Gustav Emilsson

Competitive binding-based optical DNA mapping for fast identification of bacteria - multi-ligand transfer matrix theory and experimental applications on Escherichia coli.

We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50–160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens.

Dual-Wavelength Surface Plasmon Resonance for Determining the Size and Concentration of Sub-Populations of Extracellular Vesicles

Accurate concentration determination of subpopulations of extracellular vesicles (EVs), such as exosomes, is of importance both in the context of understanding their fundamental biological role and of potentially using them as disease biomarkers. In principle, this can be achieved by measuring the rate of diffusion-limited mass uptake to a sensor surface modified with a receptor designed to only bind the subpopulation of interest. However, a significant error is introduced if the targeted EV subpopulation has a size, and thus hydrodynamic diffusion coefficient, that differs from the mean size and diffusion coefficient of the whole EV population and/or if the EVs become deformed upon binding to the surface. We here demonstrate a new approach to determine the mean size (or effective film thickness) of bound nanoparticles, in general, and EV subpopulation carrying a marker of interest, in particular. The method is based on operating surface plasmon resonance simultaneously at two wavelengths with different sensing depths and using the ratio of the corresponding responses to extract the particle size on the surface. By estimating in this way the degree of deformation of adsorbed EVs, we markedly improved their bulk concentration determination and showed that EVs carrying the exosomal marker CD63 correspond to not more than around 10% of the EV sample.

Plasmonic Metasurfaces with Conjugated Polymers for Flexible Electronic Paper in Color

A flexible electronic paper in full color is realized by plasmonic metasurfaces with conjugated polymers. An ultrathin large-area electrochromic material is presented which provides high polarization-independent reflection, strong contrast, fast response time, and long-term stability. This technology opens up for new electronic readers and posters with ultralow power consumption.

Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules

The rapid spread of antibiotic resistance – currently one of the greatest threats to human health according to WHO – is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics.

Biosensing using plasmonic nanohole arrays with small, homogenous and tunable aperture diameters

Plasmonic nanohole arrays are widely used for optical label-free molecular detection. An important factor for many applications is the diameter of the apertures. So far nanohole arrays with controllable diameters below 100 nm have not been demonstrated and it has not been systematically investigated how the diameter influences the optical properties. In this work we fine-tune the diameter in short range ordered nanohole arrays down to 50 nm. The experimental far field spectra show how the wavelength of maximum extinction remains unaffected while the transmission maximum blue shifts with smaller diameters. The near field is visualized by numerical simulations, showing a homogenous enhancement throughout the cylindrical void at the transmission maximum for diameters between 50 and 100 nm. For diameters below 50 nm plasmon excitation is no longer possible experimentally or by simulations. Further, we investigate the refractive index sensing capabilities of the smaller holes. As the diameter was reduced, the sensitivity in terms of resonance shift with bulk liquid refractive index was found to be unaltered. However, for the transmission maximum the sensitivity becomes more strongly localized to the hole interior. By directing molecular binding to the bottom of the holes we demonstrate how smaller holes enhance the sensitivity in terms of signal per molecule. A real-time detection limit well below one protein per nanohole is demonstrated. The smaller plasmonic nanoholes should be suitable for studies of molecules confined in small volumes and as mimics of biological nanopores.

Switchable Plasmonic Metasurfaces with High Chromaticity Containing Only Abundant Metals

Plasmonic color generation offers several advantages but is also limited by the cost and availability of noble metals like gold. In this work, we present color-tunable metasurfaces with high chromaticity and reflectivity consisting of an aluminum mirror, a dielectric spacer, and a plasmonic nanohole array in copper. Copper is shown to be an excellent alternative to gold when properly protected from oxidation and makes it possible to generate a wide RGB gamut covering 27% of the standard RGB. By patterning the metasurfaces into microscale pixel triplets, color photos can be well reproduced with high resolution over wafer-sized areas. Further, we demonstrate active modulation of the reflected intensity using an electrochromic conductive polymer deposited on top of the nanostructures by screen printing. This technology opens up for ultrathin and flexible reflective displays in full color, that is, plasmonic electronic paper, compatible with large-scale sustainable production.

Superior LSPR substrates based on electromagnetic decoupling for on-a-chip high-throughput label-free biosensing

Localized surface plasmon resonance (LSPR) biosensing based on supported metal nanoparticles offers unparalleled possibilities for high-end miniaturization, multiplexing and high-throughput label-free molecular interaction analysis in real time when integrated within an opto-fluidic environment. However, such LSPR-sensing devices typically contain extremely large regions of dielectric materials that are open to molecular adsorption, which must be carefully blocked to avoid compromising the device readings. To address this issue, we made the support essentially invisible to the LSPR by carefully removing the dielectric material overlapping with the localized plasmonic fields through optimized wet-etching. The resulting LSPR substrate, which consists of gold nanodisks centered on narrow SiO2 pillars, exhibits markedly reduced vulnerability to nonspecific substrate adsorption, thus allowing, in an ideal case, the implementation of thicker and more efficient passivation layers. We demonstrate that this approach is effective and fully compatible with state-of-the-art multiplexed real-time biosensing technology and thus represents the ideal substrate design for high-throughput label-free biosensing systems with minimal sample consumption.

Polymer brushes in solid-state nanopores form an impenetrable entropic barrier for proteins

Polymer brushes are widely used to prevent the adsorption of proteins, but the mechanisms by which they operate have remained heavily debated for many decades. We show conclusive evidence that a polymer brush can be a remarkably strong kinetic barrier towards proteins by using poly(ethylene glycol) grafted to the sidewalls of pores in 30 nm thin gold films. Despite consisting of about 90% water, the free coils seal apertures up to 100 nm entirely with respect to serum protein translocation, as monitored label-free through the plasmonic activity of the nanopores. The conclusions are further supported by atomic force microscopy and fluorescence microscopy. A theoretical model indicates that the brush undergoes a morphology transition to a sealing state when the ratio between the extension and the radius of curvature is approximately 0.8. The brush-sealed pores represent a new type of ultrathin filter with potential applications in bioanalytical systems.

Fabrication and Characterization of Plasmonic Nanopores with Cavities in the Solid Support

Plasmonic nanostructures are widely used for various sensing applications by monitoring changes in refractive index through optical spectroscopy or as substrates for surface enhanced Raman spectroscopy. However, in most practical situations conventional surface plasmon resonance is preferred for biomolecular interaction analysis because of its high resolution in surface coverage and the simple single-material planar interface. Still, plasmonic nanostructures may find unique sensing applications, for instance when the nanoscale geometry itself is of interest. This calls for new methods to prepare nanoscale particles and cavities with controllable dimensions and curvature. In this work, we present two types of plasmonic nanopores where the solid support underneath a nanohole array has been etched, thereby creating cavities denoted as ‘nanowells’ or ‘nanocaves’ depending on the degree of anisotropy (dry or wet etch). The refractometric sensitivity is shown to be enhanced upon removing the solid support because of an increased probing volume and a shift of the asymmetric plasmonic field towards the liquid side of the finite gold film. Furthermore, the structures exhibit different spectral changes upon binding inside the cavities compared to the gold surface, which means that the structures can be used for location-specific detection. Other sensing applications are also suggested.

Nanopore Membranes for Separation and Sensing

During the last 20 years, the use of nanopore membranes to separate molecules depending on their size, charge or other characteristics, have increased in interest. These more ordered and defined nanopores have several advantages compared to traditional ultrafiltration membranes and provide possibilities to combine with, e.g., both electrical and optical sensing schemes. In this chapter, we discuss some of the more common nanopore membranes and how they can be used both for separation and sensing of analytes.