Semiconductor nanocrystals (quantum dots) have been increasingly employed in measuring the dynamic behavior of biomacromolecules using fluorescence correlation spectroscopy. This poses a challenge, because quantum dots display their own dynamic behavior in the form of intermittent photoluminescence, also known as blinking. In this review, the manifestation of blinking in correlation spectroscopy will be explored, preceded by an examination of quantum dot blinking in general.

Two-photon excitation fluorescence cross-correlation spectroscopy (TPE-XCS) is a very suitable method for studying interactions of two distinctly labeled fluorescent molecules. As such, it lends itself nicely to the study of ligand-receptor interactions. By labeling the ligand with one color of fluorescent dye and the receptor with another, it is possible to directly monitor ligand binding rather than inferring binding by monitoring downstream effects. One challenge of the TPE-XCS approach is that of separating the signal due to the receptor from that of the ligand. Using standard organic fluorescent labels there is almost inevitably spectral cross talk between the detection channels, which must be accounted for in TPE-XCS data analysis. However, using quantum dots as labels for both ligand and receptor this limitation can be alleviated, because of the dot's narrower emission spectra. Using solely quantum dots as fluorescent labels is a novel approach to TPE-XCS, which may be generalizable to many pairs of interacting biomolecules after the proof of principle and the assessment of limitations presented here. Moreover, it is essential that relevant pharmacological parameters such as the equilibrium dissociation constant, K(d), can be easily extracted from the XCS data with minimal processing. Herein, we present a modified expression for fractional occupancy based on the auto- and cross-correlation decays obtained from a well-defined ligand-receptor system. Nanocrystalline semiconductor quantum dots functionalized with biotin (lambda(em) = 605 nm) and streptavidin (lambda(em) = 525 nm) were used for which an average K(d) value of 0.30 +/- 0.04 x 10(-9) M was obtained (cf. native system approximately 10(-15)). Additionally, the off-rate coefficient (k(off)) for dissociation of the two quantum dots was determined as 5 x 10(-5) s(-1). This off-rate is slightly larger than for native biotin-streptavidin (5 x 10(-6) s(-1)); the bulky nature of the quantum dots and restricted motion/orientation of functionalized dots in solution can account for differences in the streptavidin-biotin mediated dot-dot binding compared with those for native streptavidin-biotin.

Quantum dots are now extensively used as luminescent tags for biological and chemical applications; however, their propensity to display intermittent luminescence (aka blinking) has limited their use in quantitative assays. Various surface active redox agents, such as β-mercaptoethanol, have been posited as antiblinking agents to help mitigate this challenge. We have examined the effect of β-mercaptoethanol on the luminescence intermittency of CdSe/ZnS quantum dots using fluorescence correlation spectroscopy. We find that β-mercaptoethanol lowers the quantum dot brightness and therefore lowers the probability of observing blinking but does not otherwise inhibit intermittent luminescence. Based on changes in fluorescence correlation data and excited-state lifetimes, we propose that the partitioning of β-mercaptoethanol onto the quantum dot surfaces increases nonradiative de-excitation rates. This behavior appears to be independent of the quantum dot surface passivation layer or the solution contents.

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