Label-Free Fluorescent Determination of Lead (II) Using DNAzyme and Thiazole Orange

Abstract

A simple and label-free fluorescent sensor based upon a specific DNAzyme (8–17 DNAzyme) and thiazole orange (TO) for monitoring Pb²⁺ in water samples was described. The 8–17 DNAzyme was formed by an enzyme strand (17E) and a partially complementary substrate strand (17DS). Thiazole orange, the DNA intercalator, was nonfluorescent in aqueous solution while it intercalates into the 8–17 DNAzyme with a duplex structure resulting in strong fluorescence. In the presence of Pb²⁺, the 17E was activated and cleaved the 17DS into two at the ribonucleoside adenosine (rA) position, leading to the disruption of the duplex structure. This Pb²⁺-induced conformational change led to the quenching of the fluorescence of thiazole orange, thus forming the basis for the label-free biosensor. Under the optimized conditions, the developed protocol exhibited sensitive determination of Pb²⁺ with a detection limit of 0.73 nM and a linear range from 10 to 150 nM. The label-free fluorescent approach exhibited excellent selectivity, strong selectivity, and satisfactory analysis of a drinking water standard reference material and spiked river and tap water samples. Taking the advantage of the structure-switching 8–17 DNAzyme and the fluorescent intercalator of nucleic acids, this label-free sensor provides a new approach for routine screening of heavy metals based upon the DNAzyme.

Keywords:

• DNAzyme
• fluorescence
• lead (II)
• label-free analysis
• thiazole orange

Conflicts of interest

The authors declare that they have no conflicts of interest.

Additional information

Funding

This work was financially supported by the Innovation and Entrepreneurship Training Program for College Students of Jilin University (grant number S202110183164).