Cavity hairpin ThT-light nucleic acid switches: the construction of label- and enzyme-free sensing and imaging platforms

**Background:** Thioflavin T (ThT) is a classical fluorescent dye increasingly used in nucleic acid conformation (NAC) research. However, most NACs that excite ThT fluorescence (e.g., G-quadruplexes) require demanding conditions (e.g., K⁺/Na⁺ ions) or form unique structures, limiting ThT's application in sensing and imaging. This study aimed to develop a more universal, easily formed NAC—CGG-AAA mismatched cavity hairpin ThT-light nucleic acid switches (CHTLNAS)—for label- and enzyme-free sensing and intracellular imaging.

**Methods:** The authors used molecular docking (HDOCK server) to simulate ThT binding to hairpins with single, double, or triple mismatched base-pair cavities (A-A, A-G, A-C combinations). The CGG-AAA (A-C/A-G/A-G) triple mismatch showed the best docking score and receptor-ligand interactions. Fluorescence measurements were performed on a Cary Eclipse spectrophotometer (excitation 445 nm, emission 490 nm). Sequences were synthesized with varying numbers of CGG-AAA cavities, and ThT fluorescence enhancement was compared to complementary duplexes, single-stranded DNA, and G-quadruplex-forming sequences. For sensing, aptamer sequences for tetracycline (TET) and berberine (BB) were incorporated into CHTLNAS. Optimal hairpin loop length (3 bases for TET) and reaction conditions (5 min ThT intercalation, 8 min target incubation, 1:2 sequence:ThT ratio, 10 mM Tris-HCl pH 7.4) were determined. Detection performance was evaluated in spiked milk (TET) and human plasma (BB) samples. For intracellular imaging, CHTLNAS was conjugated to liposomes (HC-Lip) via cholesterol modification, loaded with ThT, and incubated with 3T3-L1 cells. Confocal laser scanning microscopy (CLSM) was used to track ThT distribution over 4–16 h. Binding affinity (Kd) was determined by nonlinear fitting of fluorescence titration curves. Melting temperatures were measured using SYBR Green I fluorescence.

**Key Results:** The CGG-AAA cavity hairpin achieved >500-fold fluorescence enhancement over spontaneous ThT fluorescence, 17–20-fold higher than ssDNA, and 2.5–5-fold higher than complementary duplex. The TET aptasensor (3H3Md-TET) showed a linear range of 1 nM–50 μM (R² = 0.99526) with a limit of detection (LOD) of 0.64 nM. Recovery in spiked milk ranged from 98.6%–105.8%. The BB aptasensor (H3Md-BB) showed a linear range of 1 nM–10 μM (R² = 0.99756) with an LOD of 0.55 nM. Recovery in spiked plasma ranged from 104.7%–106.4%. Both sensors completed detection within 10 min. Binding affinity (Kd) of CHTLNAS for ThT was ~2.938 μM, compared to ~159.6 μM for complementary duplex. In cellular imaging, HC-Lip+ThT showed clear cytoplasmic ThT signal co-localized with liposomes for up to 8 h, with Mander's overlap coefficient (M-value) >0.6. ThT nuclear entry was delayed beyond 8 h in the experimental group, whereas the control group (hairpin without cavity) showed nuclear ThT signal within 4 h and M-value >0.8 by 12 h. The ThT concentration used for imaging was 200 nM, significantly lower than typical μM levels required for other NAC-based approaches.

**Clinical Implications:** CHTLNAS provides a versatile, label- and enzyme-free platform for rapid (10 min), sensitive (pM–nM LOD) detection of small molecules like antibiotics (tetracycline) and alkaloids (berberine) in complex biological matrices (milk, plasma). This has direct applications in food safety monitoring and therapeutic drug monitoring. The ability to stably load ThT at low concentrations (nM) and prolong its cytoplasmic retention (>8 h) overcomes the high background signal and rapid nuclear accumulation that have limited ThT's use in live-cell imaging. This platform could be adapted for tracking drug delivery carriers, monitoring intracellular targets, and studying nucleic acid dynamics without the need for covalent labels or specialized conformations like G-quadruplexes.