miRNA 검출 및 유방암 진단을 위한 합금 금-니켈-금 자성막대 기반 CRISPR-SERS biosensor = CRISPR-Based SERS Biosensor Employing Alloy Au-Ni-Au Magnetic Rods for miRNA Detection and Breast Cancer Diagnosis

다국어 초록 (Multilingual Abstract)

Breast cancer is one of the most common diseases and is the first cause of cancer-related deaths among women worldwide. Generally, late detection of breast cancer symptoms leads to higher mortality rates. Thus, early diagnosis of breast cancer is imperative to boost patient’s survival rate. Traditional diagnostic methods such as RT-qPCR take a long time, making it less suitable for on-site diagnosis.
Herein, we developed a CRISPR-Based SERS biosensor for an early on-site diagnosis of breast cancer targeting miRNA-511, one of the biomarkers for breast cancer. The synergy of SERS and CRISPR/Cas technologies has further enhanced the selectivity and sensitivity of miRNA-511 detection. In this work, alloy gold-nickel-gold nanorods (Au-Ni-Au NRs) were first electrochemically deposited onto an anodic aluminum oxide (AAO) mask to be used as the nanomaterial probe. Consequently, CRISPR/Cas13 becomes activated upon its addition in the presence of target RNA via its binding to crRNA, allowing for a sensitive detection.
Accordingly, leveraging the integration of CRISPR and SERS technologies, this study presents a biosensor capable of rapidly detecting breast cancer biomarkers on-site, removing the need for complex amplification steps and simplification on the experimental process. Therefore, our developed biosensor offers the potential for detecting other biomarkers beyond breast cancer disease that can be highly useful in developing countries and in field settings where medical infrastructures is limited.

목차 (Table of Contents)

• 1. INTRODUCTION 1
• 1.1 Breast Cancer Detection 1
• 1.2 MicroRNA (miRNA) 3
• 1.3 Conventional methods for miRNA detection 4
• 1.4 CRISPR Cas13 System 7
• 1.5 Surface Enhanced Raman Scattering (SERS) 9
• 1.6 Alloy Au-Ni-Au Magnetic Rods (Alloy Au-Ni-Au MRs) 11
• 2. Materials and Methods 13
• 2.1 Chemicals and Reagents 13
• 2.2 Fabrication of Alloy Au-Ni-Au MRs 14
• 2.3 Characterization of Alloy Au-Ni-Au MRs 15
• 2.4 Working ability test 16
• 2.5 CRISPR reaction optimization 17
• 2.5.1 Coating concentration 17
• 2.5.2 Coating time 17
• 2.5.4 crRNA concentration 18
• 2.5.5 CRISPR reaction time 18
• 2.6 Performance evaluation 19
• 2.6.1 Sensitivity test 19
• 2.6.2 Selectivity test 19
• 2.6.3 Clinical test 20
• 3. Results and Discussion 21
• 3.1 Experimental scheme 21
• 3.2 Fabrication of Alloy Au-Ni-Au MRs 24
• 3.3 Working ability test and CRISPR reaction optimization 29
• 3.4 Performance evaluation 34
• 3.4.1 Sensitivity test 34
• 3.4.2 Selectivity test 35
• 3.5 Clinical test 38
• 4. Conclusion 42
• Reference 44
• 요약 56
• List of Publications 58