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Development of isothermal nucleic acid amplification-based all-in-one-pot biomarker analysis system for multi-detection of exosomal biomarker

  • 이명준 (Lee Myeong-Jun, 서강대학교 일반대학원)

초록 (요약문)

Exosome biomarker analysis is one of the areas of research that is rapidly attracting attention in the fields of modern medicine and life sciences. However, this field of research is facing some major limitations. First, the complicate isolation and enrichment process of exosome are need, because exosomes exist in very small amounts in biofluids. Second, the complexity of exosomes makes it difficult to identify and interpret biomarkers. Exosomes are produced from various cell origins, and their composition is also diverse. Therefore, in order to analyze exosomes, it is necessary to analyze multiple biomarkers through normalization by the number of exosomes that may affect the number of biomarkers. Third, the exosome biomarker are existed in sample with very low concentration that need high sensitive detection method. To overcome these limitations, here, we suggest easy, fast, high-sensitive, high-specific and multi-detectable one-pot based biosensor to analyzing exosomal biomarker, including protein and miRNA. With only a simple procedure, the developed biosensor can simply separate exosomes, amplify multiple exosome biomarker signals in an isothermal condition, and analyze signals with high sensitivity and high specificity at the same time. It is possible to precisely analyze various biomarkers of various cell-derived exosomes and further obtain normalized signals with a combination of biomarkers. The developed exosome biomarker analysis system can be applied not only to exosome biomarker analysis but also to other protein and nucleic acid analysis tools. This biosensing system is expected to be applied in conjunction with machine learning and artificial intelligence in the future to enable more accurate exosome diagnosis and analysis.

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목차

1. Introduction 1
1.1 Overview 1
1.2 Objectives of this research 3
1.3 References 8
2. One-pot multiplexed quantitative proteomic analysis of exosome by promoter/aptamer coding DNA-enriched gold nanoparticles and isothermal nucleic amplification 11
2.1 Introduction 11
2.2 Materials and methods 15
2.2.1 Materials 15
2.2.2 Cell culture and Exosome Isolation 16
2.2.3 Exosome counting 17
2.2.4 Transmission Electron Microscopy 18
2.2.5 Western Blot Analysis 18
2.2.6 Synthesis of Pr-Aptamer/Ab/Au 19
2.2.7 Immuno-Isothermal RNA Transcription 20
2.2.8 Fluorescence analysis of aptamer binding with fluorescent substance 21
2.2.9 One-pot high-sensitive simultaneous quantitative proteomic analysis of exosome by Pr-Apatamer/Ab/Au 22
2.3 Data and Results 24
2.3.1 Characterization and Quantification of Exosome 24
2.3.2 Characteristic of Pr-Aptamer/Ab/Au 29
2.3.3 Effect of in vitro transcription inhibition via DNA functionalized AuNP 38
2.3.4 Fluorescence interference test according to mixture of Pr-Aptamer/Ab/Au 40
2.3.5 Detection of CD protein using Pr-Aptamer/Ab/Au 47
2.3.6 Specificity of the detection of CD proteins using Pr-Apatamer/Ab/Au 50
2.3.7 High-sensitive simultaneous quantitative proteomic analysis of exosome 51
2.4 Conclusion 57
2.5 References 58
3. Promoter-enriched AuNPs probe for one-pot isolation of exosome and dual-detection of isothermal amplificated miRNAs by fluorescence of amplified light-up aptamer 63
3.1 Introduction 63
3.2 Materials and methods 68
3.2.1 Materials 68
3.2.2 Exosome counting 71
3.2.3 Transmission Electron Microscopy 72
3.2.4 Western Blot Analysis 72
3.2.5 Synthesis of Promoter /CD63Ab/AuNPs 73
3.2.6 Cell culture and Immuno-isolation of exosome using Promoter/CD63Ab/Au 74
3.2.7 RNA mediated single-strand DNA ligation assay using SplintR ligase 75
3.2.8 In vitro transcription assay 76
3.2.9 Fluorescence analysis of aptamer binding with fluorescent substance 76
3.2.10 RT-qPCR 77
3.2.11 One-pot high-sensitive simultaneous quantitative miRNA analysis of exosome by Pr-Apatamer/Ab/Au 78
3.3 Results and Discussion 80
3.3.1 Production and characterization of exosome from cancer and normal cell 80
3.3.2 Characteristics of Pr/CD63Ab/AuNPs and isolation of exosome using Pr/CD63Ab/AuNPs 82
3.3.3 Principle of One-pot miRNA elongation & amplification and detection system 90
3.3.4 Effect of in vitro transcription inhibition via DNA functionalized AuNP 96
3.3.5 Fluorescence signal analysis of miRNA using one-pot miRNA analysis system based on Pr/CD63Ab/AuNPs 98
3.3.6 Interference test of signal between miRNA-21 and U6 103
3.3.7 Analysis of exosomal miRNA from various type of cell 106
3.4 Conclusion 112
3.5 References 113
4. Hourglass-mimicking one-pot isothermal amplification and colorimetric detection biosensor for exosome miRNA analysis by multi-functional nanoparticle with CRISPR-Cas12a 119
4.1 Introduction 119
4.2 Materials and methods 125
4.2.1 Materials 125
4.2.2 Synthesis of Pr/CD63Ab/Au-MNPs 127
4.2.3 RNA mediated single-strand DNA ligation assay using SplintR ligase and In vitro transcription assay 128
4.2.4 CRISPR-Cas12a trans-cleavage assay 129
4.2.5 Synthesis of MENPs conjugated Au plate for CRISPR-Cas12a slicing system 130
4.2.6 Exosome counting 131
4.2.7 Transmission Electron Microscopy 132
4.2.8 Cell culture and Immuno-isolation of exosome using Promoter/CD63Ab/Au-MNP 132
4.2.9 Hourglass-mimicking one-pot isothermal amplification and colorimetric detection biosensor for exosome miRNA analysis by multi-functional nanoparticle with CRISPR-Cas12a 133
4.3 Results and Discussion 135
4.3.1 Synthesis and characterization of Au-MNPs 135
4.3.2 Characterization of Pr/CD63Ab/Au-MNPs and application in exosome isolation 139
4.3.3 Investigation of One-pot isothermal miRNA amplification and CRISPR-Cas12a detection system 144
4.3.4 Fabrication and characterization of Au plate-ssDNA-MENPs 149
4.3.5 Hourglass-mimicking one-pot isothermal amplification and colorimetric detection of miRNA-21 by Au plate-ssDNA-MENPs with CRISPR-Cas12a 153
4.3.6 Quantitative analysis of exosomal miRNA-21 from various type of cell 161
4.4 Conclusion 164
4.5 References 165
5. Conclusion and Perspective 172
5.1 Conclusion 172
5.1.1 One-pot multiplexed quantitative proteomic analysis of exosome by promoter/aptamer coding DNA-enriched gold nanoparticles and isothermal nucleic amplification 173
5.1.2 Promoter-enriched AuNPs probe for one-pot isolation of exosome and dual-detection of isothermal amplificated miRNAs by fluorescence of amplified light-up aptamer 174
5.1.3 Hourglass-mimicking one-pot isothermal amplification and colorimetric detection biosensor for exosome miRNA analysis by multi-functional nanoparticle with CRISPR-Cas12a 174
5.2 Perspective of Further study 175
5.2.1 Wax valves and gravity-driven flow control integrated microfluidic device for isothermal nucleic acid amplification 175
5.2.2 Automatic multi-detection of exosome biomarkers by one-pot isothermal amplification with heater-printed microfluidic device using hydrostatic pressure 177
Vita 179
List of Publications 180

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