Biomolecular interactions are fundamentally important for a wide variety of biological processes. Understanding the temperature dependence of biomolecular interactions is hence critical for applications in fundamental sciences and drug discovery. Micro-Electro-Mechanical Systems (MEMS) technology holds great potential in facilitating temperature-dependent characterization of biomolecular interactions by providing on-chip microfluidic handling with drastically reduced sample consumption, and well controlled micro- or nanoscale environments in which biomolecules are effectively and efficiently manipulated and analyzed. This dissertation is focused on a high-through and miniaturized differential scanning calorimeter for thermodynamic study of bio-molecules using MEMS techniques.
The dissertation firstly introduces the overall design and operation principles. This miniaturized DSC was fabricated based on a polyimide (PI) thin film. Highly temperature sensitive vanadium oxide was used as the thermistor material. A PDMS (Polydimethylsiloxane) microfluidic chamber was separately fabricated and then bonded firmly with the PI substrate by a stamp-and-stick method. Meanwhile, the micro heater design was optimized to reach better uniformity. A heating stage was constructed for fast and reliable scanning. In this study, we used syringes to deliver the 0.63 μL liquid sample into both the sample and reference chambers. All the testing processes were functionalized using the LabVIEW programs.
The sensing material was also characterized. To seek a higher temperature coefficient of resistance (TCR) and less resistive behavior, explorations about various PVD (physical vapor deposition) parameters and annealing conditions were conducted for optimization. In this research, we found vanadium oxide deposited under certain conditions leads to the highest TCR value (a maximum of 2.51%/oC). To better understand the material’s property, we also did the XRD (X-ray Diffraction), SEM (Scanning electron microscope).
The micro calorimeter was calibrated using a step thermal response. The time constant was around 3s, the thermal conductance was 0.6mW/K, and the sensitivity was 6.1V/W. The static power resolution of the device at equilibrium is 100 nW, corresponding to 250 nJ/K. These performances confirmed the design and material to be appropriate for both good thermal isolation and power sensitivity.
We demonstrated the miniaturized DSC’s performance on several different kinds of protein samples: lysozyme, and mAb (monoclonal antibody) and a DVD IgG (double variable domain immunoglobulin G). The results were found to be reasonable by comparing it with the commercial DSC’s tests.
Finally, this instrument may be ideal for incorporation into high throughput screening workflows for the relative comparison of thermal properties between large numbers of proteins when only small quantities are available. The micro-DSC has the potential to characterize the thermal stability of the protein sample with significantly higher throughput and less sample consumption, which could potentially reduce the time and cost for the drug formulation in the pharmaceutical industry. / Ph. D. / Virtually all biological phenomena depend on molecular interactions, which is either intra-molecular as protein folding/unfolding or intermolecular as in ligand binding. A basic biology problem is to understand the folding and denaturation processes of a protein: the kinetics, thermodynamics and how a protein unfolds and folds back into its native state. Both folding/unfolding and denaturation processes are associated with enthalpy changes. The thermodynamics of binding compounds helps a great deal to understand the nature and potency of such molecules and is essential in drug discovery. As a label-free and immobilization-free method, calorimetry can evaluate the Gibbs free energy, enthalpy, entropy, specific heat, and stoichiometry, and thus provides a fundamental understanding of the molecular interactions. Calorimetric systems including isothermal titration calorimeters (ITC) and differential scanning calorimeters (DSC) are the gold standard for characterizing molecular interactions.
In this research, a micro DSC is developed for direct thermodynamic study of bio-molecules. Compared with the current commercial DSC, it is on a much smaller scale. It consumes much less sample and time in each DSC measurement. It can enable comprehensive high-content thermodynamics study in the early stage of drug discovery and formulation. It also enables direct, precise, and rapid evaluation of the folding and unfolding of the large biomolecules like proteins, DNAs, and enzymes without labeling or immobilization. It can also be used as a powerful tool to study the membrane proteins, which is often impractical or impossible before.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/87777 |
| Date | 19 February 2019 |
| Creators | Yu, Shifeng |
| Contributors | Mechanical Engineering, Zuo, Lei, Behkam, Bahareh, Lu, Ming, Agah, Masoud, Cheng, Jiangtao |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | Creative Commons Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
Page generated in 0.0025 seconds