Biomimetic and Biophysical Approach to Profile Metastatic Cancer Cell Migration

Honors Research ScholarshipCancer metastasis is a complex process by which cells in a primary tumor acquire an aggressive phenotype, and travel to distant, secondary sites in the body. One aspect of cancer metastasis is cell migration toward the vascular system, called invasion. Multiple modalities of single cell invasion exist, including amoeboid migration and mesenchymal migration. Amoeboid migration is less well understood, and in particular, the forces involved in amoeboid migration have yet to be fully elucidated at a subcellular scale. Cellular traction force microscopy, or CTFM, is one method used to probe migration forces. However, this approach is largely limited to two dimensions, and is limited by the size of the pillars on the substrate. To address these limitations, we developed a system using microfluidics and DNA origami capable of real-time force measurement of cell migration on a subcellular scale with a 10 pN resolution. Microfluidic devices were made using soft lithography and replica molding in our laboratory. DNA origami were made using protocols developed by Michael Hudoba and Dr. Carlos Castro in the Nanoengineering and Biodesign Laboratory. The devices were imaged using TIRF microscopy to study dwell times of the sensors in the open and closed states, and the devices were analyzed with an AFM to determine that they are best suited for measuring shear forces. Further, the presence of streptavidin protein was found to have a significant effect on DOFS binding with a p-value less than 0.05. DOFS concentrations around 1 nM were found to provide the most coverage while minimizing structure aggregation. Thus, our microfluidic devices are able to be functionalized with DNA origami force sensors with a high degree of attachment. This platform is thus capable of measuring cell migration and adhesion forces, and future work should harness this system to create 3D maps of cell migration to gain insight into invasion.Institute for Materials ResearchSecond-Year Transformational Experience Program (STEP)A one-year embargo was granted for this item.Academic Major: Biomedical Engineerin

Room Temperature Nanoparticulate Interfacial Layers for Perovskite Solar Cells via solvothermal synthesis

We present a solvothermal synthetic route to produce monodispersed CuO nanoparticles (NPs) in the range of 5-10 nm that can be used as hole selective interfacial layer between indium tin oxide (ITO) and perovskite active layer for p-i-n perovskite solar cells by a spin casting the dispersions at room temperature. The bottom electrode interface modification provided by spherical CuO-NPs at room temperature promotes the formation of high quality perovskite photoactive layers with large crystal size and strong optical absorption. Furthermore, it is shown that the nanoparticulate nature of the CuO hole transporting interfacial layer can be used to improve light manipulation within perovskite solar cell device structure. The corresponding p-i-n CH3NH3PbI3-based solar cells show high Voc values of 1.09 V, which is significantly higher compared to the Voc values obtained with conventional PEDOT:PSS hole selective contact based perovskite solar cells

Vitronectin at sites of cell-substrate contact in cultures of rat myotubes

Affinity-purified antibodies to the serum glycoprotein, vitronectin, were used to study sites of cell-substrate contact in cultures of rat myotubes and fibroblasts. Cells were removed from the substrate by treatment with saponin, leaving fragments of plasma membrane attached to the glass coverslip. When stained for vitronectin by indirect immunofluorescence, large areas of the substrate were brightly labeled. The focal contacts of fibroblasts and the broad adhesion plaques of myotubes appeared black, however, indicating that the antibodies had failed to react with those areas. Contact sites within the adhesion plaque remained unlabeled after saponin-treated samples were extracted with Triton X-100, or after intact cultures were sheared with a stream of fixative. These procedures expose extracellular macromolecules at the cell-substrate interface, which can then be labeled with concanavalin A. In contrast, when samples were sheared and then sonicated to remove all the cellular material from the coverslip, the entire substrate labeled extensively and almost uniformly with anti- vitronectin. Extracellular molecules associated with substrate contacts were also studied after freeze-fracture, using a technique we term "post-release fracture labeling." Platinum replicas of the external membrane were removed from the glass with hydrofluoric acid to expose the extracellular material. Anti-vitronectin, bound to the replicas and visualized by a second antibody conjugated to colloidal gold, labeled the broad areas of close myotube-substrate attachment and the nearby glass equally well. Our results are consistent with the hypothesis that vitronectin is present at all sites of cell-substrate contact, but that its antigenic sites are obscured by material deposited by both myotube and fibroblast cells

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing >50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations

Sensitive and specific detection of E. coli using biomimetic receptors in combination with a modified heat-transfer method

We report on a novel biomimetic sensor that allows sensitive and specific detection of Escherichia colt (E. coli) bacteria in a broad concentration range from 10(2) up to 10(6) CFU/mL in both buffer fluids and relevant food samples (i.e. apple juice). The receptors are surface-imprinted polyurethane layers deposited on stainless-steel chips. Regarding the transducer principle, the sensor measures the increase in thermal resistance between the chip and the liquid due to the presence of bacteria captured on the receptor surface. The low noise level that enables the low detection limit originates from a planar meander element that serves as both a heater and a temperature sensor. Furthermore, the experiments show that the presence of bacteria in a liquid enhances the thermal conductivity of the liquid itself. Reference tests with a set of other representative species of Enterobacteriaceae, closely related to E. coli, indicate a very low cross-sensitivity with a sensor response at or below the noise level

Optimization of an Escherichia coli biosensor for a rapid and in situ detection in water

Escherichia coli is an indicator bacterium of fecal matter in water and some serotypes can be pathogenic for humans. Quantifying the presence of this type of coliform in water is vital in order to provide safety and quality to the integrated water cycle. Although monitoring this type of microorganism is very important, in recent years there has not been much progress in this area, since analytical techniques are expensive, slow and require qualified personnel. The recent progress in biosensors has opened up a range of possibilities that can improve bacterial detection. This project presents a retention system integrated with an immuno-based biosensor that can detect a higher than 102 concentration of this bacterium, but it is still in development stages. Some aspects to improve the system have been studied, such as the integration of the process inside the retention system or the reduction of time. In addition, aptamers have been considered as an alternative to antibodies, as they are more stable. After the tests, the time of the protocol was reduced and certain stages were successfully integrated within the system. Some other stages though, could not be satisfactorily integrated, so further work is needed to know more about interactions. Although there's more work ahead to make the process adequate, we have made a step towards obtaining a fast, in situ, sensitive biosensor that can be able to monitor the water quality in the industrial sector.Escherichia coli és un bacteri indicador de contaminació fecal i a més, alguns dels seus serotips poden ser patògens per l'ésser humà. És per això, que determinar la presència d'aquest tipus de coliform resulta vital per oferir seguretat i qualitat en el cicle integrat de l'aigua. En els últims anys no s'ha avançat gaire en les tècniques de monitorització d'aquest bacteri, ja que encara resulten cares, lentes i necessiten personal qualificat per poder utilitzar-les. El progrés dels últims anys en matèria de biosensors, ens obren un ventall de possibilitats per poder millorar el sistemes de detecció bacteriana. En aquest treball es presenta un sistema de retenció microbià integrat amb un biosensor que és capaç de detectar concentracions de fins 102 d'Escherichia coli en aigua, però que encara està en fase de desenvolupament. S'han estudiat els aspectes a millorar del sistema, com la integració del procés dins del sistema de retenció o la reducció de temps del protocol. A més, s'han considerat els aptàmers com a alternativa dels anticossos, al ser més estables. Després de les proves, s'ha pogut reduir el temps del protocol i s'han integrat certes fases dins del sistema. Algunes altres fases, no s'han pogut integrar favorablement, i es necessitaran més estudis per poder conèixer la interacció entre aquestes i el sistema. Tot i que es necessiten més assajos per que el procés sigui perfecte, s'ha avançat un pas per a que a la llarga s'obtingui un biosensor que es pugui utilitzar in situ, sigui sensible, ràpid i faci possible la monitorització de la qualitat de l'aigua en el sector industrial.Escherichia coli es una bacteria indicadora de contaminación fecal y además, algunos de sus serotipos pueden ser patógenos para el ser humano. Es por eso, que medir la presencia de este tipo de coliforme, resulta vital para ofrecer seguridad y calidad en el ciclo integrado del agua. La monitorización de este tipo de organismo es muy importante, pero en los últimos años no se ha avanzado mucho en esta materia ya que las técnicas resultan caras, lentas y se necesita personal cualificado para su desarrollo. Los progresos de los últimos años en materia de biosensores abren un abanico de posibilidades para poder mejorar los sistemas detección bacteriana. En este trabajo, se presenta un sistema de retención microbiana acoplado con un biosensor, capaz de detectar concentraciones de hasta 102 de E.coli, aunque todavía está en fases de desarrollo. Se han estudiado los aspectos a mejorar del sistema, como la integración del proceso dentro del sistema de retención o la reducción del tiempo del protocolo. Además, se han considerado los aptámeros como alternativa a los anticuerpos usados, ya que resultan ser más estables. Después de las pruebas realizadas, se ha reducido el tiempo del protocolo y se han integrado ciertas fases. Algunas fases del protocolo, no han se han podido integrar favorablemente dentro del sistema, y se necesitan más estudios para poder conocer la interacción entre éstas y el sistema. Aunque se necesita más trabajo para que el proceso sea perfecto, se ha avanzado un paso para que a la larga se obtenga un biosensor que se pueda usar in situ, sea sensible, rápido y haga posible la monitorización de la calidad del agua en el sector industrial.Objectius de Desenvolupament Sostenible::6 - Aigua Neta i Sanejamen

Application of hydrophobic fluorinated silicon oxide nanoparticle coating on electrodynamic screen (EDS) films for enhancing self-cleaning function of solar collectors

Optical surfaces, which are exposed to outdoor environmental conditions, are susceptible to dust deposition. Sunlight incident on the surface of photovoltaic (PV) modules is attenuated by the dust layer accumulation on the front surface of the modules. Energy-yield decrease by dust layer accumulation, called soiling losses, can be 5 to 40 percent annually unless the modules are cleaned frequently. Cleaning the optical surface with water is generally used in solar plants, which causes an unsustainable demand for freshwater in semi arid and desert areas. Adhesion of soil on the front surface of solar collectors plays a major role in the cleaning process. For low water cleaning, the module surface is often coated with an anti-soiling coating. If the surface is made hydrophilic, water can wet the surface most effectively making the cleaning process efficient, but requiring a low amount of water. If the surface is made hydrophobic, the surface energy is decreased reducing the adhesion force; the cleaning can be performed even with a lesser amount of water or with an application of an external removal force, such as wind. Major problems with the application of anti-soiling coatings are their environmental durability, poor adhesion of the coating on the surface and low resistance to abrasion. Since the removal of dust still requires spraying of water to the PV modules, the coatings get removed within a short period. Similarly, another dust removal method that does not require water for cleaning PV modules, is the Electrodynamic Screen (EDS) film, which consists of series of parallel transparent electrodes embedded within two transparent dielectric films and laminated on the surface of the solar collectors. When the electrodes are activated film by applied voltage pulses there will be a traveling electric field on the EDS surface, then the dust particles on the film surface gets charged electrostatically and are removed by Coulomb forces applied by the electric field. The EDS film application is an elective removal of the dust particles from the PV module surface without requiring any water or mechanical forces. However, the applied Coulomb force cannot overcome the dust adhesion force when the particles are smaller than 2 m in diameter or if the capillary adhesion forces are present because of the high humidity environment. The objective of the thesis is to decrease the particle adhesion forces of the EDS film surface by the application of a hydrophobic coating so that the dust removal efficiency is enhanced for moving small particles. To improve EDS film based dust-removal performance it is necessary to have the coating non-conductive and optically transparent and hydrophobic for the effective application electrostatic removal mechanisms. The hydrophobic coating would reduce the surface energy and hence the dust adhesion force, assisting the self-cleaning effect, which would not require any water consumption. Since water-based cleaning is no longer needed for dust removal when the EDS film surface is modified with hydrophobic surface properties, the durability of the coating would improve. We describe here a synthesis of a hydrophobic fluorinated silicon oxide nanoparticle coating on the EDS films for enhancing the self-cleaning function of solar collectors. We used dip-coating method to apply a single-layer hydrophobic silicon oxide nanoparticle film with a large static water contact angle on the EDS film surface. silicon oxide was prepared by the sol-gel method using Tetraethylorthosilicate (TEOS) and ammonium hydroxide as a precursor anda catalyst. The suspension was treated with 3-aminopropyltriethoxysilane (APS) and 1H,1H,2H,2H-perfluorodecyltriethoxysilane (FAS-17) to modify the surface of the coating, which greatly improved the hydrophobicity, and thus gave the coating the desired self-cleaning property. We used polyurethane (PU) as a binder between the substrate and coating to enhance the durability of the lm in outdoors applications. Experimental data on the optical transmittance, adhesion of the hydrophobic film on the glass surface of the EDS lm, and the dust removal efficiency are presented. The average optical transmission efficiency (TE) decreased from 93.43 to 89.78 percent and the output power restoration (OPR) of the solar panels laminated with EDS film with SH coating increased from 98.3 to 99.19 percent. Possible improvements to the hydrophobic coating process and its durability and future research needs are discussed