Theoretical and experimental development of a ZnO-based laterally excited thickness shear mode acoustic wave immunosensor for cancer biomarker detection

The object of this thesis research was to develop and characterize a new type of acoustic biosensor - a ZnO-based laterally excited thickness shear mode (TSM) resonator in a solidly mounted configuration. The first specific aim of the research was to develop the theoretical underpinnings of the acoustic wave propagation in ZnO. Theoretical calculations were carried out by solving the piezoelectrically stiffened Christoffel equation to elucidate the acoustic modes that are excited through lateral excitation of a ZnO stack. A finite element model was developed to confirm the calculations and investigate the electric field orientation and density for various electrode configurations. A proof of concept study was also carried out using a Quartz Crystal Microbalance device to investigate the application of thickness shear mode resonators to cancer biomarker detection in complex media. The results helped to provide a firm foundation for the design of new gravimetric sensors with enhanced capabilities. The second specific aim was to design and fabricate arrays of multiple laterally excited TSM devices and fully characterize their electrical properties. The solidly mounted resonator configuration was developed for the ZnO-based devices through theoretical calculations and experimentation. A functional mirror comprised of W and SiO2 was implemented in development of the TSM resonators. The devices were fabricated and tested for values of interest such as Q, and electromechanical coupling (K2) as well as their ability to operate in liquids. The third specific aim was to investigate the optimal surface chemistry scheme for linking the antibody layer to the ZnO device surface. Crosslinking schemes involving organosilane molecules and a phosphonic acid were compared for immobilizing antibodies to the surface of the ZnO. Results indicate that the thiol-terminated organosilane provides high antibody surface coverage and uniformity and is an excellent candidate for planar ZnO functionalization. The fourth and final specific aim was to investigate the sensitivity of the acoustic immunosensors to potential diagnostic biomarkers. Initial tests were performed in buffer spiked with varying concentrations of the purified target antigen to develop a dose-response curve for the detection of mesothelin-rFc. Subsequent tests were carried out in prostate cancer cell line conditioned medium for the detection of PSA. The results of the experiments establish the operation of the devices in complex media, and indicate that the acoustic sensors are sensitive enough for the detection of biomolecular targets at clinically relevant concentrations.Ph.D.Committee Chair: William D Hunt; Committee Member: Bruno Frazier; Committee Member: Dale Edmondson; Committee Member: Marie Csete; Committee Member: Peter Edmonson; Committee Member: Ruth O'Rega

Development of new biomaterials based on liquid crystalline phases of cellulose derivatives

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do Grau de Mestre em Engenharia BiomédicaIn the present work, new biomaterials based on liquid crystalline phases of cellulose derivatives were prepared and characterized. The possibility to monitor growth and differentiation of stem cells could be an interesting use for the HPC films studied in this thesis. Additionally, the application of HPC films as an anti-adhesive substratum in biomedical applications could also be interesting. For that purpose, two solutions of hydroxypropylcellulose (HPC) in dimethylacetamide (DMAc) were prepared. An isotropic 30% solution (HPCi) and an anisotropic 60% chiral nematic solution (HPCa) were obtained and submitted to a chemical cross-linking treatment by adding 10% (w/w) of the crosslinking agent (1,6 diisocyanatohexane, 98%). HPC films were then prepared with the help of a calibrated ruler moving at a controlled rate (v = 6 mm.s-1). The non-biological characterization of these films included physico-chemical and structural characterization. HPCi and HPCa film characterization was performed by optical microscopy, determination of the gel fraction, mechanical properties, contact angle measurements, infrared spectroscopy (FTIR), x-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The biological experimental characterization included cytotoxicity evaluation, cell adhesion, morphology, proliferation and viability assays. The band texture of HPCa films, formed during the shear, was easily observed using polarized light transmission microscopy. The mechanical anisotropy of HPCa films was also easily identified. FTIR and XPS showed that the surface chemistry of both films is similar, as well as the contact angles measured on both surfaces. SEM results showed a characteristic topography on the HPCa surface, which was verified and quantified by AFM. The interaction of these biomaterials with human mesenchymal stem cells (hMSC) was also investigated. HPC films were also tested for in vitro cytotoxicity and were found to be non-cytotoxic. The results showed that the percentage of adherent cells on HPC films was almost zero and that cells were not able to proliferate on these substrates. MSCs became round and aggregated on both HPC films, but remained aggregated and viable. This study has shown that it is possible to prepare sterile and biocompatible HPC films with potential use as coatings for anti-adhesive purposes in medical devices

Cell instructive Liquid Crystalline Networks for myotube formation

Development of biological tissues in vitro is not a trivial task and requires the correct maturation of the selected cell line. To this aim, many attempts were done mainly by mimicking the biological environment using micro/nanopatterned or stimulated scaffolds. However, the obtainment of functional tissues in vitro is still far from being achieved. In contrast with the standard methods, we here present an easy approach for the maturation of myotubes toward the reproduction of muscular tissue. By using liquid crystalline networks with different stiffness and molecular alignment, we demonstrate how the material itself can give favorable interactions with myoblasts helping a correct differentiation. Electrophysiological studies demonstrate that myotubes obtained on these polymers have more adult-like morphology and better functional features with respect to those cultured on standard supports. The study opens to a platform for the differentiation of other cell lines in a simple and scalable way

All-carbon multi-electrode array for real-time in vitro measurements of oxidizable neurotransmitters

We report on the ion beam fabrication of all-carbon multi electrode arrays (MEAs) based on 16 graphitic micro-channels embedded in single-crystal diamond (SCD) substrates. The fabricated SCD-MEAs are systematically employed for the in vitro simultaneous amperometric detection of the secretory activity from populations of chromaffin cells, demonstrating a new sensing approach with respect to standard techniques. The biochemical stability and biocompatibility of the SCD-based device combined with the parallel recording of multi-electrodes array allow: i) a significant time saving in data collection during drug screening and/or pharmacological tests over a large number of cells, ii) the possibility of comparing altered cell functionality among cell populations, and iii) the repeatition of acquisition runs over many cycles with a fully non-toxic and chemically robust bio-sensitive substrate.Comment: 24 pages, 5 figure