A STUDY OF BIOSENSORS: NOVEL APPLICATION AND NOVEL ELECTRODE by PO-YUAN (PAUL) LIN Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Chung-Chiun Liu Academic Advisor: Dr. James D. McGuffin-Cawley Department of Materials Science and Engineering CASE WESTERN RESERVE UNIVERSITY May, 2013 0 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Po-Yuan (Paul) Lin Candidate for the Doctor of Philosophy degree*. (signed) Prof. James D. McGuffin-Cawley (chair of the committee) Prof. Chung-Chiun Liu Prof. Mark R. DeGuire Prof. Pirouz Pirouz (date) March 7, 2013 *We also certify that written approval has been obtained for any proprietary material contained therein. i Table of Content Table of Content ....................................................................................................................... ii List of Tables ............................................................................................................................ iv List of Figures ............................................................................................................................ v Abstract ................................................................................................................................... viii Part I: Background and Introduction ............................................................................... 1 Prostate Cancer ................................................................................................................................. 1 Diagnosis Methods of Prostate Cancer ...................................................................................... 3 Stages of Prostate Cancer and Treatments .............................................................................. 8 Biomarker Candidates .................................................................................................................... 9 Novel Prostate Cancer Biomarker, Alpha-Methylacyl-CoA Racemase .......................... 13 AMACR Assay and Biosensor ...................................................................................................... 16 Methods of AMACR Detection ................................................................................................................. 16 Pristanic Acid ................................................................................................................................... 20 Amperometric Biosensor ............................................................................................................. 26 Fabrication of Biosensor .............................................................................................................. 37 Ir-C Biosensor .................................................................................................................................. 42 Experiments ............................................................................................................................ 44 Enzyme Activity ............................................................................................................................... 44 ACOX3 Reaction Time ................................................................................................................... 45 H2O2 Measurement ......................................................................................................................... 46 AMACR Measurement .................................................................................................................... 47 AMACR in Blood Serum ................................................................................................................. 48 Hospital Samples ............................................................................................................................ 49 Results and discussion ........................................................................................................ 51 H2O2 Measurement ......................................................................................................................... 51 AMACR Measurement in PBS Solution ..................................................................................... 55 ACOX3 Reaction Time ................................................................................................................................ 58 AMACR Concentration Measurement .................................................................................................. 60 SLC27A2 for the Reduction of Incubation Time ................................................................... 62 AMACR Concentration Measurement in Serum .................................................................... 64 Hospital Samples ............................................................................................................................ 67 Conclusion ............................................................................................................................... 70 Future Research ..................................................................................................................... 71 ii Part II: Background and Introduction ............................................................................ 73 Hydrogen Peroxide (H2O2) .......................................................................................................... 73 Applications of H2O2 ...................................................................................................................... 74 Role of H2O2 in the Detection of Diseases ............................................................................... 75 Platinum ............................................................................................................................................ 76 Rotating Disk Electrode (RDE) ................................................................................................... 81 Transmission Electron Microscope (TEM) ............................................................................. 84 Energy-Dispersive X-ray Spectroscopy (XEDS) .................................................................... 85 Preparation of Pt/Ru Nanoparticle Catalyst ......................................................................... 86 Material Characterization ............................................................................................................ 87 Electrochemical Testing ............................................................................................................... 87 Results and Discussion ........................................................................................................ 89 Structure ............................................................................................................................................ 89 Electrochemical Characterization ............................................................................................ 92 Conclusion ............................................................................................................................ 102 Future work ......................................................................................................................... 103 References ............................................................................................................................ 104 iii List of Tables Table 1 Risk factors relevant to prostate cancer probability [7] ......................................... 3 Table 2 Side effects and complications of transrectal ultrasound-guided biopsy of prostate[19] ............................................................................................................ 6 Table 3 Questions ............................................................................................................... 9 Table 4 Candidates of biomarkers for prostate cancer detection[27-29] .......................... 12 Table 5 AMACR real-time RT-PCR results in peripheral blood specimens[30] ............. 14 Table 6 Sensitivity and average of standard deviation ..................................................... 66 Table 7 Gleason scores and their representations ............................................................. 66 Table 8 Population description and mean AMACR levels by patient group .................... 68 Table 9 Sensitivities of Pt, Ru and Pt-Ru at ηtotal = +0.2V and +0.4V versus Ag/AgCl 100 Table 10 Average standard deviation of Pt, Ru and Pt/Ru at ηtotal = +0.2V and +0.4V versus Ag/AgCl reference electrode ................................................................ 100 iv List of Figures Figure 1 Prostate location and structure[1] ......................................................................... 1 Figure 2 Age-specific rates of prostate cancer globally. (data taken in 2002)[5] .............. 2 Figure 3 Transrectal ultrasound-guided prostate biopsy[20] .............................................. 6 Figure 4 NIH grants from 1986 to 2009 in biomarker related fields[26] ......................... 10 Figure 5 Enzyme lowering activation energy to accelerate a reaction[41] ....................... 18 Figure 6 Schematic representation of the mechanism of an enzymatic reaction .............. 20 Figure 7 Pathway of pristanic acid formation from phytol.[46] ....................................... 22 Figure 8 Activation of fatty acid (acyl-CoA)