Comparison of infrared-excited up-converting phosphors and europium nanoparticles as labels in a two-site immunoassay

Research in the field of immunoassays and labels used in the detection has been recently focused on particulate reporters, which possess very high specific activity that excludes the label as a sensitivity limiting factor. However, the large size and shape of the particulate labels may produce additional problems to immunoassay performance. The aim of this work was to study with two identical non-competitive two-site immunoassays whether up-converting phosphor (UCP) particles are comparable in performance with europium(III) chelate-dyed nanoparticles as particulate labels. In addition we strived to verify the common assumption of the photostability of up-converting phosphor particles supporting their potential applicability in imaging. Detection limits in two-site immunoassay for free prostate-specific antigen (free-PSA) were 0.53 ng L⁻¹ and 1.3 ng L⁻¹ using two different up-converting phosphors and 0.16 ng L⁻¹ using europium(III) nanoparticle. Large size distribution and non-specific binding of up-converting phosphor particles caused assay variation in low analyte concentrations and limited the analytical detection limit. The non-specific binding was the major factor limiting the analytical sensitivity of the immunoassay. The results suggests the need for nanoscaled and uniformely sized UCP-particles to increace the sensitivity and applicability of up-converting phosphor particles. Anti-Stokes photoluminescence of up-converting phosphor particles did not photobleach when measured repeatedly, on the contrary, the time-resolved fluorescence of europium nanoparticles photobleached relatively rapidly.