The detection of particular genetic sequences aids in the early detection and diagnosis of disease; permits monitoring of the health and state of the natural environment; and informs forensic investigations. To date, gene detection has relied on enzymatic amplification followed by optical readout. Though these technologies have advanced dramatically, the instruments and assays are costly and lack portability. The work presented herein addresses an urgent challenge: molecular diagnostics at the point-of-need. This work reports the first electronic chip capable of analyzing - directly, without amplification, and with clinically-relevant sensitivity - multiple genes of interest present in a clinical sample. It reports a dramatic acceleration in sample-to-answer times, with clinically actionable findings in minutes where legacy techniques take hours or days. The key to the sensitivity and speed of the biosensors reported herein lies in their architecture and morphology on multiple lengthscales. It is proven that hybridization-based assays employing a nucleic probe attached to a solid surface can only achieve efficient performance when displayed on a nanotextured surface. It is also discovered that these same sensing elements must reach tens of micrometers into solution to achieve rapid, sensitive detection of nucleic acids in clinical samples. As a result, the materials integrated onto the sensing chip reported herein are engineered on multiple lengthscales - from the nanometers to the tens of micrometers. Engineering is done through a combination of low-cost, convenient top-down photolithographic patterning; combined with hierarchically-designed bottom-up growth of electrodeposited sensing elements. The capstone of this work is a chip that distinguishes among different types of bacteria in an unpurified sample. The chip gives accurate answers in under half an hour when detecting bacteria at a level of 1.5 colony-forming-unit (cfu) per microliter. These speeds and sensitivies enable the application of this technology in point-of-need assays for infectious disease detection. Ultimately, the work showcases the power of bringing together techniques and principles from materials chemistry, biochemistry, applied physics, and electrical engineering to the solution of an important problem relevant to human health.

Electron-excited surface plasmon resonance (eSPR) is investigated for potential use in biosensors. Optical SPR sensors are commercially available at present and these sensors are extremely sensitive, but have the tendency to be relatively large, expensive, and ignore the potentials of microelectronic technology. By employing the use of various microelectronic and nanotechnology principles, the goal is to eventually design a device that exploits the eSPR phenomenon in order to make a sensor which is siginificantly smaller in size, more robust, and cheaper in cost.

Oligo(ethylene glycol) groups have been used as substituents in poly(p-phenyleneethynylene)s (PPEs) to provide solubility, and to boost quantum yield. Properties such as water-solubility and increased quantum yield in aqueous solution make these conjugated systems promising for biosensory applications. In this thesis, a PPE containing a branched ethylene glycol side chain is synthesized as part of a polymer array for glycan biosensing. I also report that the same side chain can be put to use in a red-emissive polymer to lend water solubility. Another monomeric unit, containing ethylene glycol chains, is incorporated into a PPE to create an ampiphilic polymer. The versatility of these polymers allows them to be used for a variety of purposes, some of which will be described herein.

<p>Microcontact printing using elastomeric stamps is a technique used in finding new and efficient ways to produce biodetection chips. Microcontact printed, with poly(dimetylslioxane) (PDMS) stamps, patterns of antibodies have been evaluated using fluorescence microscopy, imaging ellipsometry and atomic force microscopy. Fluorescent conjugated polyelectrolytes form non-covalent molecular complexes with Immunoglobulin-γ type antibodies, antigen binding to the tagged antibody result in spectroscopic shifts. Four different conjugated polyelectrolytes (POWT, POMT, PTT, PTAA) in complex with human serum albumin antibodies (aHSA) have been tested with fluorescence spectroscopy. Complexes of POWT and aHSA gave rise to thelargest wavelength shift when exposed to human serum albumin.</p><p>Several types of commercially available fluorescent antibodies and antigens were used to test the specificity of microcontact printed antibodies to different antigen solutions. Using fluorescence microscopy it could not be shown that printed antibody patterns promote specific adsorption of corresponding antigen. It is proposed however that changed surface characteristics of the substrate due to PDMS residues transferred during printing is the main driving force behind antigen adsorption.</p><p>POMT - poly (3-[(s)-5-amino-5-methoxylcarboxyl-3-oxapentyl]-2,5-thiophenylenehydrochloride)</p><p>POWT - poly (3-(s)-5-amino-5-carboxyl-3-oxapentyl]-2,5-thiophenylenehydrochloride)</p><p>PTAA - polytiophene acetic acid</p><p>PTT - poly (3-[2,5,8-trioxanonyl] thiophene)</p>

The genome sequencing programs have identified hundreds of thousands of genetic and proteomic targets for which there are presently no ascribed functions. The challenge for researchers now is to characterize them, as well as identify and characterize their natural variants. Historically, this has meant studying each individual target separately. However, due to the recent development of multi-analyte microarray devices, these characterizations can be performed in a combinatorial manner in which a single experiment provides information on thousands of targets at a time. In the past decade, microarray technology has settled in on two major designs. The first entails spotting individual receptor types onto a functionalized glass substrate. This is a simple and inexpensive process; however, due to the limited resolution of the mechanical devices used to do the spotting, the densities of these arrays are relatively low. Moreover, receptor preparation requires substantial time and effort. The second variety of microarray uses photolithographic techniques adapted from the semi-conductor industry to chemically synthesize the receptor elements in situ on the sensing surface. Because lithographic patterning is spatially very precise, these arrays achieve very high densities, with as many as one million features per square centimeter. Although these arrays obviate the necessity for laborious "off chip" probe preparation, they are expensive to produce and are limited to two types of receptors (oligonucleotides and peptides). This dissertation presents the development work performed on a hydrogel-based biosensor platform which provides a high density and low cost alternative to the two aforementioned designs. The array features are fabricated lithographically from a liquid pre-polymer doped with biologically active sensing elements at sizes as small as 50[micrometer]. Each of the feature types is uniquely shaped, which enables the features to be mass-produced in batches, pooled together and then assembled into randomly ordered arrays using highly-parallelized self-assembly techniques. The three-dimensional hydrogel features accommodate a wide variety of sensing elements, such as enzymes, antibodies and cells, which cannot be deployed using the traditional designs. This dissertation presents methods developed to integrate cellular and oligonucleotide sensing elements into the hydrogel features which preserve their biological activity and optimize the sensor's performance. / text

Microcontact printing using elastomeric stamps is a technique used in finding new and efficient ways to produce biodetection chips. Microcontact printed, with poly(dimetylslioxane) (PDMS) stamps, patterns of antibodies have been evaluated using fluorescence microscopy, imaging ellipsometry and atomic force microscopy. Fluorescent conjugated polyelectrolytes form non-covalent molecular complexes with Immunoglobulin-γ type antibodies, antigen binding to the tagged antibody result in spectroscopic shifts. Four different conjugated polyelectrolytes (POWT, POMT, PTT, PTAA) in complex with human serum albumin antibodies (aHSA) have been tested with fluorescence spectroscopy. Complexes of POWT and aHSA gave rise to thelargest wavelength shift when exposed to human serum albumin. Several types of commercially available fluorescent antibodies and antigens were used to test the specificity of microcontact printed antibodies to different antigen solutions. Using fluorescence microscopy it could not be shown that printed antibody patterns promote specific adsorption of corresponding antigen. It is proposed however that changed surface characteristics of the substrate due to PDMS residues transferred during printing is the main driving force behind antigen adsorption. POMT - poly (3-[(s)-5-amino-5-methoxylcarboxyl-3-oxapentyl]-2,5-thiophenylenehydrochloride) POWT - poly (3-(s)-5-amino-5-carboxyl-3-oxapentyl]-2,5-thiophenylenehydrochloride) PTAA - polytiophene acetic acid PTT - poly (3-[2,5,8-trioxanonyl] thiophene)

Hydrogen sulfide (H2S) is an important biomolecule for its role in mediating redox homeostasis and signaling biological processes. The study of biological sulfide is currently impeded by a lack of tools available that adequately address the questions currently facing the field. The most pressing of these questions are: how does H2S signal biological processes. To produce tools for studying H2S, chemiluminescent scaffolds were designed to study both H2S producing enzymes and directly measure free H2S. Additionally, small molecule organic persulfides were synthesized and characterized in order to study the properties and reactivity of H2S signaling species. By creating methods to directly measure biological H2S and creating model systems to investigate the active signaling species, the biological reactivity of H2S can be better understood. The luminescent methods for detecting H2S were developed in order to avoid photodecomposition inherent with fluorescent methods while still providing a spectroscopic readout for performing measurements in cells. D-cysteine concentrations can be measured using luciferin bioluminescence, and utilized to back out the H2S producing activity of DAO. Free H2S was measured using luminol derived chemiluminescence. The luminol scaffolds were studied in depth to determine what makes an H2S probe selective for H2S in order to inform the design of future H2S probes. Sulfide signaling processes were investigated using organic persulfide model systems. We found that under reducing conditions persulfides liberate free H2S, and that under basic conditions they decompose. The decomposition pathway is governed by substitution at the -carbon, which dictates the steric accessibility of the inner sulfur atom to act as an electrophile. Persulifdes do not react with acids, and are easily tagged by electrophiles to form disulfides. Persulfides are sufficiently reducing to generate NO from nitrite, facilitating cross-talk between multiple signaling species. This cross talk is mediated by formation of perthionitrite, which may function as an independent signaling species.

abstract: Over the past fifty years, the development of sensors for biological applications has increased dramatically. This rapid growth can be attributed in part to the reduction in feature size, which the electronics industry has pioneered over the same period. The decrease in feature size has led to the production of microscale sensors that are used for sensing applications, ranging from whole-body monitoring down to molecular sensing. Unfortunately, sensors are often developed without regard to how they will be integrated into biological systems. The complexities of integration are underappreciated. Integration involves more than simply making electrical connections. Interfacing microscale sensors with biological environments requires numerous considerations with respect to the creation of compatible packaging, the management of biological reagents, and the act of combining technologies with different dimensions and material properties. Recent advances in microfluidics, especially the proliferation of soft lithography manufacturing methods, have established the groundwork for creating systems that may solve many of the problems inherent to sensor-fluidic interaction. The adaptation of microelectronics manufacturing methods, such as Complementary Metal-Oxide-Semiconductor (CMOS) and Microelectromechanical Systems (MEMS) processes, allows the creation of a complete biological sensing system with integrated sensors and readout circuits. Combining these technologies is an obstacle to forming complete sensor systems. This dissertation presents new approaches for the design, fabrication, and integration of microscale sensors and microelectronics with microfluidics. The work addresses specific challenges, such as combining commercial manufacturing processes into biological systems and developing microscale sensors in these processes. This work is exemplified through a feedback-controlled microfluidic pH system to demonstrate the integration capabilities of microscale sensors for autonomous microenvironment control. / Dissertation/Thesis / Ph.D. Bioengineering 2012

Les bactéries sont les organismes les plus abondants dans le monde. Des études sur les bactéries peuvent être bénéfiques pour la recherche médicale, la qualité des ressources en eau et l'industrie alimentaire. La détection et le marquage fluorescent est une des méthodes les plus utilisées pour des objectifs bioanalytiques. Dans la recherche de marqueurs luminescents et stables, des nouvelles nanoparticules fluorescentes et auto-stabilisées à base de polymères (FNPs, 60 nm) et des chaînes de polymères fluorescents (FPCs, 5nm) ont été développées. Dans un premier chapitre, une méthodologie pour insérer ces FNPs dans la bactérie E.coli a été développée. Pour contrôler si les FNPs sont en effet internalisé, nous avons développé un protocole basé sur l'extinction de luminescence des FNPs par le bleu de méthylène. Dans un second chapitre, les biotines conjuguées de FNPs peuvent être utilisées pour étudier les protéines membranaires spécifiques. En utilisant un lien streptavidine-biotine, un "sandwich" est formé pour construire un pont entre des particules, des anticorps spécifiques et des bactéries. Les images de fluorescence SPR et les images SEM ont démontré l'interaction de la biotine conjuguée de FNPs avec la bactérie E.coli. Dans un troisième chapitre, les chaînes de polymères fluorescents de couleur verte (GFPCs) peuvent facilement entrer dans des bactéries E.coli. Les GFPCs peuvent marquer le cyctoplasme mais pas l'ADN. Les chaînes de polymères fluorescents de couleur rouge (RFPCs) peuvent marquer facilement et efficacement la membrane de bactérie E.coli. Les deux FPCs sont extrêmement brillantes et non toxiques, les chaînes sont solubles dans l'eau. Ce sont de nouveaux matériaux fluorescents pour le marquage interne et externe des bactéries. Dans le dernier chapitre, il est démontré que les FANPs sont sensibles au pH et peuvent être utilisées pour mesurer la croissance de la bactérie E.coli. Les nano-objets détectent rapidement et précisément la croissance des cellules. En effet, leur fluorescence est sensible au changement de pH résultant du métabolisme cellulaire. De plus, ces particules permettent une surveillance en continu d'un grand nombre d'échantillons pour des applications de criblage à haut débit. Les nanomatériaux présentés dans ce manuscrit sont des outils prometteurs pour les applications en biocapteurs et bioimagerie en raison de leur luminosité/brillance et photostabilité élevées ainsi que les possibilités de post-fonctionnalisation. / Bacteria are the most abundant organisms in the world. Investigations and studies on bacteria can be beneficial to medical research, water resources research and food industry. Fluorescent sensing and labeling are commonly used for bioanalytical purposes. In the quest for very bright and stable labels, novel polymer-based, self-stabilized, fluorescent nanoparticles (FNPs, 60 nm) and fluorescent polymer chains (FPCs, 5 nm) have been developed. In the first part, a methodology to insert these FNPs into E.coli bacteria was developed. To control if the FNPs are indeed internalized, we developed a protocol based upon FNPs luminescence quenching by methylene blue. In the second part, a "sandwich" system is built. By using a streptavidin-biotin link, a bridge between particles (FNP), specific antibodies and bacteria is built. SPR, fluorescent images and SEM images demonstrated the interaction of biotin conjugated FNPs with E.coli bacteria. In the third part, interactions of fluorescent polymer chains with bacteria are investigated. Green fluorescent polymer chains (GFPCs) can easily enter into E.coli bacteria. GFPCs can label the cytoplasm but not the DNA. Red fluorescent polymer chains (RFPCs) can label the membrane of E.coli bacteria easily and efficiently. Both FPCs are highly water-soluble, bright and non-toxic, they are novel fluorescent labels for internal and external biological labeling of bacteria. In the last part, it is demonstrated that pH sensitive FANPs can be used to measure the growth of E.coli. They detect rapidly and accurately bacterial growth by signaling the change of pH resulting from cellular metabolism. Moreover, these particles allow for continuous monitoring a large number of samples for high-throughput screening applications. The studied fluorescent nanomaterials are promising tools for biosensing and bioimaging applications due to their brightness, high photostability and rich functionalisation ability.

In the past decade, the field of biosensing has experienced an incredible pace of development, due to the compelling need for accurate and reliable tools for characterization of biomolecular kinetics. Specifically, label-free kinetic measurements are the most direct method for studying molecular binding, for example to establish the efficacy of drug-receptor interactions. For this reason, researchers in the pharmaceutical industry rely heavily on label-free detection for drug and antibody screening. Meanwhile, in the biosafety industry and healthcare, there is great demand for screening tools that can target biothreats, in order to accurately recognize the presence of toxins and pathogens with high sensitivity in diverse samples, such as bodily fluids, food and drinking water. This research topic has become particularly relevant during the recent pandemic, where vaccine development was carried out side by side with quantification and characterization of single viral particles. Here, we introduce a versatile biosensing platform capable of characterizing virtually any type of target compound, down to the single molecule level. For this work, we have improved the Interferometric Reflectance Imaging Sensor (IRIS) to perform accurate measurements of the binding kinetics of analytes ranging in molecular weight from less than 1kDa (small molecules) to more than 1MDa (biological nanoparticles). For the first time, we demonstrate multiplexed kinetic binding characterization of small molecules to surface immobilized antibody probes, as well as detection and phenotyping of large and complex analytes, on the same platform. The IRIS platform utilizes the optical interference signal produced by thinly layered substrates in order to precisely measure the thickness of a transparent film atop a silicon chip. In the context of this work, dynamic characterization of a wide range of biomolecular and nanoparticle targets was made possible by a multidimensional optimization, in order to improve both the sensitivity and the dynamic range of the instrument. Analysis of low molecular weight compounds required a significant increase in signal to noise ratio, which was achieved through averaging, as well as complete elimination of background solution effects ('bulk effect’). Additionally, the best surface chemistry for each application was identified by a new technique which consists of immobilizing capture probes on a multiplexed array of active polymers functionalized on the same sensor surface, allowing for simultaneous side-by-side comparison of their performance. Surface chemistry plays a huge role in kinetic measurements, in terms of probe functionality, steric hindrance, charge distribution and diffusion effects. Finally, imaging optics, illumination wavelength, and thickness of the silicon dioxide film were optimized to perform detection and phenotyping of large analytes, such as extracellular vesicles (EVs) and antibody-conjugated gold nanoparticles (mAb-GNPs). Results obtained from numerical simulations allowed for selection of the best experimental parameters for each application. Experimentally, mAb-GNPs were utilized to produce a real-time sandwich lateral flow assay. In this context, we demonstrated how the improved IRIS platform can bridge the gap between single-particle detection ('digital’ configuration) and bulk reflectance measurements ('analog’ configuration), creating a new 'hybrid' system (h-IRIS), which only requires minimal hardware adjustments to easily switch from one modality to the other. This brought a substantial improvement in sensitivity, improving the limit of detection by three orders of magnitude and enabling single-molecule level measurements. Finally, future system optimization ideas are presented to achieve even higher accuracy and further extend the range of target analytes.