor of Engineering / Director, University of Notre Dame

MicroRNA (miRNA) profiling in liquid biopsy and single-cell assays by the current microarray, digital PCR and rtPCR technologies are plagued with false positives and negatives, particularly at low copy numbers. A main reason for such inaccuracies is that these technologies are not sufficiently selective for different miRNAs that differ by only a few bases and whose dissociation constants for primer/probe hybridization are nearly identical (Egatz et al,
Biomicrofluidics, 10: 032902(2016)).

We report a new highly selective (PCR-free) solid-state nanopore miRNA quantification platform for liquid biopsy and single-cell assays. A single ion-track nanopore in a PET membrane is asymmetrically etched into a conic geometry and is coated with a high-permittivity dielectric layer by Atomic Layer Deposition (Yan et al, J Chem Phys, 138: 044706(2013)).  The surface modified conic nanopore allows for high throughput molecular translocation (100 Hz vs 1 Hz for protein nanopores) and selective delay of single-stranded (ss) nucleic acids compared to their hybridized double-stranded (ds) duplex (mean translocation time of 100 ms vs 1 ms with 5% overlap in the two distributions). The delay is due to enhanced van der Waal attraction between exposed rings of the ss-nucleic acids, with delocalized electrons, with the high-permittivity coating. Individual translocation events can be recorded for a mixture of ss- and ds-nucleic acids numbering between 100 to 100,000. Whether the translocating molecule of each event is an ss miRNA or its ds duplex can be discerned with 95% confidence.

We integrate this nanopore sensor on a biochip with an upstream Surface Acoustic Wave exosome lysing module (Taller et al., LabChip, 15: 1656(2015)) and a membrane module that can separate miRNA from proteins, genomic DNA and longer mRNA (Sun et al, LabChip, 16: 1171(2016); Slouka et al, Talanta, 145: 35(2015); Slouka et al, Annual Rev of Analytical Chem, 7:317(2014)). We capture specific miRNAs from the fractionated population with complementary probes linked to Au
coated surfaces. Selective capture is enhanced with electric and hydrodynamic forces that can discriminate single-base mutation (Marczak et al, Biosensors and Bioelectronics, 86: 840(2016); Cheng et al, LabChip, 10:828(2010); Basuray et al, ACSNano, 3:1823 (2009)). The resulting duplexes are subsequently released with 100% yield by
electrochemical cleavage within an appropriate voltage window, without releasing the target-free probes.  This two-step fractionation and isolation steps then allow the downstream solid-state nanopore to quantify specific miRNA duplexes.

Testing of this platform with cell culture media and spiked serum samples will be reported.