Nanostructured metallic film plasmonics: fabrication and biosensing applications

The research presented in this dissertation is interdisciplinary in nature. It covers the areas of micro- and nanofabrication, chemistry, materials science, and biological sensing. The running theme of the dissertation is the fabrication of micro- and nanostructures for use in plasmonic devices to aid in the optical detection of biomolecules. Phase I of the research focused on a bimetallic nanostructured (nanoslit) film to aid in improving the sensitivity in comparison to pure gold films. Phase II of the research investigated nanoledge structures (stair-step features) for their ability to trap biomolecules and aid in surface plasmon resonance sensing. Phase III of the research examined how to produce a fluidic dam, a microstructure with an overcut sidewall profile, which could aid in separating biological entities from the proteins of interest. Phase IV of the research assessed the use of the fluidic dam and nanoledge structures for detection of Troponin T, a biomarker used in the diagnosis of heart attacks. Phase V of the research focused on the design and microfabrication of a plasmonic device, which could study how surface plasmon resonance influences a photocurrent generated by immobilizing photosystem I in a nanoslit structure

State-of-the-Art of (Bio)Chemical Sensor Developments in Analytical Spanish Groups

(Bio)chemical sensors are one of the most exciting fields in analytical chemistry today. The development of these analytical devices simplifies and miniaturizes the whole analytical process. Although the initial expectation of the massive incorporation of sensors in routine analytical work has been truncated to some extent, in many other cases analytical methods based on sensor technology have solved important analytical problems. Many research groups are working in this field world-wide, reporting interesting results so far. Modestly, Spanish researchers have contributed to these recent developments. In this review, we summarize the more representative achievements carried out for these groups. They cover a wide variety of sensors, including optical, electrochemical, piezoelectric or electro-mechanical devices, used for laboratory or field analyses. The capabilities to be used in different applied areas are also critically discussed

Carbohydrate-carbohydrate interaction provides adhesion force and specificity for cellular recognition and adhesion

Carbohydrates at the cell surface have been proposed as mediators in cell-cell recognition events involved in embryogenesis, metastasis, and other proliferation processes by calcium-dependent carbohydrate to carbohydrate interactions. They are the most prominently exposed structures on the surface of living cells, and with flexible chains and many binding sites are ideal to serve as the major players in initiating these cellular events. However, biological relevance of these type interactions is often questioned because of the very low affinity binding of single carbohydrate molecules and that they manifest themselves only through the contact of a large number of molecules tightly arranged in the membrane. Weak interactions are considerably more difficult to study and only a few biologically significant examples of direct carbohydrate-carbohydrate interactions have been reported, e.g. pioneering work showing glycosphingolipid self-interactions through multivalent interaction of Lewis X epitopes. However, there are no reports on the existence of specific proteoglycan self-interactions through carbohydrate-carbohydrate interactions in cellular recognition system, as it has been done with glycosphingolipids. Here, we used sponges, organisms on which the first proteoglycan-mediated cell-cell recognition in the animal kingdom was demonstrated, as a model system to study carbohydrate-mediated cellular recognition. We show that the interaction between single oligosaccharides from surface proteoglycans is relatively strong and comparable to protein-carbohydrate interactions, highly specific, and dependent on Ca2+-ions. 200 kDa glycans from the core protein of Microciona prolifera cell surface proteoglycans have been previously shown to mediate homotypic Microciona proteoglycan-proteoglycan interactions. Here, 200 kDa glycans from four different sponge species: Microciona prolifera, Halichondria panicea, Suberites fuscus and Cliona celata were purified and investigated for species-specific interactions. Selective recognition of glycans by live cells was studied to confirm the existence of glycan-glycan recognition system in biologically relevant situations. Mature sponge cells have the ability to reaggregate species-specifically and form homogenous aggregates on a shaker at the right shear forces in the presence of physiological 10 mM Ca2+. Live cells were allowed to aggregate with glycan-coated beads similar in size to small sponge cells in the presence of calcium. They specifically recognized beads coated with their own glycans and did not mix but separated from beads coated with glycans isolated from different species. The glycan-glycan recognition assay was developed to mimic species-specific cellcell recognition in sponges. 200 kDa glycans immobilized onto beads similar in size to small sponge cells assembled species-specifically in the presence of physiological calcium, at the same shear forces as in cell-cell aggregation. Glycans coated on beads aggregated with glycans from the same species coated on beads, and separated from glycans from other species. The glycan density necessary for specific live cellcell recognition in sponges is 828 molecules/μm2. In our studies, the glycan density necessary for specific glycan-coated bead was very similar: ~810 molecules/μm2. Mature live cells demonstrated specific recognition of 200 kDa glycans during selective-binding to glycans coated on surfaces in the presence of calcium. They strongly adhered to glycans from their own surface proteoglycans coated onto a solid polystyrene phase, while the binding to glycans from different proteoglycans was 3 - 5 times lower. Moreover, homotypic adhesion to glycan-coated plates enhanced sponge cell differentiation and formation of mineral skeleton (spicules). Larval cells, after settlement and spreading of larvae, can fuse species-specifically in nature. In our studies, live larval cells recognized and adhered specifically to glycans purified from adhesion proteoglycans from their "mother sponge". They showed almost no interaction with glycans from other species. As in cell-glycan adhesion assays, highly species-specific adhesion of 200 kDa glycans to glycan-coated surfaces could be observed in the presence of physiological calcium. Tested glycans bound strongly to glycans from the same species and showed up to a six fold reduction in binding to glycans from other species. Atomic force microscopy (AFM) was performed to measure for the first time adhesion forces between single glycan molecules obtained from different surface proteoglycans. Measurements revealed equally strong adhesion forces in the range of several hundred piconewtons (pN) between glycan molecules as between proteins and glycans measured in another recognition system. Moreover, statistically significant differences (p value < 0.01) were seen between homotypic (glycans from the same species) and heterotypic (glycans from different species) interactions. Moreover, the polyvalent character of binding characterized mainly interactions between glycans from the same species. This indicates that not only the higher adhesion force per binding site as such but also the higher amount of multiple interactions between glycans from the same species versus mixture of glycans from different species guaranteed the specificity of the glycan-mediated recognition. These findings confirm for the first time the existence of specific glycan-glycan recognition system between cell surface proteoglycans. We propose that these cell's outermost surface structures serve as important players in initiating the very first contacts between cells through highly species-specific and flexible carbohydratecarbohydrate interactions

Carbohydrate-carbohydrate interaction provides adhesion force and specificity for cellular recognition and adhesion

Carbohydrates at the cell surface have been proposed as mediators in cell-cell recognition events involved in embryogenesis, metastasis, and other proliferation processes by calcium-dependent carbohydrate to carbohydrate interactions. They are the most prominently exposed structures on the surface of living cells, and with flexible chains and many binding sites are ideal to serve as the major players in initiating these cellular events. However, biological relevance of these type interactions is often questioned because of the very low affinity binding of single carbohydrate molecules and that they manifest themselves only through the contact of a large number of molecules tightly arranged in the membrane. Weak interactions are considerably more difficult to study and only a few biologically significant examples of direct carbohydrate-carbohydrate interactions have been reported, e.g. pioneering work showing glycosphingolipid self-interactions through multivalent interaction of Lewis X epitopes. However, there are no reports on the existence of specific proteoglycan self-interactions through carbohydrate-carbohydrate interactions in cellular recognition system, as it has been done with glycosphingolipids. Here, we used sponges, organisms on which the first proteoglycan-mediated cell-cell recognition in the animal kingdom was demonstrated, as a model system to study carbohydrate-mediated cellular recognition. We show that the interaction between single oligosaccharides from surface proteoglycans is relatively strong and comparable to protein-carbohydrate interactions, highly specific, and dependent on Ca2+-ions. 200 kDa glycans from the core protein of Microciona prolifera cell surface proteoglycans have been previously shown to mediate homotypic Microciona proteoglycan-proteoglycan interactions. Here, 200 kDa glycans from four different sponge species: Microciona prolifera, Halichondria panicea, Suberites fuscus and Cliona celata were purified and investigated for species-specific interactions. Selective recognition of glycans by live cells was studied to confirm the existence of glycan-glycan recognition system in biologically relevant situations. Mature sponge cells have the ability to reaggregate species-specifically and form homogenous aggregates on a shaker at the right shear forces in the presence of physiological 10 mM Ca2+. Live cells were allowed to aggregate with glycan-coated beads similar in size to small sponge cells in the presence of calcium. They specifically recognized beads coated with their own glycans and did not mix but separated from beads coated with glycans isolated from different species. The glycan-glycan recognition assay was developed to mimic species-specific cellcell recognition in sponges. 200 kDa glycans immobilized onto beads similar in size to small sponge cells assembled species-specifically in the presence of physiological calcium, at the same shear forces as in cell-cell aggregation. Glycans coated on beads aggregated with glycans from the same species coated on beads, and separated from glycans from other species. The glycan density necessary for specific live cellcell recognition in sponges is 828 molecules/μm2. In our studies, the glycan density necessary for specific glycan-coated bead was very similar: ~810 molecules/μm2. Mature live cells demonstrated specific recognition of 200 kDa glycans during selective-binding to glycans coated on surfaces in the presence of calcium. They strongly adhered to glycans from their own surface proteoglycans coated onto a solid polystyrene phase, while the binding to glycans from different proteoglycans was 3 - 5 times lower. Moreover, homotypic adhesion to glycan-coated plates enhanced sponge cell differentiation and formation of mineral skeleton (spicules). Larval cells, after settlement and spreading of larvae, can fuse species-specifically in nature. In our studies, live larval cells recognized and adhered specifically to glycans purified from adhesion proteoglycans from their "mother sponge". They showed almost no interaction with glycans from other species. As in cell-glycan adhesion assays, highly species-specific adhesion of 200 kDa glycans to glycan-coated surfaces could be observed in the presence of physiological calcium. Tested glycans bound strongly to glycans from the same species and showed up to a six fold reduction in binding to glycans from other species. Atomic force microscopy (AFM) was performed to measure for the first time adhesion forces between single glycan molecules obtained from different surface proteoglycans. Measurements revealed equally strong adhesion forces in the range of several hundred piconewtons (pN) between glycan molecules as between proteins and glycans measured in another recognition system. Moreover, statistically significant differences (p value < 0.01) were seen between homotypic (glycans from the same species) and heterotypic (glycans from different species) interactions. Moreover, the polyvalent character of binding characterized mainly interactions between glycans from the same species. This indicates that not only the higher adhesion force per binding site as such but also the higher amount of multiple interactions between glycans from the same species versus mixture of glycans from different species guaranteed the specificity of the glycan-mediated recognition. These findings confirm for the first time the existence of specific glycan-glycan recognition system between cell surface proteoglycans. We propose that these cell's outermost surface structures serve as important players in initiating the very first contacts between cells through highly species-specific and flexible carbohydratecarbohydrate interactions

Fundamentals of SARS-CoV-2 Biosensors

COVID-19 diagnostic strategies based on advanced techniques are currently essential topics of interest, with crucial roles in scientific research. This book integrates fundamental concepts and critical analyses that explore the progress of modern methods for the detection of SARS-CoV-2

Light-Induced Interactions using Optical Near-Field Devices

Optical near-fields are generated when light passes through components with wavelength, or subwavelength features. The near-fields generated at the surfaces of devices are often neglected, in part because the far-fields have more applications and are more readily accessible. Near-fields, as one might expect, occur very close to the surface of the material through which the light passes. However, near-fields present an interesting method of overcoming Rayleigh\u27s diffraction limit. For example, the evanescent field at the surface of a prism or ultrathin fibre rapidly decays, but can exist in sub-diffraction limited areas. Similarly, the field generated by a subwavelength aperture or a plasmonic particle can have local field distributions with minute dimensions, allowing one to confine light to areas otherwise unattainable, extremely close to the surface of the material in question. By exploiting this aspect of optical near-fields we apply them to problems in atom and particle trapping.Our main focus is on ultrathin optical fibres. These fibres differ from telecommunications fibre due to their lack of cladding material and their wavelength-scale dimensions. These two factors combine to produce a significant evanescent field at their waist. This field is readily accessible and can be used to trap particle or atoms through the optical forces which arise in such light-matter interactions. We can also use such devices to passively collect light which is emitted into the available guided mode. Here, we demonstrate how an ultrathin fibre can be used as a probe to determine the temperature of a cold atom cloud.Ultrathin fibres, while extremely useful, have some limiting factors related to the strength and distribution of their evanescent fields. To improve upon the design, we also investigated how one can nanostructure an optical fibre using focussed ion beam milling techniques or combine optical fibres with gold dimer arrays to produce localised field enhancements. We used nanostructured fibres to trap 100 and 200 nm dielectric spheres within the structured region. Various numerical techniques were employed to characterise both the nanostructured fibre and the plasmonic-enhanced fibre.Aside from optical fibres, we also briefy discuss how an array of Fresnel microlenses can be packaged with other atom chip designs to produce a device which could trap atoms microns away from a gold surface. We discuss the theory and fabrication technique for such a Fresnel microlens array atom chip.Okinawa Institute of Science and Technology Graduate Universit

Research Experience for Undergraduates Program, 2011

Supported by the National Science Foundation