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WIREs Nanomed Nanobiotechnol
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Labeling of neuronal receptors and transporters with quantum dots

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Abstract The ability to efficiently visualize protein targets in cells is a fundamental goal in biological research. Recently, quantum dots (QDots) have emerged as a powerful class of fluorescent probes for labeling membrane proteins in living cells because of breakthrough advances in QDot surface chemistry and biofunctionalization strategies. This review discusses the increasing use of QDots for fluorescence imaging of neuronal receptors and transporters. The readers are briefly introduced to QDot structure, photophysical properties, and common synthetic routes toward the generation of water‐soluble QDots. The following section highlights several reports of QDot application that seek to unravel molecular aspects of neuronal receptor and transporter regulation and trafficking. This article is closed with a prospectus of the future of derivatized QDots in neurobiological and pharmacological research. WIREs Nanomed Nanobiotechnol 2012, 4:605–619. doi: 10.1002/wnan.1186 This article is categorized under: Diagnostic Tools > In Vitro Nanoparticle-Based Sensing Diagnostic Tools > In Vivo Nanodiagnostics and Imaging

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Photophysical properties of quantum dots. (a) Aberration‐corrected Z‐STEM image of a commercial core/shell QD655 (Life Technologies). It can be seen that the core/shell QDot is actually an elongated, bullet‐shaped 3D object rather than a sphere. (Reprinted with permission from Ref 7. Copyright 2007 Elsevier B.V.) (b) Absorption and emission spectra of a series of CdSe nanocrystals. The size of the QDot core determines the absorption and emission spectra of QDots. (Reprinted with permission from Ref 14. Copyright 2001 Nature Publishing Group) (c) A series of UV‐illuminated CdSe nanocrystals ranging in size from ∼2 to ∼6 nm. (d) Time‐lapse image series of FITC‐ and QD‐labeled HEK‐293 cells. In contrast to traditional fluorophores, QDots are characterized by excellent photostability, which enables long‐term monitoring of biological processes. (Reprinted with permission from Ref 20. Copyright 2011 American Chemical Society) (e) Fluorescence intermittency or ‘blinking’ in the emission spectrum of a single QDot. This QDot property can be used as a criterion to distinguish single nanocrystals from aggregates.

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Fluorescence displacement assay based on ligand‐conjugated QDots for the drug discovery of allosteric antidepressants. (a) Targeted hSERTs bind to the QDot‐tagged ligands, forming complexes that increase fluorescent signal along the membrane. When exposed to a potential drug that induces a conformational change in the binding site, the QDot‐tagged ligands are displaced resulting in a decrease in fluorescence intensity. (b) Representative time‐lapse fluorescent images show the effect of paroxetine on ligand‐hSERT displacement in the presence of PBS buffer (control), 10 µM, and 20 µM paroxetine. (Reprinted with permission from Ref 65. Copyright 2011 American Chemical Society)

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Labeling of dopamine transporter (DAT) with ligand‐conjugated QDots in live HeLa cells. (left) streptavidin‐conjugated QDots were used to label DATs previously exposed to a biotinylated, PEGylated cocaine analog. (upright) chemical structure of the DAT ligand used in the study. (A1) QDot labeling of membrane DATs in a live HeLa cell. (B1) QDot‐bound DATs underwent acute redistribution from the plasma membrane to intracellular compartments as a result of protein kinase C (PKC) activation. (Reprinted with permission from Ref 20. Copyright 2011 American Chemical Society)

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QDot labeling of NGF receptors using streptavidin–biotin assembly. NGF‐conjugated QDots were used to investigate retrograde, vesicular NGF transport in living DRG neurons. (a) Schematic drawing of a QDot–NGF bound to dimerized TrkA receptors (left) and addition of QDot–NGF to the DA compartment of the three‐chamber DRG neuron culture (right). DA, distal axon; PA, proximal axon; CB, cell body. (b) Representative live fluorescence images of DRG neuron axons or cell bodies 2 h after the addition of 4 nM QDot–NGF to the DA chamber. QDot–NGF seems to bind all axons in distal axon chamber. However, only a small portion of the cell bodies and proximal axons are shown to have QDot fluorescence, reflecting the fact that not all cell bodies extend their axons into the distal axon compartment. (Reprinted with permission from Ref 79. Copyright 2007 National Academy of Sciences, USA)

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Quantum dot solubilization approaches. The as‐synthesized QDots can be rendered soluble in water via ligand exchange (top), wherein native hydrophobic surfactants are replaced with bifunctional hydrophobic capping ligands; via encapsulation in an insert silica shell (middle); or via amphiphilic polymer encapsulation (bottom), wherein native surfactants are retained and integrated into the polymer shell.

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Ligand‐conjugated QDots for SERT labeling in living cells. (a) Fluorescence images of SERT expressing HEK cells labeled with serotonin ligand functionalized QDots. Fluorescence labeling of SERT in the membrane is clearly visible (left) while cells preincubated with high‐affinity SERT inhibitor paroxetine prior to the labeling shows no sign of fluorescence (right), indicating the QDot labeling specificity. (b) Electrophysiological currents elicited by serotonin or serotonin ligand functionalized QDots. The current induced by serotonin is typical. In contrast, the current induced by serotonin ligand functionalized QDots is characteristic of currents induced by SERT antagonists. (Reprinted with permission from Ref 42. Copyright 2002 American Chemical Society)

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Indirect Qdot labeling methodology. Indirect Qdot labeling typically involves either a streptavidin–biotin interaction between biotinylated probes and streptavidin‐conjugated Qdots (a), or a noncovalent recognition of a target‐bound primary antibody with secondary antibody‐conjugated Qdots (b).

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Direct conjugation methodology. Schematic diagram of the chemical reactions that occur during covalent coupling of bioprobes to the QDot surface by using EDC/NHS (top), SMCC (middle), and SPDP (bottom) reagents.

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