Fluorescence resonance energy transfer (FRET)-based biosensors have been designed to fluorometrically detect everything from proteolytic activity to receptor-ligand interactions and structural changes in proteins. While a wide variety of fluorophores have demonstrated effectiveness in FRET probes, several potential sensor components are particularly notable. Semiconductor quantum dots (QDs) are attractive FRET donors because they are rather bright, exhibit high quantum yields, and their nanoparticulate structure enables the attachment of multiple acceptor molecules. Fluorescent proteins (FPs) are also of particular interest for fluorescent biosensors because design elements necessary for signal transduction, probe assembly, and device delivery and localization for intracellular applications can all be genetically incorporated into the FP polypeptide. The studies described in this thesis elucidate the important parameters for concerted QD-FP FRET probe design. Experimental results clarify issues of FRET pair selection, probe assembly, and donor-acceptor distance for the multivalent systems. Various analysis approaches are compared and guidelines asserted based on the results. To demonstrate the effectiveness of the QD-FP FRET probe platform, a ratiometric pH sensor is presented. The sensor, which uses the intrinsic pH-sensitivity of the FP mOrange to modulate the FP/QD emission ratio, exhibits a 20-fold change in its ratiometric measurement over a physiologically interesting pH range, making it a prime candidate for intracellular imaging applications.

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

A novel capture matrix utilizing human serum albumin (HSA) and streptococcal Protein G (PG), which possesses an albumin binding domain (ABD), was used to immobilize antibodies for improved bacterial capture efficiency in immunoassays. Enzyme linked immunosorbent assays (ELISA) were used to characterize and optimize a specific protocol for the HSA-PG capture matrix; which revealed several critical factors that should be considered. The Fc binding domain, on PG, should have high affinity for the species of capture antibody used in the assay. Goat and rabbit species antibodies bound strongly to the Fc binding domain of PG. Displacement of the capture antibody, by the detector antibody should be avoided to reduce background signals. The Fc binding domain on PG should have equivalent or lower affinity for the detector antibody, when compared to the capture antibody. Goat species antibody, used as a detector antibody, did not displace the same-species capture antibody. ELISA analysis showed detection of Escherichia coli O157:H7 cells at 1.0 x 104 CFU/ml using HSA-PG and goat antibody raised against Escherichia coli O157:H7; unlabeled antibody was used for capture while HRP labeled antibody was used for detection. Studies were performed on an automated fiber optic biosensor, RAPTOR, which was used for the rapid detection of pathogens. Biosensor assays showed detection of E. coli O157:H7 at 1.0 x 10³ CFU/ml in PBS and 1.0 x 105 CFU/ml in homogenized ground beef supernatant. Capture efficiency of the HSA-PG capture matrix was studied using the biosensor and GFP-E. coli O157:H7. The amount of cells captured was less than one percent of the sample concentration. This limit of detection and capture efficiency was comparable to the streptavidin-biotin capture matrix.

There is no rapid diagnostic technique at medical examiners' offices to determine if a decedent is infected with Mycobacterium tuberculosis. Present diagnostic testing requires at least one month for results. A biosensor-based sandwich immunoassay for the detection of M. tuberculosis was developed in this study. M. tuberculosis polyclonal antibody was used for target antigen capture and detection in the immunoassay. Live attenuated M. tuberculosis (ATCC 25177) suspended in phosphate-buffered saline with 0.1% Tween 20 was used as the antigen in the detection assay. The Analyte 2000 was the initial biosensor platform. Initial testing was of Freund's adjuvant complete. M. tuberculosis was detected 50% of the time at 1,000,000 CFU/ml and 100% of the time at 10,000,000 CFU/ml and 100,000,000 CFU/ml. Live attenuated M. tuberculosis was also tested using the Analyte 2000 biosensor. Detection was obtained 87.5% of the time at 1,000,000 CFU/ml and 100% of the time at 10,00 0,000 CFU/ml and 100,000,000 CFU/ml. The RAPTOR, an automated, portable instrument, was then tested as the fiber optic biosensor platform. Positive biosensor detection was obtained 75% of the time at cell concentrations of 1,000,000 CFU/ml, 95% of the time at 10,000,000 CFU/ml, and 99% of the time at 100,000,000 CFU/ml. Live attenuated M. tuberculosis suspended in PBST and seeded into decedent lung tissue was tested using the RAPTOR. Positive detection was obtained 21% of the time at cell concentrations of 1,000,000 CFU/ml, 86% of the time at 10,000,000 CFU/ml and 100% of the time at 100,000,000 CFU/ml. Antibody specificity studies using ELISA were performed to determine the anti-M. tuberculosis antibody's cross reactivity with microorganisms other than M. tuberculosis. M. tuberculosis actively growing in the lung of an individual is found at levels of 10,000,000 to 1,000,000,000 CFU in the lesions of the lung. This study determined that the RAPTOR biosensor assay was capable of detecting the presence of M. tuberculosis in lung tissue homogenate within three hours when the concentration of M. tuberculosis was 10,000,000 to 1,000,000,000 CFU/ml.

The development of analytical tools for investigating biological pathways on the molecular level has provided insight into diseases and disorders. However, many biological analytes such as glucose and inositol phosphate(s) lack the optical or electrochemical properties needed for detection, making molecular sensing challenging. Scintillation proximity assay (SPA) does not require analytes to possess such properties. SPA uses radioisotopes to monitor the binding of analytes to SPA beads. The beads contain scintillants that emit light when the radiolabeled analytes are in close proximity. This technique is rapid, sensitive and separation-free. Conventional SPA beads, however, are large relative to the cells and made of hydrophobic organic polymers that tend to aggregate or inorganic crystals that sediment rapidly in aqueous solution, thus limiting SPA applications. To overcome these problems, polystyrene core-silica shell nanoparticles (NPs) doped with pTP and dimethyl POPOP were fabricated to produce scintillation NPs that emit photons in the blue region of visible light. The developed scintillation particles are approximately 250 nm in diameter (i.e. 200 nm of core diameter and 10-30 nm of shell thickness), responsive to β-decay from tritium (³H) and have sufficient stability in the aqueous media. DNA hybridization-based SPA was performed to determine whether the scintillation NPs could be utilized for SPA applications. A 30-mer oligonucleotide was immobilized on the polystyrene core-silica shell NPs to give approximately 7.6 x 10³ oligonucleotide molecules per NP and ³H-labeled complementary strand was annealed to the immobilized strand. At the saturation point, increases in scintillation signal due to oligonucleotide binding to the NPs were about 9 fold compared to the control experiments in which no specific binding occurred, demonstrating that the scintillation NPs can be utilized for SPA. Along with the improved physical properties including smaller size and better stability in the aqueous system, the developed scintillation NPs could be potentially useful as biosensors in cellular studies.

The use of smartphone for the detection of biological constituents is becoming a useful tool as a point-of-care (POC) device and diagnostics. When combined with microfluidic paper analytic devices (μPAD) and particle immunoassays, we have the ability to detect bacterial pathogens with sensitivity and specificity. Environmental conditions as well as variability in smartphone imaging and the cellulose in paper microfluidics however can sometimes easily interfere with the detection of small signal changes. Combining this issue with the detection of pathogens in blood (our model biological sample of interest) becomes difficult with such a platform because of the complexity of the sample matrix. However, in this research we take a novel approach at utilizing polystyrene’s auto-fluorescence and the high energy of UV LEDs in a particle immunoassay in order to increase our signal change. We first characterized how the smartphone actually responds to UV light (275-385 nm) with respect to the RGB components in its images. We were then able to determine a favorable response using the 385 nm UV LED. The detection of green fluorescence by polystyrene particles was possible by analyzing the smartphone’s image in the green channel. There was a significant difference in signal change with blood samples including polystyrene versus just blood samples with a normalized signal intensity change of 2.5 (150%). The detection of polystyrene fluorescence was translated into a field deployable prototype, where preliminary trials showed promising results in detecting Escherichia coli in blood with a current limit of detection of 50 CFU/ml. With further experimentation and optimization the limit of detection could be improved to 10 CFU/mL, making it a very useful tool in the detection of blood borne pathogens to prevent complications with onset bacteremia and the more serious cases of sepsis. This assay platform could provide an easy to use solution with detection in a short time (assay time of 1 min) compared to the lengthy blood culture monitoring or biomarker detection.

Semiconductor nanocrystals, otherwise known as Quantum dots (Q dots), are a new type of fluorophore that demonstrates many advantages over conventional organic fluorophores. These advantages offer the opportunity to improve known immunofluorescent methods and immunofluorescent biosensors for rapid and portable bacterial detection in water. The detection of the micro organism Escherichia coli O157:H7 by attenuation of a fluorophore’s signal in water was evaluated alone and in the presence of another bacterial species. A sandwich immunoassay with antibodies adhered to SU-8 as a conventional comparison to our novel attenuation detection was also conducted. The assays were tested for concentration determination as well as investigation for cross reactivity and interference from other bacteria and from partial target cells. In order to immobilize the capture antibodies on SU-8, an existing immobilization self-assembly monolayer (SAM) for glass was modified. The SAM was composed of a silane ((3-Mercaptopropyl) trimethoxysilane (MTS)) and hetero-bifunctional cross linker (N-γ-maleimidobutyryloxy succinimide ester (GMBS)) was utilized in this procedure. The SU-8 surface was activated using various acids baths and oxygenated plasma, and it was determined that the oxygenated plasma yielded the best surface attachment of antibodies. The use of direct fluorophore signal attenuation for detection of the target E. coli resulted in the lowest detectable population of 1x10¹ cfu/mL. It was not specific enough for quantitative assessment of target concentration, but could accurately differentiate between targeted and non-targeted species. The sandwich immunofluorescent detection on SU-8 attained the lowest detectable population of 1x10⁴ cfu/ml. The presence of Klebsiella pneumoniae in solution caused some interference with detection either from cross reactivity or binding site blocking. Partial target cells also caused interference with the detection of the target species, mainly by blocking binding sites so that differences in concentration were not discernable. The signal attenuation not only had a better lowest detectable population but also had less measurement error than the sandwich immunoassay on SU-8 which suffered from non-uniformed surface coverage by the antibodies.

An important research area in medicine is molecular diagnostics of cancers and infectious diseases, which can be diagnosed, managed and treated more effectively with genetic information. We have developed an integrated sample to answer bacterial detection platform combining a simple, universal bacterial lysis approach and sensitive nanomaterial electrochemical biosensors. Lysis is rapid and effective at releasing intercellular nucleic acid targets. The platform was directly challenged with unpurified lysates and successful at determining the presence of clinically relevant concentrations within 30min from sample to answer. Another important aspect of biosensor development is the development of cheap and efficient methods for manufacturing nanostructured microelectrodes. Previously, we have used costly silicon wafers for fabrication. Here we explored alternate inexpensive materials for fabrication including printed circuit boards, plastics and glass. We show that plain borosilicate glass is effective for templated bottom-up fabrication, with comparable performance to expensive silicon based nanostructured microelectrodes. Current state-of-the-art readout of many biomarkers is hampered by serially addressing arrays of low cost biosensors, without the use of high cost active electronics. Here we have developed a new concept, solution-based electrochemical circuits, which makes highly multiplexed sensing feasible on the surface of low-cost, glass chips. This method utilizes the idea that physical separation of liquid on an insulator can result in electrochemical isolation. Using this we can reduce the number of outputs to 2√n, where n would be the number of serially connected sensors. We use urinary tract infections as a model system and prove that we can accurately detect species and antimicrobial resistance in multiplexed formats at clinically relevant concentrations.

The food processing industry requires alternative methods for detecting the foodborne pathogen Listeria monocytogenes that are cheaper and faster than the current methods. Conventional antibodies and their fragments have been used as biorecognition elements in sensors before, but their use is hindered by high production cost and relative instability. These issues are resolved by VHH fragments, derived from the heavy chain-only antibodies found in Camelidae. VHHs are inexpensive to produce, and are more resistant to environmental stressors. This work describes the isolation of phage-displayed VHHs that recognize recombinant Internalin B, a virulence factor characteristic of L. monocytogenes. Clone R303 was chosen for further characterization, and shown to bind full-length Internalin B. Furthermore, immobilized R303 was shown to capture L. monocytogenes cells. This panel of VHHs, particularly R303, can be utilized by colleagues within the Sentinel Bioactive Paper Network to make a viable biosensor for L. monocytogenes. / Sentinel Bioactive Paper Network

An important research area in medicine is molecular diagnostics of cancers and infectious diseases, which can be diagnosed, managed and treated more effectively with genetic information. We have developed an integrated sample to answer bacterial detection platform combining a simple, universal bacterial lysis approach and sensitive nanomaterial electrochemical biosensors. Lysis is rapid and effective at releasing intercellular nucleic acid targets. The platform was directly challenged with unpurified lysates and successful at determining the presence of clinically relevant concentrations within 30min from sample to answer. Another important aspect of biosensor development is the development of cheap and efficient methods for manufacturing nanostructured microelectrodes. Previously, we have used costly silicon wafers for fabrication. Here we explored alternate inexpensive materials for fabrication including printed circuit boards, plastics and glass. We show that plain borosilicate glass is effective for templated bottom-up fabrication, with comparable performance to expensive silicon based nanostructured microelectrodes. Current state-of-the-art readout of many biomarkers is hampered by serially addressing arrays of low cost biosensors, without the use of high cost active electronics. Here we have developed a new concept, solution-based electrochemical circuits, which makes highly multiplexed sensing feasible on the surface of low-cost, glass chips. This method utilizes the idea that physical separation of liquid on an insulator can result in electrochemical isolation. Using this we can reduce the number of outputs to 2√n, where n would be the number of serially connected sensors. We use urinary tract infections as a model system and prove that we can accurately detect species and antimicrobial resistance in multiplexed formats at clinically relevant concentrations.