科研之友
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摘要
A RNA aptamer modified to DNA (N1) that could bind to the target of Ni2+ was found that could bind specifically to Hg2+ ion with typical secondary structure. Thereafter, several DNA aptamers with a certain conformation of sequence mutation and splice were designed in order to increase the property of Hg2+ binding, and Aptamer-based fluorescence assay for the Hg2+ ion determination was developed. The addition of the Hg2+ to a mixture containing the duplex of a fluorophore labeled aptamer and a quencher-modified antisense nucleic acid (Q2) would force the release of Q2 from labeled aptamer, which was spontaneously accompanied by the increase of fluorescence intensity and made Hg2+ in quantitative analysis. It was shown that stable labeled aptamer DNA and Q2 hydrogen bonds formed at concentration ration of 1: 3 (labeled aptamer and Q2, respectively), under the condition of 5 min denaturation in 94 degrees C and 30 min renaturation in room temperature (25 degrees C) in the fluorescence assay preparation. For aptamer N1, the fluorescence assay results showed that linear response toward Hg2+ concentration with fluorescence quenching system ranged from 1. 25 mg/L to 20 mg/L, with the limit of detection 0. 625 mg/L. In order to improve the sensitivity of aptamer N1, 4 oligonucleotides (N4, N5, N6, N7) were designed and synthesized based on the structure of N1. The results showed that N5 had the best affinity and specificity to Hg2+ which displayed the linear range for the Hg2+ concentration detection was 0. 156 mg/L to 2. 5 mg/L with a detection limit of 78 mu g/L.
