Temperature-dependant [sic] smart bead adhesion: a versatile platform for biomolecular immobilization in microfluidic devices

Thesis (Ph. D.)--University of Washington, 2003.Microfluidic systems for chemical and biochemical analysis promise increased process portability, speed, and efficiency as well as decreased waste. One important aspect of microfluidic design is the incorporation of immobilized biomolecules in microfluidic devices. Of the methods that have been proposed for the microfluidic immobilization of biomolecules, few allow for reversible immobilization or end-user control over the immobilization process. The absence of these capabilities limits device flexibility and reusability.Presented in this dissertation is a method for achieving the reversible, controlled immobilization of biomolecules in microfluidic devices. This method is based on so called "smart beads": latex nanobeads coated with the temperature-responsive smart polymer poly(N-isopropylacrylamide) (PNIPAAm). PNIPAAm exhibits lower critical solution temperature (LCST) behavior: soluble in aqueous solutions at room temperature, but becoming hydrophobic and reversibly aggregating at temperatures greater than ∼32 UpsilonC. Similarly, 100 nm diameter polystyrene latex beads coated with PNIPAAm form a suspension that flows easily through a microfluidic channel constructed from poly(ethylene terephthalate) (PET) at room temperature, but when the temperature of the channel is elevated above the polymer LCST, the beads aggregate and adhere to the channel walls. This adhesion is stable in the presence of flow, and reversible upon return of the channel temperature to below the polymer LCST. By co-modifying PNIPAAm-coated beads with biotinylated poly(ethylene glycol), we have made possible the controlled, reversible immobilization of diverse biomolecules.Experiments preliminary to the development of the smart bead system described in this dissertation include the study of a PNIPAAm-based bioseparation scheme as well as the investigation of the behavior of PNIPAAm in microfluidic channels. Smart bead applications include an affinity chromatography system, in which the biotinylated beads serve as a chromatographic matrix for the separation of streptavidin from a flow stream, and an immunoassay system, in which beads modified with an antibody for the drug digoxin serve as the substrate for a competitive digoxin immunoassay. Further prospective applications of the smart bead system, such the facilitation of multiple parallel bioseparations in a single microfluidic channel, are also presented

Liposomes with Double-Stranded DNA Anchoring the Bilayer to a Hydrogel Core

Liposomes are important biomolecular nanostructures for handling membrane-associated molecules in the lab and delivering drugs in the clinic. In addition to their biomedical applications, they have been widely used as model cell membranes in biophysical studies. Here we present a liposome-based model membrane that mimics the attachment of membrane-resident molecules to the cytoskeleton. To facilitate this attachment, we have developed a lipid-based hybrid nanostructure in which the liposome bilayer membrane is covalently anchored to a biocompatible poly­(ethylene) glycol (PEG) hydrogel core using short double-stranded DNA (dsDNA) linkers. The dsDNA linkers connect cholesterol groups that reside in the bilayer to vinyl groups that are incorporated in the cross-linked hydrogel backbone. Size exclusion chromatography (SEC) of intact and surfactant-treated nanoparticles confirms the formation of anchored hydrogel structures. Transmission electron microscopy (TEM) shows ∼100 nm nanoparticles even after removal of unanchored phospholipids. The location of dsDNA groups at the hydrogel-bilayer interface is confirmed with a fluorescence assay. Using DNA as a linker between the bilayer and a hydrogel core allows for temperature-dependent release of the anchoring interaction, produces polymer nanogels with addressible hybridization sites on their surface, and provides a prototype structure for potential future oligonucleotide drug delivery applications

Temperature Sensing in Modular Microfluidic Architectures

A discrete microfluidic element with integrated thermal sensor was fabricated and demonstrated as an effective probe for process monitoring and prototyping. Elements were constructed using stereolithography and market-available glass-bodied thermistors within the modular, standardized framework of previous discrete microfluidic elements demonstrated in the literature. Flow rate-dependent response due to sensor self-heating and microchannel heating and cooling was characterized and shown to be linear in typical laboratory conditions. An acid-base neutralization reaction was performed in a continuous flow setting to demonstrate applicability in process management: the ratio of solution flow rates was varied to locate the equivalence point in a titration, closely matching expected results. This element potentially enables complex, three-dimensional microfluidic architectures with real-time temperature feedback and flow rate sensing, without application specificity or restriction to planar channel routing formats