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Label-free DNAzyme for highly sensitive detection of multiple biomolecules in real samples through target-triggered catalytic cleavage reactions with auramine O’s discriminated fluorescence emission

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Abstract

A universal enzyme strand (E-DNA) recyclable L-histidine (L-His), melamine (MA), and cisplatin (CP) biosensor was fabricated on the basis of a target-specific RNA-cleaving DNAzyme with specific auramine O (AuO) dye instead of thioflavin T. In this strategy, the substrate strand (S-DNA) of the RNA-cleaving site was constructed as an intramolecular stem-loop structure, and a GT-rich sequence was imprisoned in the double-stranded stem which inhibits the formation of stable G-quadruplex (G4cpx) with AuO. The presence of L-His initiates a catalytic reaction for cleaving the RNA site of the S-DNA hydrolytically releasing the GT-rich portion, which subsequently combines with AuO and forms a G4cpx for enhanced fluorescent signal. The subsequent addition of MA uncoils the G4cpx to form T-MA-T dsDNA, or addition of CP unwound the G4cpx to form CP-DNA leading to an intensive decrease of AuO emission. Remarkably, the liberated L-His can ultimately cause several rounds of cleavage, and the liberated E-DNA can catalyze the subsequent reaction with the other S-DNA. The use of L-His and E-DNA repeatedly induces S-DNA cleavage and intensifies the emission signal. The results show that the proposed biosensor is extremely sensitive to L-His, MA, and CP with a detection limit of 0.98, 10, and 3.4 nM respectively. To the best of our knowledge, the utilization of AuO as the G4cpx inducer and stabilizer for L-His, MA, and CP detection in real milk and urine samples has never been reported.

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Acknowledgements

The authors acknowledge the “Selective Excellence Research Initiative Fund SRMIST-2021” from the Department of Chemistry, SRM Institute of Science and Technology, Tamil Nadu—603 203, India.

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  1. Department of Chemistry, SRM Institute of Science and Technology, Kattankulathur, 603203, Tamil Nadu, India

    Arunjegan Amalraj & Panneerselvam Perumal

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  1. Arunjegan Amalraj
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Correspondence to Panneerselvam Perumal.

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Amalraj, A., Perumal, P. Label-free DNAzyme for highly sensitive detection of multiple biomolecules in real samples through target-triggered catalytic cleavage reactions with auramine O’s discriminated fluorescence emission. Anal Bioanal Chem 414, 4021–4037 (2022). https://doi.org/10.1007/s00216-022-04061-z

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  • DOI: https://doi.org/10.1007/s00216-022-04061-z

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