Microfluidic System for Biochemical Applications

• Author / Creator

  Zhao, Yufeng

• The last decades witnessed the rapid growth and advancement of research on microfluidic systems for chemical and biological analysis. There remain many important biochemical challenges that need to be solved with novel technologies. This thesis looks at how to apply several microfluidic techniques to several different biochemical problems. For example, one of the key biological problems in directed evolution of biomolecules is that large numbers of cells need to be processed with many repeated steps. A microfluidic cell sorter offers a simple and automated system for cell-based directed evolution. A key challenge in clinical diagnosis especially for point of care analysis is the need to clean up and prepare samples so the matrix does not cause problems in the analysis. Creating a generic clean up system requires we be able to put sample into a device and obtain a cleaned up sample solution as an output. Centrifugal microfluidic system provide this possibility. As a vehicle for development of microfluidic applications, we selected directed evolution of fluorescent proteins (FP) as a target for improvement by a microfluidic cell sorter, and metabolite assays in serum for a test of centrifugal microfluidic sample preparation devices for a “dirty sample in-clean sample out” system. To explore microfluidic fluorescent-activated cell sorting (μFACS) system, we first present characterization of such a system with a single point detector, which was developed for screening FPs on the basis of the brightness. The sorting performance is studied for optimal collection efficiency. The expression level of single cells and its effect on directed evolution by the μFACS are evaluated. Since the μFACS was designed to only screen for the brightness of FPs, other critical parameters of FPs can hardly be improved in the process of directed evolution. We thus introduce a second-generation microfluidic cell sorter with two-point detection system to improve the performance of directed evolution. The new system employs two detectors at two different places to examine the analyte-induced fluorescence change of a FP-based Ca2+ sensor. Both the brightness and fluorescence change are screened in the new system, which demonstrates superior performance in directed evolution compared to the system with single-point detector. Two rounds of directed evolution lead to a variant with improved brightness and g Ca2+-dependent fluorescence change. The Ca2+ sensor is further engineered by rational design and a series of sensors with various Ca2+ affinities are developed for intracellular Ca2+ imaging. In the second segment of the thesis, we develop a centrifugal microfluidic device to clean small volume (5 μL) human serum sample for metabolomics analysis. Sample filtration, drying and mixing are realized on a centrifugal disc. Protein precipitation by methanol and solid phase extraction by C18 beads and silica nanoparticles are utilized to remove proteins and lipids from the sample. The prepared samples are transferred to a solid-matrix laser desorption ionization (SMALDI)-chip which segregates salts from electrolyte background by crystallization during sample drying and then serves as substrate for mass spectrometry analysis. The on-chip assays are optimized to obtain high signal to noise ratio of analyte signals with low background peaks. The reproducibility of the on-chip preparation is evaluated and potential influential factors are discussed. Three ionic metabolites, glutamic acid, aspartic acid and citric acid, are quantitatively analyzed by standard addition method and the results are consistent with samples prepared by ultrafiltration method.

• Subjects / Keywords

  • metabolomics
  • fluorescent protein
  • directed evolution
  • microfluidic

• Graduation date

  Fall 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3SQ8R068

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.