Abstract

My dissertation work integrates the techniques of microfabrication, micro/nanofluidics, and bioanalytical chemistry to develop miniaturized devices for healthcare applications. Semiconductor processing techniques including photolithography, physical and chemical vapor deposition, and wet etching are used to build these devices in silicon and polymeric materials. On-chip micro-/nanochannels, pumps, and valves are used to manipulate the flow of fluid in these devices. Analytical techniques such as size-based filtration, solid-phase extraction (SPE), sample enrichment, on-chip labeling, microchip electrophoresis (µCE), and laser induced fluorescence (LIF) are utilized to analyze biomolecules. Such miniaturized devices offer the advantages of rapid analysis, low cost, and lab-on-a-chip scale integration that can potentially be used for point-of-care applications.The first project involves construction of sieving devices on a silicon substrate, which can separate sub-100-nm biostructures based on their size. Devices consist of an array of 200 parallel nanochannels with a height step in each channel, an injection reservoir, and a waste reservoir. Height steps are used to sieve the protein mixture based on size as the protein solution flows through channels via capillary action. Proteins smaller than the height step reach the end of the channels while larger proteins stop at the height step, resulting in separation. A process is optimized to fabricate 10-100 nm tall channels with improved reliability and shorter fabrication time. Furthermore, a protocol is developed to reduce the electrostatic interaction between proteins and channel walls, which allows the study of size-selective trapping of five proteins in this system. The effects of protein size and concentration on protein trapping behavior are evaluated. A model is also developed to predict the trapping behavior of different size proteins in these devices. Additionally, the influence of buffer ionic strength, which can change the effective cross-sectional area of nanochannels and trapping of proteins at height steps, is explored in nanochannels. The ionic strength inversely correlates with electric double layer thickness. Overall, this work lays a foundation for developing nanofluidic-based sieving systems with potential applications in lipoprotein fractionation, protein aggregate studies in biopharmaceuticals, and protein preconcentration. The second project focuses on designing and developing a microfluidic-based platform for preterm birth (PTB) diagnosis. PTB is a pregnancy complication that involves delivery before 37 weeks of gestation, and causes many newborn deaths and illnesses worldwide. Several serum PTB biomarkers have recently been identified, including three peptides and six proteins. To provide rapid analysis of these PTB biomarkers, an integrated SPE and µCE device is assembled that provides sample enrichment, on-chip labeling, and separation. The integrated device is a multi-layer structure consisting of polydimethylsiloxane valves with a peristaltic pump, and a porous polymer monolith in a thermoplastic layer. The valves and pump are fabricated using soft lithography to enable pressure-based sample actuation, as an alternative to electrokinetic operation. Porous monolithic columns are synthesized in the SPE unit using UV photopolymerization of a mixture consisting of monomer, cross-linker, photoinitiator, and various porogens. The hydrophobic surface and porous structure of the monolith allow both protein retention and easy flow. I have optimized the conditions for ferritin retention, on-chip labelling, elution, and µCE in a pressure-actuated device. Overall functionality of the integrated device in terms of pressure-controlled flow, protein retention/elution, and on-chip labelling and separation is demonstrated using a PTB biomarker (ferritin). Moreover, I have developed a µCE protocol to separate four PTB biomarkers, including three peptides and one protein. In the future, an immunoaffinity extraction unit will be integrated with SPE and µCE to enable rapid, on-chip analysis of PTB biomarkers. This integrated system can be used to analyze other disease biomarkers as well.

Degree

PhD

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

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