Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format

Spheroids-on-a-chip: Recent advances and design considerations in microfluidic platforms for spheroid formation and culture

© 2018 Elsevier B.V. A cell spheroid is a three-dimensional (3D) aggregation of cells. Synthetic, in-vitro spheroids provide similar metabolism, proliferation, and species concentration gradients to those found in-vivo. For instance, cancer cell spheroids have been demonstrated to mimic in-vivo tumor microenvironments, and are thus suitable for in-vitro drug screening. The first part of this paper discusses the latest microfluidic designs for spheroid formation and culture, comparing their strategies and efficacy. The most recent microfluidic techniques for spheroid formation utilize emulsion, microwells, U-shaped microstructures, or digital microfluidics. The engineering aspects underpinning spheroid formation in these microfluidic devices are therefore considered. In the second part of this paper, design considerations for microfluidic spheroid formation chips and microfluidic spheroid culture chips (μSFCs and μSCCs) are evaluated with regard to key parameters affecting spheroid formation, including shear stress, spheroid diameter, culture medium delivery and flow rate. This review is intended to benefit the microfluidics community by contributing to improved design and engineering of microfluidic chips capable of forming and/or culturing three-dimensional cell spheroids

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

Particle-modified Surface Plasmon Resonance Biosensor

Surface plasmon resonance (SPR) biosensors have attracted great attention in scientific research in the past three decades. Extensive studies on the immobilisation of biorecognition elements have been conducted in pursuit of higher sensitivity, but trialled formats have focussed on a thin layer modification next to the plasmon film, which usually requires in situ derivatization. This thesis investigates an ‘off-chip’ immobilisation strategy for SPR biosensing using silica particles and considers the implications of a particle-modified evanescent field on the signal amplitude and kinetics, for an exemplar affinity binding between immobilised IgG and its anti-IgG complement. Submicron silica particles were synthesized as carriers for the bio-recognition elements. They were then immobilised to form a sub-monolayer on the gold film of an SPR biosensor using two methods: thiolsilane coupling and physical adsorption aided by mechanical pressure. The bio-sensitivity towards an antigen/antibody interaction was lower than an SPR biosensor with an alkanethiolate SAM due to the difference in ligand capacity and position in the evanescent field. The binding kinetics of antigen/antibody pair was found to follow the Langmuir model closely in a continuous flow configuration but was heavily limited by the mass transport from the bulk to the sensor surface in a stop-flow configuration. A packed channel configuration was designed with larger gel particles as ligand carriers, packed on top of a gold film to create a column-modified SPR biosensor. This sensor has comparable bio-sensitivity to the previous sub-monolayer particle-modified systems, but the binding and dissociation of the analyte was heavily dependent on mass transport and binding equilibria across the column. A bi-directional diffusion mechanism was proposed based on a two-compartment mass transport model and the expanded model fitted well with the experimental data. The column-modified sensor was also studied by SPR imaging and analyte band formation was observed and analysed. Using the lateral resolution, a multiplexing particle column configuration was explored, and its potential in distinguishing a multicomponent analyte.Agency for Science Technology and Research, Singapor