Detection of pesticide residues on apples using surface-enhanced Raman spectroscopy A Thesis SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Tuo Chen IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in FOOD SCIENCE Adviser: Dr. Theodore P. Labuza June, 2014 © Tuo Chen 2014 Acknowledgements I am deeply indebted to the people who have assisted me in completing my thesis successfully. I would like to give my deepest sense of gratitude to Dr. Ted Labuza, for his incredible support throughout my master’s studies. This thesis would not have been written without him. I want to thank Dr. Baraem Ismail, Dr. Christy Haynes and Dr. Lili He for being my graduate committee and giving valuable advice on improving this thesis. I gratefully acknowledge the constant encouragement, support and guidance of Dr. Lili He throughout this project, who used to be the post-doctoral in our lab and is now an assistant professor at U Mass. I could not accomplish my work without her support. Lili has been a wonderful example to learn from, in all facts of life. I would also like to thank Dr. Qinchun Rao for his assistance in my research. I learned a lot from the work we did together, from technical skills to scientific thinking. Special thanks goes to Dr. Josy John, Thomas Rodda, Browyn Deen, Alyssa Pagel and the other lab members. It is a great pleasure to work with all of you. Last but not least, I would like to thank U.S. Department of Agriculture to grant this project, which provided me great chance to get involved in this challenging project. i Dedication This thesis is dedicated to my father Bingdi Chen and my mother Chili Yang. ii Abstract Pesticides are an integral part of agriculture, while increasing use leads to residues in/on agricultural products. Federal monitoring and enforcement action is dependent on the technical capability to detect pesticides. However, current methods are elaborate, time- consuming and not cost-effective. In the first part of this work, a rapid and simple surface-enhanced Raman spectroscopy (SERS) method coupled with a surface swab method for recovery and quantitative detection of thiabendazole (TBZ) on apple surfaces was developed, optimized and validated. The whole apple surface was swabbed and the swab was vortexed to release the pesticides. After that, silver dendrites (AgD) were used to bind the pesticide for Raman measurement. The limit of detection of TBZ in methanol was 0.01µg/mL, (10ppb), while the Environmental Protection Agency (EPA) limit is 5µg/gram apple- weight (5 ppm). The concentration of the recovered TBZ was predicted using a partial least square model. The recovery from the surface swab method was calculated to be 59.7% to 76.6% for intentional contamination at 0.1, 0.3, 3 and 5 ppm (µg/g apple-weight) level, respectively. The final accuracy of the swab-SERS method was calculated to be between 90.0% and 115.4%, after corrected by the releasing factor (66.6%). iii In the second part, a new approach was proposed to detect acetamiprid using an aptamer- based SERS method. The acetamiprid aptamer was chosen from the literature, thoilated and conjugated onto AgD. To block the unbounded surface on the substrate surface after aptamer immobilization, bovine serum albumin (BSA), 2-mercaptoethanol (ME) 6- mercaptohexanol (MCH) and were investigated as blocking agents. MCH and ME cannot fully block the surface when encountered with interference. The typical peaks from acetamiprid did not show on the aptamer-blocking agent-acetampired spectra when using BSA as blocking agents. The aptamer and blocking agent immobility on AgD should be further investigated and the method should be further modified. Last, the swab method was further developed and validated using UV-visible spectroscopy as a reference method. A standard curve was established based on the absorbance at 245 nm at different concentrations from 0 ppm to 1000 ppm. The assay standard curve well fit the five-parameter logistic model (r2 =0.995). The concentration of acetamiprid in the extracts was determined using this standard curve. The recovery rate of refined surface swab method is 90.6 % ± 1.4 % (n=5). This assay has a low intra- and inter-assay coefficient of variation (CV < 5 %). iv Table  of  Contents   List  of  Tables  ............................................................................................................................  ix   List  of  Figures  ............................................................................................................................  x   Chapter  1  Introduction  .........................................................................................................  1   Chapter  2  Review  of  Literature  ..........................................................................................  5   Introduction  the  Key  Apple  Pesticides  ......................................................................................  5   TBZ  .........................................................................................................................................................................  5   Acetamiprid  ........................................................................................................................................................  7   Federal  Agencies  Duty  for  Pesticide  Monitoring  ....................................................................  8   The  U.S.  Department  of  Agriculture  (USDA)  ........................................................................................  8   Food  and  Drug  Administration  (FDA)  ....................................................................................................  9   U.  S.  Environmental  Protection  Agency  (EPA)  .................................................................................  10   Pesticide  Detection  Techniques  ................................................................................................  10   Current  Detection  Approaches  for  Pesticides  ..................................................................................  10   New  Pesticide  Detection  Approach  ......................................................................................................  11   Surface-­‐Enhanced  Raman  Spectroscopy  ................................................................................  15   History  ...............................................................................................................................................................  15   Mechanisms  ....................................................................................................................................................  16   SERS  enhancers/  silver  dendrites  (AgDs)  .........................................................................................  16   Applications  of  SERS  in  food  science  ...................................................................................................  18   Challenges  in  the  SERS  detection  method  .........................................................................................  20   v Aptamers  ...........................................................................................................................................  20   Aptamer  in  vitro  selection  .........................................................................................................................  21   Advantages  and  limitations  of  aptamers  ............................................................................................  22   Immobilization  of  aptamer  onto  nano  surface  substrates  .........................................................  23   Aptamer-­‐based  assays  for  food  analysis  ............................................................................................  24   Chapter  3  Surface  Swab  Capture  Method  Coupled  by  SERS  ....................................  26   Materials  and  Methods  .................................................................................................................  26   Materials  ...........................................................................................................................................................  26   Establishment  of  a  calibration  model  of  TBZ  in  methanol  .........................................................  28   Determination  of  the  limit  of  detection  of  the  SERS  method  for  TBZ  in  methanol  ..........  30   Development  of  the  surface-­‐swab  SERS  method  ............................................................................  31   Validation  of  the  swab-­‐SERS  method  on  the  apple  surface  ........................................................  34   Results  and  Discussion  .................................................................................................................  36   LOD  of  SERS  for  TBZ  in  methanol  ..........................................................................................................  36   Establishment  of  calibration  model  and  validation  model  .........................................................  43   Validation  of  the  swab-­‐SERS  method  on  the  apple  surface  ........................................................  45   Chapter  4  Aptamer  Based  SERS  Detection  Method  ....................................................  47   Materials  and  Methods  .................................................................................................................  47   Materials  ...........................................................................................................................................................  47   Aptamer  and  AgD  preparation  ...............................................................................................................  47   Blocking  the  non-­‐specific  binding  on  the  AgD  surface  .................................................................  49   vi Detecting  acetamiprid  using  aptamer-­‐AgD  complex  ....................................................................  49   Improvement  of  the  surface  swab  method  .......................................................................................  50   Raman  instrumentation  and  Data  analysis  .......................................................................................  53   Results  and  Discussion  .................................................................................................................  54   Blocking  non-­‐specific  binding  on  AgD  surface  ................................................................................  54   Determine  acetamiprid  using  aptamer-­‐AgD  complex  ..................................................................  59   Improvement  of  the  surface  swab  method  .......................................................................................  62   Chapter  5  Conclusion  ...........................................................................................................  68   Summary  of  Methods  ....................................................................................................................  68   The  Swab-­‐SERS  method  .............................................................................................................................  68   The  Aptamer  based  SERS  method  .........................................................................................................  69   Future  Studies  .................................................................................................................................  70   Feasibility  of  the  portable  Raman  instrument  .................................................................................  70   Multi-­‐residual  detection  development  ................................................................................................  70   Selection  of  the  blocking  agent  and  immobilized  the  aptamer  on  AgD  ................................  71   Chapter  6  References  Cited  ................................................................................................  72   Appendix-­‐A  Average  spectra  of  negative  control  .......................................................  81   Appendix-­‐B  Selected  data  of  swab  optimization  ........................................................  82   Appendix-­‐C  PCA  plot  of  different  concentrations  TBZ  solution  ............................  86   Appendix-­‐D  Selected  data  of  releasing  factor  .............................................................  89   vii Appendix-­‐E  The  swab-­‐SERS  method  validation  data  ................................................  90   Appendix-­‐F  Raw  data  of  UV-­‐vis  acetamiprid  detection  ............................................  91   Appendix-­‐G  Selected  data  analysis  results  of  refined  swab  method  ...................  94   viii