1：Experimental Design and Method Selection:

The study designed 11 modified HCR systems based on an optimized HCR system with simple modifications to incorporate AATT sequences for fluorophore binding. Agarose gel electrophoresis and fluorescence measurements were used to monitor HCR progress and fluorescence enhancement.

2：Sample Selection and Data Sources:

Unmodified hairpin oligodeoxyribonucleotides (hpODNs) H1 and H2 (36 nts) with AATT sequences were used. Trigger ODNs with various concentrations and mismatches were tested. Fluorophores Hoechst 33258 and QCy-DT were employed.

3：List of Experimental Equipment and Materials:

Hoechst 33258 (purchased from Sigma-Aldrich), QCy-DT (synthesized and purified), ODNs (purchased from Eurofins genomics), agarose gel, SYBR gold stain, gel imager (FAS-BG LED Box, NIPPON Genetics), fluorescence spectrophotometer (F-7000, HITACHI High-Technologies Corporation).

4：Experimental Procedures and Operational Workflow:

HCR was performed in 5X SSCT hybridization buffer at 25°C. Hairpin ODNs were heated to 95°C for 5 min and cooled to room temperature. Equal volumes of H1, H2, fluorophore, and trigger ODN were mixed, and fluorescence was measured over time. Agarose gel electrophoresis was conducted with 3% agarose gel in 0.5X TBE buffer, stained with SYBR gold, and imaged.

5：5X TBE buffer, stained with SYBR gold, and imaged. Data Analysis Methods:

5. Data Analysis Methods: Fluorescence intensity and spectra were measured using a fluorescence spectrophotometer. Gel images were quantified with Image J program. Detection limits were estimated from fluorescence titration data.