Title Page
Contents
ABSTRACT 9
Chapter Ⅰ. DNA-presenting-gold chips for electrochemical analysis of intercalation 12
1.1. Introduction 12
1.2. Materials and Methods 15
1.2.1. Materials 15
1.2.2. Fabrication of duplex DNA presenting monolayers on gold chip 15
1.2.3. Intercalation 16
1.2.4. Thermal DNA duplex stability 16
1.2.5. Electrochemistry 16
1.3. Results and discussions 18
1.3.1. Characterization of ferrocene-modified DNA-presenting electrode 18
1.3.2. Inspecting DNA duplex stretching by intercalation 21
1.3.3. Inspecting DNA stability increase by intercalation 30
1.4. Conclusion 33
Chapter Ⅱ. DNA-presenting-magnetic particles for HCV-1b cDNA detection using mass signal amplification 34
2.1. Introduction 34
2.2. Materials and Methods 37
2.2.1. Materials 37
2.2.2. Preparation of loop DNA-modified MPs 37
2.2.3. Preparation of photo-mass tag tethered hairpin DNA 37
2.2.4. HCR amplification 38
2.2.5. LDI-TOF MS analysis 38
2.2.6. Gel electrophoresis 38
2.3. Results and discussion 40
2.3.1. Feasibility and validation of isothermal HCR amplification combined with LDI-TOF MS and the photo-cleavable mass tag 40
2.3.2. Optimization of assay condition for HCR amplification 45
2.3.3. Detection of HCV-1b target in various concentrations 49
2.4. Conclusion 51
References 52
Abstract (in Korea) 57
Table 1.1. Oligonucleotides for intercalation analysis used in this study. 17
Table 1.2. Number of Ferrocene per unit area of duplex DNA presenting monolayers at various ratios of 3-mercapto-1-propanol (OH) and 3-mercaptopropionic acid (COOH). 19
Table 1.3. Area below the ferrocene peak and relative peak area in the absence and presence of intercalator according to temperature. 32
Table 2.1. Oligonucleotides for HCR used in this study. 39
Figure 1.1. Schematic illustration for the immobilization of ferrocene tethered DNA duplex and electrochemical analysis after intercalator treatment. 14
Figure 1.2. DPV responses for the DNA duplex-modified electrode at various ratios of the self assembled monolayer consisting of 3-mercapto-1-propanol (OH) and 3-... 20
Figure 1.3. (A) Schematic illustration of DNA duplex stretching according to the location of the Fc. (B) DPV responses of the MB and Fc for each condition (a-d). 22
Figure 1.4. DPV responses obtained from the DNA duplex after treatment of MB at various concentrations. 24
Figure 1.5. DPV responses above 75 µM concentration of MB. 25
Figure 1.6. Time-dependent DPV responses after treatment of MB. 26
Figure 1.7. DPV responses obtained from the DNA duplex after treatment of EtBr at various concentrations. 28
Figure 1.8. Time-dependent DPV responses after treatment of EtBr. 29
Figure 1.9. (A) Schematic illustration of confirming thermal stability increase by intercalator. (B) DPV responses in the absence (dotted line) and presence (solid line) of... 31
Figure 2.1. Schematic illustration for the detection of target DNA using hairpin mediated isothermal hybridization chain reaction combined with LDI-TOF MS and the... 36
Figure 2.2. Verification of the feasibility of the strategy. (A) Schematic diagram for target-initiated amplification by way of hybridization chain reaction. (B) Native gel... 41
Figure 2.3. Schematic diagrams for tethering amine-modified DNA using EDC/NHS chemistry. (A) Immobilization of the loop DNA on magnetic particles. (B) Conjugation... 43
Figure 2.4. Validation of photocleavage in LDI-TOF MS analysis. (A) Mass spectra of photo-mass tag using AuNPs as a matrix. (m/z 522.149 [M]⁺, m/z 545.133 [M+Na]⁺, m/z... 44
Figure 2.5. The intensity ratio (I/IS) at various concentrations of loop DNA ranging from 0.01 nmole to 0.04 nmole in the presence of internal standard (C₆EG₃OH). 46
Figure 2.6. The intensity ratio (I/IS) at various concentrations of HP DNA ranging from 0.8 nmole (20 eq) to 4.0 nmole (100 eq) when 0.04 nmole loop DNA was used... 47
Figure 2.7. The intensity ratio (I/IS) according to the incubation time from 0.5 h to 2.0 h in the presence of internal standard (C₆EG₃OH). 48
Figure 2.8. Representative mass spectra according to various target DNA concentrations ranging from 0 pmole to 10 pmole. S/N means signal to noise of the... 50