(651i) Optimized  and Tested Zein Film for Utilization As an Effective SERS Sensor

Surface Enhanced Raman spectroscopy (SERS) is a technique of growing interest to the research community. Through the implementation of nanophotonic structures and nanoparticles, SERS sensors have the ability to increase the Raman signal by 10⁸ times [1]. Zein is a prolamine protein from corn. Its film forming capabilities make it a prime candidate for a number of different applications. One of these applications is as a biodegradable, SERS sensor. Gizem et al. found that a zein based sensor could increase the raman signal of analytes 10⁴ times. However, the limit of detection for the sensor was above that which could be used as an appropriate sensor for food allergens and toxins [2]. This study worked to optimize and test a zein substrate for this particular application.

The first phase of the work was to optimize the formulation for the zein film. Zein films solubilized in solvent alone create a very brittle film. Therefore, a number of different additives are incorporated into the film matrix to optimize the various properties of the films for particular applications. In this case, we wanted a flexible yet sturdy film that would promote a high fidelity for imprinted nanostructures. To achieve this optimization, both oleic acid (OA) plasticizer and glutaraldehyde crosslinking agent (CL) were included into the film formulation at different ratios. All films were formed with a 70% ethanol solvent with a 1:5 w of zein/volume ratio. Mono and diglyceride emulsifier was also incorporated at 1:0.05 w OA/w. Besides looking at the fidelity of nanostructures with scanning electron microscopy (SEM) the films were also analyzed with various characterization techniques that looked at the physical properties (indentation and rheology), chemical properties (Fourier Transform Infrared Spectroscopy (FTIR) and FT-Raman), and surface properties (water contact angle and atomic force microscopy) of the films.

The physical properties showed that both plasticizer and crosslinking content have a strong impact on the zein films. Indentation shows that an increase in both plasticizer and crosslinking content decrease the Vickerâ??s hardness of the material while increasing the indentation area. The rheology showed a deviation between the formulations for both gelation time and reaction rate. The 0.8:1 OA films stayed together, forming a â??maramaldeâ? type texture in 60 hours, while the 1.2:1 OA films took over 100 hours to create the same texture. The 1:1 OA films split, having the 4% CL films coincide with the high OA films and the 8% agree with those of the 0.8:1 OA. The rate at which the film solutions reached the â??marmaladeâ? state followed a first order reaction for lnGâ?? vs. time. However, at the high oleic acid concentration, the Pearson correlation coefficient shows a deviation from the first order model.

The chemical properties described a mechanism for crosslinking that became increasingly ineffective with high plasticizer content. The main peak difference within the FT-Raman spectra came from a peak located at 881 cm^-1. This particular peak corresponds to a rise in C-N-C stretching vibration [3]. In order for this sort of bonding to increase within crosslinked films, the glutaraldehyde must react with an amine group. This sort of reaction is supported by the FTIR spectra and a decrease in both the Amide II band at 1540 cm^-1 and the peak at 1080 cm^-1. The former corresponds to the N-H bending of amide groups [3], while the ladder is responsive to N-H stretching of primary amines [4]. Both of these peaks decrease in intensity with an increase in crosslinking concentration. A peak at 970 cm^-1 also arises within the FTIR spectra with crosslinking. This peak is strengthened by out of phase C-O-C ring vibrations. Concluding that while the glutaraldehyde is creating C-N-C bridges between molecules, it is also oligomerizing and forming hemiacetal rings. The degree of oligomerization increases with plasticizer content.

The surface properties only showed a significant change in topography and SEM. As the films became more crosslinked the topography consisted of much larger features than the films with only plasticizer. The SEM images demonstrated that highest oleic acid ratio was too malleable to produce high fidelity nanostructures, while the low OA films had too high of an affinity to the lithographic medium to be effectively removed from the mold. It was at the midrange plasticizer content and the 4% and 8% crosslinking ratios that created the best films for this application. However, even with this change in topography and affinity to the lithographic mold, the water contact angle measurements showed no difference between the formulations.

The final phase of the research utilized the optimized formulations to produce and test a SERS sensor. Each of the zein films were molded with inverted nanopyramidal structures and coated with gold to create the SERS enhancement. The surfaces were further altered with the incorporation of gold nanoparticles a top of the nanophotonic structures. The sensor was tested both with rhodamine 6g and with gliadin, the protein responsible for the allergic response to gluten.

In conclusion this study focused on the application of zein corn protein films as a substrate for a SERS sensor. The zein film formulation was optimized with the inclusion of both oleic acid plasticizer and glutaraldehyde crosslinking, and then characterized with a wide variety of techniques. The 2 formulations that gave the highest fidelity of nanostructures were then chosen to be tested as a SERS sensor with the utilization of both Rhodamine 6G and gliadin protein. The study found this to be a successful application for zein films.

1. Cunningham, Brian. â??[Illinois] ECE 416 Surfance Enhanced Raman Spectroscopy I,â? 2013.
2. Gezer, P.G., G.L. Liu, and J.L. Kokini. 2016. Development of a biodegradable sensor platform from gold coated zein nanophotonic films to detect peanut allergen, Ara h1, using surface enhanced raman spectroscopy. Talanta: 150, 224-232.
3. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules. Accessed April 6, 2016. https://books.google.com/books/about/The_Handbook_of_Infrared_and_ Raman_Chara.html?id=bYWNSi6abvwC.
4. Larkin, Peter.Infrared and Raman Spectroscopy, 213â??15. Oxford: Elsevier, 2011. http://www.sciencedirect.com/science/article/pii/B978012386984510014X.