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Protein Separation with Self-Assembled Nanoparticle Beds: Mechanism and Separation Performance

  • Author / Creator
    Azim, Mohammad Alaul
  • This thesis reports the separation behavior of SDS-protein complexes in colloidal self-assembled (CSA) nanoparticle beds, and the processes of stabilization of CSA beds for high voltage separation. First, the variation of electrophoretic mobility with molecular weight (6.5-66 kDa) of SDS-protein complexes and with particle size (150-690 nm) was evaluated using a classical, modified Ogston sieving model for protein separation for a random pore gel structure, and using the modified Giddings analysis developed by Wirth for uniform pores structure. The results show improved fits (R2 = 0.993-0.997) and better predictive interpolations of the molecular weight on unknowns using the Wirth/Giddings model for a uniform pore structure. The quality of fit of the single pore size model indicates that it is meaningful to characterize these structures as having a predominantly single pore size. A CSA bed of native silica nanoparticles is not stable for high voltage application. Two different approaches have been demonstrated to stabilize the CSA beds in this work. By reducing the depth of the CSA beds by half or more, three to four times the electric field stability (from ≤ 320 V/cm for 20 µm thick bed to ̴ 2250 V/cm for 4 µm thick bed) is achieved. Narrowing the capillary bore greatly stabilized the particle beds, even for smaller particle assemblies. The improved separation performance of narrow bore capillaries increases the particle bed performance in protein separation. But unfortunately the narrow bore chips are still not stable for long term repetitive use. These kind of chips are suitable for up to 5-6 runs at highest stable voltage. Base catalyzed sol-gel polymerization introduces cross links between the particles to entrap them, and solves the problem. But the acid catalyzed gel filled the pores and made the microchip unusable. The CSA bed entrapped by gel can be used for at least a week (10-15 runs /day) and the reproducibility of fabrication is high (yield = 90%). This study demonstrates that gel entrapped particles bed can be used for high performance SDS-protein separation up to several days without significant deviation of chip performance. The results demonstrate that this simple and inexpensive fabrication process for microchips with a CSA bed has high potential in improving protein analysis, compared to competing technologies such as conventional gels.

  • Subjects / Keywords
  • Graduation date
    2016-06
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3V698V0M
  • 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
    English
  • Citation for previous publication
    • A version of Chapter-3 has been published in the Proceeding of 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences as, “Highly Stabilized Colloidal Self Assembled Nanoparticle bed in Micro-channels for High Performance Size Based Protein Separation’’ Mohammad Azim, Abebaw B. Jemere and D. Jed Harrison, MicroTAS 2015; Gyeongju, South Korea ; 1978-1980
    • A part of Chapter-5 has been published in Proceeding of 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences as, “Colloidal self-assembled nanoparticles sieves with orthosilicate cross linking for protein separation in microchips and retardation coefficient for on chip protein sizing”, Mohammad Azim, Abebaw B. Jemere and D. Jed Harrison, MicroTAS 2014; San Antonio, USA; 2387-2389
    • * A version of Chapter-2 has been published in Proceeding of 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences as, “Colloidal self-assembled nanoparticles sieves with orthosilicate cross linking for protein separation in microchips and retardation coefficient for on chip protein sizing”, Mohammad Azim, Abebaw B. Jemere and D. Jed Harrison, MicroTAS 2014; San Antonio, USA; 2387-2389 Also, a version of Chapter-2 has been submitted to Electrophoresis as “Evaluation of Protein Separation Mechanism and Pore Size Distribution in Colloidal Self-Assembled Nanoparticle Sieves for On-Chip Protein Sizing.” Mohammad Azim, Ali Malekpourkoupaei, Wenmin Ye, Abebaw B. Jemere and D. Jed Harrison. (Authors contributions are described in preface)
    • A part of Chapter-4 has been published in the Proceeding of 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences as, “Highly Stabilized Colloidal Self Assembled Nanoparticle bed in Micro-channels for High Performance Size Based Protein Separation’’ Mohammad Azim, Abebaw B. Jemere and D. Jed Harrison, MicroTAS 2015; Gyeongju, South Korea ; 1978-1980
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
  • Supervisor / co-supervisor and their department(s)