Fluorescence correlation spectroscopy using quantum dots: advances, challenges and opportunities Romey F. Heuff, Jody L. Swift and David T. Cramb* Received 22nd November 2006, Accepted 12th February 2007 First published as an Advance Article on the web 2nd March 2007 DOI: 10.1039/b617115j 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. 1. Introduction Semiconductor nanocrystals (quantum dots) are on the verge of revolutionizing the field of biophotonics. The extreme brightness and photostability of quantum dots have made single biomolecule imaging almost trivial. 1,2 The term ‘‘quan- tum dot’’, was coined to represent the reduced dimensionality of nanoscopic crystals made up of 100s to 1000s of unit cells, with core diameters ranging from 1.5 nm–6 nm. At this length scale, the confinement energy of the exciton (electron/hole pair) is stronger than the Coulombic interaction, resulting in quantization of the electronic energy. For optical transitions, excitons are generated through the absorption of light. An extensive and informative review of the optical properties of single quantum dots is given in ref. 3. The optical gap of a quantum dot is size-tunable in the visible to near infrared region of the electromagnetic spectrum. After excitation, emission of a photon occurs in the absence of other relaxation pathways. Conveniently, the dots have broadband absorption, making simultaneous excitation of a selection of different color dots using a single wavelength light source possible. Moreover, they have narrower emission profiles than organic dye molecules, facilitating easier spectral separation of the fluorescence signal. Thus, quantum dots have great potential for multiplexing in biological assay applications, as well as in imaging. Quantum dots have been successfully used in long-term biological imaging with little or no cytotoxic effects. 4,5 To date, quantum dots have been used for optical coding of mammalian cells, 6,7 in the formation of biosensors 8 as diag- nostic tools for cancer 9,10 and in the development of rapid screening techniques using multiplexed hybridization detec- tion of DNA sequences. 11 They have been used as donors for fluorescence resonance energy transfer (FRET) 12 and as fluoro- phores in fluorescence lifetime imaging (FLIM) 13 because of their long lifetimes (10s of ns) compared to organic fluoro- phores (1–5 ns). Near infrared dots have been used to map sentinel lymph nodes, 14 and dots in the visible region have tracked the diffusion of individual glycine receptors at various synaptic locations 15 and so on. 16 The chemical research of quantum dots has involved devel- oping luminescent core materials and then adding a higher bandgap shell to the core surface. The resultant core/shell quantum dots were found to have superior optical properties. One of the most widely studied core/shell quantum dots is composed of CdSe/ZnS. CdSe/ZnS quantum dots are made in hydrophobic highly coordinating organic solvents such as trioctyl phosphine oxide (TOPO) 17 and in order to render them biologically compatible they need further surface mod- ifications for water solubility and target specificity. 18 A sche- matic diagram of a core/shell quantum dot is displayed in Fig. 1. The ability to solubilize CdSe/ZnS dots in aqueous media by use of an amphiphilic polymer or lipid coating while main- taining a high quantum yield 19 has led to their commercializa- tion [Quantum Dot Corp and Evident Technologies]. A peptide coating 20 can also render the dots water-soluble, but this formulation is not presently available commercially. Once water solubilized, dots can be further functionalized to target specific cellular surfaces or sites using receptor–ligand inter- actions. 21 Nowhere have the narrow spectral features of quantum dots shown more potential for applications than in fluorescence Fig. 1 Schematic diagram of a CdSe/ZnS core/shell quantum dot which possesses a passivation layer. The exploded views reveal increasing molecular detail. Department of Chemistry, University of Calgary, 2500 University Dr NW, Calgary, AB, Canada T2N 1N4. E-mail: [email protected]; Fax: +1 403 289 9488; Tel: +1 403 220 8138 1870 | Phys. Chem. Chem. Phys., 2007, 9, 1870–1880 This journal is c the Owner Societies 2007 INVITED ARTICLE www.rsc.org/pccp | Physical Chemistry Chemical Physics Downloaded by University of Calgary on 03 July 2012 Published on 02 March 2007 on http://pubs.rsc.org | doi:10.1039/B617115J View Online / Journal Homepage / Table of Contents for this issue
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Fluorescence correlation spectroscopy using quantum dots: advances,
challenges and opportunities
Romey F. Heuff, Jody L. Swift and David T. Cramb*
Received 22nd November 2006, Accepted 12th February 2007
First published as an Advance Article on the web 2nd March 2007
DOI: 10.1039/b617115j
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.
1. Introduction
Semiconductor nanocrystals (quantum dots) are on the verge
of revolutionizing the field of biophotonics. The extreme
brightness and photostability of quantum dots have made
single biomolecule imaging almost trivial.1,2 The term ‘‘quan-
tum dot’’, was coined to represent the reduced dimensionality
of nanoscopic crystals made up of 100s to 1000s of unit cells,
with core diameters ranging from 1.5 nm–6 nm. At this length
scale, the confinement energy of the exciton (electron/hole
pair) is stronger than the Coulombic interaction, resulting in
quantization of the electronic energy. For optical transitions,
excitons are generated through the absorption of light. An
extensive and informative review of the optical properties of
single quantum dots is given in ref. 3. The optical gap of a
quantum dot is size-tunable in the visible to near infrared
region of the electromagnetic spectrum. After excitation,
emission of a photon occurs in the absence of other relaxation
pathways. Conveniently, the dots have broadband absorption,
making simultaneous excitation of a selection of different
color dots using a single wavelength light source possible.
Moreover, they have narrower emission profiles than organic
dye molecules, facilitating easier spectral separation of the
fluorescence signal. Thus, quantum dots have great potential
for multiplexing in biological assay applications, as well as in
imaging.
Quantum dots have been successfully used in long-term
biological imaging with little or no cytotoxic effects.4,5 To
date, quantum dots have been used for optical coding of
mammalian cells,6,7 in the formation of biosensors8 as diag-
nostic tools for cancer9,10 and in the development of rapid
screening techniques using multiplexed hybridization detec-
tion of DNA sequences.11 They have been used as donors for
fluorescence resonance energy transfer (FRET)12 and as fluoro-
phores in fluorescence lifetime imaging (FLIM)13 because of
their long lifetimes (10s of ns) compared to organic fluoro-
phores (1–5 ns). Near infrared dots have been used to map
sentinel lymph nodes,14 and dots in the visible region have
tracked the diffusion of individual glycine receptors at various
synaptic locations15 and so on.16
The chemical research of quantum dots has involved devel-
oping luminescent core materials and then adding a higher
bandgap shell to the core surface. The resultant core/shell
quantum dots were found to have superior optical properties.
One of the most widely studied core/shell quantum dots is
composed of CdSe/ZnS. CdSe/ZnS quantum dots are made in
hydrophobic highly coordinating organic solvents such as
trioctyl phosphine oxide (TOPO)17 and in order to render
them biologically compatible they need further surface mod-
ifications for water solubility and target specificity.18 A sche-
matic diagram of a core/shell quantum dot is displayed in Fig.
1. The ability to solubilize CdSe/ZnS dots in aqueous media by
use of an amphiphilic polymer or lipid coating while main-
taining a high quantum yield19 has led to their commercializa-
tion [Quantum Dot Corp and Evident Technologies]. A
peptide coating20 can also render the dots water-soluble, but
this formulation is not presently available commercially. Once
water solubilized, dots can be further functionalized to target
specific cellular surfaces or sites using receptor–ligand inter-
actions.21
Nowhere have the narrow spectral features of quantum dots
shown more potential for applications than in fluorescence
Fig. 1 Schematic diagram of a CdSe/ZnS core/shell quantum dot
which possesses a passivation layer. The exploded views reveal
increasing molecular detail.
Department of Chemistry, University of Calgary, 2500 University DrNW, Calgary, AB, Canada T2N 1N4. E-mail: [email protected];Fax: +1 403 289 9488; Tel: +1 403 220 8138
1870 | Phys. Chem. Chem. Phys., 2007, 9, 1870–1880 This journal is �c the Owner Societies 2007
INVITED ARTICLE www.rsc.org/pccp | Physical Chemistry Chemical Physics
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experiment, where blinking probabilities are reported
over 10�3–102 s.35,44,57,58
7. Closing remarks
There are several intriguing possibilities for FCS to add to the
understanding and mitigation of quantum dot blinking. More
autocorrelation analysis in the time range 1 ms–1 ms of sur-
face-bound ensemble quantum dots would be illuminating.
The one study thus far38 reported mildly decaying autocorre-
lation data in this time range for uncapped CdS quantum dots.
FCS detection of mobile quantum dot blinking will provide a
mechanism to report on the changes in quantum dots’ re-
sponses to their environment. For example, our ongoing
studies involve using FCS to measure blinking in solutions
of varying ionic strength. Changing the quantum dots’ local
electronic environment should affect blinking, if ionization of
the dots is involved in the blinking process. Additionally, it is
clear that surface chemistry plays a major role in quantum dot
blinking.28,29,71 The addition of surface active agents to the
quantum dot solutions should affect blinking behavior as
measured by FCS. It will be interesting to determine whether
or not there is a difference between reducing and oxidizing
surfactants on blinking. Finally, it may be possible to send
quantum dots into dark states electrochemically,34 the kinetics
of which should be measurable using FCS.
Acknowledgements
The authors acknowledge the financial support of NSERC
through an AGENO grant and the Alberta Ingenuity Fund for
a graduate scholarship (JLS). We also acknowledge helpful
discussions with Professor Cecile Fradin (McMaster).
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