Browsing by Subject "Lab on a chip"
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Item Femtosecond laser nanoaxotomy lab-on-a-chip for in-vivo nerve regeneration studies(2010-12) Guo, Xun, doctor of mechanical engineering; Ben-Yakar, AdelaSurgery of axons in C. elegans using ultrafast laser pulses, and observing their subsequent regrowth opens a new frontier in neuroscience, since such research holds a great potential for the development of novel therapies and cures to neurodegenerative diseases. In order to make the required large-scale genetic screenings in C. elegans possible and thus obtain statistically significant biological data, an automated laser axotomy system needs to be developed. Microfluidic devices hold the promise of improved throughput by integrating different functional modules into a single chip. The first step to developing a microfluidic device for laser axotomy is to devise an on-chip worm trapping method, which maintains a high degree of immobilization to sever axons without using anesthetics. In this thesis, we present a novel method that uses a thin, deflectable PDMS membrane that individually traps worms in a microfluidic device. Axons can successfully be severed with the same accuracy as those using conventional paralyzing techniques. This device also incorporates recovery chambers for housing worms after surgery and for time-lapse imaging of axonal regrowth without the repeated use of anesthetics. Towards accomplishing an automated, high-throughput laser axotomy system, we developed an improved microfluidic design based on the same mechanical immobilization technique. This second generation device allows for serially processing of a large quantity of worms rapidly using a semi-automated system. Integrated to the opto-mechanical platform, a software program utilizing image processing techniques is developed. This semi-automated program can automatically identify the location of worms, their neuronal cell bodies, focus on the axons of interest, and align the laser beam with the axon via a PID based viso-servo feedback algorithm. Statistic data demonstrate that there is no significant difference in axonal reconnection rates between surgeries performed on-chip and using anesthetics. To improve flow control, a three-dimensional novel microfluidic valve structure is designed and fabricated. This novel valve structure allows for a complete sealing of the flow channel, without degrading optical conditions for imaging and laser ablation in the trapping area. Finally, we developed a prototypical microfluidic assembly that will eventually be able to interface a well-plate to automatically deliver population of worms from individual wells to the automated chip for axotomy. This interface consists of a microfluidic multiplexer to significantly reduce the number of solenoid valves needed to individually address each well.Item Programmable bio-nano-chip immunosensor for multiplexed detection of ovarian cancer biomarkers(2011-12) Raamanathan, Archana; McDevitt, John Thomas; Crooks, Richard M. (Richard McConnell)Ovarian cancer is a high mortality disease where early stage detection may have significant survival benefits. Promising next-generation non-invasive, biomarker-based screening modalities involve longitudinal monitoring of serum biomarkers and multi-marker panel detection. Here, rapid, sensitive, precise and multiplexable diagnostic platforms can facilitate biomarker validation along with early detection and screening, and this work attempts to exploit the programmable bio-nano-chip (p-BNC) immunosensor to address these specific translational needs in ovarian cancer. First, the p-BNC was adapted for Cancer Antigen 125 (CA125) quantitation, the current FDA standard, with prominent implications in novel early detection and screening modalities. Antibody pairs binding to distinct epitopes on CA125 were identified and the p-BNC operating variables (incubation times, flow rates and reagent concentrations) were attuned to deliver optimal analytical performance (inter- and intra-assay precision of 1.2% and 1.9% and Limit-of-Detection (LOD) 1.0 U/mL), competitive with current gold standards, but with a short analysis time of 43 minutes. Further validation of the system with advanced stage patient sera (n=20) demonstrated good correlation with 'gold standard' ELISA (R² = 0.97). Next, the p-BNC was adapted for concomitant analysis of CA125 and Human Epididymis Protein 4 (HE4), a novel multiplexed biomarker panel for early detection and screening. The HE4 immunoassay was developed to perform optimally with the 'rate determining' CA125 assay. Cross-reactivity analysis demonstrated high specificity multiplexing. The dose-response curves for the multiplexed CA125 and HE4 immunoassays were congruous with their singleplex counterparts with respective LODs of 0.51 U/mL and 4.18 pM and a total analysis time of 44 minutes. A small pilot scale clinical study was conducted to discriminate between surgically confirmed patient sera (n=8) and corresponding age-matched healthy controls (n=8) utilizing the multiplexed p-BNC, interpreted with a risk of ovarian malignancy algorithm. Successful discrimination was achieved between the groups with Receiver Operating Characteristic (ROC) curve AUC (Area Under the Curve) values of 1.00, 0.984 and 1.00 respectively for CA125, HE4 and the composite marker combination. Taken together, the analytical and clinical performance, multiplexing capabilities and the short turn-around times on the p-BNC offer methodological advancements over current gold standard techniques, indicating strong promise for ovarian cancer diagnostics.