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Total Integrated Sample Preparation for Microfluidic Immunoassays in Complex Biological Matrices

  • Author(s): Apori, Akwasi Asare
  • Advisor(s): Herr, Amy E
  • et al.
Abstract

A high-throughput protein analysis platform with integrated sample preparation is developed to address the identified technology gaps in biomarker validation, clinical and point-of-care diagnostics. The goals of the technology are to automate and integrate protein sample preparation with electrokinetic separations, implement immunoassays capable of processing raw biological fluids, and perform high-throughput protein assays targeted for disease diagnosis.

Integration of multiple functions is a hallmark of microfluidic tools. Efforts to fully incorporate required sample preparation steps within electrokinetic assays would benefit from a surge in technology development. On-chip fluorescence labeling, analyte enrichment, buffer exchange, reagent-sample mixing and matrix protein depletion are essential to realize the "sample-to-answer" paradigm of lab-on-a-chip systems for targeted proteomics--which hold the key to rigorous validation of potentially high impact biomarkers relevant for drug discovery, clinical diagnostics, and patient therapy and monitoring. Integration of sample preparation techniques would also expand the protein analysis application space to point-of-care assays in low resource settings.

Presently, introduction of in-situ fabrication methods has enabled advances in integration of multiple reaction zones into microfluidic geometries. Fabrication is performed at the microscale using chemically and physical functionalized polyacrylamide gels photo-patterned on microfluidic chips. These zones facilitate precise control of assay performance parameters including sample resolution, background noise, dynamic range, binding kinetics, and dispersion among others while taking advantage of microscale interactions to reduce assay time, cost, and reagent consumption while increasing sensitivity, and portability.

In this dissertation several novel techniques for on-chip proteomics and immunoassays with integrated sample preparation are introduced for the analysis of serum or proximal fluids. These methods are applied to develop laboratory medicine and point-of-care assays for the following applications: 1) immunosubtraction polyacrylamide gel electrophoresis for detection of protein mobility and binding specificity, 2) on-chip fluorogenic labeling integrated with native electrophoresis for mass isoform detection, 3) low molecular weight compound sandwich fragment antibody assays for detecting drugs of abuse, 4) integrated enrichment and labeling of proteins facilitated with size-exclusion membranes, 5) cerebrospinal fluid rhinorrhea detection for emergency room screening of traumatic brain injury, and 6) rapid single-step electrophoretic and isotachophoretic immunoassays for serum Hepatitis C confirmatory diagnostics. The unifying theme of all techniques presented herein is the precise electrokinetic control and manipulation of sample and reagents on-chip to automate and expedite time consuming and manual labor intensive benchtop immunoassays based on diffusion driven interactions.

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