Biomedical Sciences ETDs

Kevin Cushing

Publication Date

7-1-2013

Abstract

This dissertation describes the development of elastomeric capture microparticles (ECµPs) and their use with acoustophoresis to perform affinity capture assays. ECµPs that function as negative acoustic contrast particles were developed by crosslinking emulsion-based droplets composed of commercially available silicone precursors followed by functionalization with avidin/biotin reagents. The size distribution of the ECµPs was very broad or narrow depending on the emulsion system that was used during the synthesis process. Elastomeric particles exhibited a very broad size distribution when a bulk-emulsion process was used; however, when microfluidic systems were utilized, their size distribution became comparatively narrow. The functionalization of elastomeric particles was accomplished by the non-specific adsorption of avidin protein followed by bovine serum albumin (BSA) blocking and bio-specific adsorption of a biotinylated-capture antibody. Polydisperse ECµPs were functionalized to bind prostate specific antigen (PSA) or IgG-phycoerythrin (PE) in aqueous media (buffer, plasma, blood); whereas monodisperse ECµPs were functionalized to bind a high density lipoprotein in the aqueous media. Polydisperse ECµPs functionalized to bind PSA in a physiological buffer (PBS pH 7.4) demonstrated nanomolar detection using flow cytometry analysis; whereas ECµPs functionalized to bind IgG-PE demonstrated picomolar detection in 10% porcine plasma. ECµPs have a specific density of ~1.03 and are more compressible than their surrounding aqueous media; which allowed the ECµPs to exhibit negative acoustic contrast properties under an ultrasonic acoustic standing wave field. The negative acoustic contrast property of ECµPs was advantageously utilized in an IgG-PE assay conducted in 0.1% whole porcine blood. The ligand-bound ECµPs suspended in the diluted blood sample were flowed through an acoustofluidic device where the application of an ultrasonic acoustic standing wave field focused the ligand-bound ECµPs to pressure antinodes and the positive acoustic contrast blood cells to the central pressure node of the microchannel. As a result of laminar flow, focused ligand-bound ECµPs and blood cells were flowed into properly aligned outlet channels at the downstream trifurcation, where they where collected separately off-chip. The cell-free fraction containing ligand-bound ECµPs was analyzed using flow cytometry; where the detection of IgG-PE was in the picomolar range. This approach has potential applications in the development of rapid assays that detect the presence of low concentrations of biomarkers in a number of biological sample types.

Keywords

Elastomeric Particles, Flow Cytometry, Acoustophoresis, PDMS, polydimethylsiloxane, silicone, acoustofluidics, biomarkers, sorting, separations

Sponsors

National science foundation; National institute of health

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Edwards, Bruce

Second Committee Member

Lopez, Gabriel

Third Committee Member

Petsev, Dimiter

Fourth Committee Member

Freyer, James

Fifth Committee Member

Graves, Steven

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