Quantum Dot Bioconjugates Enable Order(s)-of-Magnitude Enhancement of Immunoglobulin E Detection Sensitivity.

Improving the sensitivity of immunoglobulin E (IgE) detection is crucial because the concentrations of allergen-specific IgE antibodies are often very low, particularly at early sensitization stages or in patients with weak immune responses. Bright, photostable detection labels are therefore essential for allergen microarrays designed for multiplexed IgE detection. However, conventional organic fluorophores suffer from low brightness and rapid photobleaching, thereby limiting microarray analytical performance. In contrast, fluorescent quantum dots (QDs) are exceptionally bright and photostable, enabling prolonged signal accumulation and offering new opportunities for ultrasensitive IgE detection. Here, we present a comprehensive comparative study of allergochips employing conventional conjugates of streptavidin with an organic dye (DyLight550-Str) or QDs (QD565-Str) for simultaneous detection of allergen-specific IgE antibodies covering a broad panel of allergens. Although the configuration of the commercial scanner used for recording the microarray fluorescence intensity was not optimized for QD fluorescence, the QD565-Str bioconjugate enabled the identification of the IgE antibodies in approximately 70% of samples where they had been previously identified using the DyLight550-Str reporter. In addition to microarray immunoassays, the optical properties and photobleaching behavior of both bioconjugates were investigated under conditions relevant to routine microarray diagnosis using diode lasers operating at excitation wavelengths of 398, 447, and 532 nm. Optical characterization showed that the shortest-wave visible spectral band (398 nm) was optimal for excitation of QD565-Str in a wide concentration range, with a fluorescence intensity approximately 2.2-fold higher than that of DyLight550-Str under its optimal excitation conditions (at 532 nm). The QD565-Str bioconjugates exhibited orders of magnitude greater photostability in the allergochip, enabling long-term fluorescence signal accumulation, whereas DyLight550-Str underwent pronounced photobleaching under the same conditions. Overall, the total fluorescence signal generated by QD565-Str on the allergochip was more than an order of magnitude stronger than that of the DyLight550-Str conjugate. These findings demonstrate strong potential for the use of QD bioconjugates in the development of a next-generation ultrasensitive IgE-detecting allergochip platform for multiplexed allergy diagnosis.