Date of Award
Master of Science (MS)
John D. Brennan
We report on a new solid-phase colorimetric bioassay format based on enzyme-catalyzed enlargement of gold nanoparticles (AuNP) that are coentrapped with the enzyme in a sol-gel based silica material. Biocatalysed hydrolysis of the enzyme substrate, acetylthiocholine (ATCh) leads to formation of thiocholine, which in turn reduces Au(III) onto the entrapped nanoparticles, producing particle growth and a concomitant increase in color intensity that can be correlated to the amount of substrate or inhibitor present in test solutions. The entrapped AuNP cannot leach from the silica material, leading to a solid-phase assay that has the potential to be integrated into a portable biosensing platform that can utilize visual detection of a color change as a simple readout. Toward this end, we cast an AChE-doped silica material onto a 40%MTMS/TMOS functionalized paper substrate to form the portable biosensor. Treatment with 40%MTMS/TMOS affords a more uniform paper surface allowing for more reproducible enzyme loading and better control over liquid penetration. Our results show that the assay is sufficiently sensitive to allow for detection of Paroxon to 1 μM. For automated sensor fabrication, we investigated piezoelectric inkjet printing of enzyme doped silica sols. A unique sandwich type fabr ication technique was devised whereby sol-gel materials (at stable pH) and the bio-materials were printed separately and in layers so that AChE was sandwiched between layers of silica to form the sensor. For these biosensors, a standard Ellman assay was used to monitor AChE activity with the cationic polymer, PVAm (polyvinylamine, 1.5 MDa), employed to trap and preserve the colored anionic product molecules (5-thio-2-nitrobenzoate) over a finite region. With this novel approach, a series of Paraoxon and Aflatoxin BI levels were assessed and it was determined that these compounds can be detected to 100 nM and 1 nM respectively, in ca. 5 min. The paper-based, colorimetric sensing platforms presented in this thesis are simple, cost-effective, portable, user-friendly, and need neither sophisticated instruments nor additional handling steps. Detection can be made by eye or by digital camera and image analysis software.
Luckham, Roger Elliot, "DEVELOPMENT OF SOL-GEL-BASED BIOACTIVE PAPER SENSING PLATFORMS TOWARD NEUROTOXIN DETECTION" (2009). Open Access Dissertations and Theses. Paper 4235.
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