I den här rapporten undersöks hur en vävd textil kan fungera som ett hjälpmedel i analys av vätskor, såsom förorenat vatten eller blod från människor och djur. Det finns i dagsläget ett stort forskningsområde, kallat mikrofluidik, som behandlar förflyttning av vätska i kanaler på mikrometerstora ytor, där det här projektet till viss del kan hjälpa forskningen på området att komma framåt ytterligare ett steg.   Undersökningen har genomförts med hjälp av tester i laboratorium där en väv i polyeten, med kanaler i Coolmax® (polyester) för att transportera vätskan har använts. En mängd olika testomgångar med olika fokus, har genomförts: test i bitar med raka kanaler, test där wickingen avbrutits med hjälp av sax, test där wickingen har pausats på olika sätt för att sedan startas på nytt samt ett mindre antal tester där försök till styrning av vätskan. Syftet har varit att undersöka huruvida alla sex utvalda vätskor (metylenblått, mjölk, nötblod, olivolja, Poly(3,4-ethylenedioxythiophene) och syntetisk urin) har en förmåga att wicka och om det finns skillnader mellan hur långt vätskorna förflyttar sig.   Wickingtesterna har genomförts i både horisontellt och vertikalt läge, detta för att se om och i så fall hur mycket det skiljer, gällande hur långt en vätska flödar i kanalen. Det som framkommit i projektet är att alla vätskorna hade en förmåga att wicka. Metylenblått förflyttade sig längst i horisontellt läge medan urin förflyttades längst i vertikalt läge. Nötblodet förflyttade sig kortast sträcka i både horisontellt och vertikalt läge. Det som däremot har varit svårt att fastställa är vad skillnaderna egentligen beror på. Baserat på matematiska formler för wicking har det konstaterats att vätskornas kontaktvinkel bör ha betydelse, men detta har dessvärre inte kunnat undersökas i det här projektet. / This thesis examines how a woven textile can act as an aid in the analysis of fluids, such as contaminated water or blood from humans and animals. There is currently a large research area, called microfluidics, which deals with the movement of fluid in channels on micrometer-sized surfaces, where this project can, to some extent, fill some gaps and open for further questions in other parts.   The study has been carried out by using laboratory tests where a polyethylene weave, with channels in Coolmax® (polyester) for transporting the liquid has been used. Several different test rounds with a little different focus have been carried out: test in straight pieces, tests where the wicking has been interrupted by scissors, tests where the wicking has been paused and then restarted, and a smaller number of tests where attempts to control and navigate the fluid has been tested. The purpose has been to investigate whether all six selected fluids (methylene blue, milk, blood from bovine, olive oil, poly (3,4-ethylenedioxythiophene) and synthetic urine) have the ability of wicking and if there are differences between the fluids, and how far they reach.   The wicking tests have been carried out in both horizontal and vertical positions, to see if and if so, how much it differs, how far a fluid reaches. What emerged from the project is that all the liquids had the ability to wick. Methylene blue was the fluid that moved furthest in the horizontal position while urine moved furthest in the vertical position. The blood from bovine moved the shortest distance in both horizontal and vertical positions. What has, however, been difficult to determine is what the differences really depend on. Based on mathematical formulas for wicking, it has been found that the contact angle of the liquids should be important, but this have not been investigated in this project.

The commonly used drug diclofenac is an important environmental anthropogenic pollutant. Currently, detection of diclofenac is mainly based on chemical and physical methods. Here we describe a yeast biosensor that drives the diclofenac-dependent expression of a recombinant fluorescent protein from the authentic promoter of the PDR5 gene. This key component of the pleiotropic drug response encodes a multidrug transporter that is involved in cellular detoxification. We analyse the effects on diclofenac sensitivity of artificial PDR5 promoter derivatives in wild-type and various yeast mutant strains. This approach enabled us to generate sensor strains with elevated drug sensitivity.

The commonly used drug diclofenac is an important environmental anthropogenic pollutant. Currently, detection of diclofenac is mainly based on chemical and physical methods. Here we describe a yeast biosensor that drives the diclofenac-dependent expression of a recombinant fluorescent protein from the authentic promoter of the PDR5 gene. This key component of the pleiotropic drug response encodes a multidrug transporter that is involved in cellular detoxification. We analyse the effects on diclofenac sensitivity of artificial PDR5 promoter derivatives in wild-type and various yeast mutant strains. This approach enabled us to generate sensor strains with elevated drug sensitivity.

The SCP-RICM assay employs the measurable surface energy (or adhesive work W_adh) of a micrometer-sized polymeric sphere (soft colloidal probe, SCP) interacting with a glass chip using reflection interfer-ence contrast microscopy (RICM). Depending on those two interacting surfaces' nature and functional-ization, the SCP will deform, creating a contact area with the hard glass chip. This contact area is clearly distinguishable from the sphere’s interference ring pattern and can be measured. The adhesive surface energy W_adh can be calculated from the size of the contact area. An immobilization can be overcome by choosing a two-component analyte-dependent interaction, here presented for the copper (Cu) detection. The detection of Cu was chosen as a proof-of-concept system. However, detecting metal ions is an essential endeavor because, in excessive amounts, they present a severe threat to health and the environment. The copper-dependent interaction of the yeast chaperones yCox17 (also Cox17) and ySco1 (also Sco1) were chosen as the two-component analyte-dependent interaction. The chaperones partic-ipate in vivo in the formation of the electron transport chain of S. cerevisiae and interact in the mito-chondrial inner membrane to transfer one Cu(I) ion from Cox17 to Sco1. It was necessary to immobilize one protein to the SCPs and one to the chip surface, to transfer the copper chaperones' interaction into the SCP-RICM assay core detection components. The unique self-assembling characteristics of the class I hydrophobin Ccg-2 from N. crassa were used to immobilize one interaction partner to the chip surface. Class I hydrophobins are known for the formation of re-sistant and uniform layers at hydrophilic/hydrophobic interfaces. Initial SCP-RICM assay measurements with Sco1Δ95_a-SCPs and the Cox17_c-chips indicate that copper detection using the proposed mechanism is possible (Figure 39-3). Measurements can be differentiated between 0 and 0.1 mM Cu(I) concentration in solution. Further screening of concentrations be-low 0.1 mM is still necessary. The presented proof-of-principle system for the indirect detection of copper shows copper-dependent behavior. These positive results give rise to many more options to use the SCP-RICM assay as an indirect detection system. The application range of the SCP-RICM assay could be enlarged for different analytes such as other heavy metals, bacteriophages, biomarkers, et cetera, and is relevant for fields from medicine to environmental monitoring.:TABLE OF CONTENT Table of Content I List of Figures VII List of Tables IX List of Abbreviations XI 1 Introduction 1 1.1 Biosensors 1 1.2 Analytical Detection Methods: Copper 2 1.3 SCP-RICM Assay 3 1.3.1 Sensor Chip Surface 4 1.3.2 Soft Colloidal Probes 5 1.3.3 Reflection Interference Contrast Microscopy 6 1.4 Hydrophobins 9 1.4.1 Structure and Functions of Hydrophobins 9 1.4.2 Ex vivo Applications of Hydrophobins 11 1.4.3 Class I Hydrophobin: Ccg-2 12 1.5 Mitochondrial Respiratory Chain 14 1.5.1 Copper Transport in Yeast 14 1.5.2 S. cerevisiae Sco1 protein 18 1.5.3 S. cerevisiae Cox17 protein 21 1.6 SCP-RICM Assay for Copper Detection 23 1.7 Aim of the Study 24 2 Materials and Methods 25 2.1 Laboratory Equipment 25 2.1.1 Devices 25 2.1.2 Chemicals 26 2.1.3 Consumables 28 2.1.4 Antibodies 29 2.1.5 Enzymes 30 2.1.6 Molecular Weight Standards 30 2.1.7 DNA Oligonucleotides 31 2.1.8 Plasmids and Vectors 32 2.2 Microorganisms 33 2.2.1 Strains 33 2.2.2 Cultivation of Microorganisms 34 2.2.3 Preparation of Electrocompetent E. coli Cells 36 2.2.4 Preparation of E. coli Glycerol Stocks 36 2.3 Protein Design 37 2.4 Molecular Cloning Methods 38 2.4.1 Vector Template Preparation 38 2.4.2 Agarose Gel Electrophoresis 40 2.4.3 DNA Extraction from Agarose Gels 41 2.4.4 Polymerase Chain Reaction 41 2.4.5 DNA Restriction Digest 42 2.4.6 DNA Dialysis 43 2.4.7 Ligation of DNA Fragments 43 2.4.8 Isolation of DNA from E. coli 44 2.4.9 DNA Sequencing 45 2.4.10 Transformation of E. coli via Electroporation 45 2.5 Protein Detection and Quantification 46 2.5.1 SDS PAGE 46 2.5.2 Coomassie Staining 50 2.5.3 Western Blot Analysis 51 2.5.4 Immunological Detection 51 2.5.5 Protein Quantification: Lowry Assay 52 2.5.6 Protein Quantification: Bradford Assay 53 2.5.7 Protein Quantification: NanoDrop Measurement 53 2.6 Protein Purification and Storage 54 2.6.1 Expression Analysis of Recombinant Proteins 54 2.6.2 Solubility Analysis 54 2.6.3 Protein Purification by Ni2+ Affinity Chromatography 55 2.6.4 Quantification of Purified Proteins 64 2.6.5 Dialysis of Purified Proteins 65 2.7 Glass Surface Functionalization 65 2.7.1 Glass Surface Preparation 66 2.7.2 Hydrophobin and Fusion Protein-Based Coating 66 2.7.3 Contact Angle Measurement 67 2.7.4 DRoPS Test 67 2.7.5 Atomic Force Microscopy 67 2.8 SCP Functionalization 68 2.8.1 Functionalization of SCPs with Proteins 68 2.8.2 Validation of SCP Functionalization with FITC Staining 69 2.9 SCP-RICM Assay and Its Analysis 69 3 Results 73 3.1 Generation of Recombinant Fusion Proteins 73 3.1.1 Sco1 and Sco1∆95 73 3.1.2 Cox17 84 3.1.3 Ccg-2 88 3.1.4 Overview: Optimization of Expression and Purification of Recombinant Proteins 90 3.2 His-Tag Cleavage 92 3.3 Chip Surface Functionalization 94 3.3.1 Optimization of the Glass Chip Preparation 94 3.3.2 Macroscopic Properties of the Functionalized Chip Surface 95 3.3.3 AFM Measurements 102 3.3.4 Theoretical Package of Hydrophobin Ccg-2 on the Chip Surface 103 3.4 SCP Functionalization 104 3.4.1 SCP Functionalization and FITC Staining 104 3.4.2 Theoretical Package of Proteins on SCPs 106 3.5 SCP-RICM Assay 107 4 Discussion and Further Prospectives 113 4.1 Discussion: SCP-RICM Assay and Protein-Protein Interaction 113 4.2 Outlook and Further Prospects 119 4.2.1 Heterologous Protein Expression and Purification: Methods, Cleavage and Refolding 119 4.2.2 Further Analysis of Chip Surface Functionalization 124 4.2.3 Alternative Chip Surface Functionalization Methods 126 4.2.4 SCP-RICM Assay: Data Acquisition and Evaluation 128 4.2.5 SCP-RICM Assay: Copper Detection 130 4.2.6 Exploiting the SCP-RICM Assay using Protein-Protein Interactions 131 4.2.7 Exploiting the SCP-RICM Assay with Alternative Interactions 133 5 Summary 137 6 Bibliography 141 7 Appendix 165 7.1 Sequences of Protein Constructs 165 7.1.1 Sequences of the Protein Construct Cox17_a 165 7.1.2 Sequences of the Hydrophobin-Cox17 Fusion Protein Cox17_b 165 7.1.3 Sequences of the Hydrophobin-Cox17 Fusion Protein Construct Cox17_c 166 7.1.4 Sequences of the Protein Construct Sco1_a and Sco1Δ95_a 167 7.1.5 Sequences of the Hydrophobin-Sco1 Fusion Protein Constructs Sco1_b and Sco1Δ95_b 169 7.1.6 Sequences of the Hydrophobin-Sco1 Fusion Protein Constructs Sco1_c and Sco1Δ95_c 171 7.1.7 Sequences of the Hydrophobin Ccg-2 173 7.2 pET-28b(+): Plasmid Map 173 7.3 Nickel Removal During Dialysis 175 7.4 DGR Assay 176 7.5 SCP diameter 179 Acknowledgements 181 Declaration of Authorship 183

The aim of this work was to functionalize silicon dioxide surfaces with biochemical molecules in such a way that biorecognition of target molecules in solution will be possible. By introducing a tool set of different molecules and characterization methods, a more universal approach towards various biosensor setups is presented. This includes on the one hand preparation of the biosensor surfaces to allow further molecule attachment via their reactive functional groups. Secondly, the selection of chemical molecules providing suitable counterparts for abundant functional groups of potential receptors is discussed. Two detection schemes are introduced – based on an antibody to detect the antibiotic amoxicillin and aptamers to detect thrombin. The antibody was implemented in an inverse competition assay to probe such small target molecules. Antibiotic residues are often present in wastewater. Aptamers, so-called artificial antibodies, were selected as they provide many advantages over antibodies. As a model system, two different thrombin binding aptamers were chosen which allowed to perform sandwich assays as well. The protein thrombin plays an important role in the blood coagulation cascade. To probe the individual modification steps, different techniques for analysis were applied. Surface micropatterning was introduced to improve recognition of modified areas and fluorescence-to-background ratios resulting in a thrombin detection limit down to 20 pM. One important goal was the integration in ion-sensitive field-effect transistor devices. Aptamers are small in size which might enable a higher sensitivity of these devices compared to the use of antibodies because of the Debye layer thickness. As a final step, first measurements towards silicon nanowire based field-effect transistor biosensors were carried out on devices with bottom-up and top-down fabricated nanowires using both proposed receptor-analyte combinations. The potential of these devices as portable sensors for real-time and label-free biosensing is demonstrated. / Ziel dieser Arbeit war es Siliziumdioxidoberflächen so mit biochemischen Molekülen zu funktional- isieren, dass die biologisch spezifische Erkennung von Zielmolekülen in Lösung möglich wird. Hier wird eine Auswahl an geeigneten Molekülen und Charakterisierungsmethoden für einen vielseitigen Ansatz gezeigt, der auf verschiedene Biosensorsysteme anwendbar ist. Das beinhaltet zum Einen die Präparation der Biosensoroberflächen, so dass die Moleküle über reaktive funktionelle Gruppen angebunden werden können. Als zweites ist die Auswahl der chemischen Moleküle wichtig, da diese die passenden Gegenstücke zu potentiellen funktionellen Gruppen der Rezeptoren darstellen. Zwei verschiedene Detektionsvarianten werden eingeführt – Antikörper gegen das Antibiotikum Amoxicillin und Aptamere gegen Thrombin. Der Antikörper wurde in einen inversen Wettbewerbsassay integriert um einen solch kleinen Ana- lyten detektieren zu können. Rückstände von Antibiotika sind häufig in Abwässern zu finden. Ap- tamere, sogenannte künstliche Antikörper, weisen gegenüber Antikörpern viele Vorteile auf. Als ein Modellsystem wurden zwei unterschiedliche Thrombin bindende Aptamere verwendet, was auch die Durchführung von Sandwich Assays ermöglichte. Das Protein Thrombin spielt eine wichtige Rolle bei der Blutgerinnung. Um die einzelnen Modifikationsschritte zu untersuchen, wurden verschiedene Charakterisierungsmethoden angewendet. Die Mikrostrukturierung der Funktionalisierung erleichterte die Erkennung der modifizierten Flächen und verbesserte das Fluoreszenz-zu-Hintergrund Verhältnis. Das führte zu einer Detektionsgrenze von 20 pM für Thrombin. Ein wichtiges Ziel dieser Arbeit war die Integration der Funktionalisierung in einen ionen-sensitiven Feldeffekttransistor. Die kleinen Aptamere könnten dabei aufgrund der geringen Debye-Schichtdicke bei diesen Sensoren eine höhere Sensitivität als mit Antikörpern ermöglichen. Zuletzt wurden erste Messungen hin zu Silizium Nanodraht basierten Feldeffekttransistor Biosen- soren mit beiden untersuchten Rezeptor-Analyt-Kombinationen durchgeführt. Sowohl die Chips mit bottom-up als auch mit top-down gewachsenen Nanodrähten zeigen dabei ihr Potential als handliche Sensoren zur markerfreien Detektion in Echtzeit.

Whole-cell sensors represent an emerging branch in biosensor development since they obviate the need for enzyme/antibody purification and provide the unique opportunity to assess global parameters such as genotoxicity and bioavailability. Yeast species such as Saccharomyces cerevisiae are ideal hosts for whole-cell sensor applications. However, current approaches almost exclusively rely on analyte-induced expression of fluorescent proteins or luciferases that imply issues with light scattering and/or require the supply of additional substrates. In this study, the yeast α-factor mating pheromone, a peptide pheromone involved in cell-cell communication in Saccharomyces cerevisiae, was utilized to create the whole-cell sensor read-out signal, in particular by employing engineered sensor cells that couple the response to a user-defined environmental signal to α-factor secretion. Two novel immunoassays - relying on hydrophobin-based surface engineering - were developed to quantify the α-factor. Hydrophobins are amphiphilic fungal proteins that self-assemble into robust monolayers at hydrophobic surfaces. Two recombinant hydrophobins, either lacking (EAS) or exposing the α-factor pheromone (EAS-α) upon self-assembly, were used to functionalize polystyrene supports. In a first approach (competitive immunoassay), pheromone-specific antibodies initially bound to the functionalized surface (due to the α-factor exposed by the hydrophobin layer) were competitively detached by soluble α-factor. In a second approach, the antibodies were first premixed with pheromone-containing samples and subsequently applied to functionalized surfaces, allowing for the attachment of antibodies that still carried available binding sites (inverse immunoassay). Both immunoassays enabled quantitative assessment of the yeast pheromone in a unique but partially overlapping dynamic range and allowed for facile tuning of the assay sensitivity by adjustment of the EAS-α content of the hydrophobin layer. With a limit of detection of 0.1 nM α-factor, the inverse immunoassay proved to be the most sensitive pheromone quantification assay currently available. Due to the high stability of hydrophobin monolayers, functionalized surfaces could be reused for multiple consecutive measurements. Favorably, both immunoassays proved to be largely robust against the changes in the sample matrix composition, allowing for pheromone quantification in complex sample matrices such as yeast culture supernatants. Hence, these immunoassays could also be applied to study the pheromone secretion of wild-type and engineered Saccharomyces cerevisiae strains. Additionally, a proof-of-concept whole-cell sensor for thiamine was developed by combining the hydrophobin-based immunoassays with engineered sensor cells of Schizosaccharomyces pombe modulating the secretion of the α-factor pheromone in response to thiamine. Since this read-out strategy encompasses intrinsic signal amplification and enables flexible choice of the transducer element, it could contribute to the development of miniaturized, portable whole-cell sensors for on-site application.

Antibodies are the gold standard molecular recognition elements and a cornerstone of molecular biology. They are used in immunoassays to precisely measure a specific analyte, but certain targets are especially challenging. Difficult targets include small molecules and molecules that do not induce an immune response. Aptamers are short oligonucleotides that can form 3-dimensional structures and bind targets with high specificity. Aptamers are smaller and more flexible than antibodies and could therefore solve this problem. In contrast to antibodies, aptamers are synthetically produced, so they can have affinity for molecules that do not induce an immune response. This also makes them cheaper, faster and more ethical to produce. They are also easily modified and have the ability to renature and can therefore be reused.  Our conclusions are that aptamers can outperform antibodies, especially for small molecule targets, and that the synthetic production of aptamers gives them a further advantage over antibodies. Our report compares several different types of detection methods that use aptamers and we conclude that fluorescence-based methods are the most easy to use with basic lab equipment, can be made similar to the ELISA kits in addition to giving highly sensitive detection. We describe a variety of fluorescence-based detection strategies but the optimal method will depend on the specific aptamer and target. The report also includes an ethical analysis where antibodies and aptamers are compared. This report is commissioned by Mercodia AB, a company that develops, manufactures and distributes immunoassays for biomarkers within the field of metabolic disorders. They commissioned this report in order to give an overview of how aptamers interact with their target, and also to compare aptamer-based detection strategies with sensitivity prioritized over selectivity.  This was done by literature research.

Die hier vorliegende Arbeit beschäftigt sich mit Eignung bakterieller Hüllproteine als Bindungsmatrix für die Kopplung funktionaler Moleküle mit dem Ziel, sensorische Schichten zu erzeugen. Bakterielle Hüllproteine sind biologische SAMs, anderen Oberfläche sich modifizierbare COOH-, NH2- und OH-Gruppen befinden. Die Ausbildung polymerer Strukturen erfolgt dabei in wässrigen Systemen und auf Oberflächen. Im Zuge der boomenden Entwicklung von Biosensoren werden insbesondere Biotemplate gesucht, die zwischen biologischer Komponente und Sensoroberfläche vermitteln. Bakterielle Hüllproteine stellen eine solche Zwischenschicht dar. Als Anwendungsbeispiel wurden die Proteine daher mit einem FRET-Paar und Thrombin und Kanamycin-Aptameren modifiziert. Hierbei wurden das FRET-Paar H488 und H555 an die bakteriellen Hüllproteine der beiden Haldenisolate A12 und B53 mittels EDC mit einer Modifizierungsrate von 0,54 molFarbstoff/molProtein kovalent gebunden. Bei der vorhandenen p4-Symmetrie bedeutet dies, dass ein FRET-Paar pro Einheitszelle vorhanden war. Der Nachweis eines Energietransfers zwischen den beiden am Protein gebundenen Fluoreszenzfarbstoffen H488 und H555 erfolgte mittels statischer und zeitaufgelöster Fluoreszenzmessung. Die Ergebnisse zeigten, dass ein Energietransfer nur möglich war, wenn die Proteine in polymerer Form vorlagen, unabhängig davon, ob sich die Proteine immobilisiert an einer Oberfläche oder in wässriger Lösung befanden. Mittels Variieren des Donor-Akzeptor-Verhältnisses konnte ein maximaler Energietransfer von 40 % generiert werden, wenn das Verhältnis der Fluoreszenzfarbstoffe von Donor und Akzeptor 4 betrug. Die Fluoreszenzintensität der Fluorophore wurde durch die Bindung an die Proteine nicht verringert oder gelöscht. Dies legt nahe, dass die Farbstoffe in den hydrophoben Poren immobilisiert wurden und die Poren die Fluoreszenzfarbstoffe schützen. Um weitere Aussagen über die Lage der gebundenen Fluoreszenzfarbstoffe zu erhalten, wurden die bakteriellen Hüllproteine der Stämme A12 und B53 enzymatisch verdaut und die Fragmente mittels SEC und SDS-PAGE untersucht. Dabei zeigten sich je nach Enzym und Protein unterschiedliche Bandenmuster bezüglich modifizierter und nativer Hüllproteine. Dies belegt, dass die Fluoreszenzfarbstoffe an NH2-und COOH-Gruppen der Proteine gebunden wurden und so teilweise den enzymatischen Verdau hinderten. Die SEC deutet an, dass die Fluoreszenzfarbstoffe an verschiedenen Stellen am Protein gebunden wurden. In einem zweiten Beispiel wurde das bakterielle Hüllprotein von A12 mit einem Aptamer modifiziert. Aptamere sind kurze einzelsträngige Oligonukleotide, die u.a. mittels ihrer ausgebildeten 3D-Struktur spezifisch Zielstrukturen reversibel binden können. Die hier verwendeten Aptamere binden spezifisch Thrombin und Kanamycin. Die Aptamere wurden mit Hilfe einer der beiden Vernetzer PMPI oder Sulfo-SMCC an die bakteriellen Hüllproteine kovalent gebunden. Nach dem Modifizieren der Proteine wurden diese auf entsprechenden Sensorchips immobilisiert und die Aktivität des gekoppelten Aptamers mittels Affinitätsmessungen, SPR-Spektroskopie und QCM-D-Messungen analysiert. Die Funktion des gebundenen Thrombinaptamers konnte mittels Affinitätsmessungen und QCM-D nachgewiesen werden und entspricht in beiden Fällen einer Bindung von 2 nmol Thrombin pro Quadratzentimeter. Die Funktionalität des Kanamycinaptamers sollte mittels SPR bestimmt werden, jedoch konnte keine Funktionalität des gekoppelten Kanamycinaptamers nachgewiesen werden. Alle Messungen bestätigten jedoch, dass die Bindungsmatrix aus bakteriellen Hüllproteinen keinerlei oder nur ein sehr geringes Hintergrundsignal liefert. Werden nun beide Komponenten, FRET-Paar und Aptamere, an das Protein gebunden, ist es möglich, eine sensorische Schicht zu erzeugen. Die Zielstruktur, welche detektiert werden soll, wird an das Aptamer gebunden und so in räumliche Nähe zur Sensorfläche gebracht. Stell die Zielstruktur einen Fluoreszenzlöscher dar, so wird der Energietransfer durch die räumliche Nähe des Fluoreszenzlöscher gestört. Die Detektion des Zielmoleküls erfolgt nun über die Änderung von Fluoreszenzintensitäten. Die hier vorgelegte Arbeit soll einen Grundstein legen für die Entwicklung eines solchen Sensors und insbesondere die Detektion eines Energietransfers optimieren und Schwachstellen in der Detektion nachweisen. Die systematische Untersuchung der Fluoreszenzfarbstoffe auf dem Protein ermöglichen es, in zukünftigen Arbeiten einen FRET zweifelsfrei zu detektieren. Die Modifizierung von bakteriellen Hüllproteinen von A12 mit Aptameren und die Detektion der Funktionalität der Aptamere mittels verschiedener Methoden zeigte auf, dass die bakteriellen Hüllproteine als universelle Bindungsmatrix für sensorische Moleküle dienen können, bei denen Affinitätsmessungen, SPR- oder QCM-D-Messungen genutzt werden. Besonders hervorzuheben ist, dass bakterielle Hüllproteine nahezu kein Hintergrundsignal liefern und aufgrund ihrer dünnen Monolage von etwa 6 - 9 nm die Sensitivität der Messungen nur gering beeinträchtigen.

[pt] O destino e os efeitos de produtos farmacêuticos recentemente se tornou uma questão de interesse ambiental embora pouco seja conhecido sobre sua entrada no ambiente. As drogas colinérgicas e colinesterásicas apresentam-se como um alvo importante da pesquisa ambiental relacionado a sua entrada no ambiente e suas interações físico-químicas e biológicas. Os ensaios com uso de à sondas protéicas são empregados para identificar a formação de complexos na presença de grupos específicos ou indicam a concentração de ligantes presente no ambiente onde se encontra a proteína. O emprego da enzima acetilcolinesterase (AChE) em biosensores para deterninação de inibidores é frequente porém poucos trabalhos exploram a fluorescência intrínseca das enzimas no desenvolvimento de metodologias para detectar espécies químicas ligantes. O objetivo deste trabalho foi propor novas sondas fluorescentes e formas de bioreconhecimento para a determinação de neurotoxinas em diferentes amostras. A proposta foi a de identificar sondas eficientes, de fácil obtenção e baixo custo para o desenvolvimento de métodos simples, sensíveis e seletivos. Para isso a detecção foi baseada na variação da fluorescência intrínseca das proteínas(por sua interação específica com os analitos) e na conjugação de fluoróforos extrínsecos específicos. Pode-se comprovar experimentalmente que a fração protéica obtida pode ser utilizada como sonda fluorescente na determinação dos analitos apresentados neste estudo. Nas determinações dos cinco analitos, os valores de LD e de LQ foram satisfatórios para a aplicação do método em amostras ambientais (solo) e clínicas (urina).As determinações de Gal apresentou desempenho satisfatório para atender diferentes tipos de amostras (LD = 1,3 x 10-9 mol L-1e LQ = 2,1 x 10-8 mol L-1).Os valores de LD e LQ para atropina (9,7 x 10-10 mol L-1 e 2,4 x 10-8 mol L-1, respectivamente) mostram-se adequados visto que outros sensores, como os eletroquimioluminescentes, apresentaram um valor de LQ de 1 × 10–7 mol L-1em amostras de urina. O método para os agrotóxicos metomil (LD = 9,5 x 10-10 mol L-1 e LQ = 2,2 x 10-8mol L-1); metamidofós (LD=1,5 x 10-9 mol L-1 e LQ=3,8 x 10-8 mol L-1) e metil paration (LD= 9,7 x 10-10 mol L-1 e LQ= 1,8 x 10-7 mol L-1) apresentarambom desempenho comparando-se à outros métodos descritos na literatura. A aplicação do método proposto em amostras de solo e urina (sem pré-tratamento) obtiveram recuperações a partir de 88 porcento (agrotóxicos em solo) e dos cinco analitos em urina. O método por fluorescência da sonda protéica não obteve diferença significativa do método analítico dos analitos por HPLC. A adaptação do método proposto à um sistema de biosensor com uso de uma sonda extrínseca (Tioflavina T) foi avaliado positivamente permitindo a imobilização da fração protéica em microplaca e o uso da Tioflavina T como sonda fluorescente da ligação AChE-inibidores. / [en] The fate and effects of pharmaceutical products has recently become a matter of environmental concern although little is known about its entry into the environment. Therefore, the cholinergic and cholinesterasicpresent a major target of environmental research related to his entry into the environment and their physico-chemical and biological interactions. Tests using the protein compounds as probes are employed where the optical properties of the complex protein-signal in the presence of analyte or specific groups indicating the concentration of ligand present in the environment. The use of acetylcholinesterase (AChE) activity in biosensors for inhibitors determination is common but few studies explore the intrinsic fluorescence of enzymes in the development of methods to detect chemical species ligands. The objective of this study was to propose new forms of fluorescent probes and bioaffinity for the determination of neurotoxins in different samples. The proposal was to identify probes efficient, easily accessible and low cost for the development of simple, sensitive and selective. For this the detection was based on the variation of the intrinsic fluorescence of proteins (by its specific interaction with the analytes) and the specific combination of extrinsic fluorophores. For the results presented in this plan can be affirmed that the protein fraction obtained can be used as a fluorescent probe in the determination of analytes of this study.Based on the five determinations of analyte, the values of LD and LQ are satisfactory for applying the method in environmental samples (ground) and clinical (urine). Measurements of Gal showed satisfactory performance to suit different types of samples (LD = 1.3 x 10-9mol L-1eLQ = 2.1 x 10-8mol L-1) values and LD LQ to atropine (9.7 x 10-10 mol L -1 and 2.4 x 10-8 mol L-1, respectively) appear to be suitable as other sensors, such as eletroquimioluminescentes showed a value of 1 LQ × 10-7 mol L-1 in urine samples. The method for pesticide methomyl (LD = 9.5 x 10-10 mol L-1 and LQ = 2.2 x 10-8mol L-1), methamidophos (LD = 1.5 x 10-9 mol L-1 and LQ = 3.8 x 10-8 mol L-1) and methyl parathion (LD = 9.7 x 10-10 mol L-1 and LQ = 1.8 x 10-7 mol L-1) comparing presents betterperformance to the other methods described in literature. The method proposed in soil samples and urine (no pretreatment) had recovered from 88% (ground pesticides) and five of analytes in urine. The t test comparison of means, applied to sets of determinations by fluorescence probe method and HPLC indicated the statistical equality in both recoveries. The adaptation of the proposed method a system of the biosensor with the use of an extrinsic probe (thioflavin T) has positively evaluated allowing immobilization of the protein in microplates and the use of thioflavin T as a fluorescent probe-connecting AChE inhibitors.

[ES] El interés en desarrollar biosensores de alta sensibilidad para identificar y cuantificar una amplia gama de moléculas ha aumentado notablemente durante las últimas décadas en numerosos campos de aplicación. Entre ellos probablemente el más demandado sea el diagnóstico médico, el cual ha sido impulsado por el descubrimiento de nuevos biomarcadores de enfermedades, tales como los miRNAs. No obstante, la mayoría de las técnicas existentes para realizar la detección requieren el uso de marcadores debido a la falta de sensibilidad para detectar analitos en bajas concentraciones. Las estructuras ópticas basadas en campo evanescente, donde la luz es usada para transformar interacciones bioquímicas en variaciones de las señales ópticas, constituyen una interesante alternativa para el desarrollo de este tipo de biosensores sin la necesidad de utilizar marcadores (label-free). Concretamente las estructuras fotónicas integradas en tecnología Silicon On Insulator exhiben alta sensibilidad, bajo límite de detección y alto nivel de multiplexación en aplicaciones de detección, especialmente cuando se utilizan materiales y procesos basados en silicio y compatibles con CMOS. En esta Tesis Doctoral se muestra el desarrollo de un biosensor fotónico integrado label-free para la detección de oligonucleótidos, y más concretamente biomarcadores de cáncer miRNAs. Este biosensor está basado en la combinación de estructuras de band gap fotónico y la inmovilización de sondas de tipo molecular beacon sobre su superficie. La combinación de sendos elementos de transducción y bioreconomiento ha proporcionado una elevada sensibilidad en la detección de oligonucleótidos manteniendo un footprint por debajo de 100 µm2. El uso de este biosensor fotónico ha permitido también estudiar experimentalmente una novedosa técnica de amplificación de detección. Esta técnica explota el cambio conformacional sufrido por la sonda molecular beacon tras la hibridación con su oligonucleótido complementario, permitiendo alejar una partícula/molécula de la superficie del sensor, lo cual podría ser utilizado para amplificar la respuesta de detección del sensor. Finalmente se propone una estrategia de regeneración en línea de los biosensores nanofotónicos desarrollados mediante una estrategia química basada en el uso de formamida. Esta estrategia no solo permite ahorrar tiempo sino que también reduce la variación entre las medidas obtenidas en experimentos diferentes, siendo especialmente útil cuando se testean niveles similares de analito. / [CA] L'interés en desenvolupar biosensors d'alta sensibilitat per a identificar i quantificar una àmplia gamma de molècules ha augmentat notablement durant les últimes dècades en nombrosos camps d'aplicació. Entre ells probablement el més demandat siga el diagnòstic mèdic, el qual ha sigut impulsat pel descobriment de nous biomarcadors de malalties, com ara els miRNAs. No obstant això, la majoria de les tècniques existents per a realitzar la detecció requereixen l'ús de marcadors a causa de la falta de sensibilitat per a detectar anàlits en baixes concentracions. Les estructures òptiques basades en camp evanescent, on la llum és usada per a transformar interaccions bioquímiques en variacions dels senyals òptics, constitueixen una interessant alternativa per al desenvolupament d'aquesta tipus de biosensors sense la necessitat d'utilitzar marcadors (label-free). Concretament les estructures fotòniques integrades en tecnologia Silicon On Insulator exhibeixen alta sensibilitat, baix límit de detecció i alt nivell de multiplexació en aplicacions de detecció, especialment quan s'utilitzen materials i processos basats en silici i compatibles amb CMOS. En aquesta Tesi Doctoral es mostra el desenvolupament d'un biosensor fotònic integrat label-free per a la detecció d'oligonucleòtids, i més concretament biomarcadors de càncer miRNAs. Aquest biosensor està basat en la combinació d'estructures de band gap fotònic i la immobilització de sondes de tipus molecular beacon sobre la seua superfície. La combinació d'ambdós elements de transducció i bioreconeixement ha proporcionat una elevada sensibilitat en la detecció d'oligonucleòtids mantenint un footprint per davall de 100 µm². L'ús d'aquest biosensor fotònic ha permés també estudiar experimentalment una nova tècnica d'amplificació de detecció. Aquesta tècnica explota el canvi conformacional patit per la sonda molecular beacon després de la hibridació amb el seu oligonucleòtid complementari, permetent allunyar una partícula/molècula de la superfície del sensor, la qual cosa podria ser utilitzada per amplificar la resposta de detecció del sensor. Finalment es proposa una estratègia de regeneració en línia dels biosensors nanofotònics desenvolupats mitjançant una estratègia química basada en l'ús de formamida. Aquesta estratègia no sols permet estalviar temps sinó que també redueix la variació entre les mesures obtingudes en experiments diferents, sent especialment útil quan es testen nivells similars d'anàlit. / [EN] The interest in developing highly sensitive biosensors to identify and quantify a wide range of molecules has remarkably been increasing during the last decades in numerous application fields. Among them, medical diagnosis is probably the most demanded, which has been driven by the discovery of new biomarkers of diseases, such as miRNAs. However, most of the existing techniques to perform the detection require the use of labels due to the lack of sensitivity to detect analytes at low concentrations. Evanescent-wave optical structures, where light is used to transduce biochemical interactions into variations of the optical signals, are an interesting alternative for the development of this type of biosensors allowing a label-free detection. Specifically, the planar integrated photonic structures based on Silicon On Insulator technology exhibit an extremely high sensitivity, a low detection limit and a high level of multiplexing in detection applications, especially when using materials and processes based on silicon and being CMOS compatible. This PhD Thesis is focused on the development of label-free integrated photonic biosensors for the detection of oligonucleotides, and more specifically miRNA cancer biomarkers. This biosensor is based on the combination of photonic band gap structures and the immobilization of molecular beacon probes on its surface. The combination of both transduction and biorecognition elements has provided a very high sensitivity towards the detection of target oligonucleotides while keeping a sensor footprint below 100 µm2. The use of this photonic biosensor also allowed the experimental study of a novel detection amplification technique. This technique exploits the conformational change suffered by the molecular beacon probe after hybridization with its complementary oligonucleotide, allowing the displacement of a particle/molecule away from the sensor surface, what might be used for amplifying the sensor's detection response. Finally, an online regeneration strategy for nanophotonic biosensors developed through a chemical strategy based on the use of formamide is proposed. This strategy not only saves time but also reduces the variation between measurements obtained in different experiments, being especially useful when testing similar levels of analyte. / Ruiz Tórtola, Á. (2021). Desarrollo de biosensores nanofotónicos de alta sensibilidad para la detección de biomarcadores microRNA en aplicaciones de diagnóstico médico [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172631