Bioconjugates of photon-upconversion nanoparticles with antibodies for the detection of prostate-specific antigen and p53 in heterogeneous and homogeneous immunoassays

Sensitive immunoassays for the detection of tumor biomarkers play an important role in the early diagnosis and therapy of cancer. Using luminescent nanomaterials as labels can significantly improve immunoassay performance, especially in terms of sensitivity. Lanthanide-doped photon-upconversion nanoparticles (UCNPs) are nanocrystals capable of converting near-infrared radiation into visible light, and their emission spectra can be tuned by altering the dopant ions. In this study, the bioconjugation between UCNPs and biomolecules was optimized, and different conjugates of Er- and Tm-doped UCNPs (NaYF₄:Yb³⁺,Er³⁺ and NaYF₄:Yb³⁺,Tm³⁺) were prepared for detecting prostate-specific antigen (PSA) and tumor protein p53, comparing heterogeneous and homogeneous assay formats. The heterogeneous sandwich immunoassay achieved detection limits of 1.3 pg mL^-1 for PSA and 330 pg mL^-1 for p53. The homogeneous immunoassays were based on massively parallel spectroscopy (MPS), a novel artificial intelligence-aided single-molecule approach, utilizing conjugates of two different monoclonal antibodies with Er- and Tm-doped UCNPs, respectively. The conjugates provide distinct emission spectra, with only sandwich immunocomplexes of analytes and both types of labels showing signals at the same location. MPS was suitable for observing the immunocomplexes in an aqueous dispersion using only a small sample volume. This innovative method achieved detection limits of 8.2 ng mL^-1 and 390 pg mL^-1 for PSA and p53, respectively. MPS eliminates the need for time-consuming washing steps required in heterogeneous immunoassays and is amenable to high-throughput applications.