Livet tas för givet av de flesta. Det finns däremot många som ägnar stora delar av sitt liv för att försöka lösa dess mysterier. En del av lösningen ligger i att förstå hur alla molekyler är sammanlänkade i det gigantiska nätverk som definierar den levande organismen. Under det senaste seklet har en hel del forskning utförts för att kartlägga dessa nätverk. Resultatet av dessa mödor kan vi se i de läkemedel som vi har idag och som har utvecklats för att bota eller åtminstone lindra olika sjukdomar och tillstånd. Dessvärre finns det fortfarande många sjukdomar som är obotliga (t.ex. cancer) och mycket arbete krävs för att förstå dem till fullo och kunna designa framgångsrika behandlingar. Arbetet i denna avhandling beskriver en analytisk plattform som kan användas för att effektivisera kartläggningsprocessen; protein-mikroarrayer. Mikroarrayer är ytor som har mikrometerstora (tusendels millimeter) strukturer i ett regelbundet mönster med möjligheten att studera många interaktioner mellan biologiska molekyler samtidigt. Detta medför snabbare och fler analyser - till en lägre kostnad. Protein-mikroarrayer har funnits i ungefär ett decennium och har följt i fotspåren av de framgångsrika DNA-mikroarrayerna. Man bedömer att protein-mikroarrayerna har en minst lika stor potential som DNA mikroarrayerna då det egentligen är mer relevant att studera proteiner, som är de funktionsreglerande molekylerna i en organism. Vi har i detta arbete tillverkat modellytor för stabil inbindning av proteiner, som lämnar dem intakta, funktionella och korrekt orienterade i ett mikroarray format. Därmed har vi adresserat ett stort problem med protein mikroarrays, nämligen att proteiner är känsliga molekyler och har i många fall svårt att överleva tillverkningsprocessen av mikroarrayerna. Vi har även studerat en metod att tillverka mikroarrayer av proteiner bundna till strukturer, som modellerats att efterlikna cellytor. Detta är särkilt viktigt eftersom många (hälften) av dagens (och säkerligen framtidens) läkemedel är riktade mot att påverka denna typ av proteiner och att studera dessa i sin naturliga miljö är därför väldigt relevant. I ett annat projekt har vi använt protein mikroarrayer för att detektera fyra vanliga droger (heroin, amfetamin, ecstasy och kokain). Detektionen baseras på användandet av antikroppar som lossnar från platser på ytan när de kommer i kontakt med ett narkotikum. Detta koncept kan enkelt utvecklas till att detektera mer än bara fyra droger. Vi har även lyckats att parallellt mäta förekomsten av en annan typ av förening på mikroarray ytan, nämligen det explosiva ämnet trinitrotoluen (TNT). Detta visar på en mångsidig plattform för detektionen av i princip vilken typ av farlig eller olaglig substans som helst - och på en yta! Vi föreställer oss därför att möjliga tillämpningsområden finns inom brottsbekämpning, i kampen mot terrorism och mot narkotikamissbruk etc. Mikroarrayerna har i denna avhandling utforskats med optiska metoder som tillåter studie av omärkta proteiner, vilket resulterar i så naturliga molekyler som möjligt. / Life is a thing taken for granted by most. However, it is the life-long quest of many to unravel the mysteries of it. Understanding and characterizing the incomprehensively complex molecular interaction networks within a biological organism, which defines that organism, is a vital prerequisite to understand life itself. Already, there has been a lot of research conducted and a large knowledge has been obtained about these pathways over, especially, the last century. We have seen the fruits of these labors in e.g. the development of medicines which have been able to cure or at least arrest many diseases and conditions. However, many diseases are still incurable (e.g. cancer) and a lot more work is still needed for understanding them fully and designing successful treatments. This work describes a generic analytical tool platform for aiding in more efficient (bio)molecular interaction mapping analyses; protein microarray chips. Microarray chips are surfaces with micrometer sized features with the possibility of studying the interactions of many (thousands to tens of thousands) (bio)molecules in parallel. This allows for a higher throughput of analyses to be performed at a reduced time and cost. Protein microarrays have been around for approximately a decade, following in the footsteps of the, so far, more successfully used DNA microarrays (developed in the 1990s). Microarrays of proteins are more difficult to produce because of the more complex nature of proteins as compared to DNA. In our work we have constructed model surfaces which allow for the stable, highly oriented, and functional immobilization of proteins in an array format. Our capture molecules are based on multivalent units of the chelator nitrilotriacetic acid (NTA), which is able to bind histidine-tagged proteins. Furthermore, we have explored an approach for studying lipid membrane bound systems, e.g. receptor-ligand interactions, in a parallelized, microarray format. The approach relies on the addressable, DNA-mediated adsorption of tagged lipid vesicles. In an analogous work we have used the protein microarray concept for the detection of four common narcotics (heroin, amphetamine, ecstasy, and cocaine). The detection is based on the displacement of loosely bound antibodies from surface array positions upon injection of a specific target analyte, i.e. a narcotic substance. The proof-of-concept chip can easily be expanded to monitor many more narcotic substances. In addition, we have also been able to simultaneously detect the explosive trinitrotoluene (TNT) along with the narcotics, showing that the chip is a versatile platform for the detection of virtually any type of harmful or illegal compound. This type of biosensor system is potentially envisaged to be used in the fight against crime, terrorism, drug abuse etc. Infrared reflection absorption spectroscopy together with ellipsometry has been used to characterize molecular layers used in the fabrication processes of the microarray features. Imaging surface plasmon resonance operating in the ellipsometric mode is subsequently used for functional evaluation of the microarrays using a well-defined receptor-ligand model system. This approach allows simultaneous and continuous monitoring of binding events taking place in multiple regions of interest on the microarray chip. A common characteristic of all the instrumentation used is that there is no requirement for labeling of the biomolecules to be detected, e.g. with fluorescent or radioactive probes. This feature allows for a flexible assay design and the use of more native proteins, without any time-consuming pretreatments.

This thesis demonstrates the application of biosensor technology for molecular biology investigations, utilizing a surface plasmon resonance based optical device for mass sensitive detection of biomolecular interactions at a chipsurface. Oligonucleotide model systems were designed for analysis of the action of DNA manipulating enzymes. DNA ligation, DNA cleavage and DNA synthesis could be quantitatively monitored in real-time. A protocol for DNA minisequencing was also established based on prevention of chain elongation by incorporation of chain-terminators. Determinations of affinities for short oligonucleotides hybridizing to an immobilized target were performed with various sequence content, length, temperature and degree of complementarity. The decrease in affinity for hybridizations involving mismatch situations was found to be strongly dependent on the relative position of the mismatch. Interestingly, also end-mismatches were clearly detectable. The stabilization effect achieved upon co-hybridization of two adjacently annealing short oligonucleotide modules (modular primer effect) was also investigated for different module combinations and hybridization situations. The modular concept of hybridizations was subsequently demonstrated to result in enhanced Capture of single stranded PCR products. The sequence based DNA analysis, first introduced with oligonucleotide modelsystems, was extended to the scanning and screening formutations in PCR amplified DNA from clinically relevant samples. Several different formats were investigated, eitherwith the PCR products immobilized on the chip and oligonucleotides injected or vice versa. Again, mismatch discrimination could be observed for wild type and mutant specific oligonucleotides hybridizing to the targets. The experimental set-up for mutation detection was further developed by the introduction of a subtractive mismatch sensitive hybridization outside the instrument and a subsequent determination of the relative amounts of remain ingoligonucleotides with analytical biosensor monitoring of hybridizations between fully complementary oligonucleotides. In conclusion, the applied technology was found to be a suitable tool for a wide range of molecular biology applications, with emphasis on hybridization analysis and mutation detection. / QC 20100611

Immobilization of different bio- and organic molecules on solid supports is fundamental within many areas of science. Sometimes, it is desirable to obtain a directed orientation of the molecule in the immobilized state. In this thesis, the copper (I) catalyzed Huisgen 1,3-dipolar cycloaddition, referred to as a “click chemistry” reaction, was explored as a means to perform directed immobilization of small molecule ligands on gold surfaces. The aim was to synthesize alkyne- and azide-terminated alkanethiols that would form well-organized self assembled monolayers (SAMs) on gold from the commercially available substances orthoethylene glycol and bromo alkanoic acid. N-(23-azido-3,6,9,12,15,18,21-heptaoxatricosyl)-n-mercaptododekanamide/hexadecaneamide (n = 12, 16) were successfully synthesized and allowed to form SAMs of different compositions to study how the differences in density of the functional groups on the surface would influence the structure of the monolayer and the click chemistry reaction. The surfaces were characterized by different optical methods: ellipsometry, contact angle goniometry and infrared reflection-absorption spectroscopy (IRAS). The click reaction was found to proceed at very high yields on all investigated surfaces. Finally, the biomolecular interaction between a ligand immobilized by click chemistry on the gold surfaces and a model protein (bovine carbonic anhydrase) was demonstrated by surface plasmon resonance using a Biacore system.

Cancer is the main cause of death in economically developed countries and the second leading cause of death in developing countries. Along with today’s knowledge that more than two hundred different diseases lie in the category of this prognosis there is an urge for more detailed and case-specific treatments to replace the dramatic actions of available radiation- and chemotherapy, which in many cases do not make a difference between healthy and cancer cells. The transcription factor and onco-protein c-Myc has, after being extensively studied during the past decades, become a prognostic marker for almost all cancer forms known. Still, many questions remain regarding how c-Myc interacts with its many different target proteins involved in cell-cycle regulation, proliferation and apoptosis. Current cell biology states that one of the regulating proteins, hPin1, interacts with c-Myc in a phosphorylation-dependent manner which appears to direct the correct timing of c-Myc activation and degradation through the ubiquitin/proteasome-pathway. The critical phosphorylation sites, T58 and S62, are located in the Myc-Box-I (MBI) region, a highly conserved sequence strongly coupled to aggressive tumourigenesis by hotspot mutations. Interestingly, preliminary results in the Sunnerhagen group suggested that MBI alone did not bind hPin1, suggesting hPin1 targeting a site distal from the residues to be phosphorylated. In this thesis, results from Surface Plasmon Resonance (SPR) and Nuclear Magnetic Resonance (NMR) show that the docking WW-domain of hPin1 binds unphosphorylated c-Myc at a region distal from the phosphorylation site, including residues 13-34. Furthermore, SPR experiments revealed that hPin1 binds unphosphorylated c-Myc with apparently greater affinity and with much slower kinetics than phosphorylated c-Myc. Thus, hPin1 recognition and interaction with c-Myc appears not to be dependent on phosphorylation of c-Myc prior binding. The newly identified binding region of c-Myc, located N-terminal of MBI, may further increase the understanding of protein degradation control and c-Myc function. The studies presented in this thesis provide a brick in the puzzle of c-Myc and hPin1 coupled oncogenesis for further development of new therapeutic strategies.

M-DNA is a complex formed between normal double-stranded DNA and the transition metal ions Zn2+, Ni2+, and Co2+ that is favoured by an alkaline pH. Previous studies have suggested that M-DNA formation involves replacement of the imino protons of G and T bases by the transition metal ions involved in forming the complex. Owing to the conductive properties of this unique DNA conformation, it has potential applications in nanotechnology and biosensing. This work was aimed at improving existing methods and developing new methods of characterizing M-DNA. The effects of base substitutions, particularly those of G and T, were evaluated in light of the proposed structure. Differences between M-DNA conformations induced by Zn2+ and Ni2+ were also investigated with a variety of techniques and compared to the effects of Cd2+ and Mg2+ on double-stranded DNA. M-DNA formation and stability were studied with an ethidium bromide (EtBr) based assay, M-DNA induced fluorescence quenching of DNA labelled with fluorescein and a compatible quenching molecule, isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). Production of monoclonal antibodies against the conformation was also attempted but was unsuccessful. The EtBr-based assay showed Ni(II) M-DNA to be much more stable than Zn(II) M-DNA as a function of pH and in the presence of ethylenediaminetetraacetic acid. Sequence-dependency and the effect of base substitutions were measured as a function of pH. With regards to sequence, d(G)nd(C)n tracts were found to form the conformation most easily. Base substitutions with G and T analogues that lowered the pKa of these bases were found to stabilize M-DNA more strongly than other base substitutions. A combination of temperature-dependant EtBr and ITC assays showed M-DNA formation to be endothermic, and therefore entropy driven. The SPR studies demonstrated many qualitative differences between Zn(II) and Ni(II) M-DNA formation, allowed characterization of Zn2+, Ni2+, Cd2+, and Mg2+ complexes with single-stranded DNA, and provided unambiguous evidence that M-DNA formation results in very little denaturation of double-stranded DNA. Specifically, the SPR study showed Ni(II) M-DNA to be more stable than Zn(II) M-DNA in the absence of transition metal ions, but also showed that Ni(II) M-DNA required higher concentrations of Ni2+ than Zn2+ to fully form the respective M-DNA conformations. Finally, quenching studies demonstrated Zn(II) M-DNA formation over a pH range from 6.5 to 8.5 provided that a Zn2+:H+ ratio of roughly 105 was maintained. The Keq for this interaction was 1.3 x 10-8 with 1.4 H+ being liberated per base bair of M-DNA formed. These results support the proposed structural model of M-DNA, as lowering the pKa of the bases having titratable protons over the pH range studied facilitated M-DNA formation. The fact that Zn(II) M-DNA formation was observed by fluorescence quenching at any pH provided that a constant ratio of Zn2+:H+ was maintained was consistent with a simple mass-action interaction for M-DNA formation. The differences between Zn(II) and Ni(II) M-DNA formation show that although it requires a higher pH or transition metal ion concentration, Ni(II) M-DNA is more stable than Zn(II) M-DNA once formed. This difference could play an important role in applications of M-DNA which required modulation in the stability of the M-DNA conformation.

Protein-protein and protein-molecule interactions are complicated phenomena due to the tendency of proteins to change shape and function in response to their environment. Protein aggregation whether onto surfaces or in solution, can pose numerous problems in industry. Surface plasmon resonance (SPR) devices and quartz crystal microbalances (QCM) are two real-time, label free methods that can be used to detect the interactions between molecules on surfaces. These devices often employ self-assembled monolayers (SAMs) to produce specific surfaces for studying protein-protein interactions. The objective of this work was to develop methodologies utilizing SPR to better understand protein-protein and protein-molecule interactions with possible applications in the food and separation industrial sectors. A very well characterized whey protein, β-lactoglobulin (BLG), is used in numerous applications in the food industry. BLG can undergo different types of self-aggregation due changes in external environment factors such as buffer strength, pH or temperature. In this work, a hydrophilic SAM was developed and used to study the interaction and non-specific adsorption of BLG and palmitic acid (PA), a molecule which is known to bind to BLG. It was found that PA tended to reduce BLG conformational changes once on the surface, resulting in a decrease in its surface adhesion. Fluorescent excitation emission matrices (EEM’s) using a novel fluorescence probe technique were utilized to detect protein on the surface as well as conformational changes on the surface of the sensor, although the extent these changes could not be quantified. Another whey protein, α-lactoglobulin (AL), was utilized as a surrogate protein to study the adsorption of colloidal/particulate and protein matter (CPP) extracted from filtration studies of river water. A large fraction of natural organic matter (NOM), the major foulant in membrane based water filtration, is CPP and protein. Understanding the interactions between these components is essential in abating NOM membrane fouling. Several SPR methods were investigated in order to verify the interactions. A mixture of AL and CPP particles in solution prevented the non-specific adsorption of AL to the SAM surface. This change in association was then detected through SPR. Fluorescent EEM’s of the sensor surface verified that CPP and AL bound to the surface. This finding has fundamental significance in the interpretation of NOM-based membrane fouling. To better understand the mechanisms behind non-specific adsorption, a mechanistic mathematical model was developed to describe the adsorption of BLGs onto the hydrophilic SAM. The resulting model performed well in terms of predicting adsorption based on SPR data. The model incorporated the monomer-dimer equilibrium of BLG in solution, highlighting the impact of protein aggregation on non-specific adsorption mechanisms. For future studies, improvement in fluorescent FOP surface scan methodology would help identify different protein/molecules and conformations on the surface.

Plasmonic nanoparticles and their assembly have the potential to serve as a platform in practical applications such as photonics, sensing, and nano-medicine. To use plasmonic nanoparticles in these applications, it is important to understand their optical properties and find methods to control their optical response. Using polarization-sensitive dark-field spectroscopy to study self-assembled nanoparticle rings on substrates with different permittivities I show that the interaction between collective plasmon resonances and the substrate can control the spatial scattering image. Using liquid crystals as an active medium that can be controlled with an external electric field I show that the Fano resonance of an asymmetric plasmonic assembly can be actively controlled utilizing the polarization change of scattered light passing through the liquid crystal device. Furthermore, utilizing the strong electromagnetic field enhancement of coupled plasmonic “nanospikes” on the surface of gold nanoshells with a silica core, I show the use of single spiky nanoshells as surface-enhanced Raman spectroscopy substrates. Individual spiky nanoshells give surprisingly reproducible surface-enhanced Raman spectroscopy intensities with a low standard deviation compared to clusters of nanoparticles. In summary, the work presented here provides understanding of the plasmonic response for assembled nanoparticles on different substrates, illustrated a new method to actively control the optical response of plasmonic nanoparticles, and characterizes spiky nanoshells as surface-enhanced Raman scattering platform.

M-DNA is a complex formed between normal double-stranded DNA and the transition metal ions Zn2+, Ni2+, and Co2+ that is favoured by an alkaline pH. Previous studies have suggested that M-DNA formation involves replacement of the imino protons of G and T bases by the transition metal ions involved in forming the complex. Owing to the conductive properties of this unique DNA conformation, it has potential applications in nanotechnology and biosensing. This work was aimed at improving existing methods and developing new methods of characterizing M-DNA. The effects of base substitutions, particularly those of G and T, were evaluated in light of the proposed structure. Differences between M-DNA conformations induced by Zn2+ and Ni2+ were also investigated with a variety of techniques and compared to the effects of Cd2+ and Mg2+ on double-stranded DNA. M-DNA formation and stability were studied with an ethidium bromide (EtBr) based assay, M-DNA induced fluorescence quenching of DNA labelled with fluorescein and a compatible quenching molecule, isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). Production of monoclonal antibodies against the conformation was also attempted but was unsuccessful. The EtBr-based assay showed Ni(II) M-DNA to be much more stable than Zn(II) M-DNA as a function of pH and in the presence of ethylenediaminetetraacetic acid. Sequence-dependency and the effect of base substitutions were measured as a function of pH. With regards to sequence, d(G)nd(C)n tracts were found to form the conformation most easily. Base substitutions with G and T analogues that lowered the pKa of these bases were found to stabilize M-DNA more strongly than other base substitutions. A combination of temperature-dependant EtBr and ITC assays showed M-DNA formation to be endothermic, and therefore entropy driven. The SPR studies demonstrated many qualitative differences between Zn(II) and Ni(II) M-DNA formation, allowed characterization of Zn2+, Ni2+, Cd2+, and Mg2+ complexes with single-stranded DNA, and provided unambiguous evidence that M-DNA formation results in very little denaturation of double-stranded DNA. Specifically, the SPR study showed Ni(II) M-DNA to be more stable than Zn(II) M-DNA in the absence of transition metal ions, but also showed that Ni(II) M-DNA required higher concentrations of Ni2+ than Zn2+ to fully form the respective M-DNA conformations. Finally, quenching studies demonstrated Zn(II) M-DNA formation over a pH range from 6.5 to 8.5 provided that a Zn2+:H+ ratio of roughly 105 was maintained. The Keq for this interaction was 1.3 x 10-8 with 1.4 H+ being liberated per base bair of M-DNA formed. These results support the proposed structural model of M-DNA, as lowering the pKa of the bases having titratable protons over the pH range studied facilitated M-DNA formation. The fact that Zn(II) M-DNA formation was observed by fluorescence quenching at any pH provided that a constant ratio of Zn2+:H+ was maintained was consistent with a simple mass-action interaction for M-DNA formation. The differences between Zn(II) and Ni(II) M-DNA formation show that although it requires a higher pH or transition metal ion concentration, Ni(II) M-DNA is more stable than Zn(II) M-DNA once formed. This difference could play an important role in applications of M-DNA which required modulation in the stability of the M-DNA conformation.

This dissertation consists of two topics. One is a "Multi-pass Fiber Optic Surface Plasmon Resonance Sensor (SPR)" and the other is a "Nano-metallic Surface Plasmon Lens." Since both topics involved surface plasmon, the title of this dissertation is named "Surface plasmon coupled sensor and nanolens." For a multi-pass fiber optic SPR sensor, a fiber optic 4-pass SPR sensor coupled with a field-assist capability for detecting an extremely low concentration of charged particles is first demonstrated. The multipass feature increases the sensitivity by a factor equal to the number of passes. The field-assist feature forces charged particles/molecules to the SPR surface, increasing the sensitivity by an additional factor of about 100. Overall, the sensitivity exceeds the one-pass SPR device by a factor of about 400. A 10 pM concentration of 47 nm diameter polystyrene (PS) latex beads and 1 ?M concentration of salt dissolved in DI water were detected within a few seconds by the combined system. The equivalent index resolution for atomic size corresponding to ionized chlorine in salt is 10-8. This technique offers the potential for sensitive and fast detection of biomolecules in a solution. Secondly, a 44-pass fiber optic surface plasmon resonance (SPR) sensor coupled with a field-assist capability for measurement of refractive index change due to positive and negative ions is shown. The field-assist feature forces ions to the SPR surface, causing the SPR signal response to change which reflects a decrease or increase in refractive index depending on whether positive or negative ions are being attracted to the surface. This technique offers the potential for the sensitive detection of cations and anions in a solution. For a nano-metallic surface plasmon lens, we analyze the transmission of a normally incident plane wave through an Ag/dielectric layered concentric ring structure using finite difference time domain (FDTD) analysis. The dependency of the transmission efficiency on the refractive index in slit is studied. The numerical analysis indicates that the focusing beyond diffraction limit is found even at the extended focal length comparable to the distance of 7" from the exit plane using a circularly polarized coherent plane wave, ?=405 nm. Especially, compared to an Ag-only structure, the Ag/ LiNbO3 structure amplifies the transmission power by a factor of 6. Therefore, this Ag/dielectric layered lens has the potential for significantly higher resolution imaging and optical data storage.

The work presented in this dissertation suggests novel design of chemical plasmonic sensors which have been developed based on Localized Surface Plasmon Resonance (LSPR), and Surface-enhanced Raman scattering (SERS) phenomena. The goal of the study is to understand the SERS phenomena for 3D hybrid (organic/inorganic) templates and to design of the templates for trace-level detection of selected chemical analytes relevant to liquid explosives and hazardous chemicals. The key design criteria for the development of the SERS templates are utilizing selective polymeric nanocoatings within cylindrical nanopores for promoting selective adsorption of chemical analyte molecules, maximizing specific surface area, and optimizing concentration of hot spots with efficient light interaction inside nanochannels. The organic/inorganic hybrid templates are optimized through a comprehensive understanding of the LSPR properties of the gold nanoparticles, gold nanorods, interaction of light with highly porous alumina template, and the choice of physical and chemical attributes of the selective coating. Furthermore, novel method to assemble silver nanoparticles in 3D as the active SERS-active substrate has been demonstrated by uniform, in situ growth of silver nanoparticles from electroless deposited silver seeds excluding any adhesive polymer layer on template. This approach can be the optimal for SERS sensing applications because it is not necessary to separate the Raman bands of the polyelectrolyte binding layer from those of the desired analyte. The fabrication method is an efficient, simple and fast way to assemble nanoparticles into 3D nanostructures. Addressable Raman markers from silver nanowire crossbars with silver nanoparticles are also introduced and studied. Assembly of silver nanowire crossbar structure is achieved by simple, double-step capillary transfer lithography. The on/off SERS properties can be observed on silver nanowire crossbars with silver nanoparticles depending on the exact location and orientation of decorated silver nanoparticles nearby silver nanowire crossbars. As an alternative approach for the template-assisted nanostructure design, porous alumina membrane (PAM) can be utilized as a sacrificial template for the fabrication of the nanotube structure. The study seeks to investigate the design aspects of polymeric/inorganic hybrid nanotube structures with plasmonic properties, which can be dynamically tuned by external stimuli such as pH. This research suggests several different organic/inorganic nanostructure assemblies by various template-assisted techniques. The polymeric/inorganic hybrid nanostructures including SERS property, pH responsive characteristics, and large surface area will enable us to understand and design the novel chemical plasmonic sensors.