Facile Synthesis and Characterization of Fluorescent Polystyrene Nanospheres for Homogeneous Light-Initiated Chemiluminescence Immunoassay.

Homogeneous light-initiated chemiluminescence technology (LICA) is widely used in clinical diagnostics due to the advantages of high sensitivity, minimal reagent usage, and no need for washing. Luminescent microspheres receive singlet oxygen emitted by photosensitive microspheres to generate optical signals. Therefore,¹O₂-initiated luminescent nanospheres are crucial, but there are few reports on the preparation of ¹O₂-initiated luminescent nanospheres. Herein, monodisperse luminescent Eu/C-28@PS (Eps) nanospheres were prepared and optimized using chelate Eu (TTA)₃phen and 4-(2-phenyl-5,6-dihydro1,4-oxathiin-3-yl)-N, N-ditetradecylbenzenamine (C-28) as probe dye via THF/water swelling-shrinking procedure. Various swelling parameters were studied to obtain the swelling conditions that produce the minimum particle size and narrow size distribution, which shows good results in uniform particle size distribution (~ 250 nm, a PDI of 0.03), surface carboxylate content (1.18 mmol/g), and BSA loading capability (129.8 mg/g) in the case of 20 mg total probe dosage and 2 h of incubation at 40 °C using 14% THF/water mixture as a co-solvent system. The composition of the entrapped probe has a gain effect on the ¹O₂-initiated fluorescent signal and the optimal ratio of Eu (TTA)₃phen: C-28 (1:1) was obtained on a commercial analyzer using IgG and anti-human IgG as models in PBS buffer. These results indicate that monodisperse luminescent Eps nanospheres are suitable as light-initiated chemiluminescence sensors and have great application potential in early detection, screening tests, and prognostic evaluation of patients.