Safe drinking water is essential for life, yet at least two billion people around the world consume water contaminated with pathogens among other pollutants. Standard methods like polymerase chain reaction (PCR) and membrane filtration have been developed to detect enteric pathogens in water. However, these methods are limited in their accessibility due to long wait times to obtain results, and the requirements of skilled expertise, electricity, and laboratory equipment. This research has focused on addressing some of these limitations by analyzing the mechanisms of work and optimizing an indirect colorimetric nanosensor developed in previous research. The colorimetric nanosensor investigated herein relies on a competitive binding mechanism. When positively charged gold nanoparticles coated with polyethyleneimine (PEI-AuNPs) are added to a water sample containing negatively charged Escherichia coli (E. coli) and β-galactosidase (β-Gal) enzyme, the PEI-AuNPs will preferably bind to E. coli. This leaves β-Gal free in solution to hydrolyze chlorophenol red β-D-galactopyranoside (CPRG) (a substrate added to the water sample). The hydrolysis reaction of CPRG results in changing the solution color and the magnitude of this color change is a function of the amount of E. coli present in a water sample. It was hypothesized herein that the governing factor for the nanosensor functionality is the surface charge/Coulombic interactions rather than the nanoparticle composition or the type of chemical coating on the nanoparticle surface. To test the research hypotheses, positively charged nanoparticles with different compositions and chemical coatings as well as positively charged polymers were tested herein as potential detection agents for E. coli in water using the competitive binding assay reported in the literature with some modifications. This study produced three main findings that support the research hypotheses. First, gold nanoparticles (AuNPs) were not critical to the nanosensor functionality – other positively charged nanoparticles of silver and iron oxide coated with branched PEI were able to detect E. coli as low as 105 and 107 CFU/mL, respectively. Second, the branched PEI polymer itself (i.e., without a nanomaterial) detected E. coli at 107 CFU/mL. Third, in the absence of E. coli, (1-Hexadecyl) Trimethylammonium Bromide (CTAB), a positively charged polymer, inhibited the hydrolysis of CPRG by β-Gal. This inhibition suggests that other positively charged polymer types have potential applications in colorimetric detection assays that are based on the competitive binding mechanism. The observed behavior with the aforementioned sensing agents indicated that the positive charge was likely responsible for the detection of microbes using this competitive binding detection approach rather than the type of the chemical coating/agent used. These findings open possibilities for more types of recyclable and cost effective nanomaterials and polymers to be developed for detection of E. coli using this competitive binding approach. Furthermore, research is warranted for optimizing the sensing agents tested in this study to lower their detection limit and assess their recyclability.
| Identifer | oai:union.ndltd.org:CALPOLY/oai:digitalcommons.calpoly.edu:theses-3867 |
| Date | 01 June 2021 |
| Creators | Stabler, Sarah M |
| Publisher | DigitalCommons@CalPoly |
| Source Sets | California Polytechnic State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Master's Theses |
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