Rapid, sensitive and highly specific label-free fluorescence biosensor for microRNA by branched rolling circle amplification
A simple fluorescence biosensor for rapid and sensitive target microRNA (miRNA) quantification by branched rolling circle amplification (BRCA) is developed in this work. Target miRNA functions as primer to recognize and hybridize with a circle DNA template, initiating rolling circle amplification (R...
| Main Authors: | , , , |
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| Format: | Journal Article |
| Language: | English |
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2020
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| Online Access: | https://hdl.handle.net/10356/144341 |
| _version_ | 1811688606490689536 |
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| author | Ma, Qian Li, Pin Gao, Zhiqiang Li, Sam Fong Yau |
| author2 | Lee Kong Chian School of Medicine (LKCMedicine) |
| author_facet | Lee Kong Chian School of Medicine (LKCMedicine) Ma, Qian Li, Pin Gao, Zhiqiang Li, Sam Fong Yau |
| author_sort | Ma, Qian |
| collection | NTU |
| description | A simple fluorescence biosensor for rapid and sensitive target microRNA (miRNA) quantification by branched rolling circle amplification (BRCA) is developed in this work. Target miRNA functions as primer to recognize and hybridize with a circle DNA template, initiating rolling circle amplification (RCA) by Phi29 DNA polymerase. The introduction of reverse primers complementary to the RCA products enables isothermal BRCA, in which a large amount of deoxynucleotide (dNTP) were consumed and same number of pyrophosphates (PPi) were produced. In this study, a simple and non-expensively synthesized terpyridine-based Zn(II) complex is utilized as fluorescent probe for selective detection of pyrophosphate (PPi) over dNTP. The PPi generated in this isothermal amplification process efficiently chelates to this terpyridine-Zn(II) complex, forming a highly fluorescent complex, terpyridine-Zn(II)-PPi, whose fluorescence intensity is closely related with the initial target miRNA concentration. The utilization of the isothermal BRCA amplification and direct monitoring of the DNA polymerization by-product, i.e. PPi, for non-label fluorescence detection of miRNA greatly simplify this sensor procedure. This sensor shows a linear response between the fluorescence intensity and the target miRNA concentration from 50 to 500 fM with a detection limit of 25 fM. This much-simplified sensor offers a sensitive and easy-to-use platform for miRNA quantification, and hence may significantly enhance the utilisation of miRNAs as biomarkers in drug discovery, clinical diagnosis and life science research. |
| first_indexed | 2024-10-01T05:34:53Z |
| format | Journal Article |
| id | ntu-10356/144341 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T05:34:53Z |
| publishDate | 2020 |
| record_format | dspace |
| spelling | ntu-10356/1443412020-10-29T05:13:47Z Rapid, sensitive and highly specific label-free fluorescence biosensor for microRNA by branched rolling circle amplification Ma, Qian Li, Pin Gao, Zhiqiang Li, Sam Fong Yau Lee Kong Chian School of Medicine (LKCMedicine) Science::Medicine MicroRNA Terpyridine Zinc A simple fluorescence biosensor for rapid and sensitive target microRNA (miRNA) quantification by branched rolling circle amplification (BRCA) is developed in this work. Target miRNA functions as primer to recognize and hybridize with a circle DNA template, initiating rolling circle amplification (RCA) by Phi29 DNA polymerase. The introduction of reverse primers complementary to the RCA products enables isothermal BRCA, in which a large amount of deoxynucleotide (dNTP) were consumed and same number of pyrophosphates (PPi) were produced. In this study, a simple and non-expensively synthesized terpyridine-based Zn(II) complex is utilized as fluorescent probe for selective detection of pyrophosphate (PPi) over dNTP. The PPi generated in this isothermal amplification process efficiently chelates to this terpyridine-Zn(II) complex, forming a highly fluorescent complex, terpyridine-Zn(II)-PPi, whose fluorescence intensity is closely related with the initial target miRNA concentration. The utilization of the isothermal BRCA amplification and direct monitoring of the DNA polymerization by-product, i.e. PPi, for non-label fluorescence detection of miRNA greatly simplify this sensor procedure. This sensor shows a linear response between the fluorescence intensity and the target miRNA concentration from 50 to 500 fM with a detection limit of 25 fM. This much-simplified sensor offers a sensitive and easy-to-use platform for miRNA quantification, and hence may significantly enhance the utilisation of miRNAs as biomarkers in drug discovery, clinical diagnosis and life science research. Ministry of Education (MOE) Financial support of this work by the Ministry of Education (R-143- 000-A14-114 and R-143-000-A14-133) is gratefully acknowledged. 2020-10-29T05:13:47Z 2020-10-29T05:13:47Z 2018 Journal Article Ma, Q., Li, P., Gao, Z., Li, S. F. Y. (2019). Rapid, sensitive and highly specific label-free fluorescence biosensor for microRNA by branched rolling circle amplification. Sensors and Actuators B: Chemical, 281, 424-431. doi:10.1016/j.snb.2018.10.141 0925-4005 https://hdl.handle.net/10356/144341 10.1016/j.snb.2018.10.141 281 424 431 en R-143-000-A14-114 R-143-000-A14-133 Sensors and Actuators B: Chemical © 2018 Elsevier B.V. All rights reserved. |
| spellingShingle | Science::Medicine MicroRNA Terpyridine Zinc Ma, Qian Li, Pin Gao, Zhiqiang Li, Sam Fong Yau Rapid, sensitive and highly specific label-free fluorescence biosensor for microRNA by branched rolling circle amplification |
| title | Rapid, sensitive and highly specific label-free fluorescence biosensor for microRNA by branched rolling circle amplification |
| title_full | Rapid, sensitive and highly specific label-free fluorescence biosensor for microRNA by branched rolling circle amplification |
| title_fullStr | Rapid, sensitive and highly specific label-free fluorescence biosensor for microRNA by branched rolling circle amplification |
| title_full_unstemmed | Rapid, sensitive and highly specific label-free fluorescence biosensor for microRNA by branched rolling circle amplification |
| title_short | Rapid, sensitive and highly specific label-free fluorescence biosensor for microRNA by branched rolling circle amplification |
| title_sort | rapid sensitive and highly specific label free fluorescence biosensor for microrna by branched rolling circle amplification |
| topic | Science::Medicine MicroRNA Terpyridine Zinc |
| url | https://hdl.handle.net/10356/144341 |
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