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Integration of on-chip isotachophoresis and functionalized hydrogels for enhanced-sensitivity nucleic acid detection

Significance Statement

microRNAs are short, non-coding RNAs that post-transcriptionally regulate gene expression and have emerged as diagnostic and prognostic biomarkers for cancer, diabetes, and other diseases. Techniques currently used for microRNA detection generally lack the combined sensitivity, specificity, and fast analysis time needed in clinical settings. In two publications (Anal Chem 84, 2012; Angew Chem 52, 2013), we present a two-stage microfluidic assay capable of single nucleotide-specific microRNA detection from ~500 cells with 15 minute analysis time. Our assay uses an electrokinetic focusing technique called isotachophoresis (ITP) to simultaneously preconcentrate and mix microRNAs with complementary DNA reporters. Using isotachophoresis, we achieve 10,000-fold increases in concentration and hybridization reaction rate, which allows us to dramatically reduce analysis time. Following enhanced hybridization, the system uses a functionalized in-line hydrogel to perform affinity purification of reaction products, leaving only hybridized reporters focused in isotachophoresis. Importantly, our technique leverages separate hybridization and purification stages to enhance specificity using both equilibrium thermodynamics and off-rate kinetics. We developed numerical models which capture this two-stage specificity enhancement and additionally can be used for assay design and optimization. Our first publication presents a proof-of-concept study of this assay under low-stringency conditions and using idealized samples. In our second publication, we introduce modifications to enable high-stringency analysis and demonstrate this novel technique by quantifying let-7a microRNA concentration with single-base-pair stringency using real total RNA samples from mammalian tissues and cell culture.

 

Integration of On-Chip Isotachophoresis and Functionalized Hydrogels for Enhanced-Sensitivity Nucleic Acid Detection- Global Medical Discovery

 

 

 

 

 

 

 

 

 

 

 

 

Garcia-Schwarz G, Santiago JG. Anal Chem. 2012 Aug 7;84(15):6366-9.

Department of Mechanical Engineering, Stanford University, Stanford, California 94305, USA.

Abstract

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, isotachophoresis 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.

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