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Electrochemical strategy for ultrasensitive detection of microRNA based on MNAzyme-mediated rolling circle amplification on a gold electrode
- Source :
- Microchimica Acta. 183:3061-3067
- Publication Year :
- 2016
- Publisher :
- Springer Science and Business Media LLC, 2016.
-
Abstract
- The authors describe an electrochemical strategy for ultrasensitive and specific detection of microRNA (miRNA). It is based on both multicomponent nucleic acid enzyme (MNAzyme) amplification and rolling circle amplification (RCA). In the presence of target miRNAs, partial enzyme A (partzyme A) and partial enzyme B (partzyme B) are assembled to form active MNAzymes. Once formed, the MNAzymes catalyze the cleavage of the hairpin substrates to liberate biotinylated fragments which hybridized with the capture probes immobilized on a gold electrode. The RCA is then initiated to form a product that binds many detection probes. Finally, the amperometric signal (best acquired at a working voltage of 0.22 V vs. Ag/AgCl) is obtained by employing the streptavidinylated alkaline phosphatase as the enzyme. This biosensor has a 1.66 fM detection limit, and a dynamic range that extends from 10 fM to 1 nM. It displays specificity down to single mismatch discrimination of target miRNAs and good reproducibility. It was successfully applied to the determination of miRNA in total RNA samples extracted from human breast adenocarcinoma MCF-7 cells.
- Subjects :
- Detection limit
Streptavidin
Chemistry
010401 analytical chemistry
010402 general chemistry
01 natural sciences
Combinatorial chemistry
Amperometry
0104 chemical sciences
Analytical Chemistry
chemistry.chemical_compound
Biochemistry
Rolling circle replication
Biotinylation
Nucleic acid
Differential pulse voltammetry
Biosensor
Subjects
Details
- ISSN :
- 14365073 and 00263672
- Volume :
- 183
- Database :
- OpenAIRE
- Journal :
- Microchimica Acta
- Accession number :
- edsair.doi...........16abffb8a955b81e162a5829d30acdff