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Enhanced colorimetric detection of nucleolin by rolling circle amplification and DNAzyme for cancer diagnosis

  • 명지운 (서강대학교 일반대학원)

초록

Exosomal protein is a promising cancer detector in that it increases the accuracy of detection; that is, it does not need to be separated from exosomes because it is located on the surface of it. Therefore, many methods have been developed to analyze and quantify exosomal protein such as enzyme linked immunosorbent assay (ELISA) and western blot Assay. However, as the exosomal protein is difficult to be profiled due to its low concentration, the demand of amplifying it has emerged. In this regard, we substituted nucleolin, one of the exosomal protein biomarker for cancer, with the same amount of DNA, which can be easily amplified. Furthermore, it was amplified and detected using one of the simplest ways which are Rolling Circle Amplification (RCA) and DNAzyme. The result shows a limit of detection as low as 0.1 nM which is 100 times greater than that of conventional ELISA. Our system consists of antibody conjugated magnetic micro particle and detecting sequence which contains nucleolin aptamer and primer for amplification. The particles are magnetically separated after capturing nucleolin and hybridizing with detecting sequence. Then, the remaining detecting sequence, which is the same amount of nucleolin, undergoes RCA with hemin. As a result, because the circular template DNA used in RCA is designed to create a G-quadruplex structure that forms DNAzyme with hemin, DNAzyme proportional to the amount of nucleolin will be formed. Colorimetric response was obtained by reacting with 3,3′,5,5′-Tetramethylbenzidine (TMB) and used to detect nucleolin.

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목차

1. Introduction 2

2. Materials and Methods 4
2.1 Materials and reagents 4
2.2 Preparation and characterization of antibody-conjugated MMPs 5
2.3 Preparation of circular DNA templates 5
2.4 Nucleolin Detection 5

3. Results and discussions 7
3.1 Detection principle of the proposed system 7
3.2 Characterization of antibody-conjugated MMPs 9
3.3 Feasibility verification and optimization of the present system 11
3.3.1 Sandwich ELISA: Verification of antibody-nucleolin-detecting sequence hybridization 11
3.3.2 Preparation of single-stranded circular DNA 13
3.3.3 RCA 17
3.3.4 G-quadruplex structure and hemin DNAzyme 19
3.3.5 Feasibility of DNAzyme by RCA product 21
3.4 Detection performance of the presented system 23

4. Conclusion 25

5. Reference 26

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