Novel on-chip isotachophoresis assays for nucleic acid extraction and analysis

J.G. Santiago
Stanford University, US

Keywords: MEMS, microfluidics, lab-on-a-chip, nucleic acid extraction

Summary:

Microfluidics research is growing in complexity and variety, even as the technology matures and is commercialized. Yet there remains a glaring weakness common to most microfluidic systems: The lack of automated sample preparation and its integration with downstream assays. This is particularly true for selective extraction and preconcentration of nucleic acids from complex biological samples such as blood, urine, and cultured cells. This talk will summarize research at Stanford toward meeting this unmet challenge. We are developing on-chip isotachophoresis (ITP) processes which selectively focus target analytes from a complex mixture, while rejecting unwanted molecules which may inhibit hybridization or amplification. We will present extraction of various target nucleic acids including DNA from blood and bacterial rRNA from urine. We will also present examples of integrating ITP sample preparation with downstream assays. We have integrated RNA extraction with sequence-specific quantitation using molecular beacons and alternately by combining fluorescent DNA probes with functionalized, photopatterned hydrogels. We use ITP to purify and preconcentrate target and probe molecules by >10,000x into order 10 pl reaction volumes. We show specific and sensitive detection of target sequences in order 2 minutes with little or no off-chip sample preparation, and without target amplification. We have demonstrated profiling of microRNA from total RNA, and quantitation of bacterial 16S rRNA from infected urine lysate.