While 3D printing technologies first appeared in the 1980s, prohibitive costs, limited materials, and the relatively small number of commercially available printers confined applications mainly to prototyping for manufacturing purposes. As technologies, printer cost, materials, and accessibility continue to improve, 3D printing has found widespread implementation in research and development in many disciplines due to ease-of-use and relatively fast design-to-object workflow. Several 3D printing techniques have been used to prepare devices such as milli- and microfluidic flow cells for analyses of cells and biomolecules as well as interfaces that enable bioanalytical measurements using cellphones. This review focuses on preparation and applications of 3D-printed bioanalytical devices.

Clear plastic fluidic devices with ports for incorporating electrodes to enable electrochemiluminescence (ECL) measurements were prepared using a low-cost, desktop three-dimensional (3D) printer based on stereolithography. Electrodes consisted of 0.5 mm pencil graphite rods and 0.5 mm silver wires inserted into commercially available 1/4 in.-28 threaded fittings. A bioimaging system equipped with a CCD camera was used to measure ECL generated at electrodes and small arrays using 0.2 M phosphate buffer solutions containing tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate ([Ru(bpy)3]2+) with 100 mM tri-n-propylamine (TPA) as the coreactant. ECL signals produced at pencil graphite working electrodes were linear with respect to [Ru(bpy)3]2+ concentration for 9-900 μM [Ru(bpy)3]2+. The detection limit was found to be 7 μM using the CCD camera with exposure time set at 10 s. Electrode-to-electrode ECL signals varied by ±7.5%. Device performance was further evaluated using pencil graphite electrodes coated with multilayer poly(diallyldimethylammonium chloride) (PDDA)/DNA films. In these experiments, ECL resulted from the reaction of [Ru(bpy)3]3+ with guanines of DNA. ECL produced at these thin-film electrodes was linear with respect to [Ru(bpy)3]2+ concentration from 180 to 800 μM. These studies provide the first demonstration of ECL measurements obtained using a 3D-printed closed-channel fluidic device platform. The affordable, high-resolution 3D printer used in these studies enables easy, fast, and adaptable prototyping of fluidic devices capable of incorporating electrodes for measuring ECL.

The adrenal medulla is the inner part of adrenal glands located above each kidney, that produces catecholamines. Neuroblastoma and pheochromocytoma are the most prevalent malignancies of the adrenal medulla. Quantitative diagnosis of urinary catecholamines using HPLC-coupled Mass detectors is the current method for the diagnosis of neuroblastoma and pheochromocytoma. There are two major problems with this approach, (i) Because the catecholamines concentrations have short half-life (10-100 s), a series of urine tests must be performed throughout 24hr, detecting each catecholamine separately, is inconvenient and time-consuming; (ii) mass detectors are expensive, bulky, and require highly skilled personal. Vanillylmandelic (VMA), and homavanillic acid (HVA) are the by-products of catecholamines and are emerging alternative biomarker for catecholamines due to their high stability. Here, we developed a rapid, sensitive, miniaturized, and cheaper sensing platform for simultaneous quantifications of dopamine (DA), VMA, and HVA, with the aid of iron single-atom catalysts (Fe-SACs), based electrochemical sensor. SACs are atomically distributed metal atoms that have a maximum atomic utility rate of nearly 100%, compared to 30% for traditional metal nanoparticles. MXene sheets are employed to stabilize Fe-SACs, where, the exposed lone pairs of MXene serve as sites covalently linking high-energy single Fe atoms. MXene/Fe-SACs were synthesized by treating Ti3C2TxMXene with Iron chloride via freeze-drying followed by annealing. The successful formation of the material was verified by state-of-the-art characterizations. The MXene/Fe-SACs show superior electrocatalytic performance to the commonly used Fe- nanomaterials. Then, it was coated on the electrode surface and used to analyze DA, VMA, and HVA simultaneously via cyclic voltammetry (CV) and square-wave voltammetry (SWV). Under optimized conditions, the MXene/Fe-SACs electrochemical sensor showed detection limits as low as 1 nM and a linear range between 1 nM-100 μM for DA, LOD of 5 nM & linear range of 10 nM-100 μM VMA, and LOD of 10 nM & linear range of 20 nM-100 μM HAV. The method proved successful in detecting biomarkers in (spiked) synthetic urine and human serum. Furthermore, the method was successfully demonstrated in the determination of DA release from PC12 live cells, suggesting the wide practical use of SACs in sensing catecholamines-related metabolites.

Oxytocin (OXT), a neuropeptide synthesized in the paraventricular nucleus (PVN) of the hypothalamus, functions to increase the precedence of social stimuli and promote the development of a wide range of social behaviors. However, whether OXT has a predicting role in social reward has yet to be examined. In this study, we developed a genetically encoded, scalable OXT sensor named OXTR-iTango2 and applied this technique to define the role of OXT in learned social behaviors. OXTR-iTango2 enables the combination of light- and ligand- dependent gene expression both in vitro and in vivo neural systems. In order to study the predictive role of OXT during expected socially rewarding experiences, we first conditioned animals to a social environment, and then selectively labeled OXT-sensitive ventral tegmental area dopamine (VTA-DA) neurons when animals encountered a conditioned stimulus that stood to predict a familiar social reward. Recurrent exposure to the same social stimulus normally lowered the degree of social interaction, but this reduced interaction was not observed when OXT-sensitive DA neurons were optogenetically inhibited. Thus, our findings support the notion that OXT plays a role beyond promoting social interactions, leading for a new proposed hypothesis that OXT mediation also leads to active avoidance of mundane social interactions. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

The microcantilever has become a ubiquitous tool for surface science, chemical sensing, biosensing, imaging, and energy harvesting, among many others. It is a device of relatively simple geometry with a static and dynamic response that is well understood. Further, because of it's small size, it is extremely sensitive to small external perturbations. These characteristics make the microcantilever an ideal candidate for a multitude of sensing applications. In this thesis dissertation we use the microcantilever to conduct numerous physical measurements and to study fundamental phenomena in the areas of fluid dynamics, turbulence, and biology. In each area we use the cantilever as a sensitive transducer in order to probe fluctuating forces. In micro and nanometer scale flows the characteristic length scale of the flow approaches and is even exceeded by the fluid mean free path. This limit is beyond the applicability of the Navier-Stokes equations, requiring a rigorous treatment using kinetic theory. In our first study, we conduct a series of experiments in which we use a microcantilever to measure gas dissipation in a nanoscopically confined system. Here, the distance between the gas molecules is of the same order as the separation between the cantilever and the walls of its container. As the cantilever is brought towards the wall, the flow becomes confined in the gap between the cantilever and the wall, affecting the resonant frequency and dissipation of the cantilever. By carefully tuning the separation distance, the gas pressure, and the cantilever oscillation frequency, we study the flow over a broad range of dimensionless parameters. Using these measurements, we provide an in-depth characterization of confinement effects in oscillating nanoflows. In addition, we propose a scaling function which describes the flow in the entire parameter space and which unifies previous theories based on the slip boundary condition and effective viscosity. In our next study, we seek to gain a better understanding of the transition to turbulence in a channel flow. We use a cantilever embedded in the channel wall to perform two sets of experiments: first, we study transition to turbulence triggered by the natural imperfections of the channel walls and second, we study transition under artificially added inlet noise. Our results point to two very different paths to turbulence. In the first case, wall effects lead to an extremely intermittent transitional flow and in the second case, broadband fluctuations originating at the inlet lead to less intermittent flow that is more reminiscent of homogeneous turbulence. The two experiments result in random flows in which high-order moments of near-wall fluctuations differ by orders of magnitude. Surprisingly however, the lowest order statistics in both cases appear qualitatively similar and can be described by a proposed noisy Landau equation. The noise, regardless of its origin, regularizes the Landau singularity of the relaxation time and makes transitions driven by different noise sources appear similar. Our results provide evidence of the existence of a finite turbulent relaxation time in transitional flows due to the persistent nature of noise in the system. In our last study, we turn to biologically-driven fluctuations from bacterial motion. Recent studies suggest that the motion of living bacteria could serve as a good indicator of bacteria species and resistance to antibiotics. To gain a better understanding of these fluctuations, we measure the nanomechanical motion of bacteria adhered to a chemically functionalized silicon microcantilever. A non-specific binding agent is used to attach E. coli to the surface of the device. The motion of the bacteria couples efficiently to the cantilever well below its resonance frequency, causing a measurable increase in its mechanical fluctuations. We vary the bacterial concentration over two orders of magnitude and are able to observe a corresponding change in the amplitude of fluctuations. Additionally, we administer antibiotics (Streptomycin) to kill the bacteria and observe a decrease in the fluctuations. A basic physical model is used to explain the observed spectral distribution of the mechanical fluctuations. These results lay the groundwork for understanding the motion of microorganisms adhered to surfaces and for developing micromechanical sensors for rapid bacterial identification and antibiotic resistance testing.

Au cours de ma thèse, j'ai développé un procédé de fabrication spécifique capable de produire un biocapteur qui combine deux techniques de biodétection différentes, la réponse plasmonique basée sur la résonance de plasmon de surface localisée (LSPR) et la réponse électrochimique. Les méthodes et les résultats qui sont présentés dans ce manuscrit ont été définis pour converger vers un dispositif fluidique unique combinant ces deux approches de détection différentes. Afin de trouver la configuration permettant l'excitation des résonances plasmoniques, la géométrie des nanocavités MIM (métal/isolant/métal) en réseau de lignes interdigitées a été optimisée par des simulations électromagnétiques. La fabrication par nanoimpression douce assistée UV (SoftUV-NIL) a été optimisée et, finalement, la caractérisation optique de ces nanocavités a été comparée avec succès aux simulations théoriques. Parallèlement à la réalisation de ce dispositif nanostructuré, des dispositifs électrochimiques fluidiques plus simples qui intègrent des microélectrodes classiques ont également été développés. L'objectif était d'abord de développer une chimie innovante pour le couple « biotine/streptavidine » et de comprendre ensuite comment les paramètres fluidiques peuvent affecter l'efficacité de capture des biomolécules. Ce manuscrit se termine par une discussion sur le rôle des paramètres fluidiques concernant l’efficacité de la biodétection sur la base de la théorie de Squires. / During my thesis, I worked on the development of a specific fabrication process able to produce a device that combines two different biodetection techniques, plasmonic response based on Localized Surface Plasmon Resonance (LSPR) and electrochemical response. Methods and results that are presented in this manuscript were defined to converge towards a unique fluidic device combining these two different sensing approaches. This device integrates interdigitated array of MIM nanocavities. In order to find the easier working configuration allowing the excitation of plasmonic resonances, their geometry has been optimized through electromagnetic simulations. The fabrication of these dual devices has been optimized based on Soft-UV NIL and, finally, optical characterization of these nanocavities has been successfully compared with theoretical simulations. In parallel to this challenging goal, simpler fluidic electrochemical devices that integrate conventional microelectrodes have also been developed. The goal was first to develop an innovative chemistry for the couple biotin/streptavidin and secondly to learn how fluidic parameters can affect the capture efficiency of molecules. This manuscript ends with a discussion on the role of the fluidic parameters on the biodetection efficiency based on the theory of Squires.

abstract: My research centers on the design and fabrication of biomolecule-sensing devices that combine top-down and bottom-up fabrication processes and leverage the unique advantages of each approach. This allows for the scalable creation of devices with critical dimensions and surface properties that are tailored to target molecules at the nanoscale. My first project focuses on a new strategy for preparing solid-state nanopore sensors for DNA sequencing. Challenges for existing nanopore approaches include specificity of detection, controllability of translocation, and scalability of fabrication. In a new solid-state pore architecture, top-down fabrication of an initial electrode gap embedded in a sealed nanochannel is followed by feedback-controlled electrochemical deposition of metal to shrink the gap and define the nanopore size. The resulting structure allows for the use of an electric field to control the motion of DNA through the pore and the direct detection of a tunnel current through a DNA molecule. My second project focuses on top-down fabrication strategies for a fixed nanogap device to explore the electronic conductance of proteins. Here, a metal-insulator-metal junction can be fabricated with top-down fabrication techniques, and the subsequent electrode surfaces can be chemically modified with molecules that bind strongly to a target protein. When proteins bind to molecules on either side of the dielectric gap, a molecular junction is formed with observed conductances on the order of nanosiemens. These devices can be used in applications such as DNA sequencing or to gain insight into fundamental questions such as the mechanism of electron transport in proteins. / Dissertation/Thesis / Doctoral Dissertation Physics 2019

Many drugs and environmental chemicals, such as antibiotics and chemotherapeutic agents, are nephrotoxic (toxic to the kidney) and are a common cause of acute kidney injury and chronic kidney disease. Conventional tissue models for assessment of drug-induced nephrotoxicity rely on animals or simple cell culture models, which lack tissue characteristics of the human kidney required to accurately predict a drug’s effect in clinical trials. Microfluidic kidney-on-chips can generate tissue with improved human relevance compared to traditional models, however, generally lack high-throughput and multiparametric data collection capabilities for evaluation of nephrotoxic drug exposures. Standard data collection techniques remain limited to fluorescent imaging or colorimetric assays that often focus on single endpoints, are invasive due to the addition of labels, and fail to capture dynamic changes in tissue function. Additionally, conventional toxicological readouts rely on bulk measures of injury, such as cell death, which are less sensitive than sub-lethal changes in cell function and morphology that occur prior to cell death. Due to the challenges above, there is a need for new measurement approaches that enable collection of kinetic, multi-parametric, and sub-lethal readouts of injury in kidney-on-chip systems. In this work, we developed and characterized several measurement approaches for evaluation of tissue function in kidney-on-chip systems and assessment of drug-induced nephrotoxicity. In chapter 2, we developed a novel optical-based oxygen sensing technique for measurement of sub-lethal mitochondrial dysfunction in an array of kidney-on-chips. In chapter 3, we investigated an approach for simultaneous transepithelial electrical resistance (TEER) sensing and flow control to enable near-continuous monitoring of tissue barrier function under different flow conditions. In chapter 4, we demonstrated the use of different data collection modalities, including multiple sensors, fluorescent imaging, and colorimetric-based assays, to generate multi-parametric readouts for evaluation of drug-induced nephrotoxicity in kidney-on-chips. / 2024-05-24T00:00:00Z

[pt] Investigamos as propriedades ópticas de sistemas compostos por nanoilhas de ouro (Au) funcionalizadas com biotina, na superfície de monolitos de xerogéis de sílica (SiO2), visando desenvolvere uma plataforma para biossensores baseados na Ressonância de Plasmon de Superfície Localizada (LSPR), devido à sua elevada sensibilidade a alterações no ambiente químico próximo. Os xerogéis foram sintetizados por meio de um processo de sol-gel de catálise em duas etapas. Uma solução de água deionizada, etanol e tetraetil ortossilicato foi misturada sob agitação magnética a frio, com uma proporção molar de 8,5:3,5:1, e soluções de ácido clorídrico e hidróxido de amônio foram utilizadas como catalisadores para hidrólise e condensação, respectivamente, em moldes de polipropileno. Os géis foram envelhecidos nos moldes e convertidos em xerogéis por secagem a 600 graus C em um forno. Foi depositado um filme de Au de 20 nm na superfície dos xerogéis por sputtering. Em seguida, os xerogéis de sílica com filme de ouro (Au@SiO2) foram submetidos a um tratamento térmico em forno elétrico, para criar as nanoilhas de Au. Observou-se uma mudança de coloração, de azul para rosa, característica das AuNPs. Em seguida, os sistemas AuNP@SiO2 xerogéis foram funcionalizados em dois passos consecutivos com cisteamina (CA) e N-hidroxissuccinimidobiotina (NHSB) para a detecção de avidina em meio aquoso. O par biotina-avidina é um sistema amplamente conhecido para testar biossensores, devido à sua alta especificidade e sensibilidade muito baixa. O processo de funcionalização foi monitorado por absorbância óptica UV-vis para cada passo. Soluções aquosas com concentrações de avidina (10(-6) M, 10(-7) M, 10(-8) M, 10(-9) M e 10(-10M) foram utilizadas para testar a detecção e a sensibilidade. Observou-se um deslocamento médio de 52 nm na absorbância de todas as concentrações testadas, indicando que este sistema é promissor para aplicações em biossensores plasmônicos. / [en] In this study, the optical properties of systems composed of biotin-functionalized gold nanoislands on the surface of silica xerogel monoliths (SiO2) were investigated, aiming to develop an inorganic and inert solid platform for biosensors based on Localized Surface Plasmon Resonance (LSPR) due to their high sensitivity to changes in the nearby chemical environment. The silica xerogels were synthesized through a two-step catalytic sol-gel process, where a solution of deionized water, ethanol, and tetraethyl orthosilicate was mixed under cold magnetic stirring, with a molar ratio of 8.5:3.5:1, and solutions of hydrochloric acid and ammonium hydroxide were used as catalysts for hydrolysis and condensation, respectively, in polypropylene molds. Later, the gels were aged in molds and converted to xerogels by drying at 600 C degrees in an oven. To create the gold nanoislands, a thin film of 20 nm Au was deposited on one of the top surfaces of the xerogels by sputtering, and then the Au@SiO2 xerogels were subjected to heat treatment in an electric furnace. A color change of the samples from blue to pink was observed, characteristic of gold nanoparticles. Then, the AuNP@SiO2 xerogel systems were functionalized in two consecutive steps with cysteamine (CA) and N-hydroxysuccinimide-biotin (NHSB) for the detection of avidin in an aqueous solution. The biotin-avidin pair is a well-known system for testing biosensors, due to its high specificity and very low sensitivity. The functionalization process was monitored by UV-Vis optical absorbance for each step. Aqueous solutions with concentrations of avidin (10(-6) M, 10(-7) M, 10(-8) M, 10(-9) M and 10(-10) M) were used to test detection and sensitivity. An average shift of 52 nm was observed in the absorbance spectrum of all tested concentrations, indicating that this system is a promising structure for plasmonic biosensor applications.

RNA-cleaving DNAzymes (RCDs) are a class of functional nucleic acids that can bind various targets ranging in size from small molecules to large proteins, which results in activation of cleavage activity. The activation of RCDs results in the cleavage of a ribonucleotide site in an otherwise all-DNA substrate, leading to two cleavage fragments. In this work, a previously identified DNAzyme that binds to a protein biomarker endogenous to Helicobacter pylori (J99) crude extracellular matrix was evaluated for coupling to an isothermal amplification method termed rolling circle amplification (RCA) as a way to improve the originally reported detection limit. Three RCD constructs were designed with the goal of generating a cleavage fragment that could act as a primer to initiate RCA. The first method used the original HP DNAzyme, which liberated a short cleavage fragment that could be used as a primer. However, the primer fragment was rapidly digested by the bacterial matrix, preventing RCA. A second method evaluated use of a circularized substrate and separate RCD to generate a primer, however this system was not capable of generating a cleavage fragment. A final method redesigned the original RCD to move the substrate region from the 3’ to the 5’ end of the RCD, causing the longer RCD-containing fragment to be the primer for RCA. In this case, target-triggered cleavage was observed and the resulting primer was sufficiently resistant to digestion to allow its use as a primer for RCA. Preliminary characterization of the rearranged RCD showed that it retained selectivity similar to the original RCD, but that the cleavage rate was slower. In addition, the RCA based reaction, while successful, did not produce improved detection sensitivity relative to unamplified assays. Methods to further improve RCA performance are discussed for future work. / Thesis / Master of Science (MSc)