Development of DNA biosensor based on the optical properties of gold nanoparticles

다국어 초록 (Multilingual Abstract)

The object of this thesis is to develop new homogenous detection system based on the gold nanoparticles optical properties. In this work, a gold nanoparticle-based detection methodology for sensitive and specific DNA-based diagnostic application is described. A sandwich format of Au-NPs/DNA/MMP was fabricated, the target DNA were captured and separated by taking advantage of MMPs while the Au-NPs modified with oligonucleotide detection sequences plays a role of the recognizer and singal producer. Therefore, the quantitative information of target analyte is translated into a colorimetric signal which can be easily quantitatively measured by a low-cost UV-vis spectrophotometric analysis.
Quantitative detection of specific viral DNA has become a pressing issue for the earlier clinical diagnosis of viral infectious diseases. Therefore, we reported a simple, sensitive, and inexpensive quantitative approach for DNA detection based on the autocatalytic Au deposition of gold nanoprobes via the surface reduction of AuCl-4 to Au0 on their surface in the presence of ascorbic acid (AA) and cetyltrimethylammonium bromide (CTAB). On this basis, signal enhancements in the absorbance intensity and kinetic behavior of gold enlargement in the aqueous phase have been well-investigated and explained for the selection of analytical parameters. To achieve high sensitivity, magnetic particles conjugated with capture probes (PMPs) were employed for the collection of gold nanoprobes. After denaturated by ion a pH 11 solution, the amplified signals of gold nanoprobes, which is proportional to the concentration of the target DNA, could easily be confirmed by a UV-vis scanning spectrophotometer. Limit of detection could be obtained as low as 1.0 fM by this simple method.
Due to the much lower stability of mismatched DNA strands caused by unstable duplex structures under relative low salt concentration solutions, hybridization efficiency under different buffers was well investigated, and thus, the optimized salt concentration allowed for discrimination of single-mismatched DNA from complementary targets. The results indicated this to be a very simple and economic strategy to detect single-mismatched DNA strands.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Theoretical backgrounds 4
• 2.1. Biosensor 4
• 2.2. Gold nanoparticles(Au-NPs) 5
• 2.2.1. Manufacture of gold colloids 6
• 2.2.2. Effects from gold nanoparticles quality 9
• 2.2.3. Choice of gold particle size 11
• 2.2.4. Manufacturing gold conjugates 13
• 2.2.5. Stabilizing the conjugate 14
• 2.2.6. Catalytic growth of Au nanoparticles 14
• 2.3. Magnetic beads 15
• 2.3.1. Introduction of magnetic particles (MPs) 15
• 2.3.2. Application of MPs in biodetection 16
• 2.4. Ultraviolet-visible spectroscopy 18
• 2.4.1. Applications 18
• 2.4.2. Beer-Lambert law 19
• 2.4.3. Ultraviolet-visible spectrum 22
• 2.5. Transmission electron microscopy (TEM) 22
• 2.5.1. Background 23
• 2.5.2. Limitations 24
• 2.6. Breast cancer suppressor gene (BRCA1) 24
• 2.7. Nucleic acid hybridization detection 25
• 3. Materials and methods 28
• 3.1. Materials 28
• 3.2. Methods 30
• 3.2.1. Preparation of gold seeds 30
• 3.2.2. Preparation of growth solution 30
• 3.2.3. Preparation of OEG-stabilized gold nanoprobes 31
• 3.2.4. Preparation of PMPs capture probes 32
• 3.2.5. DNA hybridization and denaturation procedure 32
• 3.2.6.Determination of salt concentration on the hybridization efficiency 33
• 4. Results and discussion 34
• 4.1. Systhesis and characterization of Au-NPs 34
• 4.2. Characterization of gold nanoprobes 35
• 4.3. Quantitative detection of DNA by nanogold enlargement in aqueous solution 37
• 4.3.1 Growth of gold nanoprobes in aqueous solution 37
• 4.3.2. Investigation of the kinetics behavior for nanogold growth 41
• 4.3.3. DNA detection using the catalytic gold nanoprobes in solution phase 43
• 4.3.4. Investigation of non-specific recognition of non-cognate DNA in the detection platform 46
• 4.4. Single mismatched DNA detection by using salt-inducing hybridization 47
• 4.4.1. Evaluation of gold nanoprobes during washing steps 49
• 4.4.2. Investigation of DNA hybridization event under different salt concentration conditions 50
• 4.4.3. Discrimination of single mismatched DNA using salt-inducing hybridization 52
• 4.4.4. Determination of the detection range and nonsepecific recognition. 54
• 4.5. Conclusions 57
• Abstract 59
• References 61