Available at: http://digitalcommons.calpoly.edu/theses/765
Date of Award
MS in Biomedical Engineering
Biomedical and General Engineering
The field of microfluidics is expanding into what is known as paper microfluidics. This uses a paper platform rather than materials (i.e. PDMS, PMMA) that are commonly used in microfluidics research. Current devices require an expensive manufacturing process and external sources to power the device. Such devices are not practical in low resource environments. As a consequence, it is the goal of this Thesis to develop a three-dimensional, multiplexed assay chip using nitrocellulose membranes. This device comprises of multiple layers of nitrocellulose membranes with defined fluidic channels. The multiple layers are bound together using double backed tape, and imbedded between the layers are conjugate reagents. In the detection region both antibodies and aptamers were evaluated.
The fiberglass pad where conjugate reagents would be contained, were initially saturated in dye. As sample was inputted into the three-dimensional chip, the fluid path could be visualized. Without the use of the conjugate pad the chip’s four detection regions showed detection within one minute of one another. However, the addition of this fibrous pad skewed time points dramatically. The hypothesis that a three-dimensional chip could be designed to detect different biomarkers in a multi-analyte sample was satisfied. However, simultaneous detection was only possible if the conjugate pad was either neglected or, possibly, a different material was used.
Additionally, current lateral flow assay technologies, another research area that paper microfluidics spawns from, use antibodies in order to capture biomarkers in sample and provide visual signal to the user. However, antibodies are sensitive to denaturation with pH and temperature, whereas aptamers can withstand much more extreme environmental conditions. A two-dimensional nitrocellulose chip was designed to compare antibodies and aptamers as capture reagents to detect VEGF, using colloidal gold as a particle to visualize detection. Both monoclonal and polyclonal anti-VEGF antibodies were used and showed no signal. On the other hand, the anti-VEGF aptamer produced a visual signal when conjugated to biotin on its 5’ end. This data was further validated by a separate project analyzing the binding kinetics of the antibody and the aptamer using Surface Plasmon Resonance. Therefore, the hypothesis that aptamers could be used as a possible capture reagent in a paper microfluidic chip for the detection of VEGF was satisfied.