No description available.

No description available.

Microfluidics have been created to acquire, operate, and process complex fluids in extremely tiny volumes with high efficiency and high speed, and without the requirement for an experienced operator. In addition, microfluidic systems also enable miniaturization and incorporation of different complex functions, which can help bring intricate diagnostic tools out of the laboratories. Ideally, these systems should be inexpensive, precise, reliable, robust, and well-suited to the medical diagnostic systems. Most of the microfluidic devices reported previously were based on devices made of polydimethylsiloxane (PDMS). PDMS is a material that dissolves in many common organic solvents. Meanwhile, it is also prone to absorb small molecules like the proteins, which is detrimental to a stable and reliable result. Current work focuses on bioassays that are badly needed in our life and these bioassays are addressed based on microfluidic platform with different materials. The translation of microfluidic technology into large scale implementations highly relies on new materials that address the limitations of PDMS. Firstly, we fabricated two different microfluidic platforms for rapid antimicrobial susceptibility testing (AST). One was made of hydrogel, and the bacterial cells were cultured on the top of the device; the other was of polypropylene (PP), and bacterial cells were cultured inside the microchannels. Meanwhile, we developed a novel "barcode" sensor, a microscope-free method for cell accumulation and cell counting, as the downstream of the PP-based chips. As a result, AST can be accomplished simply through an application on a mobile phone rather than using an expensive and sophisticated microscope. Secondly, we presented a self-contained paper-based system for lead(II) ion detection based on G-quadruplex-based luminescence switch-on assay, comprising a novel type of paper-based chip and a matching portable device. Different from the reported paper-based devices, the paper substrate we chose was art paper, which is used for printing magazines. This type of paper could prevent the absorption of liquid into the paper matrix and hold the liquid in place for a period of time; and it could also be used for temporary liquid containing like a plastic substrate (such as polypropylene (PP) and polystyrene (PS)), but the surface of the paper is inherently hydrophilic. In such a design, liquid drops are suspended on the surface of the device in designed reservoirs, rather than absorbed into the paper; when the chip is tilted, the liquid drops will move to other reservoirs according to the guidance of channels defined on the surface. To differentiate it from reported μPAD devices that are fabricated with water-permeable paper, we name this new type of paper-based devices suspending-droplet mode paper-based microfluidic devices (SD-μPAD). Different from the conventional μPADs that use capillary force to drive liquid, our SD-μPADs uses wetting and gravity as driving force. To fabricate the superhydrophobic pattern on the paper device, we developed a new microcontact printing-based method to produce inexpensive and precisely patterned superhydrophobic coating on paper. The coating material is poly(dimethylsiloxane) (PDMS), a hydrophobic and transparent silicone that has long been used for fabricating microfluidic devices. Importantly, the negative-relief stamp we used is made of Teflon, a non-stick polymer, so that the PDMS-coated paper could be peeled from the stamp flawlessly. After such fabrication process, the stamped area of the paper is coated with a textured PDMS layer that is decorated with arrays of micropillars, which could provide superhydrophobic effect and most effectively hold the droplets in place; the remaining area of the paper is still hydrophilic. As a demonstration of this new design, we developed a method using the reaction characteristics of iridium(III) complex for rapid, onsite detection of lead(II) ions in liquid samples. As the reagents have already been loaded onto the paper device during fabrication, the only reagent the users need to add is water. Because of the large Stokes shift of the iridium(III) complex probe, inexpensive optical filters can be employed, and we were able to make an inexpensive, battery-powered compact device for routine portable detection using a smartphone as a detector, allowing the rapid analysis and interpretation of results on site as well as the automatic dissemination of data to professional institutes, including tests even in poor rural areas in developing countries. Thirdly, we upgraded our suspending-droplet mode paper-based microfluidic device (SD-μPAD), which is used for the detection of lead(II) ions in liquid solution. The reason is that our paper-based SD chips are not suitable for long reaction process (> 20 min) detection of biomolecules due to the potential permeation and contaminating problems of art papers. Hence, we chose polypropylene (PP), a hydrophobic, cheap, and thermal stable material (< 110°C), as the material for the fabrication of the SD microfluidic chip. We established a convenient, low-cost, portable and reliable platform for monitoring VEGF165 accurately, which can be applied for point-of-care (POC) testing. In this project, we also employed the label-free oligonucleotide-based luminescence switch-on assay on the microfluidic platform, which possesses the advantages of high sensitivity and high selectivity. Based on the detection of VEGF165 in a three-step reaction process, we adopted a new design for the droplet transfer throughout the channels. This design could migrate the droplet through the chambers via controlling the orientation of the chip, which systematically combined the superhydrophobic force of the coating, the gravity of the droplet and the surface tension between PP and droplet. Therefore, traditional micro pump could be avoided and the total cost for the device could be substantially reduced. In addition, we developed an automatic, matched and portable device for the detection of VEGF165, which assembled by a rotatable chip holder, a UV lamp, a filter, and a camera. Finally, we developed a new whole Teflon membrane-based chip for the aptamer screening. Our article "Whole-Teflon microfluidic chips" introduced the fabrication of a microfluidic device entirely using Teflon materials, one group of the most inert materials in the world. It was a successful and representative introduction of new materials into the fabrication of microfluidic devices, which show dramatically greater anti-fouling performance. However, even such device was inadequate for current purpose, as it is rigid and lacks convenient valve control functions for particle suspensions used in systematic evolution of ligands by exponential enrichment (SELEX). For this project, we propose a SMART screening strategy based on a highly integrated microfluidic chip. This new type of whole-Teflon devices, which are made of flexible Teflon membranes, offering convenient valving control for the whole SELEX process to be performed on chip and fulfilling the anti-fouling requirement in the meantime. The SELEX cycles including positive and negative selections could be automatically performed inside tiny-size microchambers on a microchip, and the enrichment is real-time monitored. The selection cycles would be ended after the resulted signal of the aptamers with high specificity reached a plateau, or no target aptamer is captured after a number of cycles of enrichment. Owning to the antifouling property of the chip materials, the loss of the sample is tremendously reduced. The SMART platform therefore is not only free of complicated manual operations, but also high-yield and well reproducible over conventional methods

Introduction: Elevated levels of tissue factor positive (TF +) microparticles (MPs) are observed in plasma from a variety of patients with an increased risk of thrombosis. We and others have described the measurement of TF activity in MPs isolated from plasma. The aim of this study was to investigate the effects of pre-analytical and analytical variables on TF activity of MPs isolated from blood of healthy volunteers either untreated or treated ex vivo with bacterial lipopolysaccharide. Materials and methods: We evaluated the following parameters: use of different centrifugation speeds to isolate the MPs; comparison of TF activity of MPs isolated from platelet poor plasma versus platelet free plasma; effect of freeze/thaw on MP TF activity; and comparison of the MP TF activity assay with the measurement of TF protein by ELISA or flow cytometry. Results: MPs prepared from platelet poor plasma by centrifugation at 20,000 × g or 100,000 × g for 15 minutes had similar levels of TF activity. However, significantly less TF activity was found in MPs isolated from platelet free plasma compared with platelet poor plasma. Interestingly, freeze/thawing of the plasma showed donor to donor variation in MP TF activity, with a moderate increase in some individuals. Conclusion: TF + MPs can be quantitatively isolated from platelet poor or platelet free plasma by centrifugation at 20,000 × g for 15 minutes. Measurement of MP TF activity in plasma may be used to detect a prothrombotic state in patients with various diseases.

No description available.

Nucleic acid amplification is responsible for pushing the limit-of-detection of molecular diagnostic assays to unprecedented levels. We developed an assay based on protein-responsive programmable dynamic DNA assembly (PRPDA) to detect proteins via an intermediate process involving nucleic acids for taking advantage of nucleic acid amplification strategies. PRPDA has previously been designed for sensitive protein analysis in fluorescent assay formats. To further push the detection limit and to achieve assay miniaturization and multiplexing, we sought to combine PRPDA with electrochemical readout. We were able to achieve LOD of 1 pM by employing wrinkled gold electrode for the PRDA protein detection scheme. Which is 2800 times improvement compare to the 2.8 nM demonstrated by fluorescent transduction. / Thesis / Master of Applied Science (MASc)

About 10% of hyperthyroid cats have a normal total T4 (TT4), requiring further testing to make the diagnosis. Thyroid stimulating hormone (TSH) is measured using the "canine" assay (TSH-CLIA, Immulite 2000 by Siemens) as the only assay currently available. However, this assay cannot differentiate between subnormal and low-normal TSH concentrations in cats due to poor specificity (70-85%). A novel feline-optimized TSH assay (TSH-BAW, Truforma by Zomedica) was recently developed. It allows differentiation between euthyroid and hyperthyroid cats. However, the effect of non-thyroidal illness (NTI) on TSH-BAW has not been evaluated. Our objectives included the comparison of serum TSH concentration using both the TSH-CLIA and TSH-BAW assays among hyperthyroid cats, cats with NTI, and healthy cats, and the evaluation of the sensitivity and specificity of the TSH-BAW for diagnosis of FHT. This prospective cross-sectional study was performed on 102 client-owned cats, including 37 hyperthyroid, 33 healthy, and 32 NTI cats. The following thyroid hormones were measured in all cats: TT4, TSH with both assays (Immulite 2000 and Truforma). Hyperthyroidism was confirmed by thyroid scintigraphy. Euthyroidism was confirmed by repeating TT4 measurement at least three months after enrollment (if available) to rule out subclinical hyperthyroidism. Cats with NTI were further divided based on the severity of their illness. Serum TSH was compared among groups using Kruskal-Wallis followed by Dunn's procedure, and compared among NTI severity scores using the Fisher's Exact test. Significance was set at P <0.05. The sensitivity and specificity of TSH-BAW for detecting hyperthyroidism are 78% (62-90%) and 97% (84-100%), respectively. The median TSH is significantly different between hyperthyroid cats and healthy and NTI cats with both assays (P<0.01). The TSH was not different between the latter euthyroid groups (P=0.87 and P=0.29). Eight (21.6%) hyperthyroid cats have a normal TSH-BAW but undetectable TSH-CLIA. Twelve (4 healthy, 8 NTI) euthyroid cats (18.5%) have an undetectable TSH-CLIA with only two (1 healthy, 1 NTI) (3%) having an undetectable TSH-BAW. The proportion of cats with a suppressed TSH is higher with severe illnesses with the TSH-CLIA only. In conclusion, the TSH-BAW has a high specificity, identifies normal TSH in healthy cats more often, and appears to not be affected by NTI. It can be a useful tool for the diagnosis of feline hyperthyroidism. However, a low-normal TSH cannot be used to rule out hyperthyroidism. / Master of Science / Hyperthyroidism leads to elevation of the thyroid hormone total T4 (TT4). About 10% of hyperthyroid cats have a normal TT4, requiring further testing to make the diagnosis. Another thyroid hormone, thyroid stimulating hormone (TSH), could be used like it is with people. In feline medicine, it is measured using the "canine" assay (TSH-CLIA, Immulite 2000 by Siemens) as the only assay currently available. However, this assay cannot differentiate between subnormal and low-normal TSH concentrations in cats due to poor specificity (70-85%). A novel feline-optimized TSH assay (TSH-BAW, Truforma by Zomedica) was recently developed. It allows differentiation between euthyroid and hyperthyroid cats. However, the effect of non-thyroidal illness (NTI) on TSH-BAW has not been evaluated. Our objectives included the comparison of serum TSH concentration using both the TSH-CLIA and TSH-BAW assays among hyperthyroid cats, cats with NTI, and healthy cats, and the evaluation of the sensitivity and specificity of the TSH-BAW for diagnosis of FHT. The study was performed on 102 client-owned cats, including 37 hyperthyroid, 33 healthy, and 32 NTI cats. The following thyroid hormones were measured in all cats: TT4, TSH with both assays (Immulite 2000 and Truforma). Cats with NTI were further divided based on the severity of their illness. Serum TSH was compared among groups using Kruskal-Wallis followed by Dunn's procedure, and compared among NTI severity scores using the Fisher's Exact test. Significance was set at P <0.05. The sensitivity and specificity of TSH-BAW are 78% and 97%, respectively. The median TSH is significantly different between hyperthyroid cats and healthy and NTI cats with both assays. The euthyroid cats (healthy and NTI cats) were not different. Eight (21.6%) hyperthyroid cats have a normal TSH-BAW (not normal in the face of hyperthyroidism) but undetectable TSH-CLIA. The proportion of euthyroid cats with a suppressed TSH (not normal in the face of euthyroidism) is higher with the TSH-CLIA compared to the TSH-BAW. Only with the TSH-CLIA, the proportion of NTI cats with a suppressed TSH is higher than healthy cats, and is higher with severe illnesses. In conclusion, the TSH-BAW has a high specificity, identifies normal TSH in healthy cats more often, and appears to not be affected by NTI. It can be a useful tool for the diagnosis of feline hyperthyroidism. However, a low-normal TSH cannot be used to rule out hyperthyroidism.

A new rapid computer assisted micro-titer plate interferon assay system was developed and characterized for use in high capacity clinical and research applications. The biological aspect of the assay was a modification of the assay methods of Finter, Armstrong and McManus. It was an application of spectrophotometric quantification of the reduction of viral cytopathic effect (CPE) as reflected by neutral red dye uptake by viable cells. A computer program was developed for the extrapolation of raw data to reference interferon units.

With the current state of climate change and world peak oil on the horizon, it is important to focus our research efforts on alternative sources of energy. Ethanol obtained from the digestion of biomass (bioethanol) and oil harvesting from algae (biodiesel) are two promising fields of study for transportation fuel production. However, in their current state of development, neither option is capable of reasonably replacing the transportation fuel demand for this country. The land demand needed is too large for either process to become a viable option, albeit the land demand for biodiesel is considerably smaller than that of bioethanol. Therefore, when moving forward with alternative transportation fuel, harvesting oil from algae is a more promising option. Therefore, I investigated oil producing green algae to better understand algal growth, the algal cytoskeleton, and tried to establish a methodology to genetically manipulate algae. I developed a microgrowth assay in order to investigate algal growth and proliferation, while at the same time using considerably less material and space. This assay can directly monitor algal growth in response to media contents, and overcomes many of the limitations of existing microassays due to its use of solid media agar and fluorescent imaging. I also investigated algal genetic manipulation with the intention of creating a standard operating procedure, which could lead to further investigation of how to increase lipid output and increase lipid harvesting cycles through studying lipid production and cell division. Electroporation and PEG mediated transformation were the two chief methods investigated for nuclear transformation. Lastly, I performed an algal kinesin phylogenetic study to characterize the currently available algal kinesin superfamily, providing insight to proteins that are important for cell division as well as other functions within this superfamily. Kinesins 5, Kinesin 7s Class II and Class V, and Kinesin 14 Class I were identified to be important for algal cell division, while Kinesin 8, 12, 11, and some orphan kinesins will require further investigation due to their unknown plant function. Overall, this research provides a foundation for future algal studies required for optimal oil production necessary for a more sustainable future.

Indiana University-Purdue University Indianapolis (IUPUI) / Apolipoprotein M (apoM) is necessary for the formation of lipid-poor preβ-HDL particles, the initial precursor of HDL and acceptors of cholesterol efflux from peripheral cells. An assay to quantify apoM in serum is not widely-available, hampering the efforts to further understand apoM and to develop therapeutic methods to increase circulating levels of apoM. An antibody-free, high throughput mass spectrometry (MS)-based assay was developed to quantitatively measure apoM from a variety of species including human, mouse, and rat. Apolipoproteins were enriched by selectively binding to Liposorb, an affinity resin, followed by enzymatic digestion. This peptide mixture was separated by HPLC coupled in-line with tandem MS/ MS. Signal intensities from the MS/ MS fragmentation of apoM-specific peptides were measured simultaneously in a targeted method spanning many commonly used species. The same amount of purified human apolipoprotein A-IV uniformly labeled with 15N was spiked into all samples and was used as an internal standard to correct for any variation in sample handling and recovery. Assay variability and accuracy was statistically validated in a three-day spike recovery experiment to determine the working range of the assay. The concentration range for quantification of apoM using this assay was 11.2-500 nM, whereas average concentration of human apoM measured from a large sampling (n>100) was 370 nM. This assay was used to measure changes in apoM in mouse serum from a pre-clinical study that was designed to evaluate the effects of a microsomal triglyceride transfer protein (MTTP) inhibitor. All measured lipoproteins and apolipoproteins showed a dose-dependent decrease in concentration and the response of apoM closely followed the response of HDL. In a clinical application of the assay, apoM was measured in human serum to evaluate the effects of two cholesterol-lowering compounds, a statin drug and an experimental PPAR-α agonist. ApoM levels did not change with PPAR-α agonist or combination treatments, but significantly decreased with atorvastatin. The measurement of apoM provided additional information on the effects of these drug treatments that previously could not be measured. The availability of a quantitative assay for apoM provides a valuable tool in the development of cardio-protective therapeutics and understanding the mechanisms of these drugs. / Monarch LifeSciences, Eli Lilly and Company