(244e) Low-Loss on-Chip Sample Pretreatment: Depletion Isotachophoretic Isolation of DNAs from Inhibitor-Rich Samples By an Ionic Transistor | AIChE

(244e) Low-Loss on-Chip Sample Pretreatment: Depletion Isotachophoretic Isolation of DNAs from Inhibitor-Rich Samples By an Ionic Transistor


Sun, G., University of Notre Dame
Senapati, S., University of Notre Dame
Chang, H. C., Year
Low-copy number DNA detection from clinical samples is plagued by analyte loss during the various personnel-intensive sample pretreatment steps like fractionation, centrifugation and dialysis to isolate the target molecules and to remove the inhibitors or interfering agents for PCR amplification or the particular biochemical assay. In order to eliminate the undesired sample handling and simplify the sample pretreatment steps, we report an on-chip sample pretreatment device to isotachophoretically isolate DNA molecules from arbitrary inhibitor-rich samples. DNA molecules with a particular length can be concentrated and purified, without confining barriers, at a specific on-chip location for easy transfer to on-chip PCR amplification units and downstream assays. 

Our sample pretreatment device realizes depletion isotachophoresis by a microfluidic ionic transistor. The microchannel for DNA isolation is gated by an ion-selective nanoporous membrane. Because of the charge selectivity of the ion-selective membrane, ion concentration depletion occurs in the microchannel upon the application of a normal gating electric field across the membrane1. By configuring the gating and draining voltages applied on the ionic transistor, we can achieve a gating voltage-sensitive but stationary depletion front length in the microchannel. Such a stationary depletion front offers a stable electric field jump to isotachophoretically isolate charged molecules, like DNAs, at a particular location selected by the gating voltage. 

By continuously loading samples into the ionic transistor, we can isolate negatively charged DNA molecules at the stationary depletion front from samples with an arbitrary composition and volume. Inhibitors with positive charges or with low electrophoretic mobility can be removed by the electric field and the loading flow simultaneously, without the need of centrifugation and filtration. By real-time fluorescence imaging, we show that DNA molecules with different lengths, ranging from 30 bases to 400 bases, are isolated into separated bands with a near 90% isolation yield. The DNA concentration in each isolated band is also enhanced by two orders of magnitude. We further prove that the pretreatment device removes PCR inhibitors, including Calcium ions and Hemoglobin, by successfully performing on-chip PCR of the isolated DNAs. Integrating the pretreatment device with the on-chip PCR amplification and an ion current nanomembrane sensor developed in our lab for DNA detection2, we expect to achieve multiplex nucleic acid sensing with fM sensitivity directly from serum and urine samples.

[1] Chang et al, Annu Rev Fluid Mech, 44, 401-426 (2012)

[2] Slouka et al, Annu Rev Anal Chem, 7, 317 (2014)