The work presented herein describes efforts to synthesize and characterize cyanide-bridged molecular compounds with high-spin ground states. This investigation focused primarily on the assembly of hexacyanometallate units with convergent cationic metal complexes that are coordinated to capping ligands. In this manner, a family of related compounds was developed that serve as models for understanding the role of magnetic exchange interactions and anisotropy in nanomagnetic materials. The work presented in Chapter II describes the successful incorporation of the [Fe(CN)6]3- building block into planar geometries with nuclearities ranging from three to ten metal centers. In Chapter III, this methodology was optimized to yield two pentanuclear FeIII/NiII clusters, namely, the trigonal bipyramidal unit, {[Ni(tmphen)2]3[Fe(CN)6]2}, and the extended square, {[Ni(bpy)2(H2O)][Ni(bpy)2]2-[Fe(CN)6]2}. Magnetic measurements on pure phases of these samples revealed that each system exhibits ferromagnetic coupling between the L.S. FeIII and NiII centers, but neither exhibits slow paramagnetic relaxation effects down to T=2K. In Chapter IV, this chemistry was extended to the [Mn(CN)6]3-building block in order to increase magnetic exchange coupling and anisotropy in this cluster type, efforts that resulted in the isolation of the molecule, {[Mn(tmphen)2]3[Mn(CN)6]2}. This cluster exhibits intramolecular antiferromagnetic exchange interactions between the Mn centers which lead to an S=11/2 ground state and a negative ZFS value (D=-0.348 cm-1), parameters that support the experimental observation of Single-Molecule Magnet (SMM) behavior at low temperatures. A detailed investigation of the physical and structural properties of {[Co(tmphen)2]3[Fe(CN)6]2} in Chapters V and VI led to the realization that the cluster exhibits sensitivity to temperature and humidity. The molecule exists in three different electronic isomeric forms in the solid state and undergoes a charge-transfer induced spin-transition (CTIST) under the influence of temperature. The results presented in Chapter VI describe the behavior of this same cluster in solution, the highlight of which is the discovery that water reacts with the cluster to form a fourth electronic isomer. Finally, it is described in Chapter VII that this Co/Fe trigonal bipyramidal unit can be used as a building block for systematically incorporating three metal types into a family of 1-D chain and cluster compounds.

Ce travail de thèse décrit (i) la synthèse de complexes hétérométalliques d'ions 3d et 4f à partir de précuseurs de Mn, Fe et Co, de sels de lanthanides et de ligands organiques et (ii) l'étude de leurs structures et propriétés. 41 complexes polynucléaires ont été synthétisés dans le cadre de ce travail. Les structures moléculaires de tous les composés ont été déterminées par diffraction des rayons X. Les propriétés magnétiques de 22 complexes ont été étudiées, dont quatre montrent une relaxation lente de leur aimantation considérée comme la signature d'un comportement de molécule-aimant. L'activité catalytique du complexe {Mn4Dy6Li2} calciné a aussi été étudiée et s'est avérée efficace pour l'oxydation du monoxyde de carbone. L'étude systématique de complexes isostructuraux de lanthanides a montré que l'incorporation d'ions 4f peut introduire de l'anisotropie magnétique et que l'ion DyIII est généralement le meilleur candidat pour le ciblage de molécules-aimants hétérométalliques 3d- 4f.

In later years polymer replication techniques have become a frequently employed fabrication method for microfluidic and micro-optical devices. This thesis describes applications and further developments of microstructures replicated in polymer materials. A novel method for homogenous amplified single-molecule detection utilizing a microfluidic readout format is presented. The method enables enumeration of single biomolecules by transforming specific molecular recognition events at nanometer dimensions to micrometer-sized DNA macromolecules. This transformation process is mediated by target specific padlock probe ligation, followed by rolling circle amplification (RCA) resulting in the creation of one rolling circle product (RCP) for each recognized target. Throughout this transformation the discrete nature of the molecular population is preserved. By hybridizing a fluorescence-labeled DNA detection oligonucleotide to each repeated sequence of the RCP, a confined cluster of fluorophores is generated, which makes optical detection and quantification possible. Spectral multiplexing is also possible since the spectral profile of each RCP can be analyzed separately. The microfluidic data acquisition process is characterized in detail and conditions that allow for quantification limited only by Poisson sampling statistics is established. The molecular characteristics of RCPs in solution are also investigated. Furthermore a novel thermoplastic microfluidic platform is described. The platform allows for observation of the microchannels using high magnification optics and also offers the possibility of on-chip cell culture and the integration of mechanical actuators. A novel fabrication process for the integration of polymer micro-optical elements on silicon is presented. The process is used for fabrication of a micro-optical system consisting of a laser and a movable microlens making beam steering possible. Such a micro-scanning system could potentially be used for miniaturized biochemical analysis.

Padlock probes and proximity ligation are two powerful molecular tools for detection of nucleic acids and proteins, respectively. Both methods result in the formation of DNA reporter molecules upon recognition of specific target molecules. These reporter molecules can be designed to include tag sequences that can be analyzed by techniques for nucleic acid analysis. Herein, I present a dual-tag microarray (DTM) platform that is suitable for high-performance analyses of DNA reporter molecule libraries, generated by padlock and proximity probing reactions. The DTM platform was applied for analysis of mRNA transcripts using padlock probes, and of cytokines using proximity ligation. The platform drastically improved specificity of detection, and it allowed precise measurements of proteins and nucleic acids over wide dynamic ranges. The thesis also presents two techniques for multi-probe analyses of biomolecules: the triple-specific proximity ligation assay (3PLA) for protein analyses, and the spliceotyping assay for mRNA analyses. 3PLA allows highly specific measurements of as little as hundreds of target protein molecules by interrogating three target epitopes simultaneously. In spliceotyping the exon composition of individual transcripts are represented as a series of tag sequences in DNA reporter molecules, via a series of target-dependent ligation reactions. Next, the splicing patterns along individual transcripts can be revealed by amplified single molecule detection and step-wise decoding.

Optische Pinzetten stellen neuartige Werkzeuge in der Biophysik dar, die sich durch eine außerordentliche Präzision auszeichnen. Im Rahmen der vorliegenden Arbeit werden verfeinerte Bildanalysetechniken vorgestellt und neu entwickelt, die es erlauben, die Position eines Mikropartikels mit einer zeitlichen Auflösung von 0,017 s und einer Genauigkeit von ±2nm in lateraler und in axialer Richtung zu bestimmen. Dies ermöglicht eine Kraftauflösung von bis zu ±50 fN. Damit sind die Voraussetzungen für die Untersuchung von Rezeptor/Ligand-Wechselwirkungen auf der Ebene einzelner Bindungsereignisse gegeben. In der vorliegenden Arbeit werden die Wechselwirkungen zwischen den phosphorylierungsspezifischen Antikörpern HPT-101, HPT-104 und HPT-110 und Tau-Peptiden mit verschiedenen Phosphorylierungsmustern sowie zwischen DNA und den Proteinen TmHU und oPrPC untersucht. Die Wechselwirkungen zwischen Tau-Peptiden und Antikörpern werden jeweils anhand ihrer Bindungshäufigkeit sowie der Verteilung der Abrisskräfte charakterisiert. Mit einem aus der Literatur bekannten, theoretischen Modell werden folgende Bindungsparameter bestimmt: Lebenszeit der unbelasteten Bindung, charakteristische Länge und freie Aktivierungsenergie der Dissoziation. Im Einklang mit Ergebnissen einer immunochemischen Messung werden spezifische Wechselwirkungen zwischen HPT-101 und dem biphosphorylierten Tau-Peptid sowie zwischen HPT-104 bzw. HPT-110 und den Peptiden, die eine Phosphorylierung an Thr231 bzw. Ser235 enthalten, beobachtet. Zusätzlich ermöglicht die Einzelmolekülmethode auch eine detaillierte Charakterisierung der unspezifischen Wechselwirkungen mit den Tau-Peptiden, welche das jeweilige spezifisch erkannte Phosphorylierungsmuster nicht beinhalten. Der zweite Teil der Arbeit befasst sich mit dem Einfluss der Proteine TmHU und oPrPC auf einen einzelnen, mit konstanter Kraft gehaltenen DNA-Strang. Der zeitliche Verlauf der TmHU-induzierten Kondensationsreaktion wird bei Kräften zwischen 2 pN und 40 pN sowie in Abhängigkeit von der Proteinkonzentration untersucht. Bei kleinen Kräften ist eine Verkürzung in zwei Phasen auf bis zu 30% der Konturlänge zu beobachten. Unter zusätzlicher Einbeziehung der Ergebnisse einer SMD-Simulation sowie einer rasterkraftmikroskopischen Untersuchung kann die erste Phase der Verkürzung einer primären Anbindung von TmHU zugeordnet werden. Die zweite Reaktionsphase entspricht hingegen vermutlich der Ausbildung einer Überstruktur. Die Wechselwirkung von oPrPC mit DNA wird zusätzlich mit einer kombinierten Anordnung aus Nanokapillare und optischer Pinzette untersucht. Dabei zeigt sich, dass ein Protein/DNA-Komplex ausgebildet wird, der eine negative Oberflächenladung aufweist und sich in seinem Volumen und seiner Ladung von reiner DNA unterscheidet. Allerdings hat oPrPC keinen Einfluss auf den Ende-zu-Ende-Abstand bzw. die Elastizität der DNA.

Improved methods are needed to interrogate the genome and the proteome. Methods with high selectivity, wide dynamic range, and excellent precision, capable of simultaneously analyzing many biomolecules are required to decipher cellular function. This thesis describes a molecular and microfluidic toolbox designed with those criteria in mind. It also presents a tool for graphical representation of nucleic acid sequences. Proximity ligation is a novel protein detection method that requires dual and proximate binding of two oligonucleotide-tagged affinity reagents to a protein or protein complex in order to elicit a signal. The responses from such recognition reactions are the formation of specific nucleic acid reporter molecules that are subsequently amplified and quantitatively detected. A scalable microfluidic platform suitable for fluorescence detection, cell culture, and actuation is also described. The platform uses rapid injection molding to produce microstructures in thermoplastic materials. By applying a thin layer of silica to the structures, a lid made of silicone rubber coated onto a thermoplastic support can be covalently bonded to generate enclosed channels. A method is presented for precise biomolecule counting, termed “amplified single-molecule detection”. The method preserves the discrete nature of biomolecules, converting specific molecular recognition events to fluorescence-labeled micrometer-sized objects that are enumerated in microfluidic channels. I also present a novel microarray-based detection method. To attain high selectivity and a wide dynamic range, the method is based on dual recognition with enzymatic discrimination and amplification. Upon target recognition in solution, DNA probes are subjected to thousand-fold amplification in solution, followed by selective detection on arrays and another hundred-fold amplification of reporter molecule created from the first amplification reaction. Lastly, I describe a novel graphical representation of nucleic acid sequences using TrueType fonts that can be of value for visual inspection of DNA sequences and for teaching purposes

Nonequilibrium work theorems, such as the Jarzynski equality and the Crooks fluctuation theorem, allow one to use nonequilibrium measurements to determine equilibrium free energies. For example, it has been demonstrated that the Crooks fluctuation theorem can be used to determine RNA folding energies. We used single-molecule manipulation with an atomic force microscope to measure the work done on poly(dA) as it was stretched and relaxed. This single-stranded nucleic acid exhibits unique base-stacking transitions in its force-extension curve due to the strong interactions among A bases, as well as multiple pathways. Here we showed that free energy curves can be determined by using the Crooks fluctuation theorem. The nonequilibrium work theorem can be used to determine free energy curves even when there are multiple pathways.

Nonequilibrium work theorems, such as the Jarzynski equality and the Crooks fluctuation theorem, allow one to use nonequilibrium measurements to determine equilibrium free energies. For example, it has been demonstrated that the Crooks fluctuation theorem can be used to determine RNA folding energies. We used single-molecule manipulation with an atomic force microscope to measure the work done on poly(dA) as it was stretched and relaxed. This single-stranded nucleic acid exhibits unique base-stacking transitions in its force-extension curve due to the strong interactions among A bases, as well as multiple pathways. Here we showed that free energy curves can be determined by using the Crooks fluctuation theorem. The nonequilibrium work theorem can be used to determine free energy curves even when there are multiple pathways.

Nonequilibrium work theorems, such as the Jarzynski equality and the Crooks fluctuation theorem, allow one to use nonequilibrium measurements to determine equilibrium free energies. For example, it has been demonstrated that the Crooks fluctuation theorem can be used to determine RNA folding energies. We used single-molecule manipulation with an atomic force microscope to measure the work done on poly(dA) as it was stretched and relaxed. This single-stranded nucleic acid exhibits unique base-stacking transitions in its force-extension curve due to the strong interactions among A bases, as well as multiple pathways. Here we showed that free energy curves can be determined by using the Crooks fluctuation theorem. The nonequilibrium work theorem can be used to determine free energy curves even when there are multiple pathways.

Nonequilibrium work theorems, such as the Jarzynski equality and the Crooks fluctuation theorem, allow one to use nonequilibrium measurements to determine equilibrium free energies. For example, it has been demonstrated that the Crooks fluctuation theorem can be used to determine RNA folding energies. We used single-molecule manipulation with an atomic force microscope to measure the work done on poly(dA) as it was stretched and relaxed. This single-stranded nucleic acid exhibits unique base-stacking transitions in its force-extension curve due to the strong interactions among A bases, as well as multiple pathways. Here we showed that free energy curves can be determined by using the Crooks fluctuation theorem. The nonequilibrium work theorem can be used to determine free energy curves even when there are multiple pathways.