Functionalized Bead Based Microchip for Immunoassay and Virus Immuno-affinity Chromatography

  • Author / Creator
    Zhang, Le
  • We demonstrate the functionalization of a highly ordered porous molecular sieving matrix created by colloidal self-assembly (CSA) of 2 micrometer diameter silica particles in microfluidic chips, for highly efficient immuno-capture of viruses. By tuning the particle size, with appropriate surface chemistry, we can easily match the pore size which could maximize biological species-particle wall collision, leading high capture efficiency. The ordered uniform lattice of pores, which are 15 % of the size of the particles used, should prove ideal for the capture of viruses (on the 50-150 nm scale in size). We report on characterization of capture beds with 300 nm pores. These beds were used to detect fluorescein using immobilized anti-fluorescein on the bed, and also to capture and detect type-5 adenovirus in suspension, and in infected cells using appropriate antibodies. We demonstrate the performance of the concept using a fully packed column for fluorescein immunoassay. We used 2 μm carboxylated silica particles self-assembled into a 6 mm long-bed, modified with EDC/NHS, and immobilized anti-fluorescein antibody or type-5 adenovirus-recognizing antibody. An electric field was applied to utilize electro-osmotic flow as the solvent pumping force. A 4.51 nM fluorescein solution was captured by immuno-affinity using anti-fluorescein antibody, and then released with an eluent to an empty downstream analysis region to give a very large signal, providing a positive control. Similar experiments were performed with type-5 adenovirus, demonstrating detection at a concentration of 8.3 × 103 viral particles (VP) per milliliter. Adenovirus in celllysate was also detected in this microchip, with the lowest concentration detectable at 1.5 × 103 PFU/mL. Combination of advanced biological detection methods with microfluidics based extraction and concentration techniques has tremendous potential for realization of a portable, cost effective and sensitive pathogen detection system. Taking advantage of the unique fluid flow characteristics of CSA structures on an even smaller scale than employed here, faster, more sensitive and more economical capture and pre-concentration techniques for diagnostic assays for a wider range of analytes can be developed.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • 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.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Chemistry
  • Supervisor / co-supervisor and their department(s)
    • D. Jed Harrison, Department of Chemistry
  • Examining committee members and their departments
    • Steven H. Bergens, Department of Chemistry
    • Mark T. McDermott, Department of Chemistry