fig2

Figure 2. Validation of dual antibody co-localization on BsGNPs. (A) Schematic illustrating the dual-preparation strategy to verify co-localization of trastuzumab and pertuzumab on the same gold nanoparticle. Two complementary BsGNP formulations were generated: one containing PE-labeled trastuzumab with unlabeled pertuzumab, and the other containing FLU-labeled pertuzumab with unlabeled trastuzumab; (B) Fluorescence intensity measured in the PE channel, showing a strong signal exclusively for BsGNPs bearing PE-labeled trastuzumab; (C) Fluorescence intensity measured in the FLU channel, showing a specific signal exclusively for BsGNPs bearing FLU-labeled pertuzumab. Unlabeled BsGNPs exhibit negligible signal in both channels (B and C); (D) Representative FLIM images of (i) unlabeled BsGNPs, (ii-iii) single-labeled BsGNPs (PE-labeled or FLU-labeled, respectively), and (iv) dual-labeled (FLU/PE) BsGNPs. Fluorescent puncta correspond to antibody-functionalized nanoparticles. Scale bar = 500 µm; color bar indicates fluorescence lifetime (ns). (E) Fluorescence lifetime (FLT) and fluorescence intensity (FI) analyses. Characteristic single-component lifetimes for singly-labeled BsGNPs and multicomponent lifetimes in dual-labeled BsGNPs were detected within the same regions of interest, confirming co-localization of both fluorophores and thus both antibodies on individual nanoparticles. ² estimation criteria for all FLT values in our data analysis model were less than 15. Results are shown as the mean ± SD of three independently prepared samples (with the value for each sample calculated as the mean of five consecutive measurements). AU: Arbitrary units; ns: nanoseconds; BsGNP: bispecific gold nanoparticle; PE: phycoerythrin; FLU: fluorescein; SD: standard deviation.