Protein biomarkers circulating in blood hold the promise of improved diagnosis, prognosis and follow-up of treatment of disease via minimally invasive procedures. For the discovery and validation of such biomarkers, methods are needed that can facilitate parallel, highly specific and in-depth analysis of the blood proteome. The work presented in this thesis intends to develop and apply such assays, building on the concept of the proximity ligation assay (PLA). In paper I, I present an easy and non-expensive alternative for the conjugation of oligonucleotides to antibodies via biotin-streptavidin-biotin interaction. This approach can be used when large sets of antibodies and/or oligos need to be validated for their performance as probes in PLA reactions. In paper II, a solid-phase variant of PLA (SP-PLA) for the detection and quantification of proteins in blood is presented. SP-PLA exhibited an improved limit of detection compared to commercial ELISA assays by two orders of magnitude. In addition SP-PLA exhibited a broader dynamic range by at least one order of magnitude and required only 5 μl of sample, rendering the method very well suited for analyses of precious bio-banked material. Last but not least, SP-PLA was used to validate the diagnostic potential of GDF-15 as a biomarker for cardiovascular disease in a set of cardiovascular disease patients and healthy controls. Paper III discusses the development of a multiplex SP-PLA (MultiPLAy) for the simultaneous detection of 36 proteins in just 5 μl of sample. MultiPLAy exhibited an improved LOD when compared to state-of-the-art bead-based sandwich assays. Most importantly, we observed only a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that much higher levels of multiplexing will be possible. The assay was used to identify putative biomarkers in sample cohorts of colorectal cancer (CRC) and cardiovascular disease (CVD). Subsequent multivariate analysis revealed previously known diagnostic biomarkers. Furthermore, we successfully applied next-generation sequencing as a readout for the protein assays, allowing for the first time digital recording of protein profiles in blood. In paper IV, we investigated the suitability of prostasomes as blood biomarkers in patients with prostate cancer using a newly developed PLA assay (4PLA) that utilizes five binders for the detection of complex target molecules. The assay successfully detected significantly elevated levels of prostasomes in blood samples from prostate cancer patients prior to radical prostatectomy, compared to controls and men with benign biopsy results.

abstract: According to sources of the Centers for Disease Control and Prevention, approximately 1.7 million traumatic brain injury (TBI) cases occur annually in the United States. TBI results in 50 thousand deaths, nearly 300 thousand hospitalizations and 2.2 million emergency room visits causing a $76 billion economic burden in direct and indirect costs. Furthermore, it is estimated that over 5 million TBI survivors in the US are struggling with long-term disabilities. And yet, a point-of-care TBI diagnostic has not replaced the non-quantitative cognitive and physiological methods used today. Presently, pupil dilation and the Glasgow Coma Scale (GCS) are clinically used to diagnose TBI. However, GSC presents difficulties in detecting subtle patient changes, oftentimes leaving mild TBI undiagnosed. Given the long-term deficits associated with TBIs, a quantitative method that enables capturing of subtle and changing TBI pathologies is of great interest to the field. The goal of this research is to work towards a test strip and meter point-of-care technology (similar to the glucose meter) that will quantify several TBI biomarkers in a drop of whole blood simultaneously. It is generally understood that measuring only one blood biomarker may not accurately diagnose TBI, thus this work lays the foundation to develop a multi-analyte approach to detect four promising TBI biomarkers: glial fibrillary acidic protein (GFAP), neuron specific enolase (NSE), S-100β protein, and tumor necrosis factor-α (TNF-α). To achieve this, each biomarker was individually assessed and modeled using sensitive and label-free electrochemical impedance techniques first in purified, then in blood solutions using standard electrochemical electrodes. Next, the biomarkers were individually characterized using novel mesoporous carbon electrode materials to facilitate detection in blood solutions and compared to the commercial standard Nafion coating. Finally, the feasibility of measuring these biomarkers in the same sample simultaneously was explored in purified and blood solutions. This work shows that a handheld TBI blood diagnostic is feasible if the electronics can be miniaturized and large quantity production of these sensors can be achieved. / Dissertation/Thesis / Doctoral Dissertation Biomedical Engineering 2017

abstract: Peptide microarrays are to proteomics as sequencing is to genomics. As microarrays become more content-rich, higher resolution proteomic studies will parallel deep sequencing of nucleic acids. Antigen-antibody interactions can be studied at a much higher resolution using microarrays than was possible only a decade ago. My dissertation focuses on testing the feasibility of using either the Immunosignature platform, based on non-natural peptide sequences, or a pathogen peptide microarray, which uses bioinformatically-selected peptides from pathogens for creating sensitive diagnostics. Both diagnostic applications use relatively little serum from infected individuals, but each approaches diagnosis of disease differently. The first project compares pathogen epitope peptide (life-space) and non-natural (random-space) peptide microarrays while using them for the early detection of Coccidioidomycosis (Valley Fever). The second project uses NIAID category A, B and C priority pathogen epitope peptides in a multiplexed microarray platform to assess the feasibility of using epitope peptides to simultaneously diagnose multiple exposures using a single assay. Cross-reactivity is a consistent feature of several antigen-antibody based immunodiagnostics. This work utilizes microarray optimization and bioinformatic approaches to distill the underlying disease specific antibody signature pattern. Circumventing inherent cross-reactivity observed in antibody binding to peptides was crucial to achieve the goal of this work to accurately distinguishing multiple exposures simultaneously. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2014

Com a evolução tecnológica há nos dias de hoje um aumento de dispositivos eletrônicos presentes ao nosso redor. Com o passar dos anos diversas funcionalidades vêm sendo agregadas a estes inclusive com maior poder de processamento. Em particular, para sistemas embarcados houve um crescimento da quantidade de sensores para diversos propósitos. Seguindo esta tendência, na área de saúde também houve um aumento significativo de aparelhos e dispositivos de monitoramento, tais como glicosimetros, oxímetros, monitoramento de pressão e batimento cardíaco por exemplo, que através de sensores realizam transdução dos dados pertinentes ao parâmetro envolvido. Este trabalho apresenta a pesquisa e o desenvolvimento de um sistema embarcado com a propriedade de multiplexação de sensores, ou seja, foi desenvolvido um dispositivo microcontrolado o qual visa multiplicar a capacidade de monitoramento de analitos, conseguindo analisar múltiplos sensores para um mesmo experimento. Ao decorrer deste desenvolvimento foram utilizados quatro sensores dispostos simetricamente em um béquer, os dados são coletados e tratados de forma sequencial e individual. Inicialmente utilizamos um sistema embarcado com um microcontrolador (PIC 18F2550) que é responsável por digitalizar a informação e pela conexão via terminal USB. Posteriormente um microprocessador (Raspberry Pi Zero, placa embarcada) fez-se necessário devido ao melhor processamento de dados. Os sensores aqui estudados tratam-se de sensores químicos, que são introduzidos a uma célula eletroquímica, onde se encontram um eletrodo de referência (Prata em uma solução de Cloreto de Prata) e os outros quatro filmes finos que irão compor o sistema multiplexado. Para este estudo em específico o material escolhido para fabricação dos filmes finos foi um polímero condutivo, mais especificamente polianilina (PANI). Esta foi depositada sobre um substrato de oxido de estanho dopado com flúor (FTO) através da eletrodeposição. Para sensores não específicos (não imobilizados para um analito alvo) os dois sistemas embarcados apresentaram respostas satisfatórias. Prosseguindo com o estudo e usando filmes finos para analitos biológicos (ureia e glicose) o microcontrolador não conseguiu separar os sinais de cada filme fino. Apenas o sistema com a Raspberry Pi obteve sucesso, devido a maior resolução no conversor analógico para digital e sua maior capacidade de processamento para determinar com uma maior precisão os valores obtidos. O sistema pode ser facilmente expandido para um maior número de sensores. / With the evolution of technology there is nowadays an increase in the number of electronic devices present around us. Over the years various functionalities have been added to these devices including the increased processing power. In particular, for embedded systems there has been an increase in the number of sensors for various purposes. Following this trend, in the area of health, there has also been a significant increase in systems and monitoring devices, such as glycosimeters, oximeters, pressure monitoring and heart rate, for example, which, through sensors, transduce data pertinent to the parameter involved. This work presents the research and development of an embedded system with the property of multiplexing sensors, that is, a microcontrolled device was developed which aims to multiply the capacity of analytes monitoring, being able to analyze multiple sensors for the same experiment. During this development four sensors were used symmetrically arranged in a beaker, the data were collected and treated sequentially and individually. Initially we used an embedded system with a microcontroller (PIC 18F2550) that is responsible for scanning the information and for the connection via USB terminal. Subsequently a microprocessor (Raspberry Pi Zero, embedded board) was made necessary due to the better processing of data. The sensors studied here are chemical sensors, which are introduced to an electrochemical cell, where a reference electrode is found (Silver in a Silver Chloride solution) and the other four thin films that will make up the multiplexed system. For this specific study the material chosen for the manufacture of thin films was a conductive polymer, more specifically polyaniline (PANI). This was deposited on a substrate of fluorine-doped tin oxide (FTO) by electrodeposition. For non-specific sensors (not immobilized for a target analyte) the two embedded systems presented satisfactory responses. Proceeding with the study and using thin films for biological analytes (urea and glucose) the microcontroller failed to separate the signals from each thin film. Only the system with Raspberry Pi has been successful, due to the higher resolution in the analog to digital converter and its greater processing capacity to determine with greater precision the obtained values. The system can be easily expanded to a larger number of sensors.

This thesis work consists of two different research projects. In the first project the optimization of the Frequency Shifted Reference-Ultrawideband (FSR-UWB) is discussed. After identifying the improvement areas in the FSR-UWB scheme, we performed analysis and proposed optimized values of the restricted integration and the front-end filter. It is observed that, by integrating the received signal over the entire symbol period, excess noise is allowed into the system and thus potentially degrades the performance. We showed that by restricting the integration period we get the expected gains in an Additive White Gaussian Noise (AWGN) channel but the gains are limited for a multipath fading channel. For these limited gains, the new integration block unnecessarily complicates the receiver structure. For front-end filter optimization the system performance is analyzed using a generic filter, h(t) and it is shown that a matched filter is the optimal filter for low values of Es/N0 whereas a unity gain band pass filter is optimal for high Es/N0 values, where Es is the symbol energy and N0/2 is the power spectral density of additive white Gaussian noise. In the second project we explored a general class of waveforms that can be used as separating waveforms to provide multiple-access for FSR-UWB systems. It is shown in this section that for single user scenario binary codes selected from {−1, 1} are optimal codes that can be used to separate data and the reference signals. For multiple-user access, a class of polynomials are discussed that can be used as separating waveform as they completely eliminate MAI. It is shown in the latter part that the optimal codes for multiple-user access are the binary codes selected from {−1, 1}. These codes are selected as the row vectors of the Hadamard Matrix. Simulation supported the application of this analysis to UWB systems, with either a small number of frames or operating over channels with small delay spread.

Microarray technology provides powerful tools in the field of biomedicalresearch because it can measure molecular interactions in a highly parallelfashion. It has uses in protein, DNA or cell research, in both discovery anddiagnostic applications. Microfluidics, on the other hand, provides thenecessary tools to rapidly transport and mix small volumes of sample to amicro-sensor area. Bridging these two technologies has the potential todevelop a miniaturized, automated and ease-of-use toolbox for biologicalanalysis. However, the integration of microfluidics with microarrays is notstraightforward, as if a robust and leak-tight seal between the microarray andthe microfluidic channels. Current sealing methods are either impractical,such as mechanical clamping, or not compatible with proteins, such as heat orplasma bonding or gluing. Moreover the former methods create a permanentseal that, once applied prevents the microfluidic structure to be removed later.This work focuses on developing a microfluidic add-ons ("Biosticker") that canbe robustly sealed with protein microarrays with maintained biologicalactivity, but at the same time easily removed to allow for multiple stickersapplied in a sequence or scanning of the microarray in a standard reader. Thefeatures of the novel Biostickers are made possible by the use ofOff-stoichiometry thiol-ene-epoxy (OSTEmer) polymers. In this thesis, wedesign and fabricate Biostickers for rapid integration with pre-spottedmicroarrays and experimentally verify how these micropatterned Biostickerscan be used to significantly facilitate multiplexed assays, by avoiding the useof beads. / Microarray-tekniken är ett kraftfullt verktyg inom biomedicinsk forskningeftersom den kan mäta miljontals molekylära interaktioner parallellt. Den haranvändningsområden i protein-, DNA- eller cellforskning, både i forskningoch diagnostik. Mikrofluidik, å andra sidan, ger de nödvändiga verktygen föratt snabbt transportera och blanda små provvolymer till en sensoryta. Genomatt kombinera dessa två teknologier finns potential att utveckla enminiatyriserad, automatiserad och lättanvänd verktygslåda för biologiskanalys. Emellertid är integrationen av mikrofluidik med mikroarrayer inteenkel, då ytorna är känsliga, kanalerna mycket små men tätningen måste varaperfekt. De vanligast förekommande förseglingsmetoder är antingenopraktiska, som mekaniskt tryck eller så är de inte kompatibla med proteiner,som t.ex. värme- eller plasmabondning. Dessutom syftar de flestaförseglingsmetoder mot att skapa en permanent försegling som vidanvändning förhindrar mikrofluidikstrukturer från att tas bort i ett senareskede, tex. vid avläsning i en skanner. Detta arbete fokuserar på att utvecklamikrostrukturerade plastartiklar ("Biosticker") innehållande kanaler ochkaviteter. Dessa Biosticker kan på ett robust och läckfritt sätt kansammanfogas med proteinmikroarrayer utan att påverka den biologiskaaktivitetet men samtidigt kunna avlägsnas för att tillåta flera Biostickersapplicerade i en sekvens eller scanning i en mikroarrayläsare. Dessafunktioner möjliggörs genom av så kallade icke-stökiometrska-tiol-ene-epoxipolymerer (OSTEmer) används som material. I den här avhandlingenutvecklas och tillverkas Biostickers för snabb integrering medproteinmikroarrayer. Det verifieras även experimentellt hur dessa Biostickerskan användas för att underlätta genomförandet av sk. multiplexadeprotinanalyser.

A multiplexed method for performing MS/MS on multiple ions simultaneously in a miniature rectilinear ion trap (RIT) mass spectrometer has been developed. This method uses an ion encoding procedure that relies on the mass bias that exists when ions are externally injected into an RIT operated with only a single phase RF applied to one pair of electrodes. The ion injection profile under such conditions ions is Gaussian-like over a wide range of RF amplitudes, or low mass cutoff (LMCO) values, during ion accumulation. We show that this distribution is related to ion m/z and is likely caused by ions having an optimal range of pseudo-potential well depths for efficient trapping. Based on this observation, precursor ion intensity changes between two different injection LMCO values can be predicted, and these ion intensity changes are found to be carried through to their corresponding product ions, enabling multiplexed MS/MS spectra to be deconvoluted. The gas-phase reactions of a series of coordinatively unsaturated [Ni(L)n]y+ complexes, where L is a nitrogen-containing ligand, with chemical warfare agent (CWA) simulants in a miniature rectilinear ion trap mass spectrometer were investigated as part of a new approach to detect CWA. Results show that the metal complex ions can react with low concentrations of several CWA simulants, including dipropyl sulfide (simulant for mustard gas), acetonitrile (simulant for the nerve agent tabun), and diethyl phosphite (simulant for nerve agents sarin, soman, tabun, and VX), thereby providing a sensitive means of detecting these compounds. The [Ni(L)n]2+ complexes are found to be particularly reactive with the simulants of mustard gas and tabun, allowing their detection at low parts-per-billion (ppb) levels. These detection limits are well below the median lethal doses for these CWAs, which indicates the applicability of this new approach, and are about two orders of magnitude lower than electron ionization detection limits on the same mass spectrometer. The use of coordinatively unsaturated metal complexes as reagent ions offers the possibility of further tuning the ion-molecule chemistry so that desired compounds can be detected selectively or at even lower concentrations. Mass spectrometry has become a tool for studying noncovalently bound complexes. Specifically, electrospray ionization mass spectrometry (ESI-MS) has found increasing use for the determination of affinity (Ka) or dissociation (Kd) constants. Direct measurement of the equilibrium components by ESI-MS is the most straightforward approach for determining binding equilibrium constants, but this approach is prone to error and has some inherent limitations. Transferring complexes from solution to the gas phase may perturb the equilibrium concentrations and/or different ionization efficiencies may cause the resulting ion signals not to reflect actual solution concentrations. Furthermore, ESI only works under a limited range of solvent conditions (i.e. low ionic strengths), which limits the broad applicability of this approach. We propose an approach based on covalent labeling in the context of metal-catalyzed oxidation (MCO) reactions that, when combined with MS, overcomes such limitations when determining metal-ligand binding constants. The MCO-MS approach will provide concurrent information regarding metal binding site and metal-protein binding affinity. Optimization of the MCO reaction through isotopic mass tags will permit enhanced identification of modified residues. Application of this method to study the affinity and binding interactions of other divalent metals with β2m are likely to provide insight into the specificity of copper for causing β2m amyloid formation.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24304号 / 生博第487号 / 新制||生||65(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 渡邊 直樹, 教授 見学 美根子, 教授 今吉 格 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Recent developments in holographic recording of volume Bragg gratings (VBGs) in photo-thermo-refractive (PTR) glass have demonstrated their utility as components in high power laser systems for spectral narrowing, transverse mode control, beam combining, and pulse stretching/compression. VBG structures are capable of diffracting incident light into a single diffraction order with high efficiency given the Bragg condition is met. The Bragg condition depends on both the wavelength and angle of the incident light making VBGs useful for filtering and manipulating both the wavelength and angular spectrum of a source. This dissertation expands upon previous research in PTR VBGs by investigating multiplexed VBGs and their applications in laser systems. Multiplexing involves the integration of several VBGs into the same volume of PTR glass. This process enables the fabrication of splitting and combining elements which have been used for high power beam combining with significantly reduced complexity as compared to other combining schemes. Several configurations of multiplexed beam combiners were demonstrated for both spectral and coherent combining systems with high power results yielding a combined power of 420 W with 96% efficiency. Multiplexing was also used to produce unique phase structures within VBGs. This effect was exploited to create extremely narrowband spectral filters called moire Bragg gratings. The technical challenges of producing moire gratings in bulk glass have revealed new insights into the use of PTR glass as a recording medium and produced devices capable of narrowband filtering of only 15 pm in the near infrared. Experiments were performed using such devices as intra-cavity laser elements for longitudinal mode selection. Investigations have also been made into increasing the level of multiplexing possible within PTR glass. These explorations included scaling the number of beam combining channels, fabrication of integrated multi-notch filters, and generated several other potentially interesting devices for future research. The summation of this work indicates a promising future for multiplexed VBGs in PTR glass.

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