백금/HRP 공동 캡슐화 나노입자의 시너지 촉매 작용을 통한 CRISPR-Cas12a 기반 DNA 돌연변이 검출 신호 향상 = Signal Enhancement of CRISPR–Cas12a-Based DNA Mutation Detection via Synergistic Catalysis by Platinum/Horseradish Peroxidase Co-encapsulated Nanoparticles

다국어 초록 (Multilingual Abstract)

Lung cancer is the leading cause of cancer-related deaths worldwide, accounting for the highest mortality rate in both men and women. As lung cancer has indiscernible initial symptoms and is often already advanced at the time of detection, early diagnosis is crucial to significantly improving survival rate. In particular, epidermal growth factor receptor (EGFR), a point mutation DNA, plays an important role in diagnosis of lung cancer. Among various detection methods for DNA biomarkers, colorimetric assays allow simple and fast visual confirmation depending on targets presence. In this study, We propose a sensitive CRISPR-Cas12a-based colorimetric biosensor to detect EGFR using enzyme balls for early diagnosis of lung cancer, enabling simple and rapid on-site diagnosis. CRISPR-Cas12a exhibits indiscriminate trans-cleavage activity on adjacent single-stranded DNA (ssDNA) upon recognition of a specific target. Therefore, we synthesized an enzyme-ball that has been capsulated with horseradish peroxidase (HRP) and platinum (Pt) nanoparticles, which enhance the colorimetric signal. Simply, this study provides a simple and rapid strategy for sensitive diagnosis of lung cancer.

목차 (Table of Contents)

• 1. Introduction 1
• 1.1 The Importance of DNA Detection 1
• 1.2 Significance of Single-Nucleotide Mutation Diagnosis 3
• 1.3 CRISPR based DNA detection 5
• 1.4 Point-of-care testing (POCT) with Colorimetric system 6
• 1.5 Enzyme and Nanozyme Catalysis for Colorimetric Detection 8
• 1.5.1 Enzymes as Natural Catalysts 8
• 1.5.2 Nanozymes as Artificial Enzyme Mimics 9
• 1.5.3 Hybrid EnzymeNanozyme Systems 11
• 1.6 Designing a DNA Detection System 12
• 2. Materials and Methods 16
• 2.1 Materials and Reagents 16
• 2.2 Synthesis and Characterization of Materials 17
• 2.2.1 Characterization 17
• 2.2.2 Synthesis of HRP@BNP@Pt 18
• 2.2.3 Fabrication of porous Au Plates 19
• 2.3 Evaluation of Material Performance 21
• 2.3.1 Evaluation of POD synergistic Activity 21
• 2.3.2 Evaluation of CRISPR reaction 21
• 2.3.3 Evaluation of restrIction Enzyme reaction 22
• 2.4 Construction of Colorimetric DNA detection Platforms 23
• 2.4.1 Preparation of Nanoprobe 23
• 2.4.2 Integration of porous Au Plates 23
• 2.4.3 Colorimetric Detection of DNA using CRISPR 26
• 2.4.4 Specific Discrimination of Mutation DNA 27
• 2.4.5 Statistical Analysis 28
• 3. Results and Discussions 29
• 3.1 Detection system characterization 29
• 3.1.1 Characterization of HRP@BNP@Pt 29
• 3.1.2 Characterization of CRISPR for WT and MT DNA 32
• 3.1.3 Design of the Probe 35
• 3.2 Evaluation of POD synergistic Activity 37
• 3.3 Evaluation of CRISPR-based colorimetric detection system 40
• 3.4 Sensitivity Test 44
• 3.4.1 Sensitive Detection for EGFR MT DNA 46
• 3.4.2 Specific EGFR MT DNA Detection by using restriction Enzymes 46
• 4. Conclusions 49
• References 52