Abstract
The emerging paradigm of lab-on-a-chip powered by microfluidics is expected to revolutionize miniaturization, automation and integration in the point-of-care centers which require quick, efficient and reproducible results. Furthermore, high throughput requirement from the life sciences laboratories have made the development of lab-on-a-chip a major area of research over the past few years. Immunoassays, which are routinely used to determine the concentrations of various analytes in life sciences laboratories, are one of the most important laboratory tests that require efficient automation and integration. These are different from other laboratory tests in the clinical laboratories such as colorimetric tests because they involve formation of antibody-antigen complexes to generate a signal that can be measured. The immunoassays also involve the application of magnetically responsive beads to increase the surface area for the reactions thereby enhance the signal. Although miniaturization was started in the early 1990’s there are no commercial devices that perform immunoassays involving magnetic beads. None of the state-of-the art commercial microfluidic technologies, which are based on continuous flow in etched microchannel, have been able to fully deliver the promised benefits of microfluidics. This is primarily due to their incompatability with common sample matrices and architectural inflexibility. Furthermore, the transport of the magnetic beads in microchannels is a difficult task in continuous mode of operation as magnetic susceptibility of the beads is rather weak and because of demagnetization of the particles. In this thesis, a droplet-based microfluidic lab-on-a-chip based on electrowetting actuation is developed to perform immunoassays using magnetic beads on human physiological samples. Biocompatibility of the electrowetting system is established by demonstrating repeatable and rapid transport of human physiological fluids such as whole blood, serum, proteins such as bovine serum albumin, antibodies for insulin and interleukin-6 and enzymatic reagents such as horseradish peroxidase (HRP), alkaline phosphatase (ALP). Various magnetic configurations for efficient attraction of the magnetic beads that assist in the washing are developed. Several parameters involved in washing magnetic beads are studied and the buffer for resuspending the beads, magnetic strength to attract the magnetic beads and concentration of beads to be used were established. An efficient protocol for washing magnetic beads on chip was developed with a bead retention efficiency of almost 100%. A complete magnetic immunoassay is performed on chip for Insulin and Interleukin-6. The least concentration of Insulin and IL-6 detectable on chip is 0.24 pg/μL and 4 fg/μL respectively. Standard curves are developed for both the analytes over a range of concentrations. The repeatability of the assays is established by performing the assay on different samples on different days and the standard error is shown to be less than 3%. Magnetic immunoassays on the droplet based lab-on-a-chip are also performed on serum for Insulin and IL-6 and it is shown that the results are comparable to data obtained via conventional laboratory analysis. This work represents the first demonstration of integrated and automated operation of a digital-microfluidic lab-on-a-chip for immunoassays involving magnetically responsive beads on clinically relevant sample matrices.
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