1：Experimental Design and Method Selection:

Synthesis of UCNP core/shell structure (NaYF4:Yb,Er,Tm/NaYF4) via thermal decomposition and coating with PMAO copolymer. Conjugation of DARPin protein to UCNP-PMAO using EDC/NHS chemistry. Characterization of nanocomplexes using photoluminescence spectroscopy, dynamic light scattering, TEM, and cytotoxicity assays. In vitro binding specificity assessed with HER2-positive (SK-BR-3) and negative (CHO) cell lines via confocal microscopy. In vivo studies included tumor xenograft models in immunodeficient mice for whole-body imaging, biodistribution analysis in organs, and comprehensive toxicity assessments (acute, chronic, immunotoxicity, allergenicity, reproductive toxicity, mutagenicity).

2：Sample Selection and Data Sources:

Human mammary adenocarcinoma SK-BR-3 cells (ATCC HTB30) and Chinese hamster ovary CHO cells (ATCC CCL-61) for in vitro studies. Balb/c nude athymic mice and hybrid mice F1 (CBA x C57Bl/6j) for in vivo studies. Tumor models established by subcutaneous injection of SK-BR-3 cells.

3：List of Experimental Equipment and Materials:

Spectrofluorimeter (CM2203, SOLAR), Zetasizer Nano ZS (Malvern), TEM LEO-912 AB OMEGA (Carl Zeiss), confocal laser scanning microscope LSM 710/710 NLO (Carl Zeiss), in-house optical whole-animal imaging system, MTT assay reagents, E. coli BL21(DE3) for DARPin expression, YCl3, Y2O3, Yb2O3, Er2O3, PMAO copolymer (Sigma-Aldrich).

4：Experimental Procedures and Operational Workflow:

UCNP synthesis involved precipitation and thermal treatment. DARPin expressed in E. coli and purified. UCNP coated with PMAO and conjugated to DARPin. Cell binding assays performed at 4°C to prevent endocytosis. Animal studies included IV injections of nanocomplexes, imaging at various time points, organ collection for histology, and toxicity evaluations following standardized protocols.

5：Data Analysis Methods:

Photoluminescence intensity used for concentration estimation. Hydrodynamic diameter and zeta potential measured via dynamic light scattering. Cytotoxicity data analyzed with SigmaPlot 8.0. In vivo data analyzed with STATSTICA v.7.0 using Student's t-test, Mann-Whitney U-test, chi-squared test, and non-parametric methods as appropriate. Image analysis performed with ImageJ 1.47v.