Solution-Phase Single Quantum Dot Fluorescence Resonance Energy Transfer

Abstract

We present a single particle fluorescence resonance energy transfer (spFRET) study of freely diffusing self-assembled quantum dot (QD) bioconjugate sensors, composed of CdSe-ZnS core-shell QD donors surrounded by dye-labeled protein acceptors. We first show that there is direct correlation between single particle and ensemble FRET measurements in terms of derived FRET efficiencies and donor-acceptor separation distances. We also find that, in addition to increased sensitivity, spFRET provides information about FRET efficiency distributions which can be used to resolve distinct sensor subpopulations. We use this capacity to gain information about the distribution in the valence of self-assembled QD-protein conjugates and show that this distribution follows Poisson statistics. We then apply spFRET to characterize heterogeneity in single sensor interactions with the substrate/target and show that such heterogeneity varies with the target concentration. The binding constant derived from spFRET is consistent with ensemble measurements.

Improved peptidyl linkers for self-assembly of semiconductor quantum dot bioconjugates

“…Although a noncovalent conjugation strategy is used here, the ratio of His 6 peptide and thus acceptor DNA acceptor per QD is also easily controlled through the molar ratio added for self-assembly with the QD as repeatedly demonstrated in Refs. [1,2,6,7,11,13,14,21,23] and discussed in detail in references [13,21,23].…”

“…The FRET analysis also accounted for the contribution and any deviation due to Poisson distribution effects on low valence during the selfassembly process as described in Ref. [13,21,23]. …”

Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles

“…adenovirus knob protein to small Au clusters (Hainfeld et al 1999), the enzyme glutathione S-transferase (Gupta et al 2008) to quantum dots or by direct binding of the polyhistidine tag to the Zn-containing inorganic surface of CdSe/ZnS quantum dots as discussed before, e.g. for maltose binding protein (Mattoussi et al 2000;Pons et al 2006a) or fluorescent proteins (Dennis & Bao 2008).…”

Bioconjugation of quantum dots: Review & impact on future application

“…However, in contrast to what is mentioned in literature they observed a decrease in avidity when more than 3 proteins were coupled to a single QD. [42] studied the distribution of histidine (His n )-tagged proteins per QD in a solution of QD-protein conjugates with a given nominal protein-to-QD ratio below saturation of the QD surface. They confirmed that in this situation the process follows a Poisson distribution.…”

“…gel electrophoresis). When the average conjugate valence exceeds 4 biomolecules per QD the effect of the Poisson distribution is minimized and can be neglected [42,43].…”