(179b) On-Chip Sample Preparation and Nucleic Acid Profiling Using Isotachophoresis

Robust, automated sample preparation and its seamless integration with other microfluidic functions remains a major unsolved challenge in diagnostic applications.  This is particularly true for extraction, purification, and preconcentration of nucleic acids from complex biological samples such as blood, urine, and cultured cells.  At Stanford, we are developing on-chip isotachophoresis (ITP) processes which selectively focus a target analyte from a complex mixture, while rejecting unwanted molecules (e.g., proteins or multivalent cations) which may inhibit hybridization or amplification.  We will present on-chip extraction of various target nucleic acids including DNA from blood lysate, rRNA from urine lysate, and microRNA from total RNA. 

We will also present a novel assay where we simultaneously extract/purify targets, and then identify and quantify specific sequences using molecular beacons.  Molecular beacons are short, sequence specific DNA probes which fluoresce upon hybridization.  We use ITP to preconcentrate both target and beacons by ~10,000x into order 10 pl reaction volumes.  We will show specific and sensitive detection of target sequences in order 2 minutes with minimal sample preparation, and without target amplification.    We will demonstrate quantitation of specific sequences of microRNA from total RNA, and of bacterial 16S rRNA from infected urine lysate.