1：Experimental Design and Method Selection:

The study designed a heterogeneous assay using PEG microgels functionalized with DNA double strand probes based on toehold-mediated strand displacement for fluorescence recovery upon target capture. The design included optimization of probe density and microgel concentration to tune sensitivity.

2：Sample Selection and Data Sources:

Synthetic oligonucleotides of hcmv-miR-US4-5p and non-specific sequences (e.g., miR-143-3p) were used, along with human serum samples to test assay performance in complex matrices.

3：List of Experimental Equipment and Materials:

Materials included PEGDMA, acrylic acid, KPS, fluorescein O-methacrylate, EDC, PVA, DMSO, sodium hydroxide, MES buffer, methacryloxyethylthiocarbonylrhodamine B, Tris buffer, DNA/RNA oligonucleotides from Metabion, human serum from Lonza. Equipment included spectrofluorometer (Horiba JobinYvon FluoroMax-4), confocal microscope (Leica SP5), dynamic light scattering instrument (Malvern Zetasizer Nano ZS), and custom image-processing software.

4：Experimental Procedures and Operational Workflow:

Microgels were synthesized via free-radical polymerization, functionalized with DNA probes using EDC coupling, and quenched with quencher strands. Assays were performed by mixing microgels with target solutions in hybridization buffer, incubating overnight, and measuring fluorescence with spectrofluorometry or confocal microscopy. Kinetics and specificity tests were conducted.

5：Data Analysis Methods:

Fluorescence data were analyzed using non-linear fitting for kinetics (one phase association equation), statistical analysis with Student's t-test, and image analysis for confocal data to calculate intensity and signal-to-noise ratios.