A low-noise ratiometric fluorescence biosensor for detection of Pb based on DNAzyme and exonuclease III-assisted cascade signal amplification.

As a common heavy metal ion with strong toxicity and wide distribution, lead ions (Pb) had great harm to the human body. In this work, a low-noise ratiometric fluorescence biosensor was developed based on Pb-dependent DNAzyme and exonuclease III (Exo III)-assisted cascade signal amplification. Firstly, the substrate chain of DNAzyme (S-DNA) was modified on the surface of magnetic beads (MBs) through the combination of biotin and streptavidin, and then the enzyme chain of DNAzyme (E-DNA) was connected to the MBs by forming a double-stranded DNA (dsDNA) with S-DNA. A hairpin DNA (HP) labelled with Cy3 and Cy5 respectively at both ends was used as a fluorescence probe. The emission peaks of Cy3 and Cy5 can appear at 562 nm and 665 nm respectively, and their fluorescence intensity ratio (F/F) was chosen as the acquisition signal. The ratiometric sensor can reduce the interference of detection environment and avoid false positive reactivity. Due to the cleavage of DNAzyme and the release of single-stranded DNA (ssDNA) in the presence of Pb, the hairpin structure of HP was opened and the FRET between two fluorophores disappeared, resulting in the strengthened signal of Cy3 and the weakened signal of Cy5. Furthermore, the ratio [Formula: see text] signal increased gradually with the increase of Pb concentration. When the concentration of Pb was in the range of 0.1-1000 nM, [Formula: see text] had a good linear relationship with [Formula: see text], the correlation coefficient (R) was 0.997, and the limit of detection (LOD) was 77 pM. The presented ratiometric fluorescence biosensor had lower LOD and wider detection range via comparing with other methods. At the same time, the sensor also obtained the satisfactory results for detection of Pb in tap water, tea, and rice flour samples. The provided ratiometric biosensor has great potential in the monitoring of various targets. A low-noise ratiometric fluorescence biosensor based on the FRET between two fluorophores was developed, and the DNAzyme and exonuclease III-assisted cascade signal amplification was used to improve the sensitivity of the method. The biosensor had a detection limit as low as 77 pM.