Heterogeneity of Upconversion Bioconjugates Revealed by Single Nanoparticle Spectroscopy

Lanthanide-doped upconverting nanoparticles (UCNPs) are promising for bioimaging and biosensing, with functionality often relying on conjugation to biomolecules, such as antibodies, for specific recognition. However, the factors governing the consistency and repeatability of nanoconjugate performance are often overlooked due to difficulties in resolving single conjugation events. Here, we present a correlative microscopy approach that combines upconversion and downshifting imaging to assess UCNP-antibody conjugates at the single-particle level. Using Alexa-647-labeled anti-spike antibodies, we quantify antibody loading via photobleaching analysis while leveraging upconversion emission to distinguish single particles from clusters. Statistical analysis reveals substantial heterogeneity among the conjugates, including unconjugated particles, single-particle conjugates, and clusters, with varied antibody numbers per UCNP, averaging up to six. We also show that structural changes in antibodies promote cluster formation. This single-particle-level strategy enables the direct quantification of nanoconjugate heterogeneity, offering valuable insights for optimizing the design and performance of UCNP-based biomedical tools.