A programmable DNA circuit based on target recycling-assisted DNAzyme combined with elemental labeling ICP-MS for p21 mRNA detection.

The strand displacement reaction (SDR) has shown considerable potential in bioanalysis due to its enzyme-free nature, predictable base-pairing behavior and excellent programmability. Herein, we developed a programmable DNA circuit with SDR-mediated target recycling-assisted DNAzyme (TRDz) for dual signal amplification, and combined it with lanthanide (Tm)-labeled inductively coupled plasma mass spectrometry (ICP-MS) for the sensitive quantification of p21 mRNA. Specifically, we elaborately programmed both the input and output of the SDR by introducing a recognition probe (H1) and a DNAzyme functional domain, achieving a dual-cycle design. This design realizes enzyme-mimetic catalytic amplification without incorporating actual enzymes, thus preserving the high programmability and chemical stability of purely DNA-based circuits. In the presence of p21 mRNA, H1 efficiently recognizes it, exposing a toehold that triggers SDR. Through the SDR process, p21 mRNA can be recycled, leading to the release of a large number of output strands (S1). Subsequently, S1 self-folds into a catalytically active DNAzyme to cleave Substrate-Tm, generating abundant Tm-labeled fragments that can be captured by DNA-functionalized magnetic beads and analyzed by ICP-MS. Benefiting from the dual amplification of target recycling and DNAzyme cycling, a desirable detection limit of 0.37 pM was achieved. The developed TRDz-ICP-MS analytical platform also exhibited reliable performance in complex biological matrices, with recovery ranging from 93% to 112% in total RNA extracted from human whole blood, and could effectively distinguish cancer patients from healthy individuals. Overall, this simple, efficient, and programmable DNA circuit provides a promising platform for the sensitive detection of clinical biomarkers.