|Ph.D Student||Xander Frank Van Kooten|
|Subject||On-chip Separation and Focusing Using Isotachophoresis|
with Large-Volume Processing and Analyte
|Department||Department of Mechanical Engineering||Supervisors||Professor Bercovici Moran|
|Dr. Govind Kaigala|
Biochemical assays are the cornerstone of in vitro medical diagnostics, as they provide a way to detect analytes such as proteins and nucleic acids, which provide information about the physiological state of an individual. However, these analytes are often present at such low concentrations that they cannot be detected by conventional assays. To overcome this, electrokinetic methods can be used to increase the local concentration of analytes. Isotachophoresis is an electrophoretic method that enables such a local concentration enhancement by stacking analytes at an electric field gradient between two distinct electrolytes.
This thesis presents methods and devices that use on-chip ITP to focus and purify analytes from large sample volumes, and presents a way to decouple electrokinetic separations from downstream analytical processes.
In commercial microchannels, ITP focuses analytes from an initial sample volume of several hundred nanoliters. This small processed volume sets an upper bound on the concentration factor, as ITP only collects analytes that are initially present within this volume. We have developed a microchannel geometry that enables focusing from large sample volumes by using a tapered channel and geometrical features that reduce dispersion. Using this chip, we demonstrate 104,000-fold focusing of DNA oligonucleotides from an initial volume of 50 μL into a 500 pL focused zone. This concentration increase accelerates hybridization reactions, enabling detection of 1 pM DNA oligonucleotides using molecular beacons, and bacteria down to 100 cfu/mL.
Large-volume processing also benefits ITP-based separation and purification. To illustrate this, we present a method that enables spatially resolved genotyping of formalin-fixed paraffin-embedded (FFPE) tissue sections. This method involves locally sampling tissue sections with a microfluidic probe, followed by large-volume ITP purification of DNA from the sampled lysate, and finally Sanger sequencing of multiple genes. Using this workflow we demonstrate genotyping of an array of sampling sites on FFPE sections, with a spatial resolution of ~200 μm, and we identify single-nucleotide mutations in human breast invasive ductal carcinoma.
Although electrophoretic techniques such as ITP provide a powerful way to separate and focus analytes on-chip, one of their key limitations is that the analytes cannot be accessed after separation. This limits their use to assays that can be performed under high external electric fields. To overcome this, we have developed a method to decouple electrophoretic separations from downstream analyses. This method uses on-demand injection of an oil phase to capture separated and focused analytes in a sub-nanoliter water-in-oil droplet. We use this method to retain concentrated analytes for tens of minutes after ITP focusing, and we show a 22-fold improvement in concentration compared to free diffusion. Finally, we demonstrate manipulation of an individual droplet containing ITP-purified DNA after amplification, by performing parallel detection reactions off-chip using a single 240 pL droplet.