1：Experimental Design and Method Selection:

The study employed a molecular imprinting technique to create synthetic receptors for HSA detection. HSA-imprinted nanoparticles were synthesized via mini-emulsion polymerization using ethylene glycol dimethacrylate (EGDMA) as a cross-linker and N-methacryloyl-L-leucine methyl ester (MALM) as a functional monomer. The nanoparticles were characterized and immobilized on SPR chips for real-time, label-free detection of HSA in aqueous and urine samples using surface plasmon resonance (SPR) technology.

2：Sample Selection and Data Sources:

HSA, hemoglobin (Hb), and transferrin (HTR) proteins were used as samples, with HSA concentrations ranging from

3：15 to 500 nM in phosphate buffered saline (PBS) pH 4 and spiked urine samples. Competitor proteins (Hb and HTR) were used at 500 nM for selectivity studies. List of Experimental Equipment and Materials:

Materials included HSA, Hb, HTR, EGDMA, MALM, poly(vinyl alcohol) (PVA), sodium dodecyl sulphate (SDS), azobisisobutyronitrile (AIBN), PBS pH

4：4, and others from Sigma-Aldrich, Merck AG, and Fluka. Equipment included homogenizer (T10, Ika Labortechnik), centrifuge (Allegra-64R Beckman Coulter), FTIR-ATR spectrophotometer (Perkin Elmer Spectrum 100), Nano Zetasizer (NanoS, Malvern Instruments), SEM (Zeiss Supra55), freeze dryer (Chris Alpha 1-2 LD plus), platinum coater (Quorum Q150R), SPRimager II (GWC Technologies), contact angle analyzer (KRüSS DSA 100), AFM (Nanomagnetics Instruments), and ellipsometer (Nanofilm EP3-Nulling Ellipsometer). Experimental Procedures and Operational Workflow:

Synthesis of MALM monomer and HSA-imprinted nanoparticles via mini-emulsion polymerization; characterization of nanoparticles using FTIR-ATR, zeta-size, and SEM; preparation of SPR chips by cleaning with piranha solution and immobilizing nanoparticles; characterization of SPR chips using AFM, contact angle, and ellipsometry; real-time SPR monitoring of HSA binding in PBS and urine with equilibration, adsorption, and desorption steps; selectivity tests with Hb and HTR; reproducibility and stability assessments over multiple cycles and months.

5：Data Analysis Methods:

Data analysis included calculation of limit of detection (LOD) and limit of quantification (LOQ) using linear regression (LOD=3S/b, LOQ=10S/b), kinetic analysis with pseudo-first-order models, isotherm modeling (Scatchard, Langmuir, Freundlich, Langmuir-Freundlich), and statistical evaluation of reproducibility and stability using relative standard deviation (RSD).