Experimentation and Multiphysical Modeling of Bioanalytical Microdevices

Bioanalytics involves quantitative measurements of complex biological samples that contain metabolites, DNA, RNA, and proteins. Efficient sample preparation for downstream analysis and sensitive detection of analytes can be achieved via bioanalytical microdevices. Fully realizing the potential of these devices requires tool characterization and bioprocess optimization, in addition to understanding device physics. Therefore, this thesis introduces multiphysical modeling and experimentation of microdevices, with applications to diabetes care and single-cell analysis. To understand the physics of viscometric glucose microsensors, this thesis presents a model of the sensor, which couples the fluid flow with vibrating diaphragms. The model is used to predict the sensor response to glucose via theory of squeeze-film damping and vibrations of pre-stressed plate. A first-principle-based model resulting from the theory can be evaluated from the device's geometric and material properties, and quantitatively determines the device response to vibrational excitations at varying glucose concentrations. Next, this thesis introduces a theoretical model for viscometric glucose microsensors that employ harmonic microcantilever oscillation in the sensing liquid. The presented model associates the unsteady Stokes equation with the motion of a bounded viscous liquid to understand the hydrodynamic impact on the cantilever. With a proper consideration of the viscosity and bounded geometry of liquid media, the model relaxes the thin-film assumption required for the diaphragm-based model, enabling an accurate representation of fluid-structure interactions based on fundamental structural vibration and fluid flow equations. Next, this thesis presents an experimental exploration of a hydrogel-based affinity microsensor for glucose monitoring via dielectric measurements. The microsensor incorporates a synthetic hydrogel that is attached to the device surface via in situ polymerization, which eliminates mechanical moving parts required in the viscometric glucose sensors. Changes in the dielectric properties of the hydrogel when binding reversibly with glucose molecules have been measured using a MEMS capacitive transducer to determine the glucose concentration. Experimental results demonstrate that in a glucose concentration range of 0–500 mg/dL and with a resolution of 0.35 mg/dL or better, the microsensor exhibits a repeatable and reversible response, and can potentially be useful for continuous glucose monitoring in diabetes care. Additionally, this thesis presents a microfluidic preprocessing method that integrates single-cell picking, lysing, reverse transcription and digital polymerase chain reaction to enable the isolation, tracking and gene expression analysis at single-cell level for individual cells. The approach utilizes a photocleavable bead-based microfluidic device to synthesize and deliver stable complementary DNA for downstream gene expression analysis, thereby allowing chip-based integration of multiple reactions and facilitating the minimization of sample loss or contamination. Finally, this thesis ends with concluding remarks and directions of future work towards continuous glucose monitoring and high-throughput single-cell genetic analysis

Structure and Dynamic Studies of the Nuclear Pore Complex at the Single-Molecule Level

Nuclear pore complexes (NPCs) are large macromolecular structures forming the only known direct route across the double bilayer membrane of the nuclear envelope. The NPC structure has been extensively explored in an effort to elucidate the mechanisms by which they control transport. Many of these studies have found the presence of a central mass or plug within the central channel of NPCs, although neither the function nor identity of the central mass were clear. Here, several techniques including electron microscopy, Förster resonance energy transfer (FRET), and high-resolution near-field scanning optical microscopy (NSOM) are utilized to specifically locate vault ribonucleoproteins to NPCs. This interaction, along with several other results, strongly suggests that vaults represent the central mass of NPCs. A single-molecule transport assay was also developed in order to record the translocation of individual fluorescent dextrans through NPCs. Comparison of the single-molecule dwell times under various conditions led to a better understanding of the specific mechanism controlling the non signal-mediated transport of cargo through NPCs

Materials and neuroscience: validating tools for large-scale, high-density neural recording

Extracellular recording remains the only technique capable of measuring the activity of many neurons simultaneously with a sub-millisecond precision, in multiple brain areas, including deep structures. Nevertheless, many questions about the nature of the detected signal and the limitations/capabilities of this technique remain unanswered. The general goal of this work is to apply the methodology and concepts of materials science to answer some of the major questions surrounding extracellular recording, and thus take full advantage of this seminal technique. We start out by quantifying the effect of electrode impedance on the amplitude of measured extracellular spikes and background noise. Can we improve data quality by lowering electrode impedance? We demonstrate that if the proper recording system is used, then the impedance of a microelectrode, within the range typical of standard polytrodes (~ 0.1 to 2 MΩ), does not significantly affect a neural spike amplitude or the background noise, and therefore spike sorting. In addition to improving the performance of each electrode, increasing the number of electrodes in a single neural probe has also proven advantageous for simultaneously monitoring the activity of more neurons with better spatiotemporal resolution. How can we achieve large-scale, highdensity extracellular recordings without compromising brain tissue? Here we report the design and in vivo validation of a complementary metal–oxide–semiconductor (CMOS)-based scanning probe with 1356 electrodes arranged along approximately 8 mm of a thin shaft (50 μm thick and 100 μm wide). Additionally, given the ever-shrinking dimensions of CMOS technology, there is a drive to fabricate sub-cellular electrodes (< 10 μm). Therefore, to evaluate electrode configurations for future probe designs, several recordings from many different brain regions were performed with an ultra-dense probe containing 255 electrodes, each with a geometric area of 5 x 5 μm and a pitch of 6 μm. How can we validate neural probes with different electrode materials/configurations and different sorting algorithms? We describe a new procedure for precisely aligning two probes for in vivo “paired-recordings” such that the spiking activity of a single neuron is monitored with both a dense extracellular silicon polytrode and a juxtacellular micro-pipette. We gathered a dataset of paired-recordings, which is available online. The “ground truth” data, for which one knows exactly when a neuron in the vicinity of an extracellular probe generates an action potential, has been used for several groups to validate and quantify the performance of new algorithms to automatically detect/sort single-units

Rationally Designed DNA Origami Carriers for Quantitative Single Molecule Detection with Nanopipettes

The ability to detect small concentrations of biomarkers in patient samples is one of the cornerstones of modern healthcare. In general, biosensing approaches employed to address this need are based on measuring signals resulting from the interaction of a large ensemble of molecules with the sensor. Here, a biosensor platform using DNA origami, featuring a central cavity with a target–specific DNA aptamer, as carriers for translocation through nanopores which enables individual biomarkers to be identified and counted to compile a sensing signal is reported. It is shown that the modulation of the ion current through the nanopore upon the DNA origami translocation strongly depends on the presence and in fact the size of a central cavity. While DNA origami without a central cavity cause a single peak in the ion current, DNA origami of the same dimensions but featuring a central cavity lead to double peaks in the ion current. This is also true for DNA origami (with and without central cavities) made of similar sized DNA but of different dimensions. It is also observed that the peak characteristics, peak amplitude and the dwell time, are different depending on the presence or absence of a central cavity. This work exploits these parameters to generate a biosensing platform capable of detecting human C–reactive protein (CRP) in clinically relevant fluids. DNA origami frames with cavities large enough to lead to clear ion current double peaks were designed and a CRP–specific DNA aptamer was introduced into the cavity. Also, upon binding of CRP, the ion current peak changes to a single peak and the peak characteristics change. Using this three–parameter classification, CRP–occupied and unoccupied carriers can be distinguished when they translocate through the nanopore. Thus CRP biosensing by computing the ratio of occupied vs total number of frames with a limit of detection of 3 nM is successfully demonstrated

Single-molecule methods for an improved understanding of biophysical interactions: from fundamental biology to applied nanotechnology

The advent of single-molecule methods has greatly extended the scale at which we are able to probe natural systems. The information that can be gained by studying biological systems on a single-molecule scale, in the absence of ensemble averaging, provides an unprecedented amount of detail about molecular interactions in real-time. Single-molecule biophysical techniques have provided unique insights into the nature of protein-DNA interactions, and have allowed for the development of novel platforms to study nano-bio interactions. In this thesis, we will describe two main sets of experiments to explore molecular interactions at the single-molecule scale. We will focus on the study of protein-DNA interactions and also the interactions between biological molecules and synthetic nanoparticles, using a variety of single-molecule techniques. Protein-DNA interactions are essential to cellular processes, many of which require proteins to recognize a specific DNA target-site. This search process is well-documented for monomeric proteins, but not as well understood for systems that require dimerization or oligomerization at the target site for activity. We present a single-molecule study of the target-search mechanism of Protelomerase TelK, a recombinase-like protein that is only active as a dimer. Interestingly, we observe that TelK undergoes 1D diffusion on non-target DNA as a monomer, as expected, but becomes immobile on DNA as a dimer or oligomer despite the absence of its target site. We further show that TelK condenses non-target DNA upon dimerization, forming a tightly bound nucleo-protein complex. Together with simulations of dimer-active protein search, our results suggest a search model whereby monomers diffuse along DNA, and subsequently dimerize to form an active complex on target DNA. These results show that target-finding occurs faster than nonspecific dimerization at biologically relevant protein concentrations. This model may provide insights into the search mechanisms of proteins that are active as multimeric complexes for a more accurate and comprehensive model for the target-search process by sequence specific proteins (SSPs). In addition to studying the target-search process of protelomerase TelK, we have also studied the molecular mechanism of TelK activity at the target site. We attempt to capture the dynamics responsible for DNA hairpin formation by TelK, and we discuss the unique features of TelK-DNA interactions that contribute to the complexity of this process. Nanomaterials have unique optical, chemical and mechanical properties that make them useful in biological applications, acting as drug and gene delivery agents, electrical and optical sensors, and cell-signaling components. Although many tools exist to characterize both biomolecules and nanomaterials, these methods are currently unable to give a detailed picture of biomolecular structure at the nano-bio interface. As a result, local electronic properties, bioavailability, toxicological effects, and basic molecular structure and conformation of biomolecules on nanoparticles remain unclear. Single-Walled Nanotubes (SWNTs) are allotropes of carbon with a cylindrical nanostructure. Though SWNTs tend to form insoluble aggregates, sonicating SWNTs with DNA forms a DNA-SWNT complex that is soluble in water. Single-stranded DNA (ssDNA) is believed to form a helical structure on the SWNT surface. This DNA-SWNT complex is not only soluble in water and does not appear to be toxic to mammalian cells, but it is also uptaken by mammalian cells via endocytosis. Therefore, there is significant interest in understanding the mechanism of SWNT encapsulation by ssDNA. However, current experimental tools have been unable to probe the structure of biomolecules on the surface of nanomaterials. Consequently, little is known about the mechanism by which ssDNA wraps SWNT, and how biomolecules interact with the resulting DNA-SWNT structure. In order to extend the range of biochemical interactions that can be detected on a SWNT surface, we have developed a variety of experimental platforms to study biological interactions on SWNT surfaces by extending several well-established single-molecule biophysics techniques to the study of nano-bio interactions. By applying single-molecule techniques to the study of the nano-bio interface, we uncover changes in the expected behavior of biomolecules. These effects include cooperative DNA hybridization, changes in the accessibility of DNA to nuclease proteins, and protein deactivation on a SWNT surface. We also uncover details of the mechanism by which ssDNA wraps SWNT to form a biologically-compatible nanoparticle-biomolecule conjugate

Development and modelling of a versatile active micro-electrode array for high density in-vivo and in-vitro neural signal investigation

The electrophysiological observation of neurological cells has allowed much knowledge to be gathered regarding how living organisms are believed to acquire and process sensation. Although much has been learned about neurons in isolation, there is much more to be discovered in how these neurons communicate within large networks. The challenges of measuring neurological networks at the scale, density and chronic level of non invasiveness required to observe neurological processing and decision making are manifold, however methods have been suggested that have allowed small scale networks to be observed using arrays of micro-fabricated electrodes. These arrays transduce ionic perturbations local to the cell membrane in the extracellular fluid into small electrical signals within the metal that may be measured. A device was designed for optimal electrical matching to the electrode interface and maximal signal preservation of the received extracellular neural signals. Design parameters were developed from electrophysiological computer simulations and experimentally obtained empirical models of the electrode-electrolyte interface. From this information, a novel interface based signal filtering method was developed that enabled high density amplifier interface circuitry to be realised. A novel prototype monolithic active electrode was developed using CMOS microfabrication technology. The device uses the top metallization of a selected process to form the electrode substrate and compact amplification circuitry fabricated directly beneath the electrode to amplify and separate the neural signal from the baseline offsets and noise of the electrode interface. The signal is then buffered for high speed sampling and switched signal routing. Prototype 16 and 256 active electrode array with custom support circuitry is presented at the layout stage for a 20 μm diameter 100 μm pitch electrode array. Each device consumes 26.4 μW of power and contributes 4.509 μV (rms) of noise to the received signal over a controlled bandwidth of 10 Hz - 5 kHz. The research has provided a fundamental insight into the challenges of high density neural network observation, both in the passive and the active manner. The thesis concludes that power consumption is the fundamental limiting factor of high density integrated MEA circuitry; low power dissipation being crucial for the existence of the surface adhered cells under measurement. With transistor sizing, noise and signal slewing each being inversely proportional to the dc supply current and the large power requirements of desirable ancillary circuitry such as analogue-to-digital converters, a situation of compromise is approached that must be carefully considered for specific application design

Advanced Approaches to High Intensity Laser-Driven Ion Acceleration

Since the pioneering work that was carried out 10 years ago, the generation of highly energetic ion beams from laser-plasma interactions has been investigated in much detail in the regime of target normal sheath acceleration (TNSA). Creation of ion beams with small longitudinal and transverse emittance and energies extending up to tens of MeV fueled visions of compact, laser-driven ion sources for applications such as ion beam therapy of tumors or fast ignition inertial confinement fusion. However, new pathways are of crucial importance to push the current limits of laser-generated ion beams further towards parameters necessary for those applications. The presented PhD work was intended to develop and explore advanced approaches to high intensity laser-driven ion acceleration that reach beyond TNSA. In this spirit, ion acceleration from two novel target systems was investigated, namely mass-limited microspheres and nm-thin, free-standing diamond-like carbon (DLC) foils. Using such ultrathin foils, a new regime of ion acceleration was found where the laser transfers energy to all electrons located within the focal volume. While for TNSA the accelerating electric field is stationary and ion acceleration is spatially separated from laser absorption into electrons, now a localized longitudinal field enhancement is present that co-propagates with the ions as the accompanying laser pulse pushes the electrons forward. Unprecedented maximum ion energies were obtained, reaching beyond 0.5 GeV for carbon C$^{6+}$ and thus exceeding previous TNSA results by about one order of magnitude. When changing the laser polarization to circular, electron heating and expansion were shown to be efficiently suppressed, resulting for the first time in a phase-stable acceleration that is dominated by the laser radiation pressure which led to the observation of a peaked C$^{6+}$ spectrum. Compared to quasi-monoenergetic ion beam generation within the TNSA regime, a more than 40 times increase in conversion efficiency was achieved. The possibility to manipulate the shape of the ion acceleration front was successfully demonstrated by use of a spherically curved target surface. Finally, the last part of the presented work is devoted to accomplishments in laser development

Development of electrochemical platforms for DNA sensing

[eng] The present doctoral thesis is framed in the research and development (R & D) project between a private biotechnology company of molecular diagnostics Genomica SAU, the Institute for Bioengineering of Catalonia (IBEC), the University of Barcelona, and the Microfluidics ChipShop Company. The main objective of the project is making, implementation and marketing of a diagnostic device for early detection of DNA sequences involved with cancer. The multi device, or lab-on-chip (LOC), consists of a central automation unit (CAU), a system in miniature of DNA amplification or chain reaction polymerase (mini-PCR), and a biosensing platform (DNA chip) that consisting of a matrix or electrochemical array. The three elements are integrated by a microfluidic system in sandwich format cartridge. For this purpose, the aim of this thesis was the creation, characterization and optimization of the biochemical recognition platform between two single strands of DNA of dissimilar lengths but with some complementary sequences for the subsequent electrochemical detection of a hybridization event between them. Then, the integration into the cartridge of above platform was done. For the creation of this platform, we chose to use a self-assembled monolayer (SAM) of thiols as biorecognition interface of the 14 DNA sequences that are part of the project. During optimization of the interface chips individual gold and various molecules were used being chosen the molecule with two arms disulfide of polyethylene glycol (PEG) and a malaimida group at the end of one of them. This linker (or MalPEG linker) reacts with the gold surface due to the dative interaction between the sulfur atoms of the disulfide and the gold atoms from the surface of the chips. At the same time, the malaimida group reacts with the thiol group of the capture probes, joining. The PEG groups function as anti-adhesion molecules. Surface plasmon resonance (SPR) and cyclic voltammetry (CV) were techniques used to characterize the substrate and the hybridization event. For the manufacture of the cartridge, this was divided into two main blocks, the biosensing or electrochemical block and PCR block. The electrochemical block is composed of 4 layers, one of 64 working electrodes and gold paths for contact with the potentiostat, another layer that defines the area of the sensors must be functionalized gold and isolating the gold surface of the tracks. The third layer is a double-sided adhesive that has a hexagonal hole working as hybridization chamber, and the last layer is a screen printing layer with the reference electrode (RE) and counter electrodes. The above layers form an electrochemical cell wherein the hybridization will occurs. Regarding the PCR block, this is a system of two layers with a type microfluidic channel kind loop and its function is to contain the solutions during the process of DNA amplification by the mini-PCR. During the integration of the optimized SAM into an electrochemical cartridge a manual and automated ways were used to immobilize the capture probes. Several tests were performed in order to obtain the best conditions and ratios between the molecules to maximize the hybridization signal during the electrochemical detection.[spa] El presente trabajo de tesis está enmarcado en un proyecto de investigación y desarrollo (I+D) entre la empresa privada Genomica S.A.U., el Instituto de Bioingeniería de Cataluña (IBEC), la Universidad de Barcelona y la empresa alemana ChipShop Microfluidics. El objetivo principal es el desarrollo, puesta a punto y comercialización de un dispositivo electroquímico de diagnóstico médico para etapas tempranas de cáncer. El objetivo de la tesis es la creación, optimización y posterior integración de una interfaz de biosensado de ADN en el dispositivo de diagnóstico, siendo pieza fundamental en el desarrollo de éste. La interfaz escogida fue una monocapa autoensamblada (SAM) que hace las veces de biosensor y que es capaz de anclar secuencias de ADN como sondas de captura y así poder detectar, selectivamente, las secuencias objetivo complementarias. El dispositivo también cuenta con un sistema microfluídico y un sistema de amplificación de ADN de reacción en cadena de la polimerasa en miniatura. La SAM esta inmovilizada en un array electroquímico que consta de 64 electrodos de trabajo que funcionan como elemento transductor de la señal electroquímica redox de los eventos de hibridación que ocurren sobre ellos. La funcionalización y puesta a punto del dispositivo se llevó a cabo inmovilizando múltiples sondas de captura después de una optimización de las concentraciones entre las diferentes partes constituyentes de la monocapa. Técnicas ópticas y electroquímicas fueron utilizadas para la caracterización de cada etapa y técnicas de fotolitografiado y de impresión por pantalla fueron utilizadas para la fabricación de los componentes del dispositivo. Finalmente, y después de algunos cambios surgidos durante el desarrollo del dispositivo, se llega a un diseño final y a las pruebas con muestras reales, proceso que aún está en etapa experimental