Assessing the stochastic intermittency of single quantum dot luminescence for robust quantification of biomolecules

Single molecule detection schemes promise that one has the ability to reach the ultimate limit of detection: one molecule. In this paper, we use the stochastic luminescence of single semiconductor nanocrystals (quantum dots, QDs) to detect and localize particles as digital counts. These digital counts can be correlated to the concentration of analytes in solution. Here, we use total internal reflection fluorescence (TIRF) microscopy to probe individual QDs immobilized on a functionalized substrate. QDs have found their niche in the bioanalytical community due to their remarkable brightness and stability. Despite their numerous outstanding photophysical properties, QDs at the single particle level display a pronounced intermittent luminescence, posing a challenge for the detection of individual particles. In this paper, we demonstrate a reliable method for detecting QDs that takes advantage of these signal fluctuations by comparing the variations in the QD's fluorescence signals against variations of the background signal. The quantitative methodology developed here results in signal-to-background ratios up to 90:1, which is at least 8-times higher than the ratios obtained using methodologies relying solely on signal integration. This enhanced signal-to-background ratio facilitates a robust thresholding process and results in femtomolar limits of detection.