1：Experimental Design and Method Selection:

The assay was designed in a competitive format using lateral flow test strips with streptavidin on the test line, biotinylated thyroxine, and antibody-conjugated magnetic nanoparticles. Methods included magnetic particle quantification (MPQ), spectral-correlation interferometry (SCI), spectral-phase interferometry (SPI), dynamic light scattering (DLS), and enzyme-linked immunosorbent assay (ELISA).

2：Sample Selection and Data Sources:

Human blood serum samples with known concentrations of free thyroxine were used, provided by Russian Cardiology Research and Production Complex. Calibration samples were prepared with different fT4 concentrations.

3：List of Experimental Equipment and Materials:

MPQ readers, SPI and SCI biosensors, DLS instrument, ELISA equipment. Materials included magnetic nanoparticles (Bio-Estapor Microspheres M1-020/50), antibodies, biotinylated thyroxine, streptavidin, nitrocellulose membranes, absorbent pads, and various chemicals from suppliers like Sigma-Aldrich and Thermo Fisher Scientific.

4：Experimental Procedures and Operational Workflow:

Conjugation of antibodies to magnetic nanoparticles using carbodiimide method; incubation of conjugates with serum samples and T4-bt; placement of test strips into samples; measurement of magnetic signal using MPQ readers; size distribution measurements with DLS; kinetic studies with SPI and SCI; specificity tests with non-target molecules.

5：Data Analysis Methods:

Data were analyzed using calibration curves (e.g., 5-parameter logistic curve for ELISA), calculation of dissociation constants from sensograms, and statistical analysis of size distributions and signal intensities.