Improved Immunoassay Sensitivity in Serum as a Result of Polymer-Entrapped Quantum Dots: 'Papaya Particles'.

Fluorescent labels are widely employed in biomarker quantification and diagnostics, however they possess narrow Stokes shifts and can photobleach, limiting multiplexed detection applications and compromising sensitivity. In contrast, quantum dots do not photobleach and have much wider Stokes shifts, but a paucity of robust surface attachment chemistries for bioconjugation has limited their uptake in biomedical diagnostics. We report a novel class of biofunctional fluorescent labels based on trapping of ∼10(4) quantum dots within a core nanoparticle. The doped particles act as scaffolds for generation of a multilayered shell consisting of a functionalized hydrophilic polymer with covalently attached receptors for analyte capture. These constructs, which conceptually resemble a papaya fruit, are chemically stable, remain monodispersed for >6 months in buffer, and show utility in immunoassay applications. Using monoclonal antibody fragments against nonstructural protein dengue NS1, an early biomarker for dengue fever, antibody immobilization capacity was 75-fold higher compared with traditional carbodiimide protein coupling. In the model dengue immunoassay, we observed a 15-fold lower limit of detection and 4-fold higher fluorescence intensity with the "papaya particles" compared to current "best-in-class" commercial reagents. Direct deployment in human serum allowed sensitive detection of different NS1 serotypes with lower limits of detection within the clinically relevant range (1-10 ng/mL), and sufficient specificity for identification of the dengue serotype was achieved for concentrations >10 ng/mL (DV1-3) and >50 ng/mL (DV4). The combination of chemical and physical stability and high binding capacity combined with the intrinsic advantages of quantum dots may enable more simple, robust diagnostic assays in the future.