A spatially confined "double-key lock" smart DNA hydrogel for dynamic detection of MicroRNA in cells.

Smart DNA hydrogels have attracted extensive attention in bioanalysis and biomedicine due to their programmable sol-gel phase transition ability and specific recognition characteristics of biomolecules. Herein, we propose a spatially confined "double-key lock" smart DNA hydrogel with temperature and target as double keys, which was constructed using poly(N-isopropylacrylamide)-acrylamide (NIPAM-AM) hydrogel as the carrier and aggregation-induced emission (AIE) type 1,1,2,2-tetra(4-carboxybenzene) ethylene (TCPE) as the fluorescence (FL) probe. This hydrogel exhibited a synergistic multi-response mechanism that combined the thermal phase transition of NIPAM-AM hydrogel, and the target recognition effect of complementary DNA strands. Upon concurrent stimulation by temperature elevation and target cDNA binding, this hydrogel underwent enhanced crosslinking, forming a denser three-dimensional network that restricted TCPE mobility and amplified its FL through aggregation. Molecular dynamics simulations further confirmed that the response mechanism involved the synergistic effect of thermodynamic equilibrium regulation and molecular configuration rearrangement. The combination of the smart DNA hydrogel and the rolling circle amplification (RCA) strategy realized the FL nucleic acid analysis of microRNA-21 (miRNA-21) as an analyte, exhibiting a wide linear response in the range of 0.1 fM to 10 nM with a limit of detection of 33.3 aM. Moreover, this platform was successfully applied to the quantitative analysis of miRNA-21 in the serum of lung cancer and gastric cancer patients, and can in situ track the secretion behavior of tumor cell derived miRNA-21. This strategy provides new insights into the multi-scale design of temperature-biomolecular double-responsive smart nanomaterials and offers a promising strategy for advanced biosensing and bioanalytical applications.