Development of microcantilever sensors for cell studies

Micro- and nano- electromechanical devices such as microcantilevers have paved the way for a large variety of new possibilities, such as the rapid diagnosis of diseases and a high throughput platform for drug discovery. Conventional cell assay methods rely on the addition of reagents, disrupting the measurement, therefore providing only the endpoint data of the cell growth experiment. In addition, these methods are typically slow to provide results and time and cost consuming. Therefore, microcantilever sensors are a great platform to conduct cell culturing experiments for cell culture, viability, proliferation, and cytotoxicity monitoring, providing advantages such as being able to monitor cell kinetics in real time without requiring external reagents, in addition to being low cost and fast, which conventional cell assay methods are unable to provide. This work aims to develop and test different types of microcantilever biosensors for the detection and monitoring of cell proliferation. This approach will overcome many of the current challenges facing microcantilever biosensors, including but not limited to achieving characteristics such as being low cost, rapid, easy to use, highly sensitive, label-free, multiplexed arrays, etc. Microcantilever sensor platforms utilizing both a single and scanning optical beam detection methods were developed and incorporated aspects such as temperature control, calibration, and readout schemes. Arrays of up to 16 or 32 microcantilever sensors can be simultaneously measured with integrated microfluidic channels. The effectiveness of these cantilever platforms are demonstrated through multiple studies, including examples of growth induced bending of polyimide cantilevers for simple real-time yeast cell measurements and a microcantilever array for rapid, sensitive, and real-time measurement of nanomaterial toxicity on the C3A human liver cell line. In addition, other techniques for microcantilever arrays and microfluidics will be presented along with demonstrations for the ability for stem cell growth monitoring and pathogen detection

Development of calix[4]arene-functionalized microcantilever array sensing system for the rapid, sensitive and simultaneous detection of metal ions in aqueous solutions

The work described in this thesis was conducted with the aim of: 1) investigating the binding capabilities of calix[4]arene-functionalized microcantilevers towards specific metal ions and 2) developing a new16-microcantilever array sensing system for the rapid, and simultaneous detection of metal ions in fresh water. Part I of this thesis reports on the use of three new bimodal calix[4]arenes (methoxy, ethoxy and crown) as potential host/guest sensing layers for detecting selected ions in dilute aqueous solutions using single microcantilever experimental system. In this work it was shown that modifying the upper rim of the calix[4]arenes with a thioacetate end group allow calix[4]arenes to self-assemble on Au(111) forming complete highly ordered monolayers. It was also found that incubating the microcantilevers coated with 5 nm of Inconel and 40 nm of Au for 1 h in a 1.0 M solution of calix[4]arene produced the highest sensitivity. Methoxy-functionalized microcantilevers showed a definite preference for Ca²⁺ ions over other cationic guests and were able to detect trace concentration as low as 10⁻¹² M in aqueous solutions. Microcantilevers modified with ethoxy calix[4]arene displayed their highest sensitivity towards Sr²⁺ and to a lesser extent Ca²⁺ ions. Crown calix[4]arene-modified microcantilevers were however found to bind selectively towards Cs⁺ ions. In addition, the counter anion was also found to contribute to the deflection. For example methoxy calix[4]arene-modified microcantilever was found to be more sensitive to CaCl₂ over other water-soluble calcium salts such as Ca(NO₃)₂ , CaBr₂ and CaI₂. These findings suggest that the response of calix[4]arene-modified microcantilevers should be attributed to the target ionic species as a whole instead of only considering the specific cation and/or anion. Part II presents the development of a 16-microcantilever sensor setup. The implementation of this system involved the creation of data analysis software that incorporates data from the motorized actuator and a two-axis photosensitive detector to obtain the deflection signal originating from each individual microcantilever in the array. The system was shown to be capable of simultaneous measurements of multiple microcantilevers with different coatings. A functionalization unit was also developed that allows four microcantilevers in the array to be coated with an individual sensing layer one at the time. Because of the variability of the spring constants of different cantilevers within the array, results presented were quoted in units of surface stress unit in order to compare values between the microcantilevers in the array

Nanomechanical measurements of fluctuations in biological, turbulent, and confined flows

The microcantilever has become a ubiquitous tool for surface science, chemical sensing, biosensing, imaging, and energy harvesting, among many others. It is a device of relatively simple geometry with a static and dynamic response that is well understood. Further, because of it's small size, it is extremely sensitive to small external perturbations. These characteristics make the microcantilever an ideal candidate for a multitude of sensing applications. In this thesis dissertation we use the microcantilever to conduct numerous physical measurements and to study fundamental phenomena in the areas of fluid dynamics, turbulence, and biology. In each area we use the cantilever as a sensitive transducer in order to probe fluctuating forces. In micro and nanometer scale flows the characteristic length scale of the flow approaches and is even exceeded by the fluid mean free path. This limit is beyond the applicability of the Navier-Stokes equations, requiring a rigorous treatment using kinetic theory. In our first study, we conduct a series of experiments in which we use a microcantilever to measure gas dissipation in a nanoscopically confined system. Here, the distance between the gas molecules is of the same order as the separation between the cantilever and the walls of its container. As the cantilever is brought towards the wall, the flow becomes confined in the gap between the cantilever and the wall, affecting the resonant frequency and dissipation of the cantilever. By carefully tuning the separation distance, the gas pressure, and the cantilever oscillation frequency, we study the flow over a broad range of dimensionless parameters. Using these measurements, we provide an in-depth characterization of confinement effects in oscillating nanoflows. In addition, we propose a scaling function which describes the flow in the entire parameter space and which unifies previous theories based on the slip boundary condition and effective viscosity. In our next study, we seek to gain a better understanding of the transition to turbulence in a channel flow. We use a cantilever embedded in the channel wall to perform two sets of experiments: first, we study transition to turbulence triggered by the natural imperfections of the channel walls and second, we study transition under artificially added inlet noise. Our results point to two very different paths to turbulence. In the first case, wall effects lead to an extremely intermittent transitional flow and in the second case, broadband fluctuations originating at the inlet lead to less intermittent flow that is more reminiscent of homogeneous turbulence. The two experiments result in random flows in which high-order moments of near-wall fluctuations differ by orders of magnitude. Surprisingly however, the lowest order statistics in both cases appear qualitatively similar and can be described by a proposed noisy Landau equation. The noise, regardless of its origin, regularizes the Landau singularity of the relaxation time and makes transitions driven by different noise sources appear similar. Our results provide evidence of the existence of a finite turbulent relaxation time in transitional flows due to the persistent nature of noise in the system. In our last study, we turn to biologically-driven fluctuations from bacterial motion. Recent studies suggest that the motion of living bacteria could serve as a good indicator of bacteria species and resistance to antibiotics. To gain a better understanding of these fluctuations, we measure the nanomechanical motion of bacteria adhered to a chemically functionalized silicon microcantilever. A non-specific binding agent is used to attach E. coli to the surface of the device. The motion of the bacteria couples efficiently to the cantilever well below its resonance frequency, causing a measurable increase in its mechanical fluctuations. We vary the bacterial concentration over two orders of magnitude and are able to observe a corresponding change in the amplitude of fluctuations. Additionally, we administer antibiotics (Streptomycin) to kill the bacteria and observe a decrease in the fluctuations. A basic physical model is used to explain the observed spectral distribution of the mechanical fluctuations. These results lay the groundwork for understanding the motion of microorganisms adhered to surfaces and for developing micromechanical sensors for rapid bacterial identification and antibiotic resistance testing

Characterization and evaluation of the microcantilever radiation detector

In this study, a microcantilever charged-particle detector was characterized and evaluated. The method employed involved sensing an electric field emanating from a small collector plate on which charge built up to charged-particle flux.Sensitivity to alpha particles was determined using frequency and damping rate parameter shifts. Alpha particle detection experiments were compared to experiments using a charged plate of fixed voltage in order to characterize response more fully and to identify differences between expected charge accumulation and actual accumulation. Changes in cantilever behavior resulting from changes in ambient environmental conditions were also studied in order to determine to what extent they would impact charged-particle detection. In particular, microcantilever tip-surface adhesion force and jump-to-contact distance were studied as a function of relative humidity, and the dynamics of the liquid neck extending between the micro cantilever tip and the surface at small separation distances were investigated. In addition, relationship between the angle of the microcantilever tip relative to the surface and the excitation of multiple resonance modes was identified and described

Ferrule-top micromachined devices: A universal platform for optomechanical sensing

Iannuzzi, D. [Promotor

Investigating the binding capabilities of triazole-calix[4]arene functionalized microcantilever sensors toward heavy metals in aqueous solution

The main objective of this work was to investigate the binding capabilities of gold-coated micro-cantilever sensors functionalized with a bimodal triazole-calix[4]arene towards select heavy metals (e.g. Hg²⁺, Fe³⁺, Ni²⁺, Zn²⁺, and Pb²⁺). The interaction between the triazole-calix[4]arene functionalized micro-cantilevers and the target analytes resulted in the formation of a differential surface stresse, which in turn, resulted in a mechanical deflection of the microcantilever. Results showed that microcantilever arrays modified with triazole-calix[4]arene were capable of detecting trace concentrations of Hg²⁺ ions as low as 10⁻¹¹ M, which is sufficiently low for most applications. Results also showed that triazolecalix[4]arene functionalized microcantilevers were capable of detecting the presence of Pb²⁺ ions in aqueous solution of Hg²⁺. A new functionalization unit was also constructed to functionalize all 8 microcantilevers in an array with different sensing layers simultaneously, thus increasing the accuracy and reliability of the experimental results

Micromechanics of oxides - From complex scales to single crystals

Protective oxide scales shield high temperature materials from corrosion, thus ensuring safety and long material life under adverse operating conditions. Cracking and spallation of such scales can lead to fatigue crack initiation and expose the material to further oxidation. It is therefore imperative to measure the fracture properties of oxides so that they can be incorporated in the life estimation models of high temperature materials. Existing models require inputs on oxide properties such as fracture strain and elastic modulus. The established measurement methods are mainly applied for thick (several microns) scales, but for many materials such as superalloys the oxides are thinner (< 1 \ub5m), and the results would be affected by the influence of substrate and residual stresses. Focused ion beam machining (FIB) enables the preparation of micro sized specimens in the size range of these scales. \ua0In this work, a modified microcantilever geometry with partially removed substrate is proposed for testing of oxide scales. Room temperature microcantilever bending of thermally grown superalloy oxide (complex oxide with an upper layer of spinel and lower layer of Cr2O3) revealed the presence of plasticity, which is attributed to the deformation of the upper cubic spinel layer and low defect density of the volume being probed. Due to difficulties in isolating Cr2O3 from the complex oxide layer, dedicated oxidation exposures are performed on pure chromium to generate Cr2O3 which is tested using the same cantilever geometry at room temperature and 600 \ub0C. Results show lower fracture strain at 600 \ub0C in comparison to room temperature and presence of cleavage type of transgranular fracture in both cases, pointing to a need for studying cleavage fracture of Cr2O3. This was analysed using microcantilever bending of single crystal Cr2O3 to identify the preferential cleavage planes. Finally, fracture toughness was also measured through microcantilever bending and micropillar splitting. \ua0Thus, it is shown that micromechanical testing is an effective tool for measuring fracture properties of oxide scales. The fracture study of Cr2O3 scales show that it is a complex process in which the crystallographic texture also plays a role. Surface energy and fracture toughness criterion was unable to explain the fracture behaviour of single crystal Cr2O3 observed from experiments. Such a comprehensive analysis can contribute towards the development of reliable models for oxidation assisted failure

Lab-On-Chip for Ex-Vivo study of morphogenesis of tip growing cells of pollen tube

The purpose of the thesis is to develop a microfluidic based lab-on-chip (LOC) platform providing an Ex-Vivotesting environment that is able to mimic certain aspects of the in vivo growth conditions of the pollen tube, a cellular protuberance formed by the male gametophyte in the flowering plants. The thesis focuses on design, fabrication, modeling and testing of various LOC devices for the study of static and dynamic behavior of pollen tubes in response to mechanical stimulation. TipChip, an LOC platform, was developed to advance both experimentation and phenotyping in cell tip growth research. The platform enabled simultaneous testing of multiple pollen tubes. Using TipChip, we were able to answer several outstanding questions regarding pollen tube biology. We found that contrary to other types of tip growing cells such as root hairs and fungal hyphae, pollen tubes do not have a directional memory. Furthermore, we explored the effect of geometry of the microfluidic cell culture on pollen tube growth. We found that changing the width of the microfluidic channels does not have a significant effect on the pollen tube growth rate, while the growth rate was increased by increasing microchannel depth. We modified the original TipChip design to ascertain identical growth conditions for sequentiallyarranged pollen tubes and to ensure even distribution of entrapment probabilities for all microchannels. The effect of different dimensions of the microfluidic network on cell trapping probability was assessed using computational fluid dynamics and verified by experimental testing. The design was optimized based on trapping probability and uniformity of fluid flow conditions within the microchannels. This thesis also presents a novel method of fabricating a high aspect ratio horizontal PDMS microcantilever-based flow sensor integrated into a microfluidic device. The performance of the flow sensor was tested by introducing various flow rates into the microfluidic device and measuring the deflection of the cantilever’s tip using an optical microscope. The thesis addresses the quantification of cellular growth force of Camellia pollen tip growing cells using FlexChip, a flexure integrated LOC on polymer. We quantified the force that pollen tube is able to exert using a microfluidic lab-on-a-chip device integrated with flexural structure. The pollen grain is trapped in the microfluidic network and the growing tube is guided against a flexible microstructure that is monolithically integrated within the microfluidic chip. The invasive growth force of growing pollen tube was calculated from the maximal bending of microstructure modelled by Finite Element Analysis (FEA). Furthermore, the effect of the mechanical obstacle on the pollen tube's growth dynamics was assessed by quantifying the shift in the peak frequency characterizing the oscillatory behavior of the pollen tube growth rate. Our detailed analysis of the pollen tube growth dynamic before and during the contact with microcantilever revealed that pollen tube growth rate was reduced by 44% during the contact with the microcantilever. Moreover, the peak of oscillation frequency of pollen tube growth rate was reduced more dramatically by 70-75%. This suggests that the pollen tube actively changes its growth pattern to cope with the mechanical obstacle. Our findings in this thesis are novel in terms of pollen biology, and we believe insights from this research will lead to a better understanding of morphogenesis of a kind of tip growing cells, namely, pollen tube

Novel miniaturised and highly versatile biomechatronic platforms for the characterisation of melanoma cancer cells

There has been an increasing demand to acquire highly sensitive devices that are able to detect and characterize cancer at a single cell level. Despite the moderate progress in this field, the majority of approaches failed to reach cell characterization with optimal sensitivity and specificity. Accordingly, in this study highly sensitive, miniaturized-biomechatronic platforms have been modeled, designed, optimized, microfabricated, and characterized, which can be used to detect and differentiate various stages of melanoma cancer cells. The melanoma cell has been chosen as a legitimate cancer model, where electrophysiological and analytical expression of cell-membrane potential have been derived, and cellular contractile force has been obtained through a correlation with micromechanical deflections of a miniaturized cantilever beam. The main objectives of this study are in fourfold: (1) to quantify cell-membrane potential, (2) correlate cellular biophysics to respective contractile force of a cell in association with various stages of the melanoma disease, (3) examine the morphology of each stage of melanoma, and (4) arrive at a relation that would interrelate stage of the disease, cellular contractile force, and cellular electrophysiology based on conducted in vitro experimental findings. Various well-characterized melanoma cancer cell lines, with varying degrees of genetic complexities have been utilized. In this study, two-miniaturized-versatile-biomechatronic platforms have been developed to extract the electrophysiology of cells, and cellular mechanics (mechanobiology). The former platform consists of a microfluidic module, and stimulating and recording array of electrodes patterned on a glass substrate, forming multi-electrode arrays (MEAs), whereas the latter system consists of a microcantilever-based biosensor with an embedded Wheatstone bridge, and a microfluidic module. Furthermore, in support of this work main objectives, dedicated microelectronics together with customized software have been attained to functionalize, and empower the two-biomechatronic platforms. The bio-mechatronic system performance has been tested throughout a sufficient number of in vitro experiments.Open Acces