Digital printing of enzymes on textile substrates as functional materials

Recently, there have been significant developments in inkjet printing for applications in various fields such as medicine, biomaterials and sensors. In this research, enzymes like horseradish peroxidase (HRP) and glucose oxidase (GOx) were directly printed by inkjet printer onto flexible textile fabric in predefined patterns to produce a functional material. The functionality of the printed enzymes (bioink) was investigated by chemical reaction after printing fresh and stored bio-ink in a digital printer. The results indicated that these enzymes can be effectively printed individually or in combination, which retains their functionality after printing. Furthermore, HRP was coupled and printed with fluorescent group, the result confirmed that the printed enzyme was still active and retained its functionality despite the printing process. Hence, the digital printing technique can be used as a novel method for producing functional textiles for advanced applications in monitoring health and security

MEMS micro-contact printing engines

This thesis investigates micro-contact printing (µCP) engines using micro-electro-mechanical systems (MEMS). Such engines are self-contained and do not require further optical alignment and precision manipulation equipment. Hence they provide a low-cost and accessible method of multilevel surface patterning with sub-micron resolution. Applications include the field of biotechnology where the placement of biological ligands at well controlled locations on substrates is often required for biological assays, cell studies and manipulation, or for the fabrication of biosensors. A miniaturised silicon µCP engine is designed and fabricated using a wafer-scale MEMS fabrication process and single level and bi-level µCP are successfully demonstrated. The performance of the engine is fully characterised and two actuation modes, mechanical and electrostatic, are investigated. In addition, a novel method of integrating the stamp material into the MEMS process flow by spray coating is reported. A second µCP engine formed by wafer-scale replica moulding of a polymer is developed to further drive down cost and complexity. This system carries six complementary patterns and allows six-level µCP with a layer-to-layer accuracy of 10 µm over a 5 mm x 5 mm area without the use of external aligning equipment. This is the first such report of aligned multilevel µCP. Lastly, the integration of the replica moulded engine with a hydraulic drive for controlled actuation is investigated. This approach is promising and proof of concept has been provided for single-level patterning

Nanoparticle Necklace Network Arrays Exhibiting Room Temperature Single-Electron Switching

A single nanoparticle is one of the most sensitive electronic devices for sensing chemicals in a gas or liquid. The conductivity of a single Au nanoparticle is significantly modulated by the binding of a molecule that alters charge by just one electron. However, the single-electron sensitivity requires cryogenic temperatures and interconnection is not easy. A patterned two-dimensional network of one-dimensional nanoparticle necklaces made up of 10 nm Au particles are fabricated and shown to exhibit similar single-electron effect at room temperature. Furthermore, the long range conductivity of over 10’s of microns makes the structure easy to self-assemble onto conventional microelectronics circuitry. A device exhibiting single-electron effect is characterized by highly non-linear current-bias behavior where at bias, V \u3e VT current rises rapidly and scales as (V/VT – 1)ζ, where ζ ≥ 1 is the critical exponent and VT is the threshold voltage. Below VT, current does not flow. Thus, VT is the switching voltage and larger ζ signifies sharper switching characteristics. While arrays of one and two dimension are well known to exhibit appreciable VT at cryogenic temperatures, at ambient temperatures the blockade effect vanishes. The unique architecture of the necklace network results in a weak dependence of VT on temperature which leads to room temperature single-electron effect. The high sensitivity of the nanoparticle necklace network array at room temperature allows coupled live cells to electronically switch, or gate, the device through cellular metabolic activity. Additionally, the critical exponent, ζ, which is a measure of how current will rise during switching, can be significantly enhanced by cementing the necklaces with the dielectric material CdS, thereby greatly increasing the switching gain and sensitivity of the device. Given robust room temperature single-electron switching, enhanced ζ values, cellular coupling capability, and natural integrability with microelectronics circuitry, nanoparticle necklace network arrays have the potential to be implemented in a wide range of applications, such as, chemical sensors, biofuel cells, biomedical devices, and data storage devices. Adviser: Ravi F. Sara

Nanotechnology in regenerative medicine: the materials side

Regenerative medicine is an emerging multidisciplinary field that aims to restore, maintain or enhance tissues and hence organ functions. Regeneration of tissues can be achieved by the combination of living cells, which will provide biological functionality, and materials, which act as scaffolds to support cell proliferation. Mammalian cells behave in vivo in response to the biological signals they receive from the surrounding environment, which is structured by nanometre-scaled components. Therefore, materials used in repairing the human body have to reproduce the correct signals that guide the cellstowards a desirable behaviour. Nanotechnology is not only an excellent tool to produce material structures that mimic the biological ones but also holds the promise of providing efficient delivery systems. The application of nanotechnology to regenerative medicine is a wide issue and this short review will only focus on aspects of nanotechnology relevant to biomaterials science. Specifically, the fabrication of materials, such as nanoparticles and scaffolds for tissue engineering, and the nanopatterning of surfaces aimed at eliciting specific biological responses from the host tissue will be addressed.Postprint (published version

Selective placement of actin filaments on protein patterned surfaces

Motors proteins are used by living organisms to convert chemical energy into mechanical energy. The human body uses such motors proteins to transport materials through cells and, in the case of the biomolecular motor system of actin and myosin, to contract muscle. By understanding how these biological motors work, artificial motors with improved function may be possible and may be engineered to work in complex biological and non-biological environments. Recent research efforts have focused on understanding how to harness the power of, and manipulate the functioning of biological motors for integration into useful nanoscale systems. One important step towards this integration is the binding of motor proteins onto substrates and the full characterization of the system. The aim of this thesis was to study the feasibility of selective immobilization of actin filament motor protein based on the bioaffinity reaction between patterned streptavidin on a substrate and biotinylated actin filaments on an aminopropyltriethoxysilane (APTES)-functionalized glass surface. Gelsolin was used to cap the barbed/positive end of actin and to link actin to biotin molecules on the functionalized surface. Results demonstrate significant binding of actin filaments on streptavidin patterned surfaces via bioaffinity immobilization. Fluorescent microscopy and image processing software were used to characterize these results. Characterization of the APTES-functionalized surface was conducted using atomic force microscopy (AFM). The relationship between actin and gelsolin capping protein was examined as well as non-specific binding control of actin filaments

Application of advanced surface patterning techniques to study cellular behavior

Surface manipulation for the fabrication of chemical or topographic micro- and nanopatterns, has been central to the evolution of in vitro biology research. A high variety of surface patterning methods have been implemented in a wide spectrum of applications, including fundamental cell biology studies, development of diagnostic tools, biosensors and drug delivery systems, as well as implant design. Surface engineering has increased our understanding of cell functions such as cell adhesion and cell-cell interaction mechanics, cell proliferation, cell spreading and migration. From a plethora of existing surface engineering techniques, we use standard microcontact printing methods followed by click chemistry to study the role of intercellular contacts in collective cancer cell migration. Cell dispersion from a confined area is fundamental in a number of biological processes, including cancer metastasis. To date, a quantitative understanding of the interplay of single cell motility, cell proliferation, and intercellular contacts remains elusive. In particular, the role of E- and N-Cadherin junctions, central components of intercellular contacts, is still controversial. Combining theoretical modeling with in vitro observations, we investigate the collective spreading behavior of colonies of human cancer cells (T24). The spreading of these colonies is driven by stochastic single-cell migration with frequent transient cell-cell contacts. We find that inhibition of E- and N-Cadherin junctions decreases colony spreading and average spreading velocities, without affecting the strength of correlations in spreading velocities of neighboring cells. Based on a biophysical simulation model for cell migration, we show that the behavioral changes upon disruption of these junctions can be explained by reduced repulsive excluded volume interactions between cells. This suggests that in cancer cell migration, cadherin-based intercellular contacts sharpen cell boundaries leading to repulsive rather than cohesive interactions between cells, thereby promoting efficient cell spreading during collective migration. Despite the remarkable progress in surface engineering technology and its applications, a combination of pattern properties such as stability, precision, specificity, high-throughput outcome and spatiotemporal control is highly desirable but challenging to achieve. Here, we introduce a versatile and high-throughput covalent photo-immobilization technique, comprising a light-dose dependent patterning step and a subsequent functionalization of the pattern via click chemistry. This two-step process is feasible on arbitrary surfaces and allows for generation of sustainable patterns and gradients. The method is validated in different biological systems by patterning adhesive ligands on cell repellent surfaces, thereby constraining the growth and migration of cells to the designated areas. We then implement a sequential photopatterning approach by adding a second switchable pattering step, allowing for spatiotemporal control over two distinct surface patterns. As a proof of concept, we reconstruct the dynamics of the tip/stalk cell switch during angiogenesis. Our results show that the spatiotemporal control provided by our “sequential photopatterning” system is essential for mimicking dynamic biological processes, and that our innovative approach has a great potential for further applications in cell science. In summary, this work introduces two novel and versatile paradigms of surface patterning for studying different aspects of cell behaviour in different cell types. The reliability of both setups is experimentally confirmed, providing new insight into the role of cell-cell contacts during collective cancer cell migration as well as the tip/stalk switch behaviour during angiogenesis

Nanofabrication for Molecular Scale Devices

The predicted 22-nm barrier which is seemingly going to put a final stop to Moore’s law is essentially related to the resolution limit of lithography. Consequently, finding suitable methods for fabricating and patterning nanodevices is the true challenge of tomorrow’s electronics. However, the pure matter of moulding devices and interconnections is interwoven with research on new materials, as well as architectural and computational paradigms. In fact, while the performance of any fabrication process is obviously related to the characteristic of the materials used, a particular fabrication technique can put constraints on the definable geometries and interconnection patterns, thus somehow biasing the upper levels of the computing machine. Further, novel technologies will have to account for heat dissipation, a particularly tricky problem at the nanoscale, which could in fact prevent the most performing nanodevice from being practically employed in complex networks. Finally, production costs – exponentially growing in the present Moore rush – will be a key factor in evaluating the feasibility of tomorrow technologies. The possible approaches to nanofabrication are commonly classified into top-down and bottom-up. The former involves carving small features into a suitable bulk material; in the latter, small objects assemble to form more complex and articulated structures. While the present technology of silicon has a chiefly top-down approach, bottom-up approaches are typical of the nanoscale world, being directly inspired by nature where molecules are assembled into supramolecular structures, up to tissues and organs. As top-down approaches are resolution-limited, boosting bottom-up approaches seems to be a good strategy to future nanoelectronics; however, it is highly unlikely that no patterning will be required at all, since even with molecular-scale technologies there is the need of electrically contacting the single elements and this most often happens through patterned metal contacts, although all-molecular devices were also proposed. Here, we will give some insight into both top-down and bottom-up without the intention to be exhaustive, because of space limitations

Fabrication of (bio)molecular patterns with contact printing techniques

[spa] Un patrón es una colección de unidades formadoras que se repiten predeciblemente en una magnitud definida. Los investigadores han utilizado patrones para garantizar la funcionalidad y repetitividad de sus estudios. Para conseguir eso, los datos obtenidos de los estudios se comparan entre varios resultados, esperando así una correlación. Dos métodos de investigación están basados en patrones: uno requiere un sustrato con unidades repetidas localizadas en un plano cartesiano definido, obteniendo una plataforma de análisis múltiple. El segundo método utiliza localizaciones definidas con diferentes áreas de prueba, creando así una plataforma de multianálisis. La miniaturización de estas pruebas permiten reducir el costo, maximizar la eficiencia e incrementar la repetitividad de los ensayos. Los micropatrones consisten en puntos de (bio)moléculas limitados en pequeñas áreas para crear zonas de reacción múltiples. Esta tecnología fue inicialmente utilizada para crear las interacciones del ADN para estudios genómicos. La técnica evolucionó para crear patrones de proteínas y actualmente se utiliza para estudios bioquímicos a gran escala y de muy alto rendimiento. Patrones de una (bio)molécula repetida a través del sustrato son fabricados rutinariamente en muchos laboratorios utilizando técnicas de impresión por contacto, por inyección u otro métodos. El cimiento de estas técnicas es transferir una (bio)molécula de una solución a un sustrato. Esta Tesis pretende expandir los métodos de creación de micropatrones por técnicas de impresión por contacto. Inicialmente se caracterizó una máquina automatizada de impresión por microcontacto para crear patrones y estudiar las variables que afectan al momento de la impresión. Se correlacionaron la presión y el tiempo de impresión para entender la morfología del patrón resultante. Igualmente se caracterizó el posicionamiento micrométrico de los patrones para crear estructuras complejas. Posteriormente, la máquina se modificó para incluir la técnica de impresión con plumas poliméricas. Esta técnica permitió crear micropatrones en superficies minúsculas. Estos micropatrones fueron luego liberados para crear micropartículas que pueden ser personalizadas para aplicaciones diversas. Finalmente, se formuló una nueva técnica de replicación de patrones de ADN desde un patrón inicial, manteniendo la información química y espacial presente en éste.[eng] For that, the obtained data is purposely compared over and over in hope that the results are comparable. Two main research approaches are based on patterns: The initial requires a single substrate with localized and repeated units to create multiple testing sites, obtaining a repeated, multi-analysis system. The second approach uses fixed localization with different testing motifs, creating a diverse multi-analysis platform. The miniaturization of these assays provides an alternative to reduce cost, maximize efficiency, and increase repeatability. Micropatterns consist on immobilized (bio)molecular motifs constrained in small areas over a solid substrate. These fixed spots provide up to thousands of reaction sites for parallel detection. Micropatterns were first developed to study the interaction between Deoxyribonucleic acid (DNA) strands and the study of the genome. Afterwards, this technology was used to create miniaturized protein patterns. Today, this technology is essential for large-scale and high-throughput biological and biochemical studies. Single-feature microarrays are routinely reproduced at many laboratories using various contact, non-contact, or alternatively methods. The foundation is to transfer a (bio)molecule in a solution onto a solid substrate obtaining a defined feature shape. This Thesis aims to expand the current contact replication techniques for microarray fabrication. Initially, an automatized microcontact printing tool was characterized to create complex patterns on a wide range of substrates. Thiols, silanes, and various biomolecules were printed on glass, silicon oxide or gold. The printing properties were explored to create a definitive protocol for further applications. The effect of the printing force and dwell time were thoroughly studied to form a mathematical expression to understand all the variables involved during contact printing. The miniscule resolution provided by the automatized tool allowed the creation of complex micropatterns with single or multiple printings steps. This tool was later upgraded and fitted with new controllers to create smaller patterns. An alternatively contact printing technique called polymer pen lithography was used to pattern the surface of specialized substrates to create micropatterns on constricted areas. The miniaturized microarrays were later liberated to create functionalized microparticles. These microparticles can be tuned for many biochemical applications, such as protein interaction studies, drug discovery or life science. Lastly, a new contact replication method was established to fabricate DNA arrays. An initial DNA master arrays was fabricated with known contact printing techniques. Then, either hybridized or in situ synthesized strands were transported to an intermediate substrate. A second hybridization or synthesis was used to transport a replica of the master array to a new substrate, maintaining the chemical and spatial information present on the original array

Fabrication and Application of Flexible Sensors

A transfer printing method was developed to transfer carbon nanotubes (CNTs) from polyethylene terephthalate (PET) film to poly(dimethyl siloxane) (PDMS) polymer. Carbon nanotubes are composed of carbon atoms arranged in a honeycomb lattice structure, which are electrically conducting. When embedded in a nonconducting polymer, carbon nanotubes impart electrical conductivity to the nanocomposite, thus forming a nanocomposite that has potential applications in highly sensitive strain and pressure sensors. Several printing methods have been studied to deposit carbon nanotubes onto PDMS, including inkjet printing. Inkjet printing is a desirable deposition method since it is low-cost, simple, and allows the processing of aqueous-based inks. However, directly inkjet printing carbon nanotubes onto PDMS has been a challenge because the printed film becomes non-uniform due to the uneven drying of the droplets. Therefore, a method of transfer printing was developed to embed carbon nanotubes uniformly in PDMS. The transfer printing method consists of first inkjet printing patterns of carbon nanotubes onto a PET film, which quickly absorbs the aqueous ink and allows uniformity of the printed carbon nanotube patterns. The next step is spin-coating PDMS on the PET film to cover the carbon nanotube patterns, followed by curing the PDMS. The following step is thermally treating the PET film to promote the transfer of carbon nanotubes to PDMS, and finally peeling off PDMS from PET film to complete the transfer of carbon nanotube patterns. The transferred patterns had widths as small as 125 µm, while the obtained PDMS thickness was as low as 27.1 µm, which enabled the fabrication of highly sensitive force and pressure sensors. The transfer printing method was employed to fabricate a two-dimensional force sensor, which was composed of lines of carbon nanotubes in the x and y directions. The transduction mechanism lies in the generation of strain on the carbon nanotube pattern. When strain is produced, the resistance of the pattern changes due to the increase or decrease of the number of conduction paths in the carbon nanotube pattern. The practical application as a two-dimensional sensor was shown by monitoring the touch force exerted by multiple objects on the sensor. Due to the flexibility and stretchability of PDMS, fabricated air pressure sensors were capable of detecting small pressure differences. The sensors were composed of a circular diaphragm containing inkjet-printed carbon nanotube patterns. When air pressure increased on one side of the diaphragm, the deflection caused a strain on the CNT line, thus changing its resistance. Pressure sensors with a diaphragm diameter of five millimeters, diaphragm thickness of 27.1 µm showed sensitivity of 10.99 percent change in resistance per kilopascal (%/kPa) and limit of detection of 3.1 Pa. The pressure sensor has potential applications in monitoring minute air pressure differences such as those generated by the breathing pattern. The application of the highly sensitive and biocompatible pressure sensor was shown through the measurement of the pressure generated by a 3D-printed respiratory system

The Fabrication and Applications of Protein Patterns Produced Via Particle Lithography

A novel particle lithography technique with the ability to pattern protein in hexagonal dot arrays was developed. The patterning method consists of a simple three-step procedure: (1) formation of a close-packed polystyrene microsphere monolayer, (2) grafting of a protein-resistant layer of poly(ethylene glycol) (PEG), and (3)selective adsorption of protein into the resulting PEG holes. The diameter and center-to-center spacing of the patterned features was varied simultaneously by changing the diameter of the spheres used in the lithographic mask or independently using a simple heating modification. A combination of the original and modified procedures was used to produce patterns of protein dots with diameters of 450 nm - 9 ìm and center-to-center spacings of 2 - 10 ìm. To demonstrate the applicability of the particle lithography technique, a fluorescent-based immunoassay was created using quantum dot bioconjugates (QDBCs). The millions of protein dot features per patterned substrate served as redundant sampling points that produced a subpicomolar detection limit. Finally, the QDBC patterns were also used to investigate the differences between neutrophil spreading on patterned and homogenously coated anti-PSGL-1 (PL1) surfaces