Enzyme-linked immunosorbent assays (ELISA) are critically important tools in fundamental research and medical diagnostic applications due to their ability to reliably detect antigenic molecules in complex biological samples. Nevertheless, their sensitivity can be insufficient for extracting the desired scientific information in challenging biological assays as well as for diagnosing several lethal diseases at an early stage. While miniaturization of the ELISA technique onto the microfluidic platform recently has allowed significant reductions in its sample volume requirement and incubation period, there is still a need for developing approaches that will enable this assay to detect important biomarkers at lower concentrations than currently possible.
We have addressed this scientific need in the present dissertation by developing microfluidic approaches to enhancing ELISA sensitivity. The first approach among these focused on incubating multiple aliquots of the sample against the assay surface to increase analyte capture on it. Careful optimization of the incubation period showed this strategy to significantly improve ELISA sensitivity without prolonging the assay time for samples of anti-mouse BSA and human TNF-α.
In the second approach, we demonstrated that analyte molecules may be significantly pre-concentrated over a bead surface by immobilizing these particles at the interface of a shallow and a deep microchannel, in conjunction with a sample flow around them via electrokinetic means. More interestingly, the use of flow pulses rather than a continuous sample transport was observed to improve the effectiveness of this pre-concentration strategy by over an order of magnitude. In this work, an 80-fold decrease in the limit of detection for the cancer marker CA 19-9 was accomplished using the pulsed flow pre-concentration method. We also developed an alternate approach to increase the surface area to volume ratio in the assay chamber for better analyte employing a polyacrylamide gel matrix within the microchannel. Again, this strategy yielded similar enhancement in ELISA sensitivity due to reduced mass transport limitations in the system allowing easier access of the capture sites by the analyte molecules.
In a different project, we also exploited the use of an electric field for speeding up analyte capture on the assay surface. This goal was accomplished by fabricating a new kind of microchip device with a gold layer deposited on its channel surface. The basic idea here was to flow in a sample solution into the channel and then focus them around the ELISA surface using an electric field inducing more antigen-antibody interactions for faster analyte capture. We successfully demonstrated a reduction in the limit of detection for mouse IgG and human TNF-α using this approach by 2 to 3 orders of magnitude. Furthermore, we successfully applied this method to sensitive detection of amyloid beta derived diffusible ligands in human cerebrospinal fluid samples for more reliable and potentially early diagnosis of Alzheimer’s disease.
In the final chapter of this dissertation, we have presented some initial work on two projects which, if completed, have the potential to further expand the capabilities of the ELISA method. The first project among these focused on the development of a quantitative approach to assessing analyte levels in biological matrices that are significantly cross-reactive with the assay surface based on the standard addition technique. The second project on the other hand, involved the design of a homogeneous ELISA technique that relied on the coupling of two enzyme labels conjugated to antibodies with affinity for different epitopes on the antigen molecule.
|Commitee:||Basile, Franco, Corcoran, Robert, Kubelka, Jan, Oakey, John|
|School:||University of Wyoming|
|School Location:||United States -- Wyoming|
|Source:||DAI-B 78/12(E), Dissertation Abstracts International|
|Subjects:||Chemistry, Analytical chemistry|
|Keywords:||ELISA sensitivity, Microfluidic|
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