Doctor of Philosophy

dissertationMonitoring and remediation of environmental contaminants (biological and chemical) form the crux of global water resource management. There is an extant need to develop point-of-use, low-power, low-cost tools that can address this problem effectively with min­ imal environmental impact. Nanotechnology and microfluidics have made enormous ad­ vances during the past decade in the area of biosensing and environmental remediation. The "marriage" of these two technologies can effectively address some of the above-mentioned needs [1]. In this dissertation, nanomaterials were used in conjunction with microfluidic techniques to detect and degrade biological and chemical pollutants. In the first project, a point-of-use sensor was developed for detection of trichloroethylene (TCE) from water. A self-organizing nanotubular titanium dioxide (TNA) synthesized by electrochemical anodization and functionalized with photocatalytically deposited platinum (Pt/TNA) was applied to the detection. The morphology and crystallinity of the Pt/TNA sensor was characterized using field emission scanning electron microscope, energy dis­ persive x-ray spectroscopy, and X-ray diffraction. The sensor could detect TCE in the concentrations ranging from 10 to 1000 ppm. The room-temperature operation capability of the sensor makes it less power intensive and can potentially be incorporated into a field-based sensor. In the second part, TNA synthesized on a foil was incorporated into a flow-based microfluidic format and applied to degradation of a model pollutant, methylene blue. The system was demonstrated to have enhanced photocatalytic performance at higher flow rates (50-200 ^L/min) over the same microfluidic format with TiO2 nanoparticulate (commercial P25) catalyst. The microfluidic format with TNA catalyst was able to achieve 82% fractional conversion of 18 mM methylene blue in comparison to 55% in the case of the TiO2 nanoparticulate layer at a flow rate of 200 L/min. The microfluidic device was fabricated using non-cleanroom-based methods, making it suitable for economical large-scale manufacture. A computational model of the microfluidic format was developed in COMSOL Multiphysics® finite element software to evaluate the effect of diffusion coefficient and rate constant on the photocatalytic performance. To further enhance the photocatalytic performance of the microfluidic device, TNA synthesized on a mesh was used as the catalyst. The new system was shown to have enhanced photocatalytic performance in comparison to TNA on a foil. The device was then employed in the inactivation of E. coli O157:H7 at different flow rates and light intensities (100, 50, 20, 10 mW/cm2). In the second project, a protocol for ultra-sensitive indirect electrochemical detection of E. coli O157:H7 was reported. The protocol uses antibody functionalized primary (magnetic) beads for capture and polyguanine (polyG) oligonucleotide functionalized sec­ ondary (polystyrene) beads as an electrochemical tag. The method was able to detect concentrations of E. coli O157:H7 down to 3 CFU/100 mL (S/N=3). We also demonstrate the use of the protocol for detection of E. coli O157:H7 seeded in wastewater effluent samples

Rapid Label-Free Detection of Pathogens by Local pH Modulation

Pathogenic bacteria present major issues for human health across the world. One of the ways to mitigate the negative impacts from contaminated food and water sources is to decrease the time required to test potentially contaminated sources. This study examined a new method of label free detection using local pH modulation to quantitatively detect bacteria. By tagging antibodies with a pH-sensitive fluorescent dye it was possible to detect the presence of bacteria bound to antibodies. Local pH can be effected by the presence of charged molecules because they attract counter ions. By utilizing the negatively charged surface of bacteria to attract counter ions in the form of hydrogen ions the local pH can be lowered, thereby lowering the fluorescence of fluorescein. By measuring fluorescence with respect to bacterial cell concentration a relationship between bacteria concentration and fluorescence can be established. It is also advantageous to know if the pathogens detected are active and alive or dead. Adding a rapidly uptaken carbon source (glucose) allows for differences between live and dead cells to be detected. This approach was tested in microtiter plates and using immunomagnetic beads as the testing platforms. Using microtiter plates concentrations of ~10^6 E. coli cells could be detected although not to a statistically significant level. The addition of glucose showed that live cells could be distinguished from UV killed cells but cell numbers could not be established. Immunomagnetic beads displayed inconclusive results indicating the need for continued experiments

Advances in Microfluidics and Lab-on-a-Chip Technologies

Advances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains such as biology research, infectious disease management, food safety, and biodefense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care. It has also introduced new functionality, enhanced sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods such as nucleic acid amplification, next-generation sequencing, high resolution melting analysis, cytogenetics, protein detection and analysis, and cell sorting. We also review microfluidic sample preparation platforms applied to molecular diagnostics and targeted to sample-in, answer-out capabilities

Biosensorsüsteem mastiiti põhjustavate bakterite kiireks ja samaaegseks määramiseks piimas

Väitekirja elektrooniline versioon ei sisalda publikatsiooneMastiit on udarapõletik, mis enamasti tekib patogeensete mikroorganismide sattumisel läbi nisajuha udaraveerandisse, olles peamine lüpsilehmade nakkushaigus. Mastiidi poolt põhjustatud kahju kogu maailma 271 miljoni piimalehma kohta on hinnanguliselt 16-26 miljardit eurot aastas. Traditsiooniliste meetoditena mastiiti tekitavate bakterite identifitseerimiseks on tänapäeval kasutusel mikrobioloogilised analüüsid, mis võtavad aega 1-2 päeva ja laboratoorsetes tingimustes tehtavad patogeenide geenianalüüsid, mille tegemiseks kulub 6 tundi. Kuna ravi edukuse tagamiseks on selle täpsuse kõrval väga oluline ka selle võimalikult operatiivne alustamine, siis on vajalik välja töötada sellised analüüsimeetodid, mis võimaldavad patogeenide identifitseerimist oluliselt kiiremini kui praegu ning mida on võimalik kasutada farmides kohapeal. Tänasel päeval sellised meetodid puuduvad. Doktoritöö käigus töötati välja biosensorsüsteem ja mõõtemetoodika kolme peamise mastiiti tekitava patogeeni – Staphylococcus aureus’e Escherichia coli ja Streptococcus uberis’e määramiseks nii eraldi kui ühtlasi ka kõikide nimetatud patogeenide koos määramiseks. Biosensorsüsteemi konstrueerimisel uuriti selle tundlikkust, tööpiirkonda, selektiivsust ja sobivust rakendamiseks keerulistes maatriksites nagu piim. Biosensori selektiivsus kindla patogeeni määramiseks teiste bakterite olemasolul oli väga hea ning seda on võimalik kasutada mitme bakteri samaaegseks määramiseks. Väljapakutud biosensorsüsteemil põhinevat mõõtesüsteemi on võimalik kasutada loomade tervise automatiseeritud kontrolliks farmis kohapeal ja seeläbi kiiresti identifitseerida juba varajases staadiumis potentsiaalne haigus. Varajane haiguse avastamine aitab alustada koheselt kiiret ja sobivat ravi, parandades seeläbi looma heaolu ja piima kvaliteeti ning vähendada tootmiskulusid ja majanduslikku kahju.Mastitis, mostly caused by bacterial infection of the mammary gland, is a major health problem of dairy cows. The resulting decrease of milk production and reduction of its quality along with medication costs and probable premature culling of animals cause essential economic burden. The total mastitis caused losses in dairy industry are estimated to be 16–26 billion € annually in view of a global population of 271 million dairy cows. For the identification of mastitis-causing pathogens, the gold standard is microbiological culturing of bacteria, which in recent years has been partially replaced by polymerase chain reaction analyses of bacterial DNA. Although reliable, these methods require hours to obtain results, not allowing effective treatment of animals and optimal milk processing. Therefore to assure timely and correct treatment of animals, there is a great need for a method applicable for rapid automatic detection of mastitis causing pathogens in milk. A rapid method and an immuno-biosensing system have been proposed and applied for the rapid multiplex detection of major mastitis causing pathogens (Staphylococcus aureus, Escherichia coli and Streptococcus uberis) in milk. The key features of the biosensor development were its selectivity, sensitivity, working range and applicability for rapid automated analyses in complex biological sample matrix like milk. The selectivity of the biosensing system in the presence of several pathogens was very good and it was applicable for the multiplex detection of different pathogens. Based on the results of the present study, the proposed biosensing system has a great potential to serve as a tool for in-line automatic monitoring of milk and animal health in dairy farms

Rapid and Sensitive Detection of Escherichia coli O157:H7 Using a QCM Sensor based on Aptamers Selected by Whole-Bacterium SELEX and a Multivalent Aptamer System

Escherichia coli O157:H7 is one of the top five pathogens contributing to foodborne diseases, causing an estimated 2,138 cases of hospitalization in the US each year. The extremely low infectious dose demands for more rapid and sensitive methods to detect E. coli O157:H7. The objective of this study is to select aptamers specifically binding to E. coli O157:H7 using whole-bacterium SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and to create a multivalent aptamer system by rolling circle amplification (RCA) with the selected aptamer sequence for sensitive detection of E. coli O157:H7 using a quartz crystal microbalance (QCM) sensor. Briefly, A total of 19 rounds of selection against live E. coli O157:H7 and 6 rounds of counter selection were performed for SELEX. One sequence S1 that appeared 16 (out of 20) times was characterized and a dissociation constant (Kd) of 10.30 nM was obtained. Using phi29 DNA polymerase, RCA reaction was performed, which produced a long ssDNA strand composed of thousands of repetitive aptamer sequences, termed as a multivalent aptamer system, on the electrode. The QCM sensor based on a multivalent aptamer system was able to quantitatively detect E. coli O157:H7. The limit of detection (LOD) of the QCM sensor was determined to be 34 CFU/ml, respectively, with the whole detection procedure in less than 40 min. The QCM sensor also showed high specificity for E. coli O157:H7 when it was cross-tested with five non-target bacteria. The QCM aptasensor in this study provided a common platform for detection of different foodborne pathogens

Advances in microfluidics and lab on a chip technologies

pre-printAdvances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains like biology research, infectious disease management, food safety and bio-defense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care (POC). They have also introduced new functionality, enhanced the sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods like nucleic acid amplification, next generation sequencing, high resolution melting analysis, cytogenetics, protein detection and analysis, and cell sorting. We also review microfluidic sample preparation platforms applied to molecular diagnostics and targeted to sample-in, answer-out capabilities

Shiga Toxin-Producing Escherichia coli (STEC) Detection Strategies with Formalin-Fixed STEC Cells

Certain pathogenic Escherichia coli known as Shiga toxin (Stx)-producing Escherichia coli (STEC) are commensals in cattle, and typically cause bloody diarrhea in humans once the Stx toxin is secreted in invaded intestinal epithelial cells. Infections with STEC cells can lead to hemolytic uremic syndrome, which is commonly associated with kidney failure. Several STEC serogroups have been declared adulterants in raw, non-intact ground meat, and future regulations could potentially lead to a higher number of STEC serogroup detection strategies for these pathogenic microorganisms. Microbiological research laboratories may benefit from formalin-fixed STEC cells for periodic (daily, weekly, monthly, among others) instrument validation/calibration by serving as a working set of known cell concentration samples and internal standard i.e. positive control. These cell concentrations may be used across laboratories in different geographical locations, within an individual laboratory, and across a broad range of detection assays (molecular as well as immuno-based). This thesis consists of three research parts: a comprehensive literature review that covers STEC incidence in foods and molecular detection techniques (chapter 1), a literature review that covers immuno-based detection strategies (chapter 2), and a research manuscript that involves the development of an internal standard and positive control with formalin-fixed STEC cells that can be used for a broad range of molecular as well immuno-based detection assays for instrument calibration and validation purposes (chapter 3)

Detection, Control and Contamination of Mycotoxins

The objective of this collection is to illustrate the most recent research on the development of novel and/or rapid methods for mycotoxin determination, and to propose new strategies for monitoring and/or reducing mycotoxin contamination. Innovative sample preparation techniques or protocols and the possibility of multiclass mycotoxin detection will be very positively considered for possible inclusion in this Special Issue. Both methods based on (bio)sensors and chromatography with various detectors (including mass spectrometry) are welcome. Applications of already published methods on new matrices without any modification will not be accepted. However, extensive studies and monitoring on the spread of contamination through the food production chain could be of interest for this collection

Configurable and Up-Scalable Microfluidic Life Science Platform for Cell Based Assays by Gravity Driven Sequential Perfusion and Diffusion

Microfluidics has the potential to significantly change the way modern biology is performed, but for this potential to be realized several on-chip integration and operation challenges have to be addressed. Critical issues are addressed in this work by first demonstrating an integrated microfluidic tmRNA purification and real time nucleic acid sequence based amplification (NASBA) device. The device is manufactured using soft lithography and a unique silica bead immobilization method for the nucleic acid micro purification column. The integrated device produced a pathogen-specific response in < 3 min from the chip-purified RNA. Further enhancements in the device design and operation that allow the on-chip integration of mammalian cell handling and culturing produced a novel integrated NASBA array. This system demonstrated for the first time that it is possible to combine on a single micro-device cell culture and real time NASBA. In order to expand the cell based assay capabilities of the integrated NASBA array and simplify the device operation novel hydrodynamics and cell sedimentation within trench structures and gravity driven sequential perfusion and diffusion mechanisms were developed. These mechanisms were characterized and implemented within an iCell array device. iCell array can completely integrate cell based assays with bio-analytical read-out. The device is highly scalable and can enable the configurable on-chip integration of procedures such as adherent and non-adherent cell-culture, cellstimulation, cell-lysis, cell-fixing, protein-immunoassays, bright field and fluorescent microscopic monitoring, and real time detection of nucleic acid amplification. The device uses on-board gravity driven flow control which makes it simple and economical to operate with dilute samples (down to 5 cells per reaction), low reagent volumes (50 nL per reaction), highly efficient cell capture (100% capture rates) and single cell protein and gene expression sensitivity. The key results from this work demonstrate a novel technology for versatile, fully integrated microfluidic array platforms. By multiplexing this integrated functionality, the device can be used from routine applications in a biology laboratory to high content screenings