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PLANT-NANOMATERIAL INTERACTION: ZEIN NANOPARTICLE INTERACTIONS WITH SOYBEAN PLANTS, ROOTS, AND LEAVES, MEASURED FLUOROMETRICALLY Department of Biological and Agricultural Engineering, Louisiana State University Kurt Ristroph, Dr. Cristina Sabliov, Dr. Carlos Astete, Dr. Jeff Davis Abstract. Delivery of pesticides in nano-form offers an effective method for protecting plants and crops from insects while also decreasing applied overall pesticide volume per acre and minimizing negative environmental impacts from chemical runoff and ecological accumulation. Specifically, nanoparticles’ ability to attach to certain surfaces and control the release of entrapped bioactives allows for precise delivery and enhanced efficacy of the bioactive. The goal of this project was to develop and test a nano-scale protein-based delivery system designed to enhance the activity of an entrapped pesticide, methoxyfenozide by releasing it in a controlled manner on the surface of leaves and roots of soybeans. In order to understand how the nanoparticles (NPs) adhere to soybean leaves, fluorescent nanoparticles were made from zein, a biodegradable protein derived from corn, that had previously been fluorescently tagged with Fluorescein Isothiocyanate (FITC). Soybean leaves were then immersed in the fluorescent nanoparticle suspension (NP concentration: 1.8 mg/mL) for 15 minutes before being washed with water for different time increments (1, 2, 3, 4, 6, and 8 hours). The treated leaves were freeze-dried for 48 hours and then processed to measure fluorescence levels. Compared to control samples, the leaves exposed to nanoparticles had dramatically higher fluorescence readings, indicating the presence of nanoparticles after washing. The tests were repeated on the roots of soybean plants. Results indicated that the zein nanoparticles have the ability to remain on soybean leaves and roots even when washed. This ability is critical for the continued release of the pesticide where needed, but poses a potential safety concern for consumers. Understanding the mechanisms of the nanoparticle-plant attachment and entrapped pesticide action is therefore necessary to ensure the safety of consumers exposed to NP-treated plants. Hypothesis Zein nanoparticles attach to the leaves of soybean plants and remain on the surface of the leaves for a time greater than that of traditional pesticides, even in the event of environmental effects such as rain. Materials and Methods Nanoparticle synthesis and characterization Zein-FITC NPs were prepared by emulsion-diffusion method. 300 mg of zein-FITC were dissolved in 9.4 ml acetone:water (8:2 volume ratio). The aqueous phase was made by adding 100 ml of water into 57.8 mg of didodecyldimethylammonium bromide (DMAB) surfactant. The organic phase was added dropwise into the aqueous phase, and the mixture was then passed through a microfluidizer (M-110P, Microfluidics, MA, USA) at 30,000 psi twice. Acetone was evaporated from the solution with a rotovapor (Buchi R-124, Buchi Analytical Inc., DE, USA) Dynamic light scattering (DLS) technique was used to determine the mean size, zeta potential and polydispersity index (PDI) of the polymeric NPs with a Zetasizer Nano (Malvern Instruments, Southborough, MA). Samples were diluted 1:20 (v/v) in HPLC-grade water, and the values of were determined using a mono-modal distribution. The morphology of the zein NPs was studied by scanning electron microscopy (SEM) using a JSM-6610LV system (Jeol, Peabody, MA) Fluorescence tests Leaves or roots were immersed in the zein-FITC NP solution (diluted 1:1 with water) at room temperature for 15 minutes. After drying, samples were immersed in water for 1, 2, 3, 4, 6, and 8 hours to wash. The washed samples and a no-wash control were then placed in a freeze drier (FreeZone 2.5 Plus, Labconco Corp., MO, USA) and dried for 48 hours. Three 5 mg samples from each time sample were placed in a centrifuge tube with 1 ml of 1-normal NaOH. The samples were immersed in an agitated water bath at 70° C for 30 minutes and then centrifuged for 15 minutes at 30,000 rpm. Following this digestion process, three 100 µl samples were taken from each centrifuge tube and measured for fluorescence. Results Leaf and root fluorescence and subsequent NP concentration (green) were found to be significantly higher than the control fluorescence, even after 8 hours of washing. The heightened fluorescence from the treated samples indicates that NPs remained on the leaf and root surfaces after different wash times. For the leaves, concentration was found to decrease for approximately 2 hours to a concentration of approximately 0.1 mg/mL. The roots’ concentrations also displayed asymptotic behavior. In each case, the treatment data were shown to be statistically different from the control data (95% confidence) using JMP software. It was thus concluded through fluorometric tests that zein NPs adhered to soybean roots and leaves. These results will contribute to the development of NP- plant attachment mechanisms, which will assist the development of models for environmental and consumer safety. Acknowledgements Louisiana Soybean and Grain Research and Promotion Board Right: roots during the 15-minute immersion in a 1:5 dilution of the zein-FITC NP solution, used for the development of a fluorescence-concentration standard curve. Below: root samples for the standard curve and for fluorescence readings after 3 and 6 hours of washing.