Defense Date

2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Pharmaceutics

First Advisor

Dr. H. Thomas Karnes

Abstract

The analysis of biological samples in clinical or research settings often requires measurement of analytes from complex and limited matrices. Immunoaffinity separations in miniaturized formats offer selective isolation of target analytes with minimal reagent consumption and reduced analysis times. A prototype capillary-based microfluidic system has been developed for immunoaffinity separations in biological matrices with laser-induced fluorescence detection of labeled antigens or antibodies. The laboratory-constructed device was assembled from two micro syringe pumps, a microchip mixer, a micro-injector, a diode laser with fused-silica capillary flow cell, and a separation capillary column. The columns were prepared from polymer tubing and packed under negative pressure with a stationary phase that consisted of biotinylated antibodies attached to streptavidin-silica beads. A custom software program controlled the syringe pumps to perform step gradient elution and collected the signal as chromatograms. The system performance was evaluated with flow accuracy, mixer proportioning, pH gradient generation, and assessment of detectability. A direct labeling/direct capture immunoaffinity separation of C-reactive protein (CRP) was demonstrated in simulated serum. CRP, a biomarker of inflammation and cardiovascular disease risk assessment, was fluorescently labeled in a one-step reaction and directly injected into the system. A quadratic calibration model was selected and precision and accuracy were reported. Parathyroid hormone was also analyzed by the direct capture approach, but displayed nonspecific binding of human plasma matrix components that limited the useful assay range. Capillary sandwich assays of CRP in human serum and cerebrospinal fluid were performed using both capture and detection antibodies. The detection antibody was labeled and purified offline to minimize signal from labeled matrix components. Four parameter logistic functions were used to model the data and precision and accuracy were evaluated. During the study, 250 nL injection volumes 2.0 µL/min flow rates were employed, minimizing sample and reagent consumption. The microfluidic system was capable of separating antigens from biological matrices and is potentially portable for patient point-of-care settings. Additionally, the flexible design of the separation capillary allows for the analysis of different clinical markers by changing the antibodies and the low assay volume requirements could lead to less invasive patient sampling techniques.LabVIEW version 7 or later is required to open the attached files.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

June 2008

PumpControl.vi (925 kB)
ReadProgram.vi (803 kB)

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