ANTIBACTERIAL, PLASMONIC, AND TOXIC PROPERTIES OF ENGINEERED NANOPARTICLES A Thesis presented to the Faculty of the Graduate School University of Missouri In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy by Trang Ha Dieu Nguyen Drs. Mengshi Lin, Azlin Mustapha Thesis Supervisors DECEMBER 2016 © Copyright by Trang Ha Dieu Nguyen 2016 All rights reserved. The undersigned, appointed by the dean of the Graduate School, have examined the thesis entitled ANTIBACTERIAL, PLASMONIC AND TOXIC PROPERTIES OF ENGINEERED NANOPARTICLES Presented by Trang Ha Dieu Nguyen, a candidate for the degree of Doctor of Philosophy, and hereby certify that, in their opinion, it is worthy of acceptance. Mengshi Lin, Ph., Food Science Program Azlin Mustapha, Ph., Food Science Program Bongkosh Vardhanabhuti, Ph., Food Science Program Chong He, Ph., Department of Statistics ACKNOWLEDGEMENTS In the heat of moment, when many ideas for the acknowledgment part of my dissertation come along, I was so excited.
This dissertation took me four and a half years and it has given me much of challenges and joyfulness. This work would not have been possible without the guidance and supports of all the following individuals. First and foremost, I would like to thank my two wonderful advisors, Drs. Mengshi Lin and Azlin Mustapha.
Lin is a facilitator and a mentor who abundantly helped and offered me invaluable assistance, supports and encouragements during my time in Mizzou. I believe he spent hundreds of hours editing my manuscripts, even in the weekends. Mustapha inspired me by her enthusiasm, energy, and excellent knowledge. I cannot fulfill my dissertation without her advice and guidance when I had problems with my experiments.
She is the person who has proven that a female scientist can balance well between her family and career! I would like to show my gratitude to my committee members, Dr. Vardhanabhuti and Dr. He through the journey. I appreciate their valuable suggestions and comments, especially Dr.
V for her co-authorship in one of my manuscripts. I also would like to express my thanks to Dr. Alexander for letting me help them in teaching assistance. I have learned from them teaching methods, curricula and appropriate manner when interacting with students.
ii I thank my past and present lab members and from different labs for being wonderful colleagues to work with. Special thanks are given to Zhang Zhong for his co-authorship and assistance. I acknowledge the Vietnam International Education Development under the Ministry of Education and Training for financial support in the first two years of my study. Finally, I owe my deepest gratitude to my family in Vietnam and my husband’s family in the US for endless love, support, and encouragement, without which I could not finish my work.
My husband and my sons Yanni and Raphael are the greatest gifts that God brought to me. Thank you all for being with me, loving me unconditionally, being my support and inspiring me every single day of my life. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS. ii LIST OF TABLES.
vii LIST OF FIGURES .1 Nanomaterials and current uses in foods and consumer products .2 Surface enhanced Raman spectroscopy (SERS) and its enhancement mechanisms 13 2.3 Applications of SERS for Food Adulterant Detection .5 Antibacterial properties of inorganic ENPs .6 Mechanisms of antimicrobial properties of inorganic ENPs .7 Physical and chemical properties of inorganic ENPS affect their antimicrobial activities .8 Cytotoxicity of inorganic NPs to human cells .9 Mechanisms of cytoxocity of ENPS .10 Physical and chemical properties of NMs associated with nanotoxicity .11 Toxicity Assessment of NPs. 48 Use of Graphene and Gold Nanorods as Substrates for Detection of Pesticides by Surface Enhanced Raman Spectroscopy .2 Materials and Methods .1 Preparation of chemicals .2 Gold film silicon substrate .3 Synthesis of gold nanorods .4 Fabrication of SERS substrates.3 Results and Discussions. 69 Use of Aminothiophenol as an Indicator for the Analysis of Silver Nanoparticles in Consumer Products by Surface-Enhanced Raman Spectroscopy (SERS) .2 Materials and Methods .2 Characterization of Ag NPs in the products .3 Determine Ag NPs in tested products .4 Conjugation of PATP onto Ag NPs .5 Detection of Ag NPs Using SERS and PATP-Ag NPs conjugation.3 Results and Discussion. 86 Toxicity of Graphene Oxide on Intestinal Bacteria, and Caco-2 Cells.2 Materials and Methods .1 Characterization of GO .2 Preparation of Bacterial strains .3 Effect of GO on the growth of E.4 Mammalian cell study .5 MTT reduction assay .6 WST-8 proliferation assay .7 Scanning electron microscopy (SEM) analysis .8 Transmission electron microscopy (TEM) analysis .3 Results and Discussion.
108 Antibacterial Properties of Selenium Nanoparticles and Their Toxicity on Caco-2 Cells .2 Materials and Methods .1 Chemicals, bacterial strains mammalian cells .2 Characterization of Se NPs .3 Preparation of bacterial strains.4 Synthesis of Se NPs .5 Effect of Se NPs on the growth of bacterial strains .6 Mammalian cell study .7 MTT reduction assay .8 WST-8 proliferation assay .9 Scanning electron microscopy (SEM) .10 Transmission electron microscopy (TEM) .3 Results and Discussion .1 Characterization of Se NPs .2 Antibacterial effects of Se NPs on pathogenic bacteria .3 Cytotoxic effect of Se NPs on Caco-2 cells. 128 Conclusions and Future Plans. 169 vi LIST OF TABLES Table Page Table 1. Food and Food-related products that claim to contain nanoparticles.
Antibacterial effect of inorganic ENPs against different microorganisms. In vitro cytotoxicity effects of graphene materials and Se NPs. Band assignment of major peak in SER spectra form three pesticides*. Limit of detection of using G-Au-AuNRS substrate for detection of azinphos- methyl, carbaryl, and phosmet.
Total concentration of silver and Ag NPs, average size, and the intensity of SERS spectra acquired from five commercial products. GO characteristics, values presented means ± SD from triplicate measurements. The zeta-potential values of selenium nanoparticles. Zeta potential values of bacteria strains.
124 vii LIST OF FIGURES Figure Page Figure 2-1. Schematic of a surface-enhanced light scattering process (Schatz and others 2006; Alonso-González and others 2012). Structure of substrates: (a) graphene-Au-AuNR (G-Au-AuNR); (b) graphene- AuNR (G-AuN); (c) Au-AuNR. SERS spectra of PATP, PATP mixed with AgNO3, PATP with 30 nm Ag NP3.
Comparisons of enhancement effects from Ag NPs. Concentration-dependent SERS spectra (part of full scale) of Ag NPs with PATP (10 mg/mL) as an indicator (A); the linear relation between Raman intensity and Ag NPs concentration (B). SERS spectra four five Ag NPs-containing dietary and antimicrobial products. Negative controls were prepared using the solvent of PATP (methanol).
Characterization of Ag NPs in the dietary supplements and antimicrobial products. UV–vis absorption spectrum of GO aqueous dispersion (A). FTIR spectrum of dried graphite oxide sample (B). TEM images of GO aqueous dispersion.
Effect of GO on the growth of E. SEM images of E. animalis without (A, C, E) and with (B, D, F) GO treatment. Cell viability of Caco-2 cells determined by the MTT assay after 24 h exposure to different concentrations of GO.
Data represent means ± SD. Means with the same letters are not significantly different (P > 0. Cell viability of Caco-2 cells determined by the WST-8 assay after 24 h exposure to different concentrations of GO. Data represent means ± SD.
Means with the same letters are not significantly different (P > 0. TEM images of Caco-2 cells (A, C) control and (B, D) treated with GO. Cell viability of Caco-2 cells determined by MTT and WST-8 assays after a 24-h exposure to GO-pretreated media. Data represent means ± SD.
Means with the same letters are not significantly different (P > 0. An UV-vis absorbance spectrum of Se NPs solution (A) and a FTIR spectrum of condensed Se NPs (B). TEM image of Se NPs. Effect of Se NP on the growth of E.
Microbial counts (CFU/mL) of S. aureus after 15 h of exposure to different concentrations of Se NPs. Data with the same letter are not significantly different (P ˃ 0. SEM images of S.
aureus without (left) and with (right) Se NP treatment. TEM images of S. aureus without (left) and with (right) Se NP treatment. Cell viability of Caco-2 cells determined by MTT assay (A) and WST-8 assay (B) after a 24-h exposure to different concentration of Se NPs.
Data represent mean ± SD. Means with the same letters are not significant different (P ˃ 0.124 x ANTIBACTERIAL, PLASMONIC AND TOXIC PROPERTIES OF ENGINEERED NANOPARTICLES Dieu-Trang Nguyen Ha Drs. Mengshi Lin, Azlin Mustapha Thesis Supervisors ABSTRACT There has been increasing application of novel nanomaterials in recent years in the area of agriculture and food science. This dissertation aims to study novel nanomaterials and investigate their applications in food safety, and to develop and use surface-enhanced Raman spectroscopy (SERS) as a rapid, simple, and sensitive analytical method to improve food safety.
There have been increasing applications of nanomaterials in various areas, which may cause human exposure and environmental pollution. Therefore, it is important to study the toxicity of different nanomaterials against bacteria and human cells. The objectives of this study were to: (1) develop new types of substrate consisting of monolayer graphene, gold film, and/or gold nanorod structures; (2) detect and measure silver nanoparticles (Ag NPs) in consumer products using SERS and aminothiophenol as an indicator molecule; (3) investigate the effect of graphene oxide (GO) on human intestinal bacteria and human intestinal cells; (4) study the antimicrobial activity of selenium nanoparticles (Se NPs) against foodborne pathogens and the toxicity of Se NPs against Caco-2 cells. A simple, fast, and efficient method was developed to fabricate new SERS substrates by coating a gold nanorod-decorated graphene sheet on silicone substrate.
The results demonstrate that GO is biocompatible and has a potential to be used in agriculture xi and food science, indicating that more studies are needed to exploit its potential applications. The data show that Se NPs can be used as an antimicrobial agent to inhibit the growth of Staphylococcus aureus in foods and can potentially be used as a chemopreventative and chemotherapeutic agent. More studies are needed to elucidate the mechanisms of Se NPs and GO’s cytotoxicity and their antibacterial properties. More research is also needed to improve the performance of SERS substrates using different materials and use them in improving food safety.
xii CHAPTER 1 Introduction 1.1 Background There have been a surge of food safety incidents and scandals in recent years, which have raised concerns from consumers about food safety issues. One of the most notorious food safety incident occurred in China about the contamination of melamine in milk in 2007 to 2008 (Chan and others 2008). In 2010, cowpea contaminated with isocarbophos, a highly toxic pesticide, was found in Hainan Province. Clenbuterol, an illegal veterinary drug, was used to feed pigs and the consumption of the pork resulting in flustered, trembling, headache, nausea, and vomiting for humans (Xue and Zhang 2013).
The bacterium Escherichia coli O104:H4 was found in fenugreek seeds from Egypt and killed and sickened many people in the European Union (Jia and Jukes 2013). Furthermore, the United States Department of Agriculture (USTA) estimated that the increased consumption of fresh produce is responsible for 48% of all the reported food illnesses in the US (Painter and others 2013). To improve food safety and minimize food incidents, it is crucial to have suitable techniques to detect food contaminants that are usually present at low concentrations in foods and other consumer products. The method should be fast, cheap, and sensitive to quickly detect contaminated foods and prevent unsafe products from reaching consumers.
Current analytical methods for the detection and quantification of food adulterants in commercial products include high performance liquid chromatography (HPLC), gas 1 chromatography mass spectroscopy (GC/MS), and other methods. These techniques have been widely used to analyze both chemical and biological contaminants in different food products. For example, Wahed and others (2016) employed HPLC to quantify formaldehyde in mango, fish, and milk.