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

Biomechanical Characterization at the Cell Scale: Present and Prospects

The rapidly growing field of mechanobiology demands for robust and reproducible characterization of cell mechanical properties. Recent achievements in understanding the mechanical regulation of cell fate largely rely on technological platforms capable of probing the mechanical response of living cells and their physico–chemical interaction with the microenvironment. Besides the established family of atomic force microscopy (AFM) based methods, other approaches include optical, magnetic, and acoustic tweezers, as well as sensing substrates that take advantage of biomaterials chemistry and microfabrication techniques. In this review, we introduce the available methods with an emphasis on the most recent advances, and we discuss the challenges associated with their implementation

Force-controlled Biomanipulation for Biological Cell Mechanics Studies

Ph.DDOCTOR OF PHILOSOPH

BioMEMS

As technological advancements widen the scope of applications for biomicroelectromechanical systems (BioMEMS or biomicrosystems), the field continues to have an impact on many aspects of life science operations and functionalities. Because BioMEMS research and development require the input of experts who use different technical languages and come from varying disciplines and backgrounds, scientists and students can avoid potential difficulties in communication and understanding only if they possess a skill set and understanding that enables them to work at the interface of engineering and biosciences. Keeping this duality in mind throughout, BioMEMS: Science and Engineering Perspectives supports and expedites the multidisciplinary learning involved in the development of biomicrosystems. Divided into nine chapters, it starts with a balanced introduction of biological, engineering, application, and commercialization aspects of the field. With a focus on molecules of biological interest, the book explores the building blocks of cells and viruses, as well as molecules that form the self-assembled monolayers (SAMs), linkers, and hydrogels used for making different surfaces biocompatible through functionalization. The book also discusses: Different materials and platforms used to develop biomicrosystems Various biological entities and pathogens (in ascending order of complexity) The multidisciplinary aspects of engineering bioactive surfaces Engineering perspectives, including methods of manufacturing bioactive surfaces and devices Microfluidics modeling and experimentation Device level implementation of BioMEMS concepts for different applications. Because BioMEMS is an application-driven field, the book also highlights the concepts of lab-on-a-chip (LOC) and micro total analysis system (μTAS), along with their pertinence to the emerging point-of-care (POC) and point-of-need (PON) applications

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

BioMEMS

As technological advancements widen the scope of applications for biomicroelectromechanical systems (BioMEMS or biomicrosystems), the field continues to have an impact on many aspects of life science operations and functionalities. Because BioMEMS research and development require the input of experts who use different technical languages and come from varying disciplines and backgrounds, scientists and students can avoid potential difficulties in communication and understanding only if they possess a skill set and understanding that enables them to work at the interface of engineering and biosciences. Keeping this duality in mind throughout, BioMEMS: Science and Engineering Perspectives supports and expedites the multidisciplinary learning involved in the development of biomicrosystems. Divided into nine chapters, it starts with a balanced introduction of biological, engineering, application, and commercialization aspects of the field. With a focus on molecules of biological interest, the book explores the building blocks of cells and viruses, as well as molecules that form the self-assembled monolayers (SAMs), linkers, and hydrogels used for making different surfaces biocompatible through functionalization. The book also discusses: Different materials and platforms used to develop biomicrosystems Various biological entities and pathogens (in ascending order of complexity) The multidisciplinary aspects of engineering bioactive surfaces Engineering perspectives, including methods of manufacturing bioactive surfaces and devices Microfluidics modeling and experimentation Device level implementation of BioMEMS concepts for different applications. Because BioMEMS is an application-driven field, the book also highlights the concepts of lab-on-a-chip (LOC) and micro total analysis system (μTAS), along with their pertinence to the emerging point-of-care (POC) and point-of-need (PON) applications

Microfluidics for the detection of Cryptosporidium

This thesis details the development of microfluidics for the label-free sorting and/ or identification of waterborne pathogens which are commonly detected in contaminated drinking-water supplies using the United States Environmental Protection Agency method 1623.1 (USEPA 1623.1). This method recovers and detects pathogens of the Cryptosporidium and Giardia species, which can cause human gastroenteritis upon ingestion. USEPA 1623.1 is employed universally in developed regions (e.g., Europe, North America, Australia, New Zealand). Specifically, this thesis describes microfluidic systems that were developed with the objective of rapidly discriminating viable (i.e., intact and apparently infectious), humanpathogenic Cryptosporidium oocysts from non-viable, human-pathogenic oocysts and/ or species which are considered non-hazardous to human health. Such a system would reduce the overall detection time and allow a more accurate assessment of the risk posed to human health. A microfluidic setup incorporating dielectrophoresis was designed and employed for the viability-based sorting and enumeration of a human-pathogenic species of Cryptosporidium. This device enabled the sorting of untreated (live) and heat-inactivated (non-viable) sub-populations of the human pathogenic Cryptosporidium parvum with over 80% efficiency. Existing Microfluidic Impedance Cytometry (MIC) and Microfluidic-enabled Force Spectroscopy (MeFS) technologies were adapted for the enumeration, detection and viability determination of human-pathogenic Cryptosporidium oocysts, plus the discrimination of Cryptosporidium species which pose a major risk to human health from those which pose little to no risk. Using MIC, it was possible to discriminate untreated and heat-inactivated C. parvum with over 90% certainty. Furthermore, populations of C. parvum, Cryptosporidium muris (low-risk, human pathogen) and Giardia lamblia (also recovered using USEPA 1623.1) were discriminated from one another with over 90% certainty. Using MeFS, it was possible to differentiate temperature-inactivated (either by freeze- or heat-treatment) C. parvum from live C. parvum with a minimum of 78% efficiency. Finally, the high-risk, human pathogenic C. parvum was discriminated from C. muris with over 85% efficiency. Upon further validation, i.e., the analysis of other Cryptosporidium species and of oocysts which have been inactivated by other means (e.g., ozonation, ultraviolet radiaton), it is hoped that water utilities will employ such method(s) to more accurately characterise the human risk associated with contaminated supplies

Thermal-AFM under aqueous environment

The aim of this thesis is to describe the work developing and demonstrating the use of Scanning Thermal Microscopy (SThM) in an aqueous electrically conductive environment for the first time. This has been achieved by using new instrumentation to allow conventional SThM probes to measure and manipulate the temperature of non-biological and biological samples. For the latter, the aqueous environment is crucial to allow in-vitro experimentation, which is important for the future use of SThM in the life sciences. SThM is known to be a powerful technique able to acquire simultaneous topographic and thermal images of samples. It is able to measure the microscopic thermal properties of a surface with nanoscale spatial resolution. However, SThM has traditionally been limited to use in vacuum, air and electrically inert liquids. The aqueous Scanning Thermal Microscopy (a-SThM) described in this thesis is an entirely novel technique that opens up a new field for thermal-AFM. The first challenge addressed in this work was the adaptation of a commercial Multimode Nanoscope IIIa AFM to permit electrical access to a SThM probe completely immersed in aqueous solutions. By employing a newly designed probe holder and electronic instrumentation, the probe could then be electrically biased without inducing electrochemical reactions. This approach permitted conventional microfabricated thermal probes to be operated whilst fully immersed in water. This innovation allowed SThM measurements under deionized (DI) water to be performed on a simple solid sample (Pt on Si3N4) and the results compared with in-air scans and accurate 3D Finite Element (FE) simulations. Once the validity of the technique was proven, its performance was investigated, including crucially the limit of its thermal-spatial resolution; this was investigated using nanofabricated solid samples (Au on Si3N4) with well-defined features. These results were compared to the FE model, allowing an understanding of the mechanisms limiting resolution to be developed. In order to demonstrate the advantages granted by the water’s superior thermal conductivity compared to air or other liquids, non-contact thermal images were also acquired using the same samples. The final part of this thesis was focused on extending SThM into the biological area; a completely new field for this technique. New results are presented for soft 4 samples: I-collagen gel and collagen fibrils, which were thermally manipulated using a self-heated SThM probe. This successfully demonstrated the possibility of using heat to alter a biological sample within a very well localised area while being operated for long time in an aqueous environment. The difference in force response originated from the AFM scans with different levels of self-heating further proved the robustness of the technique. Finally, the technique was employed to study MG-63 living cells: The SThM probe was left in contact with each cell for a pre-determined period of time, with and without self heating. The results demonstrated that only the heated cells, directly beneath the probe tip died, tallying with the highly localised temperature gradient predicted by FE analysis

Nanoprobes for Tumor Theranostics

This book reports cutting-edge technology in nanoprobes or nanobiomaterials used for the accurate diagnosis and therapy of tumors, involving a multidisciplinary of chemistry, materials science, oncology, biology, and medicine