Fluorescent Bioanalytical Platforms Based on Graphene Oxide

학위논문 (박사)-- 서울대학교 대학원 : 화학부, 2015. 2. 민달희.Detection of biomolecules that were considered as important biological targets is a crucial tool in the field of clinical diagnosis and drug discovery. In general, most of the typical methods such as gel electrophoresis, immunoassay, antibody based assay have been showed relatively low specificity and sensitivity compared to required specifications for high level bio-analysis. These systems are laborious, time/cost-consuming, and not suitable for high throughput assay. Recently, alternative approaches have been developed based on nanomaterials such as gold nanoparticles, mesoporous silica nanoparticles and carbon based materials for noticeable improvement of the sensing performance. These nanomaterials have been applied in fluorescence, electrochemistry, mass spectrometry or SERS (Surface Enhanced Raman Scattering) based biosensors. These systems also enabled more rapid, cost and time-effective analysis, compared with conventional methods, thus they can provide a means in the field of target related basic research and further applications. Herein, we report fluorescent sensing systems based on graphene oxide (GO) for quantitative analysis of various biomarkers in clinical diagnosis and drug discovery. GO, a water-soluble version of graphene, has come into the spotlight in development of bioanalytical system due to its remarkable electronic, mechanical and thermal properties. Among these properties, the general strategy in this study relies on the fluorescence-quenching capability of GO for fluorescent molecules within short distance (~ 20 nm). By using fluorescent probes or substrates, this GO-based platforms allow real-time measurement and quantitative analysis for various biomolecules such as nucleic acids and enzymes in short time. In this study, we developed 5 bioanalytical platforms which were classified into two categories according to each purpose: 1) nucleic acid detection platforms for analysis of double stranded DNA, single stranded DNA or RNA, single nucleotide polymorphism (SNP), 2) enzyme assay platforms for analysis of DNA exonuclease and protein kinase. Commonly, they could overcome the limitations of classical approaches and showed various applicability for further application. We believe that the present platforms will become an important assay tool in the field of personalized diagnostics, drug discovery and individual biomolecules related basic research.Abstract -------------------------------------------------------------------------------------- i Table of Contents ------------------------------------------------------------------------- iii List of Figures ----------------------------------------------------------------------------- vi 1. Introduction 1.1 Nanotechnology in Bio-Application -------------------------------------- 1 1.2 Graphene Oxide-------------------------------------------------------------- 2 1.3 Description of Researches 1.3.1 Direct, Sequence-Specific Detection of dsDNA Based on Peptide Nucleic Acid and Graphene Oxide without Requiring Denaturation --------------------------------------------------------- 3 1.3.2 Bovine Serum Albumin: A Simple Strategy for Practical Applications of PNA in Bioanalysis ------------------------------ 4 1.3.3 A New Strategy for Nucleic Acid Detection with High Sequence Specificity Using Graphene Oxide and Peptide Nucleic Acid --------------------------------------------------------- 5 1.3.4 A Simple DNA Exonuclease Activity Assay Based on Graphene Oxide ----------------------------------------------------------------- 6 1.3.5 Robust and Quantitative Platform for Multiplexed Protein Kinase Inhibitor Screening ---------------------------------------- 7 1.4 Reference ----------------------------------------------------------------- 8 2. Direct, Sequence-Specific Detection of dsDNA Based on Peptide Nucleic Acid and Graphene Oxide without Requiring Denaturation 2.1 Introduction ---------------------------------------------------------------- 10 2.2 Result ----------------------------------------------------------------------- 14 2.3 Conclusion ----------------------------------------------------------------- 24 2.4 Material and Methods ---------------------------------------------------- 25 2.5 Reference ------------------------------------------------------------------ 29 3. Bovine Serum Albumin: A Simple Strategy for Practical Applications of PNA in Bioanalysis 3.1 Introduction ---------------------------------------------------------------- 32 3.2 Result ----------------------------------------------------------------------- 35 3.3 Conclusion ----------------------------------------------------------------- 54 3.4 Material and Methods ---------------------------------------------------- 55 3.5 Reference ------------------------------------------------------------------ 56 4. A New Strategy for Nucleic Acid Detection with High Sequence Specificity Using Graphene Oxide and Peptide Nucleic Acid 4.1 Introduction --------------------------------------------------------------- 59 4.2 Result ----------------------------------------------------------------------- 63 4.3 Conclusion ----------------------------------------------------------------- 76 4.4 Material and Methods ---------------------------------------------------- 78 4.5 Reference ------------------------------------------------------------------ 80 5. A Simple DNA Exonuclease Activity Assay Based on Graphene Oxide 5.1 Introduction --------------------------------------------------------------- 83 5.2 Result ----------------------------------------------------------------------- 86 5.3 Conclusion ----------------------------------------------------------------- 93 5.4 Material and Methods ---------------------------------------------------- 94 5.5 Reference ------------------------------------------------------------------ 96 6. Robust and Quantitative Platform for Multiplexed Protein Kinase Inhibitor Screening 6.1 Introduction ---------------------------------------------------------------- 98 6.2 Result --------------------------------------------------------------------- 101 6.3 Conclusion ---------------------------------------------------------------- 116 6.4 Material and Methods --------------------------------------------------- 118 6.5 Reference ----------------------------------------------------------------- 120 7. Conclusion --------------------------------------------------------------------- 123 Summary in Korean (국문요약) -------------------------------------------------- 125Docto