Jerry C. Chang

Biocompatible Quantum Dots for Biological Applications

Semiconductor quantum dots are quickly becoming a critical diagnostic tool for discerning cellular function at the molecular level. Their high brightness, long-lasting, size-tunable, and narrow luminescence set them apart from conventional fluorescence dyes. Quantum dots are being developed for a variety of biologically oriented applications, including fluorescent assays for drug discovery, disease detection, single protein tracking, and intracellular reporting. This review introduces the science behind quantum dots and describes how they are made biologically compatible. Several applications are also included, illustrating strategies toward target specificity, and are followed by a discussion on the limitations of quantum dot approaches. The article is concluded with a look at the future direction of quantum dots.

Single Molecule Analysis of Serotonin Transporter Regulation Using Antagonist-Conjugated Quantum Dots Reveals Restricted, p38 MAPK-Dependent Mobilization Underlying Uptake Activation

The presynaptic serotonin (5-HT) transporter (SERT) is targeted by widely prescribed antidepressant medications. Altered SERT expression or regulation has been implicated in multiple neuropsychiatric disorders, including anxiety, depression and autism. Here, we implement a generalizable strategy that exploits antagonist-conjugated quantum dots (Qdots) to monitor, for the first time, single SERT proteins on the surface of serotonergic cells. We document two pools of SERT proteins defined by lateral mobility, one that exhibits relatively free diffusion, and a second, localized to cholesterol and GM1 ganglioside-enriched microdomains, that displays restricted mobility. Receptor-linked signaling pathways that enhance SERT activity mobilize transporters that, nonetheless, remain confined to membrane microdomains. Mobilization of transporters arises from a p38 MAPK-dependent untethering of the SERT C terminus from the juxtamembrane actin cytoskeleton. Our studies establish the utility of ligand-conjugated Qdots for analysis of the behavior of single membrane proteins and reveal a physical basis for signaling-mediated SERT regulation.

Visualization of the Cocaine-Sensitive Dopamine Transporter with Ligand-Conjugated Quantum Dots

The presynaptic dopamine (DA) transporter is responsible for DA inactivation following release and is a major target for the psychostimulants cocaine and amphetamine. Dysfunction and/or polymorphisms in human DAT (SLC6A3) have been associated with schizophrenia, bipolar disorder, Parkinsons disease, and attention-deficit hyperactivity disorder (ADHD). Despite the clinical importance of DAT, many uncertainties remain regarding the transporters regulation, in part due to the poor spatiotemporal resolution of conventional methodologies and the relative lack of efficient DAT-specific fluorescent probes. We developed a quantum dot-based labeling approach that uses a DAT-specific, biotinylated ligand, 2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (IDT444), that can be bound by streptavidin-conjugated quantum dots. Flow cytometry and confocal microscopy were used to detect DAT in stably and transiently transfected mammalian cells. IDT444 is useful for quantum-dot-based fluorescent assays to monitor DAT expression, function, and plasma membrane trafficking in living cells as evidenced by the visualization of acute, protein-kinase-C (PKC)-dependent DAT internalization.

Controlling the Reactivity of Ampiphilic Quantum Dots in Biological Assays through Hydrophobic Assembly of Custom PEG Derivatives

Modifications of the quantum dot (QD) surface are routinely performed via covalent biomolecule attachment, and poly(ethylene glycol) (PEG) derivatization has previously been shown to limit nonspecific cellular interactions of QD probes. Attempts to functionalize ampiphilic QDs (AMP-QDs) with custom PEG derivatives having a hydrophobic terminus resulted in self-assembly of these PEG ligands to the AMP-QD surface in the absence of covalent coupling reagents. We demonstrate, via electrophoretic characterization techniques, that these self-assembled PEG-QDs exhibit improved passivation in biological environments and are less susceptible to unwanted protein adsorption to the QD surface. We highlight the artifactual fluorescent response protein adsorption can cause in biological assays, and discuss considerations for improved small molecule presentation to facilitate specific QD interactions.

A Fluorescence Displacement Assay for Antidepressant Drug Discovery Based on Ligand-Conjugated Quantum Dots

The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) protein plays a central role in terminating 5-HT neurotransmission and is the most important therapeutic target for the treatment of major depression and anxiety disorders. We report an innovative, versatile, and target-selective quantum dot (QD) labeling approach for SERT in single Xenopus oocytes that can be adopted as a drug-screening platform. Our labeling approach employs a custom-made, QD-tagged indoleamine derivative ligand, IDT318, that is structurally similar to 5-HT and accesses the primary binding site with enhanced human SERT selectivity. Incubating QD-labeled oocytes with paroxetine (Paxil), a high-affinity SERT-specific inhibitor, showed a concentration- and time-dependent decrease in QD fluorescence, demonstrating the utility of our approach for the identification of SERT modulators. Furthermore, with the development of ligands aimed at other pharmacologically relevant targets, our approach may potentially form the basis for a multitarget drug discovery platform.

Visualization of lipid raft membrane compartmentalization in living RN46A neuronal cells using single quantum dot tracking.

Lipid rafts are cholesterol-enriched subdomains in the plasma membrane that have been reported to act as a platform to facilitate neuronal signaling; however, they are suspected to have a very short lifetime, up to only a few seconds, which calls into question their roles in biological signaling. To better understand their diffusion dynamics and membrane compartmentalization, we labeled lipid raft constituent ganglioside GM1 with single quantum dots through the connection of cholera toxin B subunit, a protein that binds specifically to GM1. Diffusion measurements revealed that single quantum dot-labeled GM1 ganglioside complexes undergo slow, confined lateral diffusion with a diffusion coefficient of ∼7.87 × 10(-2) μm(2)/s and a confinement domain about 200 nm in size. Further analysis of their trajectories showed lateral confinement persisting on the order of tens of seconds, comparable to the time scales of the majority of cellular signaling and biological reactions. Hence, our results provide further evidence in support of the putative function of lipid rafts as signaling platforms.

Labeling of neuronal receptors and transporters with quantum dots.

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.

Single-Quantum-Dot Tracking Reveals Altered Membrane Dynamics of an Attention-Deficit/Hyperactivity-Disorder-Derived Dopamine Transporter Coding Variant

The presynaptic, cocaine- and amphetamine-sensitive dopamine (DA) transporter (DAT, SLC6A3) controls the intensity and duration of synaptic dopamine signals by rapid clearance of DA back into presynaptic nerve terminals. Abnormalities in DAT-mediated DA clearance have been linked to a variety of neuropsychiatric disorders, including addiction, autism, and attention deficit/hyperactivity disorder (ADHD). Membrane trafficking of DAT appears to be an important, albeit incompletely understood, post-translational regulatory mechanism; its dysregulation has been recently proposed as a potential risk determinant of these disorders. In this study, we demonstrate a link between an ADHD-associated DAT mutation (Arg615Cys, R615C) and variation on DAT transporter cell surface dynamics, a combination only previously studied with ensemble biochemical and optical approaches that featured limited spatiotemporal resolution. Here, we utilize high-affinity, DAT-specific antagonist-conjugated quantum dot (QD) probes to establish the dynamic mobility of wild-type and mutant DATs at the plasma membrane of living cells. Single DAT-QD complex trajectory analysis revealed that the DAT 615C variant exhibited increased membrane mobility relative to DAT 615R, with diffusion rates comparable to those observed after lipid raft disruption. This phenomenon was accompanied by a loss of transporter mobilization triggered by amphetamine, a common component of ADHD medications. Together, our data provides the first dynamic imaging of single DAT proteins, providing new insights into the relationship between surface dynamics and trafficking of both wild-type and disease-associated transporters. Our approach should be generalizable to future studies that explore the possibilities of perturbed surface DAT dynamics that may arise as a consequence of genetic alterations, regulatory changes, and drug use that contribute to the etiology or treatment of neuropsychiatric disorders.

A Bright Light to Reveal Mobility: Single Quantum Dot Tracking Reveals Membrane Dynamics and Cellular Mechanisms

This perspective describes recent progress in single quantum dot techniques, with an emphasis on their applications in exploring membrane dynamics and cellular mechanisms. In these cases, conventional population measurements, such as fluorescence recovery after photobleaching, yield only a mean value on an ensemble or bulk collection of molecules, where the behavior of individual proteins and vehicles is missing. In recent years, the single quantum dot imaging approach has been introduced as a sub-category of single molecule fluorescent techniques to reveal single protein/vehicle dynamics in real-time. One of the major advantages of using single quantum dots is the high signal-to-noise ratio originating from their unique photophysical properties such as extraordinarily high molar extinction coefficients and large effective Stokes shifts. In addition to a brief overview on the principle of single quantum dot imaging techniques, we highlight recent discoveries and discuss future directions in the field.

Real-Time Quantum Dot Tracking of Single Proteins

We describe a single quantum dot tracking method that can be used to monitor individual proteins in the membrane of living cells. Unlike conventional fluorescent dyes, quantum dots (fluorescent semiconductor nanocrystals) have high quantum yields, narrow emission wavelengths, and excellent photostability, making them ideal probes in single-molecule detection. This technique has been applied to study the dynamics of various membrane proteins including glycine receptors, nerve growth factors, kinesin motors, and γ-aminobutyric acid receptors. In this chapter, a basic introduction and experimental setup for single quantum dot labeling of a target protein is given. In addition, data acquisition and analysis of time-lapse single quantum dot imaging with sample protocols are provided.