Optimization of Zein Based Surface Enhanced Raman Spectroscopy Biosensor for the Detection of Gliadin as a Marker for Celiac Disease
Biosensors are a growingly popular alternative to chemical sensors. With their fast detection time and portability, biosensors have the ability to be applicable in a number of different industries. The current methods for allergen and toxin detection in the food industry often require highly trained personnel and can be very time consuming. These disadvantages plus the growing desire for the consumer to make more informed choices about what they eat, shows a need for an easy to use, rapid, and specific method of detection for analytes of interest to the food industry (Alocilja and Radke 2003). In this thesis, a mostly biodegradable biosensor is proposed. The platform of the biosensor investigated is made from zein corn protein. This mostly hydrophobic protein has been previously evaluated for use as a biodegradable surface enhanced Raman spectroscopy (SERS) sensor (Gezer eta al. 2016; Gezer et al. 2016a; Gezer et al. 2016b). The sensor was able to successfully detect both the acrylamide neurotoxin (Gezer et al. 2016b) and the Ara-h1 peanut allergen protein (Gezer et al. 2016a). However, these studies left room for the development of a more enhanced zein based sensor platform. This thesis describes the construction of a zein based SERS sensor platform first through the comparison of various zein film formulation changes and then with the implementation of gold nanoparticles. At the end, the sensor was tested using the gluten allergen protein, gliadin. Through the implementation of both oleic acid (OA) plasticizer and glutaraldehyde (GDA) crosslinker, nine different formulations of zein films were tested for their ability to create an optimized platform for a SERS sensor. The overall test for this application is the fidelity, or replication, of microstructures imprinted into the films. For this thesis, the inverted micropyramidal structure topography was achieved through the process of soft lithography. The zein films were evaluated using scanning electron microscopy (SEM) to determine the degree to which the inverted micropyramidas were successfully replicated. The films were also evaluated for indentation, water contact angle, zein secondary structure changes, and color. The crosslinking and plasticization mechanisms were also monitored in solution with rheological techniques and compiled with both Fourier-transform infrared (FTIR) as well as FT-Raman spectroscopies. Both the OA and the GDA had effects on the various physical and properties of these films. At 0.8 OA, the zein molecules both aggregated and crosslinked to a high degree, but at 1.0 and 1.2 OA the plasticizer had a larger effect on the overall films. A larger storage modulus (G’) was achieved with an increase in GDA, while the same trend was observed for a decrease in OA. The zein gels were very soft due to a large amount of protein aggregation and ethanol. Through FTIR, it was evident that the GDA aldehydes react with zein amine groups to form linkages through imine bonds. Very minimal differences were found between the different formulations in terms of color, water contact angle, and zein secondary structure. Overall, the 0.8 OA ratio with 4% GDA was found to be the optimal formulation for high fidelity inverted micropyramidal structures to be used in a SERS based sensor. This optimized formulation, including both OA and GDA, was then further enhanced with the decoration of gold nanoparticles. Gold was transferred to the zein films during the imprinting process of soft lithography. A polydimethylsiloxane (PDMS) intermediate mold was used to transfer inverted micropyramidal structures along with a 20 nm gold coating to the zein films. Cysteamine was then used to attach 50 nm gold particles to the gold coated zein films. A 100 mM of cysteamine created the best dispersion of nanoparticles on the platform surface when compared to 10 mM with SEM. This divergence was further proven by the detection of Rhodamine-6G on the sensor surface. An overall enhancement of 10x was achieved with the implementation of these nanoparticles over an imprinted zein film with no gold. The gluten allergen protein gliadin was used to test the optimized film formulation with decorated gold nanoparticles. Anti-gliadin was affixed to the sensor surface with 11-MUA activated by a reaction EDC and NHS. Although, the averaged spectra showed differences in overall Raman intensity- no fingerprint peak could be identified due to the similar protein (analyte) on protein (antibody) on protein (film/sensor platform) nature of the sensor. Principal component analysis (PCA) was used to differentiate between the sensors. However, PCA was unable to scatter the data effectively for every gliadin concentration due to some processing problems with the sensor replication. This research proved that a more optimized SERS sensor could be developed with a biodegradable zein based platform. (Abstract shortened by ProQuest.)
Kokini, Purdue University.
Food Science|Engineering|Materials science
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