Mr. Timothy M. Blicharz Profile

Timothy M. Blicharz

at Tufts Univ

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Proceedings Article | 23 October 2006 Paper

Detection of inflammatory cytokines using a fiber optic microsphere immunoassay array

Timothy Blicharz, David Walt

Proceedings Volume 6380, 638010 (2006) https://doi.org/10.1117/12.686158

KEYWORDS: Fiber optics, Optical arrays, Multiplexing, Proteins, Luminescence, Diagnostics, Image segmentation, Chemistry, Statistical analysis, Microfluidics

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A multiplexed fiber optic microsphere-based immunoassay array capable of simultaneously measuring five inflammatory cytokines has been developed. Five groups of amine-functionalized 3.1 micron microspheres were internally encoded with five distinct concentrations of a europium dye and converted to cytokine probes by covalently coupling monoclonal capture antibodies specific for human VEGF, IFN-gamma, RANTES, IP-10, and Eotaxin-3 to the microspheres via glutaraldehyde chemistry. The microspheres were pooled and loaded into a 1 mm diameter fiber optic bundle containing ~50,000 individual etched microwells, producing the multiplexed cytokine immunoassay array. Multiple arrays can be created from a single microsphere pool for high throughput sample analysis. Sandwich fluoroimmunoassays were performed by incubating the probe array in a sample, followed by incubation in a mixture of biotin-labeled detection antibodies that are complementary to the five cytokines. Finally, universal detection of each protein was performed using a fluorescence imaging system after briefly immersing the array in a solution of fluorophore-labeled streptavidin. The multiplexed cytokine array has been shown to respond selectively to VEGF, IFNgamma, RANTES, IP-10, and Eotaxin-3, permitting multiplexed quantitative analysis. Ultimately, the multiplexed cytokine array will be utilized to evaluate the potential of using saliva as a noninvasive diagnostic fluid for pulmonary inflammatory diseases such as asthma.

Proceedings Article | 12 November 2005 Paper

Microsphere based saliva diagnostics

David Rissin, Christopher DiCesare, Ryan Hayman, Timothy Blicharz, David Walt

Proceedings Volume 6007, 60070X (2005) https://doi.org/10.1117/12.630781

KEYWORDS: Sensors, Diagnostics, Statistical analysis, Proteins, Biological research, Chemistry, Fiber optics, Multiplexing, Optical arrays, Calibration

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Saliva presents a minimally invasive alternative medium to blood for performing diagnostics¹. Microsphere sensors for ions, small organic molecules, and proteins are currently being developed and optical microarrays containing thousands of these sensors will be used for simultaneous multi-analyte analysis. The fiber bundle platform in use is 1mm in diameter and contains approximately 50,000 individually addressable 3.1μm fibers, each with an etched well capable of housing a single 3.1μm microsphere sensor. Micron-sized bead-based chemistries are produced in house, followed by deposition onto a fiber-optic bundle platform, allowing for multiplexed analysis. The ultimate goal is to develop a universal diagnostic system using saliva as the diagnostic medium. This platform will permit multiplexed analysis of a sample by integrating microfluidics with the optical arrays loaded with sensors capable of detecting relevant biomarkers associated with a wide range of disease states. Disease states that are currently under investigation include end stage renal disease (ESRD) and Sjoegrens Syndrome (SS).

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