Novel Biosensors And Their Applications In Related Biophysical Studies

Cryobiology and cell biomechanics are the two major branches of modern biomedical engineering research. In this Ph.D. dissertation, we have focused on problems occurred during the cryopreservation of human platelets. Two main themes (cryobiology and cell biomechanics) have been used to elucidate the research work.Cryobiology is the branch of biology that studies the effects and applications of low temperatures on biological systems. With current technological advancements in modern cryoscience, enabling technologies, such as cryopreservation and cryotherapy, have been widely and successfully incorporated in numerous cell-based medical applications including tissue engineering, regenerative medicine, assisted reproduction, cancer treatment, etc. However, how to readily and accurately characterize thermal properties of biological samples, and how to gain further and deeper insights in microscale heat transfer phenomena and thermal injury, have been a global issue and attracted major attention over the years.The first main portion of this dissertation is oriented around the research activities in advancing a microtechnology enabled thermal conductivity sensor system and through the developed microsystem, exploring the fundamental characteristics and phenomena of microscale bio-thermal transfer phenomena and cryoinjury.The first chapter elucidates in detail about the development of an improved thermal-needle approach for accurate and fast measurement of thermal conductivity of aqueous and soft biomaterials using modern microfabrication techniques. This microscopic measuring device was comprehensively characterized at temperatures from 0℃ to 40℃. Despite the previous belief, system calibration constant was observed to be highly temperature-dependent. Dynamic thermal conductivity responses to temperature and concentration variations have observed using the miniaturized single tip sensor for various concentrations of CPAs, i.e., glycerol, ethylene glycol and dimethyl sulfoxide. A dual dependence (temperature and concentration) thermal conductivity prediction model for aqueous solutions was also proposed. The estimation model is based on the modified Filippov equation coupled with the second-order polynomial. The proposed model appears to be in excellent correlation with the obtained thermal conductivity measurements of three various concentrated binary CPA solutions at a wide range of temperature conditions based on the pooled fitting. Chicken breast, chicken skin, porcine limb, and bovine liver were assayed to investigate the effect of anatomical heterogeneity on thermal conductivity using the arrayed multi-tip sensor at 20℃. Experimental results revealed distinctive differences in localized thermal conductivity, which suggests the use of approximated or constant property values is expected to bring about results with largely inflated uncertainties when investigating bio-heat transfer mechanisms and/or performing sophisticated thermal modeling with complex biological tissues. Overall, the presented micro thermal sensor with automated data analysis algorithm is a promising approach for direct thermal conductivity measurement of aqueous solutions and soft biomaterials and is of great value to cryopreservation of tissues, hyperthermia or cryogenic, and other thermal-based clinical diagnostics and treatments.The second chapter within the first section explores in depth regarding transmembrane water transportation (membrane permeability measurements) and probability of intracellular ice formation. Ice formation in living cells is a lethal event during freezing and its characterization is important to the development of optimal protocols for not only cryopreservation but also cryotherapy applications. Although the model for probability of ice formation (PIF) in cells developed by Toner et al. has been widely used to predict nucleation-limited intracellular ice formation (IIF), our data of freezing HeLa cells suggest that this model could give misleading prediction of PIF when the maximum PIF in cells during freezing is less than 1 (PIF ranges from 0 to 1). We introduce a new model to overcome this problem by incorporating a critical cell volume to modify the Toner’s original model. We further reveal that this critical cell volume is dependent on the mechanisms of ice nucleation in cells during freezing, i.e., surface-catalyzed nucleation (SCN) and volume-catalyzed nucleation (VCN). Taken together, the improved PIF model may be valuable for better understanding of the mechanisms of ice nucleation in cells during freezing and more accurate prediction of PIF for cryopreservation and cryotherapy applications.Cell biomechanics is another significant component of today’s biomedical engineering. The emphasize of cell biomechanics is to further the understanding of normal and disease physiology at cellular level through the study of constitutive relationships between mechanical factors/properties of the biological system and cell functions and/or mechanical behaviors.The second portion of this dissertation is focused on the research activities in advancing a BioMEMS-based platelet function evaluation biomedical device and the attempt in establishing a standardized protocol in assaying overall platelet function.Platelets play an important role in hemostasis by forming a thrombotic plug that seals the vessel wall and promotes vascular healing. After platelets adhere and aggregate at the wound site, their next step is to generate contractile forces through the coordination of physicochemical interactions between actin, myosin, and αIIbβ3 integrin receptors that retract the thrombus’size and strengthen its adhesion to the exposed matrix. Although platelet contractile forces (PCF) are a definitive feature of hemostasis and thrombosis, there are few approaches that can directly measure them. In this study, we describe the development of an approach to measure PCF in microthrombi using a microscopic flexible post force sensor array. Quasi-static measurements and live microscopic imaging of thrombin-activated platelets on the posts were conducted to assay the development of PCF to various hemostatic conditions. Microthrombi were observed to produce forces that monotonically increased with thrombin concentration and activation time, but forces subsided when thrombin was removed. PCF results were statistically similar on arrays of posts printed with fibronectin or fibrinogen. PCF measurements were combined with clot volume measurements to determine that the average force per platelet was 2.1 ± 0.1 nN after 60 min, which is significantly higher than what has been measured with previous approaches. Overall, the flexible post arrays for PCF measurements are a promising approach for evaluating platelet functionality, platelet physiology and pathology, the impacts of different matrices or agonists on hemostatic responses, and in providing critical information regarding platelet activity that can guide new hemostatic or thrombotic strategies.In summary, the two developed micro/nanotechnology enabled biomedical micro total analysis system, along with the standardized experimental protocols and procedures, have demonstrated not only great potential in helping to make the cryopreservation of platelets a reality, but also superior adaptability in providing more insights into many related fundamental biology studies.