Spectroscopic characterization of streptavidin functionalized quantum dots

Abstract

The spectroscopic properties of quantum dots can be strongly influenced by the conditions of their synthesis. In this work we have characterized several spectroscopic properties of commercial, streptavidin functionalized quantum dots (QD525, lot#1005-0045 and QD585, Lot#0905-0031 from Invitrogen). This is the first step in the development of calibration beads, to be used in a generalizable quantification scheme of multiple fluorescent tags in flow cytometry or microscopy applications. We used light absorption, photoexcitation, and emission spectra, together with excited-state lifetime measurements to characterize their spectroscopic behavior, concentrating on the 400-500nm wavelength ranges that are important in biological applications. Our data show an anomalous dependence of emission spectrum, lifetimes, and quantum yield (QY) on excitation wavelength that is particularly pronounced in the QD525. For QD525, QY values ranged from 0.2 at 480nm excitation up to 0.4 at 450nm and down again to 0.15 at 350nm. For QD585, QY values were constant at 0.2 between 500nm and 400nm, but dropped to 0.1 at 350nm. We attribute the wavelength dependences to heterogeneity in size and surface defects in the QD525, consistent with characteristics previously described in the chemistry literature. The results are discussed in the context of bridging the gap between what is currently known in the physical chemistry literature of quantum dots, and the quantitative needs of assay development in biological applications.

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

“…Our effort to develop calibration beads began with a study to characterize some spectroscopic properties of streptavidin functionalized quantum dots (1). An important finding from that work was the relatively low quantum yields of the Qdots relative to fluorescein.…”

“…They have very high absorption cross-sections and narrow emission bandwidths, relative to fluorescent dyes, that make them highly suitable for use as reporters for multiplexing assays 1 This work was supported by NIHK25AI 60036 (TB), NSF CTS0332315 (GPL), CA85747 (MAO), F32CA123842-01 (SKC), U54MH074425 (LAS). We thank Dr. James Jett for critically reading an earlier version of this manuscript.…”

“…The broad absorption continuum spectra, and tunability of emission energy suggests that a dot of a suitable color can be used as a calibration reporter standard on beads for a chosen fluorophore, provided all the spectroscopic parameters are known. This study is preceded by a systematic examination of some spectroscopic characteristics of commercially available quantum dots QD525, (Lot #1005-0045) and QD585, (Lot #0905-0031) (1). In that study, we determined the emission yields of the quantum dot samples over a wide range of excitation wavelengths (350-500 nm).…”

“…Within the same spectral window, anomalies associated with the QD525 photoluminescence spectra were more pronounced as the quantum yield varied from a high of ≈ 0.4 at 450 nm to a low of 0.2 at 480 nm. Size heterogeneity in the dots was believed to be a significant contributor to the variation in quantum yield (1). The data from that work are included in Table 1.…”

Quantum dots and prion proteins

“…Modification of QDs by the streptavidin proved a very successful method evaluated in numerous publications. 78,[95][96][97][98] Due to this success, streptavidin-QDs are nowadays also commercially available. In addition, biotin-functionalized QDs were developed to exploit the same interaction (Fig.…”