Abstract
In this study, we describe a novel universal and highly sensitive strategy for the electrochemiluminescent (ECL) detection of sequence specific DNA at the aM level based on Nt.BbvCI (a nicking endonuclease)-assisted target recycling amplification (TRA), rolling circle amplification (RCA) and hemin/G-quadruplex. The target DNAs can hybridize with self-assembled capture probes and assistant probes to form “Y” junction structures on the electrode surface, thus triggering the execution of a TRA reaction with the aid of Nt.BbvCI. Then, the RCA reaction and the addition of hemin result in the production of numerous hemin/G-quadruplex, which consume the dissolved oxygen in the detection buffer and result in a significant ECL quenching effect toward the O2/S2O82− system. The proposed strategy combines the amplification ability of TRA, RCA and the inherent high sensitivity of the ECL technique, thus enabling low aM (3.8 aM) detection for sequence-specific DNA and a wide linear range from 10.0 aM to 1.0 pM. At the same time, this novel strategy shows high selectivity against single-base mismatch sequences, which makes our novel universal and highly sensitive method a powerful addition to specific DNA sequence detection.
Highlights
The detection of trace amounts of sequence specific DNA has numerous important applications in clinical diagnosis [1], food safety [2] and environmental monitoring [3]
The recently developed rolling circle amplification(RCA) [12,13,14] and target DNA recycling amplification (TRA) [15,16,17] techniques demonstrate some improvement in sensitivity; they are widely used in ECL DNA sensor fabrication [18,19]
The goal of this study is to develop a new universal and highly sensitive strategy for the ECL-meditated detection of sequence-specific DNA by coupling Nt.BbvCI-assisted TRA with an RCA reaction for signal amplification and formation of hemin/G-quadruplex
Summary
The detection of trace amounts of sequence specific DNA has numerous important applications in clinical diagnosis [1], food safety [2] and environmental monitoring [3]. PCR-based methods have been established in past decade, including real-time fluorescence quantitative polymerase chain reaction (rt-PCR) [4,5,6], gene chip [7], and gene sequencing [8]. These techniques exhibit the advantages of excellent sensitivity and rapidity, but the sophisticated, expensive equipment and professional skills needed limit their practical application. Many ECL DNA sensors have been proposed as alternatives to PCR-based methods for their simplicity, precise, selectivity, relative low cost and wide dynamic range except sensitivity. The recently developed rolling circle amplification(RCA) [12,13,14] and target DNA recycling amplification (TRA) [15,16,17] techniques demonstrate some improvement in sensitivity; they are widely used in ECL DNA sensor fabrication [18,19]
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