Title Page
Contents
LIST OF ABBREVIATIONS 10
ABSTRACT 11
PART Ⅰ. PEG and PEDOT:PSS-based Conductive Hydrogel Sensors for Specifically Detection of Cathepsin D in Human Sweat 13
1. INTRODUCTION 13
2. MATERIALS AND METHODS 17
2.1. Materials 17
2.2. Preparation of conductive hydrogels 17
2.3. MTS assay of conductive hydrogels 18
2.4. Fluorescence detection by cathepsin D 18
3. RESULTS AND DISCUSSION 19
3.1. Evaluation of the durability of conductive hydrogels 19
3.2. Cell cytotoxicity of conductive hydrogels 21
3.3. Peptide synthesized for cathepsin D substrate 23
3.4. Fluorescence detection results of cathepsin D 25
4. CONCLUSION 27
PART Ⅱ. Detection of Sarcopenia biomarkers based on immunomagnetic time-resolved fluorescence assay using Eu(III) chelate and Europium nanoparticles 28
1. INTRODUCTION 28
2. MATERIALS AND METHODS 32
2.1) Materials and Instruments 32
2.2) Instruments 32
2.3) Preparation of Eu(III) chelate conjugated with anti-TNF alpha antibodies 34
2.4) Preparation of EuNPs conjugated with anti-TNF alpha antibodies 34
2.5) Magnetic bead-based Sandwich assay 35
3. RESULTS AND DISCUSSION 37
3.1) Determination of the concentration of magnetic beads using TRF reader 37
3.2) Labeling Eu(III) chelate and EuNPs with antibodies 39
3.3) Standards curves of TNF-α Immunomagnetic assay using Eu(III) chelate 42
3.4) Standards curves of TNF-α Immunomagnetic assay using EuNPs 44
3.5) Spike test for saliva and specificity of TNF-α 47
4. CONCLUSION 49
Ⅲ. REFERENCES 50
Ⅳ. ABSTRACT IN KOREAN 55
Figure 1. (a) The Swelling Behavior of Conductive Hydrogel:Water Content Wt of PEGDA Hydrogel with different concentration PEDOT:PSS (b) FTIR spectra from Conductive...[이미지참조] 20
Figure 2. Cell viability test using MTS assay after 48 hours treating 1x, 0.5x, 0.25x of each hydrogel eluate for 24 hours in serum-free media (a) Calibration curve of 10⁵ cells/well of NIH-... 22
Figure 3. HPLC analysis of peptide synthesized for cathepsin D substrate (H-DABCYL-Arg-Gly-Phe-Phe-Pro-Lys(5/6-FAM)-Cys-NH₂) (a) The chromatographic profile shows a main... 24
Figure 4. (a) Fluorescence increases over time in FRET-Peptide-bound micropatterned hydrogel after cleavage when treated with 100μM and 200μM of cathepsin D (b) Fluorescence... 26
Figure 5. The fluorescence intensity of various concentrations of Eu(III)-labeled magnetic bead 38
Figure 6. Standard curves for (a) EuNPs and (b) EuNPs labeled with TNF-α antibodies measured using a lab-made TRF reader 40
Figure 7. (a) Comparison of DLS results for particle size distribution of europium nanoparticles (EuNPs) and TNF-α antibody labeled with europium nanoparticles (EuNPs-... 41
Figure 8. The fluorescence intensity of various concentrations for generating a standard curve for Human TNF-α detection, with TNF-α volumes of (a) 100 μL ranging from 10... 43
Figure 9. The fluorescence intensity of various concentrations within the dynamic range for generating a standard curve for human TNF-α detection, ranging from 1 pg/mL to 1000... 45
Figure 10. Scanning Electron Microscopy (SEM) images of Streptavidin-coated magnetic beads at high magnifications (a) 33,000x and (b) 60,000x. Subsequent to immunoreaction,... 46
Figure 11. (a) Impact of varied saliva dilutions (20%, 50%, and 80%) on TNF-α concentration in saliva and the specificity of TNF-α detection (b) The fluorescence intensity... 48
Scheme 1. (a) Overview of Solid-phase Peptide Synthesis (b) Structural formula of peptide (H-DABCYL-Arg-Gly-Phe-Phe-Pro-Lys(5/6-FAM)-C-NH₂) 15
Scheme 2. (a) General principle of FRET based on cleavage of peptide and illustration of the change of (b) Fluorescent signals by Cathepsin D (c) Structural formula of AC-PEG-SCM 16
Scheme 3. Scheme of TRF-based Immunomagnetic Assay 31
Scheme 4. (a) Principle of a lab-made TRF reader and (b) the raw data of fluorescence intensity 33
Scheme 5. Conversion of ATBTA-Eu³⁺ to DTBTA-Eu³⁺ and the labeling reaction to amino groups of antibodies 36