Centrifugal Microfluidic System for Biochemical Applications

• Author / Creator

  Hou, Yuting

• The development of life sciences has imposed higher requirements for biochemical analysis, especially for point-of-care analysis. Polymer-made centrifugal CD microfluidic chips could fulfill the needs of biochemical analysis with their low cost, the feasibility of mass production, excellent biocompatibility, miniaturization, and automation. With recent advances, the field of centrifugal microfluidics has become more attractive than ever for the integration of complex laboratory workflows. Therefore, the research and development of centrifugal microfluidic discs are of great significance.
  We explored two possible applications of centrifugal microfluidic in the biochemical field. One disc is designed to complete a multiple-step bench-top blood sample clean-up procedure automatically where the output sample could be directly used for downstream heavy instrument analysis. Another disc is an integrated system with an on-disc readout sensor, a compact device for on-site use. All discs are fabricated with transparency PMMA and then laminates together by PSA. Both PMMA and PSA are laser cut using a commercial CO2 laser cutting machine. Reagent reservoirs are mainly fabricated on PMMA layers, while fluid transportation channels are featured on PSA layers. The thin thickness and hydrophobicity of PSA enable neatly liquid transfer and siphon valving. The pneumatic valve and siphon valve are two crucial features for realizing automation with the cooperation of centrifugal force. Also, other forces in a rotating reference system play important roles in on-disc operation like mixing.
  The first centrifugal microfluidic disc aims to prepare whole blood for small molecule detection automatically. Fresh blood is one of the most critical bio-samples that reflect the stimulant condition of the human body and is widely used in research and diagnosis. However, the complicated matrix of blood hindered the direct analysis of small molecules and make sample preparation necessary, especially for mass spectrometry detection, which is the golden rule in small molecule studies. Sample preparation procedures such as blood cell separation, protein precipitation, and lipid removal are all integrated on the disc. Although mixing is a challenge in the centrifugal system, the mixing of plasma and organic solvent for protein precipitation is realized by “shaking" mode, which takes advantage of acceleration and deceleration when spinning speed changes. The processed sample is proved to be suitable for both HILIC-MS and MALDI-MS analysis. According to the Agilent HILIC-MS protocol for analysis of 17 underivatized amino acids, all these 17 amino acids in the disc-processed blood sample are separated and analyzed by positive mode HILIC-MS using HILIC. MALDI-MS is employed during C18 and silica particle usage optimization by observing the crystallization of sample spots on GLAD film and comparing the S/N of several amino acid peaks. Also, quick estimations of specific amino acids are performed by MALDI-MS involving spiked isotopes.
  Other than automated sample preparation, developing a miniaturization integration detection system is another direction of the centrifugal microfluidic investigation. In the second work, we first introduce smart polymer-based etalon sensors to the centrifugal microfluidic system with transparency disc material PMMA. The etalon is fabricated with a sandwich structure on a thin glass slide, including two layers of Au and one middle layer of hydrogel. The top Au surface is modified with anti-progesterone(P4) DNA aptamer, which exhibits distinctive reflected color changes with the absence of different concentrations of progesterone in the sample. All required on-disc procedures to enable etalon sensoring, including sample incubation, buffer wash, and salt stimuli, are automated by three pneumatic valves and one siphon valves. Three pneumatic valves with increased burst spinning speed allow the sequential release of each preloaded reagent, including sample, wash buffer, and salt solution. At the same time, the reusable siphon takes charge of emptying the incubation chamber after each reaction. The color change degree of the etalon is inversely related to the concentration of P4 as the P4 binding DNA aptamer takes reformation that prevents polymer hydrogel collapse caused by stimuli of Na+ in the salt solution. The color change is readout by image color analysis, avoiding the usage of sophisticated optical devices like reflectance spectrometer, which further simplify the device setting and make the system applicable for on-site use.

• Subjects / Keywords

  • Centrifugal Microfluidic
  • Blood Sample
  • Amino Acids Analysis
  • Etalon Sensor
  • Hormone Detection
  • Color Sensor

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-xyg6-nb97

• 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.