Utility of Heat and Cold at Multi-scale for Point-of-care Diagnosis and Cryopreservation Applications

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Utility of Heat and Cold at Multi-scale for Point-of-care Diagnosis and Cryopreservation Applications


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The utility of heat and cold at multi-scale has led to numerous breakthroughs in biomedical research. This dissertation focuses on the point-of-care (POC) diagnosis and microscale biomaterials cryopreservation applications. Specifically, vitrification, a solidification process from liquid to glass phase by rapid cooling, is a promising ice-free cryopreservation approach to achieve high post cryopreservation viability for microscale biomaterials. However, traditional vitrification methods using the convective heat transfer for cooling and warming are limited to process small droplet sizes (i.e., pico- to nano-liter). This is due to the intrinsic constraints on the size dependent cooling and warming rate by convective heat transfer. Therefore, it remains a major challenge to process large amounts of biomaterials using the traditional vitrification approaches. POC diagnostics are designed to provide fast and simple measurements to facilitate timely medical decision making to improve clinical outcomes. Lateral flow assays (LFAs), using gold nanoparticles (GNPs) as the contrast label, have dominated POC diagnostics in the last three decades due to their low cost, simplicity, and robust operation. Nonetheless, LFAs are approximately 1000-fold lower in sensitivity than laboratory-based techniques such as enzyme-linked immunoassay (ELISA), which constrains their impact. In a positive test, the GNPs accumulate at the test line of the LFA, leading to a red color. The collective nanoscale heating of the GNPs in the LFA by laser irradiation results in a macroscale temperature change in the LFA. This temperature change was captured by the IR camera in the thermal contrast amplification (TCA) reader, leading to improved sensitivity of commercial LFAs by 8-32 folds compared to the traditional visual detection of the GNPs. To further close the gap with ELISA, designing a customized LFA for the TCA reader holds great promise. First, I studied the role of GNP sizes on the analytical performance of LFA. Analysis of transport and reaction kinetics revealed that the reaction is the rate limit term that determines the number of GNPs captured on the test line. Larger sized GNPs can carry more antibodies on the surface to enhance the reaction. In addition, thermal analysis showed that larger sized GNPs provide higher temperature increase under the same laser irradiation. Altogether, the use of 100 nm GNPs and TCA reader provides 256-fold improvement in the sensitivity compared to the traditional 30 nm GNPs and visual detection. Further, to optimize other components of the LFA for enhanced signal to noise ratio, I developed a figure of merit named binding ratio (BR). The BR represents the ratio of specific binding (i.e., signal) to non-specific binding (i.e., noise) in the LFA and can be quantitatively compared among various LFA conditions using the TCA reader, which is beyond the capabilities of the traditional methods. The BR was used to provide decisive and efficient guidance for optimizing the LFA running buffer and membrane blocking buffer. The customized TCA LFA for HIV p24 protein detection achieved 8 pg/ml detection sensitivity, which is comparable to the standard laboratory ELISA tests. Then, I applied the GNP-laser heating to improve the droplet vitrification based cell cryopreservation. To overcome the droplet size limit (i.e., pico- to nano-liter) in traditional methods that use convective heat transfer, microliter sized cell-encapsulated droplet was printed onto a cryogenic copper dish for vitrification. With the enhanced cooling by conduction, the minimal concentration of permeable CPA to achieve vitrification is reduced by > 20%, compared to the traditional convective cooling method by direct printing into LN2. In addition, laser nanowarming provides > 400 folds faster warming rate compared to the traditional convective warming method. Altogether, > 90% post cryopreservation viability using 2 M permeable CPA in 4 μL droplets was demonstrated with human umbilical cord blood stem cells (UCBSC). This improves the throughput of droplet vitrification approach from µL/min (traditional methods) to mL/min. Finally, I developed a universal cryopreservation method for Drosophila embryos (500 µm * 180 µm * 180 µm ellipsoid). We significantly improve the robustness of the embryo permeabilization, cryoprotectant agent loading and rewarming processes. We develop a cryomesh approach which allows the scale-up to process large number of embryos (> 1,000), and importantly, provides fast warming rate (> 220, 000 °C/min) that favors high survival. In addition, we demonstrate that flies retained normal sex ratio, fertility and SNP markers after successive generations of cryopreservation and months of storage in liquid nitrogen. Importantly, we successfully validated our protocol with 25 wild type and mutant strains including Drosophila stocks from the Bloomington stock center, as well as other labs. We report that > 50% embryos hatch and > 25% of the resulting larvae develop into adults (normalized survival to control embryos) after cryopreservation. We also demonstrate that low survival strains can be improved by outcrossing to mitigate the effect of genetic background. Lastly, we show that two non-specialists are able to successfully execute our protocol with consistent results, demonstrating the simplicity and robustness of the methodologies.



University of Minnesota Ph.D. dissertation. November 2020. Major: Mechanical Engineering. Advisor: John Bischof. 1 computer file (PDF); xii, 213 pages.

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Zhan, Li. (2020). Utility of Heat and Cold at Multi-scale for Point-of-care Diagnosis and Cryopreservation Applications. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/225902.

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