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Biosens Bioelectron. 2014 Jul 15;57:103-9. doi: 10.1016/j.bios.2014.01.050. Epub 2014 Feb 3.

A universal electrochemical sensing system for small biomolecules using target-mediated sticky ends-based ligation-rolling circle amplification.

Author information

1
School of Chemistry & Environment, Beihang University, Xueyuan Road #37, Haidian District, Beijing 100191, China.
2
School of Chemistry & Environment, Beihang University, Xueyuan Road #37, Haidian District, Beijing 100191, China. Electronic address: lilidong@buaa.edu.cn.
3
School of Chemistry & Environment, Beihang University, Xueyuan Road #37, Haidian District, Beijing 100191, China. Electronic address: guolin@buaa.edu.cn.

Abstract

A novel versatile electrochemical platform for ultrasensitive detection of small biomolecules was developed using ligation-rolling circle amplification (L-RCA) with analyte-mediated sticky ends. In order to achieve DNA cyclization, we designed two ss-DNA probes: the leftpart probe could form a "hairpin" structure by denaturing; the rightpart probe could also form a "hairpin" structure based on analyte-activated conformation change. Then the two probes with the same sticky ends (G-AATTC) could be ligated in the presence of Escherichia coli DNA ligase, forming a circular template for rolling circle amplification (RCA), which could be triggered by adding the primer probe and Phi29 DNA polymerase. Electrochemical impedance spectroscopy (EIS) was employed as the detection method. Overall, the proposed L-RCA-based sensing system not only exhibits excellent analytical characteristics with a detection limit of 320 pM and a linear range of 5 orders of magnitude (1 nM-10 μM), but also provides a universal design idea of L-RCA, which broadens the use of DNA amplification method and holds great promise in ultrasensitive bioassay in the future.

KEYWORDS:

Adenosine; Electrochemical; Electrochemical impedance spectroscopy (EIS); Ligation-rolling circle amplification (L-RCA); Sticky ends

PMID:
24561524
DOI:
10.1016/j.bios.2014.01.050
[Indexed for MEDLINE]
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