In the past decade, DNA origami self-assembly has been widely applied for creating customised nanostructures with base-pair precision. In this technique, the unique chemical addressability of DNA can be harnessed to create programmable architectures, using components ranging from dye or protein molecules to metallic nanoparticles. In this thesis, we apply DNA nanotechnology for developing novel light-harvesting and optical voltage sensing nano-devices. We use the programmable positioning of dye molecules on a DNA origami plate as a mimic of a light-harvesting antenna complex required for photosynthesis. Such a structure allows us to systematically analyse optimal design concepts using different dye arrangements. Complementary to this, we use the resistive-pulse sensing technique in a range of electrolytes to characterise the mechanical responses of DNA origami structures to the electric field applied. Based on this knowledge, we assemble voltage responsive DNA origami structures labelled with a FRET pair. These undergo controlled structural changes upon application of an electric field that can be detected through a change in FRET efficiency. Such a DNA-based device could ultimately be used as a sensitive voltage sensor for live-cell imaging of transmembrane potentials.

GaN nanopore arrays with pore diameters of approximately 75 nm were fabricated by inductively coupled plasma etching (ICP) using anodic aluminum oxide (AAO) films as etch masks. Nanoporous AAO films were formed on the GaN surface by evaporating an Al film onto a GaN epilayer and subsequently anodizing the aluminum. To minimize plasma-induced damage, the template was exposed to CF4-based plasma conditions. Scanning electron microscopy (SEM) analysis shows that the diameter and the periodicity of the nanopores in the GaN were directly transferred from the original anodic alumina template. The pore diameter in the AAO film can be easily controlled by tuning the anodization conditions. Atomic force microscopy (AFM), photoluminescence (PL) and micro-Raman techniques were employed to assess the quality of the etched GaN nanopore surface. Such a cost-effective method to produce nano-patterned GaN template would be useful for growth and fabrication of III-Nitrides based nanostructures and photonic band gap materials. / Singapore-MIT Alliance (SMA)

DNA nanotechnology has revolutionised our capability to shape and control three-dimensional structures at sub-nanometre length scales. In this thesis, we use DNA to build synthetic membrane-inserting channels. Porphyrin and cholesterol tags serve as membrane anchors to facilitate insertion into the lipid membrane. With atomic force microscopy, confocal imaging and ionic current recordings we characterise our DNA nanochannels that mimic their natural protein-based counterparts in form and function. We find that they exhibit voltage-dependent conductance states. Amongst other architectures, we create the largest man-made pore in a lipid membrane to date approaching the electrical diameter of the nuclear pore complex. Pushing the boundaries on the other end of the spectrum, we demonstrate the ultimately smallest DNA membrane pore made from a single membrane-spanning DNA duplex. Thereby, we proof that ion conduction across lipid membranes does not always require a physical channel. With experiments and MD simulations we show that ions flow through a toroidal pore emerging at the DNA-lipid interface around the duplex. Our DNA pores spanning two orders of magnitude in conductance and molecular weight showcase the rational design of synthetic channels inspired by the diversity of nature - from ion channels to porins.

The use of the MinION sequencer (Oxford Nanopore) was tested on samples prepared to simulate infectious samples. The tested procedure is to simulate work with a sample with an unknown pathogen. Therefore, a metagenomic approach was chosen. Three kits were tested: Rapid Barcoding Sequencing, PCR Barcoding and Premium whole genome amplification. Each kit differed in duration, difficulty to prepare and in amplification of nucleic acids. In total it was chosen eight viruses with different genome lengths and with varying types of the genome (5,6 - 152 kb, ss/ds RNA, dsDNA). Ten samples were prepared to simulate different types of infection (respiratory, gastrointestinal tract and urine), and one sample contained pure water as a negative control. Before preparation of the library with Oxford Nanopore's kits, DNase/RNase treatment was used. The viral RNA was transcribed into DNA and in chosen samples were amplificated to reach a higher concentration of nucleic acids. Rapid barcoding sequencing kit detected all selected viruses with the highest number of viral reads (4403) with a length between 100 and 250 nt and quality coverage of viral genomes. PCR Barcoding kit detected five out of eight viruses, and the number of identified reads with a length of 100-200 nt distinctly decreased. Premium whole genome...

The genus Rhodotorula includes basidiomycetous oleaginous yeast species. Rhodotorula babjevae can produce compounds of biotechnological interest such as lipids, carotenoids, and biosurfactants from low value substrates such as lignocellulose hydrolysate. High-quality genome assemblies are needed to develop genetic tools and to understand fungal evolution and genetics. Here, we combined short- and long-read sequencing to resolve the genomes of two R. babjevae strains, CBS 7808 (type strain) and DBVPG 8058, at chromosomal level. Both genomes are 21 Mbp in size and have a GC content of 68.2%. Allele frequency analysis indicates that both strains are tetraploid. The genomes consist of a maximum of 21 chromosomes with a size of 0.4 to 2.4 Mbp. In both assemblies, the mitochondrial genome was recovered in a single contig, that shared 97% pairwise identity. Pairwise identity between most chromosomes ranges from 82 to 87%. We also found indications for strain-specific extrachromosomal endogenous DNA. A total of 7591 and 7481 protein-coding genes were annotated in CBS 7808 and DBVPG 8058, respectively. CBS 7808 accumulated a higher number of tandem duplications than DBVPG 8058. We identified large translocation events between putative chromosomes. Genome divergence values between the two strains indicate that they may belong to different species.

This dissertation outlines the research and development of ground-breaking nanometer sized openings (nanopores) integrated with an on-chip optofluidic platform. This platform represents a significant advancement for single nanoparticle sensing. In this work specifically, the integrated optofluidic platform has been used to electrically and optically filter and detect single nanoparticles using ionic current blockade and fluorescence experiments. The correlation of electrical and optical signal has provided the highest sensitivity single nanoparticle measurements ever taken with integrated optofluidic platforms. The particular optofluidic platform used for this work is an antiresonant reflecting optical waveguide (ARROW). ARROW hollow and solid core waveguides are interference based waveguides that are designed to guide light in low index media such as liquids and gases. Because of this unique guiding property, ARROW hollow cores can be used to sense and analyze low concentrations of single particles. Additionally, because ARROW platforms are based upon standard silicon processing techniques and materials, they are miniature sized (~1 cm2), inexpensive, highly parallelizable, provide a high degree of design flexibility, and can be integrated with many different optical and electrical components and sources. Finally, because of the miniature, integrated nature of the ARROW platform, it has the potential to be incorporated into hand held devices that could provide quick, inexpensive, user-friendly diagnostics. The ARROW platform has been through many revisions in the past several years in an attempt to improve performance and functionality. Specifically, advanced fabrication techniques that have been used to decrease the production time, increase the yield, and improve the optical quality of ARROW platforms are discussed in the first part of this work. These advancements were all developed in order to facilitate the production of high quality integrated nanopores and ARROW platforms. The second part of this work then focuses on the actual integration of micrometer sized openings (micropores) and nanopores in the hollow waveguide section of ARROW platforms for filtering, detecting, and analyzing single nanoparticles. The successes and attempts at achieving these results are the basis of this dissertation of work.

The ability to precisely count inorganic and organic nanoparticles and to measure their size distribution plays a major role in various applications such as drug delivery, nanoparticles counting, and many others. In this work I present a simple resistive pulse method that allows translocations, counting, and measuring the size and velocity distribution of silica nanoparticles and liposomes with diameters from 50 nm to 250 nm. This technique is based on the Coulter counter technique, but has nanometer size pores. It was found that ionic current drops when nanoparticles enter the nanopore of a pulled micropipette, producing a clear translocation signal. Pulled borosilicate micropipettes with opening 50 ~ 350 nm were used as the detecting instrument. This method provides a direct, fast and cost-effective way to characterize inorganic and organic nanoparticles in a solution. In this work I also introduce a newly developed Capillary Ionic Transistor (CIT). It is presented as a nanodevice which provides control of ionic transport through nanochannel by gate voltage. CIT is Ionic transistor, which employs pulled capillary as nanochannel with a tip diameter smaller than 100 mm. We observed that the gate voltage applied to gate electrode, deposited on the outer wall of a capillary, affect a conductance of nanochannel, due to change of surface charge at the solution/capillary interface. Negative gate voltage corresponds to lower conductivity and positive gate increases conductance of the channel. This effect strongly depends on the size of the channel. In general, at least one dimension of the channel has to be small enough for electrical double layer to overlap.

The transport of a biomolecule through a nanopore occurs in many biological functions such as, DNA or RNA transport across nuclear pores and the translocation of proteins across the eukaryotic endoplasmic reticulum. In addition to the biological processes, it has potential applications in technology such as, drug delivery, gene therapy, and single molecule sensing. The DNA translocation through a synthetic nanopore device is considered as the basis for cheap and fast sequencing technology. Motivated by the experimental advances, many theoretical models have been developed. In this thesis, we explore the dynamics of driven translocation of a semiflexible polymer through a nanopore in two dimensions (2D) using Langevin dynamics (LD) simulation. By carrying out extensive simulation as a function of different parameters such as, driving force, length and rigidity of the chain, viscosity of the solvent, and diameter of the nanopore, we provide a detailed description of the translocation process. Our studies are relevant for fundamental understanding of the translocation process which is essential for making accurate nano-pore based devices.

Nanopore sequencing is the next generation ofsequencing methods which promises to deliver cheaper andmore portable genome sequencing capabilities. A single DNAor RNA strand is passed through a nanopore nested in anartificial membrane with an electric potential applied across it.The nucleotide bases of the helix then interact with the ioniccurrent in the nanopore, resulting in a unique signal that canbe translated into the correct corresponding nucleotide sequence.This project investigated whether features of the raw signal datacould be used as predictive indicators of the duration time ofeach nucleotide base in the nanopore. This is done in orderto segment the signal before translation. The training data setused came from the sequenced DNA molecules of an E. Colibacterium. Distribution candidates were fitted to a histogram ofthe duration data of the training set. Features of the currentsignal and distribution parameters were correlated in orderinvestigate if a linear predictive model could be created. Theresults indicate that the feature zero-crossings is not an optimaloption for construction of a linear model, while the large jumpsand moving variance features often generate linear patterns. The parameter of the Log-logistic distribution had the best fit withthe lowest relative root mean square deviation (rRMSD) of 2.7%. / Nanopore sequencing är nästa generationensmetod för DNA sekvensering som kommer att bidra medbilligare och mer portabla sekvenseringsmöjligheter. Metodeninnebär att en enkelsträngad DNA eller RNA molekyl passerargenom porer i nanostorlek, placerade i ett artificiellt membransamtidigt som en elektrisk potential appliceras över membranet.Nukleotiderna i genmolekylen interagerar med jonströmmen iporen, vilket resulterar i en unik signal som kan översättas tillden korresponderande sekvensen av nukleotider som passerat.Detta projekt gick ut på att undersöka om egenskaper frånsignalen kan användas som predikativa indikatorer för varaktighetensom varje nukleotid befinner sig i membranporen. Dettaför att sedan kunna segmentera signalen före översättningen tillDNA sekvensen. Träningsdata som användes är sekvenserad DNAfrån en E. Coli bakterie. Kandiderande sannolikhetsfördelningaranpassades till ett histogram som beskriver varaktigheten.Egenskaperna och parametrar från fördelningarna korreleradesför att skapa en linjär modell. Resultatet visade att antaletskärningar i x-axeln som signalegenskap inte är det optimalavalet för konstruktion av en linjär modell. Skillnaden mellan två signalvärden som är mindre än en varierbar konstant ochglidande variansen som signalegenskaper genererar ofta linjäramönster. Resultatet visade även att sannolikhetsfördelningen Loglogistichade lägst relativ medelkvadratavvikelse (rRMSD) på 2.7%. / Kandidatexjobb i elektroteknik 2021, KTH, Stockholm

Nanopore DNA sequencing is a novel method forsequencing DNA where an electronic signal is modulated bynucleotides passing through nanosized pores embedded in a mem-brane. While current state-of-the-art solutions employ recurrentneural networks to analyse the signal, temporal convolutionalnetworks have recently been shown to match or outperformrecurrent networks in signal processing tasks. In this project, weinvestigate the performance of temporal convolutional networkson a simplified version of the sequencing task, where thegoal is to predict the nucleotides passing through the pore ateach instance in time, without reconstructing the correspondingDNA sequence. The impact of several network parameters onpredictive performance is analysed to determine an optimalarchitecture. While the implemented networks are shown tobe proficient at predicting nucleotides within the pore, thecurrent implementation is unlikely to outperform state-of-the-art solutions without further improvement. / En nyligen utvecklad metod för att sekvensera DNA innefattar att en elektrisk signal moduleras genom att nukleotider passerar genom porer i nanostorlek. I kommersiella lösningar analyseras denna signal med hjälp av maskininlärning via Recurrent Neural Networks, men en variant av neruala nätverk som kallas Temporal Convolution Networks har nyligen har visat sig ha bättre prestanda jämfört med Recurrent Networks för olika typer av signalbehandlingsproblem. Målet med detta projekt är att undersöka användbarheten av Temporal Convolutional Networks för en förenklad version av DNA-sekvensering, där uppdraget endast är att identifera de nukleotider som passerar genom poren vid varje given tidpunkt, istället för att rekonstruera en komplett DNA-sekvens. För att kunna bestämma en optimal arkitektur på nätverket så undersöks effekten av flera olika parametrar. De implementerade nätverken visas ha god förmåga att klassificera nukleotider, men är troligtvis i behov av ytterligare förbättringar för att kunna konkurrera med nuvarande kommersiella lösningar. / Kandidatexjobb i elektroteknik 2020, KTH, Stockholm