Integration of on-chip isotachophoresis and functionalized hydrogels for enhanced-sensitivity nucleic acid detection.
We introduce an on-chip electrokinetic assay to perform high-sensitivity nucleic acid (NA) detection. This assay integrates electrokinetic sample focusing using isotachophoresis (ITP) with a background signal-removal strategy that employs photopatterened, DNA-functionalized hydrogels. In this multistage assay, ITP first enhances hybridization kinetics between target NAs and end-labeled complementary reporters. After enhanced hybridization, migration through a DNA-functionalized hydrogel region removes excess reporters through affinity interactions. We demonstrate our assay on microRNAs, an important class of low-abundance biomarkers. The assay exhibits 4 orders of magnitude dynamic range, near 1 pM detection limits starting from less than 100 fg of microRNA, and high selectivity for mature microRNA sequences, all within a 10 min run time. This new microfluidic framework provides a unique quantitative assay for NA detection.
Department of Mechanical Engineering, Stanford University, Stanford, California 94305, USA.
Giancarlo
G
Garcia-Schwarz
Juan G
JG
Santiago
D004247:DNA / Q000737:chemistry / Q000378:metabolism; D020100:Hydrogels / Q000737:chemistry*; D057625:Isotachophoresis; D035683:MicroRNAs / Q000032:analysis; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000032:analysis*
D020100:Hydrogels; D035683:MicroRNAs; D009696:Nucleic Acids; D004247:DNA