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Luminescent Quantum Dots

Transcript of Luminescent Quantum Dots

  • Investigating Biological Processes at the Single Molecule LevelUsing Luminescent Quantum Dots


    1Laboratoire Photons et Matie`re, CNRS UPR5, ESPCI, 10, rue Vauquelin, 75005 Paris, France; and 2Division of OpticalSciences, US Naval Research Laboratory, Washington, DC 20375, USA

    (Received 16 September 2008; accepted 12 May 2009; published online 12 June 2009)

    AbstractIn this report we summarize the progress made inthe past several years on the use of luminescent QDs to probebiological processes at the single molecule level. We start byproviding a quick overview of the basic properties ofsemiconductor nanocrystals, including synthetic routes, sur-face-functionalization strategies, along with the main attri-butes of QDs that are of direct relevance to single moleculestudies based on uorescence detection. We then detail somevaluable insights into specic biological processes gainedusing single QDs. These include progress made in probingbiomolecular interactions, tracking of protein receptors bothin vitro and in live cells, and single particle resonance energytransfer. We will also discuss the advantages offered andlimitations encountered by single QD uorescence as aninvestigative tool in biology.

    KeywordsSemiconductor, Nanocrystal, Quantum dot,

    Single molecule, Fluorescence, FRET, Biosensing, Hybrid-

    ization, Molecular motors.


    Semiconductor nanocrystals, or quantum dots(QDs), have recently emerged as a new set of uoro-phores that can enhance biological assays, uorescencedetection and imaging.11,45,53,64,65,67,69,93,95,104 This isdue to some of their unique optical and spectroscopicproperties, which are often not matched by mostconventional uorophores, such as organic dyes anduorescent proteins. QDs exhibit high uorescencequantum yields, high photobleaching thresholds, and apronounced resistance to photo- and chemical-degra-dation.45,67,69,95,104 They also exhibit narrow and tun-able emission bands along with broad excitationspectra and high extinction coefcient (~10100 timeslarger than most dyes). These properties allow the

    exibility to excite them efciently far from theiremission peaks. There has been a tremendous interestin using them to develop a variety of biological assaysand for sensor design. These include imaging of liveand xed cells, imaging of tissue, immunoassays andenergy transfer based assays. Energy transfer basedsensing has in particular expanded in the past fewyears, with sensors developed for the detection of smalland large molecule targets, hybridization interactions,and enzyme digestion.11,53,64,65,67,93,95

    Ensemble measurements are macroscopic in natureand they provide information about average propertiesof samples such as size, conformation or orientation ofproteins. In comparison, single molecule measure-ments are able to resolve molecular scale heterogene-ities and the fate of individual molecules. Fluorescencedetection applied to single molecules can allow accessto valuable molecular scale information, and it hasbecome one of the most commonly used single mole-cule techniques in biological research.18 Furthermore,recent progress in optical instrumentation and thedevelopment of highly sensitive detection tools (such asAPD and CCD) allowed easier detection and resolu-tion of uorescence from single uorophores. This hasin turn allowed the development of a variety of singlemolecule assays to study ligand-receptor binding,changes in the conformation of macromolecules (e.g.,proteins, short and large oligonucleotides), and singlemolecule diffusion and transport.18,106 Outside bio-physical research, single molecule uorescence wassuccessfully applied to individual colloidal QDs. Itallowed access to truly unique and remarkable infor-mation about the uorescence of single QDs that werenot accessible from ensemble measurements. In par-ticular, two unique properties distinguish luminescentQDs from most conventional uorophores: (1) singleQD exhibit very narrow PL spectra compared to thoseaveraged for macroscopic samples (FWHM ~ 15 nmvs. 3040 nm for ensemble spectra at room tempera-ture) and (2) the uorescence emission of isolated

    Address correspondence to Hedi Mattoussi, Division of Optical

    Sciences, US Naval Research Laboratory, Washington, DC 20375,

    USA. Electronic mail: [email protected]

    Annals of Biomedical Engineering, Vol. 37, No. 10, October 2009 ( 2009) pp. 19341959DOI: 10.1007/s10439-009-9715-0

    0090-6964/09/1000-1934/0 2009 US Naval Research Laboratory


  • nanoparticles under continuous excitation is intermit-tent in nature (blinking).24,26,49,50,74

    The unique optical properties of QDs can be par-ticularly benecial for single molecule uorescencemeasurements. Due to their large extinction coecient,narrow emission spectra, and their resistance to photo-degradation, QDs may be individually detected withhigh signal-to-noise ratios and for extended periods oftime (several minutes under sustained irradiation), afeature that is not available to traditional organic dyes.This can permit easy discrimination of dierent singleQD colors. This also makes them particularly suitablefor multiplexed single molecule uorescence imaging.As such, single molecule uorescence has emerged asone of the major aspects of employing these uoro-phores in biology.16,56

    In this report, we start with a brief description of themost eective synthetic procedures to prepare highquality colloidal nanocrystals, outline the commonlyused surface functionalization techniques, and describethe most pertinent photo-physical properties of use tobiology. We then review the progress that use of singleQD uorescence has allowed in biology, with a focuson a few representative examples where unique andvaluable insights into specic biological processes havebeen gained. These include progress made in probingbiomolecular interactions, tracking of protein recep-tors in vitro and in live cells and single particle energytransfer. We will also discuss the advantages offeredand limitations encountered by single QD uorescenceas an investigative tool in biology.



    Synthesis of Dispersible and Highly Luminescent QDs

    Luminescent QDs that have found most use inbiology are colloidal in nature, and they are preparedusing solution phase reactions. The rst solution-phasegrowth of the nanoparticles was realized within inversemicelles.38,90 However, the major advance in solution-phase synthesis took place in 1993, when Bawendi andco-workers showed that pyrolysis of organometallicprecursors can provide high quality CdSe nanocrystalsthat have crystalline cores, narrow size distribution(~10% or less) and exhibit relatively high photoemis-sion quantum yields.71 The rst demonstration of thisreaction scheme employed the rapid injection of dim-ethylcadmium (CdMe2) and trioctylphosphine selenide(TOP:Se) mixed with trioctylphosphine (TOP) into ahot (280300 C) coordinating solution of trioctyl-phosphine oxide (TOPO). One of the key aspects of

    this synthesis route was that colloidal QDs couldreproducibly be made to exhibit narrow emission withlow defect contributions and relatively high roomtemperature quantum yields (QY ~ 510%). This hasallowed performance of several viable photophysicaland structural characterizations (using for exampleabsorption, uorescence spectroscopy and scatteringtechniques).61,71,75 It has further raised the potentialfor technological applications based on QDs (e.g.,LEDs and photovoltaic devices).13,32,43,63,70

    In subsequent studies, Peng and co-workers furtherrened this reaction scheme and showed that severalprecursors which are less pyrophoric and less volatilethan CdMe2 could potentially be used for preparinghigh quality colloidal nanocrystals of CdS, CdSe, andCdTe.80,86 Other groups quickly followed up on Pengswork, and those efforts combined have outlined theimportance of impurities (usually acids coordinating tothe metal precursors) in the reaction progress. In thisroute, high purity TOPO and controlled amounts ofmetal coordinating ligands and metal precursors, suchas CdO, Cd carboxylates, phosphonates, or acetyl-acetonates, were used to synthesize various Cd-basednanocrystals, as schematically represented inFig. 1.80,86,89 The high temperature synthetic route wasalso applied to making near-IR emitting PbSe QDsinitially by Murray and co-workers, and then by othergroups, using oleic acid and Lead(II) acetate trihydrateor lead oxide.19,72,103,110

    To further take advantage of the progress made inimproving the quality of QDs achieved using hightemperature synthesis and to improve the luminescencequantum yield, researchers applied the concept of bandgap engineering developed for semiconducting quan-tum wells to the growth of colloidal nanocrystals (seeFig. 1).48 It was rst demonstrated by Guyot-Sionnnest and co-workers that overcoating CdSe QDswith a layer of ZnS could improve the PL quantumyields to values of 30%.39 Following that two com-prehensive reports by Dabbousi et al.15 and Penget al.79 detailed the complete reaction conditions forpreparing a series of CdSeZnS and CdSeCdS coreshell nanoparticles that are strongly uorescent andstable. We should add that air-stable precursors havealso been used for the overcoating reactions, followingthe reports of synthesis of CdS, CdSe nanocrystals.54,89

    One of the issues associated with overcoating QDs withZnS shell to improve the PL yield is the crystal latticemismatch between core and shell materials, which canproduce non-homogenous shell structure due to strain-induced defects. To potentially address this problem afew groups have grown multilayer shells on the nativecore (e.g., CdSeCdSZnS)105; this progressivelyadapts the crystalline lattice parameters of the ov-ercoating shell to those of the co