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Igor L Medintz

Publications and source records attributed to Igor L Medintz.

At least 19 recordsLinked to original sources
PubMed2006

Solution-phase single quantum dot fluorescence resonance energy transfer.

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.

Cadmium Compounds↗
PubMed2006

Recent progress in developing FRET-based intracellular sensors for the detection of small molecule nutrients and ligands.

Intracellular sensors for nutrients such as sugars, metabolic precursors and signaling ligands such as amino acids will help to elucidate the complex roles of these small molecules in biology. In this update, several recently developed sensors, originating from the Frommer laboratory, which combine bacterial periplasmic-binding protein-based specificity for ligand targeting with fluorescent protein-derived resonance energy transfer for signal transduction are reviewed. The insight gained from designing these sensors, along with the preliminary results gathered from their first application, serve to illustrate the impact that they can have on improving our fundamental understanding of biology.

Biopolymers↗
PubMed2006

Hydrodynamic dimensions, electrophoretic mobility, and stability of hydrophilic quantum dots.

Luminescent semiconductor quantum dots (QDs) have great potential for use in biological assays and imaging. These nanocrystals are capped with surface ligands (bifunctional molecules, amphiphilic polymers, phospholipids, etc.) that render them hydrophilic and provide them with functional properties. These coatings alters their hydrodynamic radii and surface charge, which can drastically affect properties such as diffusion within the cell cytoplasm. Heavy atom techniques such as transmission electron microscopy and X-ray scattering probe the inorganic core and do not take into account the ligand coating. Herein we use dynamic light scattering to characterize the hydrodynamic radius (R(H)) of CdSe-ZnS QDs capped with various hydrophilic surface coatings (including dihydrolipoic acid and amphiphilic polymers) and self-assembled QD-protein bioconjugates. Experiments were complemented with measurements of the geometric size and zeta potential using agarose gel electrophoresis and laser Doppler velocimetry. We find that the effects of surface ligands on the hydrodynamic radius and on the nanoparticle mobility are complex and strongly depend on a combination of the inorganic core size and nature and lateral extension of the hydrophilic surface coating. These properties are critical for the design of QD-based biosensing assays as well as QD bioconjugate diffusion in live cells.

Biosensing Techniques↗
PubMed2006

Materials for fluorescence resonance energy transfer analysis: beyond traditional donor-acceptor combinations.

The use of Förster or fluorescence resonance energy transfer (FRET) as a spectroscopic technique has been in practice for over 50 years. A search of ISI Web of Science with just the acronym "FRET" returns more than 2300 citations from various areas such as structural elucidation of biological molecules and their interactions, in vitro assays, in vivo monitoring in cellular research, nucleic acid analysis, signal transduction, light harvesting and metallic nanomaterials. The advent of new classes of fluorophores including nanocrystals, nanoparticles, polymers, and genetically encoded proteins, in conjunction with ever more sophisticated equipment, has been vital in this development. This review gives a critical overview of the major classes of fluorophore materials that may act as donor, acceptor, or both in a FRET configuration. We focus in particular on the benefits and limitations of these materials and their combinations, as well as the available methods of bioconjugation.

Fluorescence Resonance Energy Transfer↗
PubMed2006

Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates.

Proteases are enzymes that catalyse the breaking of specific peptide bonds in proteins and polypeptides. They are heavily involved in many normal biological processes as well as in diseases, including cancer, stroke and infection. In fact, proteolytic activity is sometimes used as a marker for some cancer types. Here we present luminescent quantum dot (QD) bioconjugates designed to detect proteolytic activity by fluorescence resonance energy transfer. To achieve this, we developed a modular peptide structure which allowed us to attach dye-labelled substrates for the proteases caspase-1, thrombin, collagenase and chymotrypsin to the QD surface. The fluorescence resonance energy transfer efficiency within these nanoassemblies is easily controlled, and proteolytic assays were carried out under both excess enzyme and excess substrate conditions. These assays provide quantitative data including enzymatic velocity, Michaelis-Menten kinetic parameters, and mechanisms of enzymatic inhibition. We also screened a number of inhibitory compounds against the QD-thrombin conjugate. This technology is not limited to sensing proteases, but may be amenable to monitoring other enzymatic modifications.

Amino Acid Sequence↗
PubMed2006

Designer variable repeat length polypeptides as scaffolds for surface immobilization of quantum dots.

We demonstrate the use of a series of engineered, variable-length de novo polypeptides to discretely immobilize luminescent semiconductor nanocrystals or quantum dots (QDs) onto functional surfaces. The polypeptides express N-terminal dicysteine and C-terminal hexahistidine residues that flank a variable number (1, 3, 5, 7, 14, 21, 28, or 35) of core beta-strand repeats, with tyrosine, glutamic acid, histidine, and lysine residues located at the turns. Polypeptides have molecular weights ranging from 4 to 83 kDa and retain a rigid structure based on the antiparallel beta-sheet motif. We first use a series of dye-labeled polypeptides to test and characterize their self-assembly onto hydrophilic CdSe-ZnS QDs using fluorescence resonance energy transfer (FRET). Results indicate that peptides maintain their beta-sheet conformation after self-assembly onto the QD surfaces, regardless of their length. We then immobilize biotinylated derivatives of these polypeptides on a NeutrAvidin-functionalized substrate and use them to capture QDs via specific interactions between the peptides' polyhistidine residues and the nanocrystal surface. We found that each of the polypeptides was able to efficiently capture QDs, with a clear correlation between the density of the surface-tethered peptide and the capacity for nanocrystal capture. The versatility of this capture strategy is highlighted by the creation of a variety of one- and two-dimensional polypeptide-QD structures as well as a self-assembled surface-immobilized FRET-based nutrient sensor.

Amino Acid Sequence↗
PubMed2006

TNT detection using multiplexed liquid array displacement immunoassays.

The presence of trace contamination of soil and groundwater with explosives is an ongoing concern, for which improved methods are required to facilitate their detection and quantification. This is true both for the monitoring of remediation and for site characterization. Immunosensors have been found effective for solution-phase detection of environmental contaminants. Our work utilized the Luminex100 (flow cytometer) to detect TNT in a multiplexed displacement immunoassay format. The Luminex100 can perform a multiplexed assay by discriminating between up to 100 different bead sets. We used this capability to evaluate four different TNT monoclonal antibodies, two recombinant TNT antibodies, and a control antibody simultaneously for the rapid detection of TNT and other nitroaromatics. TNT could be detected at 0.1 ppb and quantified over the range of 1.0 ppb to 10 ppm. In addition, the assay was shown to be effective in various matrixes such as lake water, seawater, and acetone extracts of soil. Seawater required dilution with two parts buffer to avoid loss of microspheres, while the acetone extracts were diluted 100-fold or more to minimize solvent affects.

Antibodies, Monoclonal↗
PubMed2006

Maltose-binding protein: a versatile platform for prototyping biosensing.

The bacterial periplasmic-binding protein (PBP) superfamily members, in particular the maltose-binding protein, have been used extensively to prototype a variety of biosensing platforms. Although quite diverse at the primary sequence level, this protein superfamily retains the same basic two-domain structure, and upon binding a recognized ligand almost all PBPs undergo a conformational change to a closed structure. This process forms the basis for most, but not all, PBP-based biosensor signal transduction. Many direct detection or reagentless sensing modalities have been utilized with maltose-binding protein for both in vitro and in vivo detection of target compounds. Signal transduction modalities developed to date include direct fluorescence, electrochemical detection, fluorescence resonance energy transfer (FRET)-based detection, surface-tethered FRET sensing, hybrid quantum dot FRET sensing, and enzymatic detection, each of which have different benefits, potential applications and limitations.

Biosensing Techniques↗
PubMed2006

Förster resonance energy transfer investigations using quantum-dot fluorophores.

Förster resonance energy transfer (FRET), which involves the nonradiative transfer of excitation energy from an excited donor fluorophore to a proximal ground-state acceptor fluorophore, is a well-characterized photophysical tool. It is very sensitive to nanometer-scale changes in donor-acceptor separation distance and their relative dipole orientations. It has found a wide range of applications in analytical chemistry, protein conformation studies, and biological assays. Luminescent semiconductor nanocrystals (quantum dots, QDs) are inorganic fluorophores with unique optical and spectroscopic properties that could enhance FRET as an analytical tool, due to broad excitation spectra and tunable narrow and symmetric photoemission. Recently, there have been several FRET investigations using luminescent QDs that focused on addressing basic fundamental questions, as well as developing targeted applications with potential use in biology, including sensor design and protein conformation studies. Herein, we provide a critical review of those developments. We discuss some of the basic aspects of FRET applied to QDs as both donors and acceptors, and highlight some of the advantages offered (and limitations encountered) by QDs as energy donors and acceptors compared to conventional dyes. We also review the recent developments made in using QD bioreceptor conjugates to design FRET-based assays.

Fluorescence Resonance Energy Transfer↗
PubMed2005

Quantum dot-based multiplexed fluorescence resonance energy transfer.

We demonstrate the use of luminescent quantum dots (QDs) conjugated to dye-labeled protein acceptors for nonradiative energy transfer in a multiplexed format. Two configurations were explored: (1) a single color QD interacting with multiple distinct acceptors and (2) multiple donor populations interacting with one type of acceptor. In both cases, we showed that simultaneous energy transfer between donors and proximal acceptors can be measured. However, data analysis was simpler for the configuration where multiple QD donors are used in conjunction with one acceptor. Steady-state fluorescence results were corroborated by time-resolved measurements where selective shortening of QD lifetime was measured only for populations that were selectively engaged in nonradiative energy transfer.

Carrier Proteins↗
PubMed2005

Decoration of discretely immobilized cowpea mosaic virus with luminescent quantum dots.

This report describes two related methods for decorating cowpea mosaic virus (CPMV) with luminescent semiconductor nanocrystals (quantum dots, QDs). Variants of CPMV are immobilized on a substrate functionalized with NeutrAvidin using modifications of biotin-avidin binding chemistry in combination with metal affinity coordination. For example, using CPMV mutants expressing available 6-histidine sequences inserted at loops on the viral coat protein, we show that these virus particles can be specifically immobilized on NeutrAvidin functionalized substrates in a controlled fashion via metal-affinity coordination. To accomplish this, a hetero-bifunctional biotin-NTA moiety, activated with nickel, is used as the linker for surface immobilization of CPMV (bridging the CPMVs' histidines to the NeutrAvidin). Two linking chemistries are then employed to achieve CPMV decoration with hydrophilic CdSe-ZnS core-shell QDs; they target the histidine or lysine residues on the exterior virus surface and utilize biotin-avidin interactions. In the first scheme, QDs are immobilized on the surface-tethered CPMV via electrostatic attachment to avidin previously bound to the virus particle. In the second strategy, the lysine residues common to each viral surface asymmetric unit are chemically functionalized with biotin groups and the biotinylated CPMV is discretely immobilized onto the substrate via NeutrAvidin-biotin interactions. The biotin units on the upper exposed surface of the immobilized CPMV then serve as capture sites for QDs conjugated with a mixture of avidin and a second protein, maltose binding protein, which is also used for QD-protein conjugate purification. Characterization of the assembled CPMV and QD structures is presented, and the potential uses for protein-coated QDs functionalized onto this symmetrical virion nanoscaffold are discussed.

Chromogenic Compounds↗
PubMed2005

Application of a homogenous assay for the detection of 2,4,6-trinitrotoluene to environmental water samples.

A homogeneous assay was used to detect 2,4,6-trinitrotoluene (TNT) spiked into environmental water samples. This assay is based on changes in fluorescence emission intensity when TNT competitively displaces a fluorescently labeled, TNT analog bound to an anti-TNT antibody. The effectiveness of the assay was highly dependent on the source of the sample being tested. As no correlation between pH and assay performance was observed, ionic strength was assumed to be the reason for variation in assay results. Addition of 10 phosphate-buffered saline to samples to increase their ionic strength to that of our standard laboratory buffer (about 0.17 M) significantly improved the range over which the assay functioned in several river water samples.

Buffers↗
PubMed2005

A hybrid quantum dot-antibody fragment fluorescence resonance energy transfer-based TNT sensor.

We demonstrate the use of luminescent QDs conjugated to antibody fragments to develop solution-phase nanoscale sensing assemblies, based on fluorescence resonance energy transfer (FRET) for the specific detection of the explosive 2,4,6-trinitrotoluene (TNT) in aqueous environments. The hybrid sensor consists of anti-TNT specific antibody fragments attached to a hydrophilic QD via metal-affinity coordination. A dye-labeled TNT analogue prebound in the antibody binding site quenches the QD photoluminescence via proximity-induced FRET. Analysis of the data collected at increasing dye-labeled analogue to QD ratios provided an insight into understanding how the antibody fragments self-assemble on the QD. Addition of soluble TNT displaces the dye-labeled analogue, eliminating FRET and resulting in a concentration-dependent recovery of QD photoluminescence. Sensor performance and specificity were evaluated.

Cadmium Compounds↗
PubMed2005

Synthesis of compact multidentate ligands to prepare stable hydrophilic quantum dot fluorophores.

We describe a simple and versatile scheme to prepare an array of heterofunctional multidentate ligands that permit strong and stable interactions with colloidal semiconductor nanocrystals (quantum dots, QDs) and render them soluble in aqueous environments. These ligands were synthesized by reacting various chain length poly(ethylene glycols) with thioctic acid, followed by ring opening of the dithiolane moiety, creating a bidentate thiol motif with enhanced affinity for CdSe-ZnS core-shell QDs. Functionalization with these ligands permits processability of the nanocrystals not only in aqueous but also in many other polar solvents. These ligands provide a straightforward means of preparing QDs that exhibit greater resistance to environmental changes, making them more amenable for use in live cell imaging and other biotechnological applications.

Biotechnology↗
PubMed2005

Can luminescent quantum dots be efficient energy acceptors with organic dye donors?

We assessed the ability of luminescent quantum dots (QDs) to function as energy acceptors in fluorescence resonance energy transfer (FRET) assays, with organic dyes serving as donors. Either AlexaFluor 488 or Cy3 dye was attached to maltose binding protein (MBP) and used with various QD acceptors. Steady-state and time-resolved fluorescence measurements showed no apparent FRET from dye to QD. We attribute these observations to the dominance of a fast radiative decay rate of the donor excitation relative to a slow FRET decay rate. This is due to the long exciton lifetime of the acceptor compared to that of the dye, combined with substantial QD direct excitation.

Carbocyanines↗
PubMed2005

Self-assembled TNT biosensor based on modular multifunctional surface-tethered components.

We demonstrate a self-assembled reagentless biosensor based on a modular design strategy that functions in the detection of TNT and related explosive compounds. The sensor consists of a dye-labeled anti-TNT antibody fragment that interacts with a cofunctional surface-tethered DNA arm. The arm consists of a flexible biotinylated DNA oligonucleotide base specifically modified with a dye and terminating in a TNB recognition element, which is an analogue of TNT. Both of these elements are tethered to a Neutravidin surface with the TNB recognition element bound in the antibody fragment binding site, bringing the two dyes into proximity and establishing a baseline level of fluorescence resonance energy transfer (FRET). Addition of TNT, or related explosive compounds, to the sensor environment alters FRET in a concentration-dependent manner. The sensor can be regenerated repeatedly through washing away of analyte and specific reformation of the sensor assembly, allowing for subsequent detection events. Sensor dynamic range can be usefully altered through the addition of a DNA oligonucleotide that hybridizes to a portion of the cofunctional arm. The modular design of the sensor demonstrates that it can be easily adapted to detect a variety of different analytes.

Biosensing Techniques↗
PubMed2005

Quantum dot bioconjugates for imaging, labelling and sensing.

One of the fastest moving and most exciting interfaces of nanotechnology is the use of quantum dots (QDs) in biology. The unique optical properties of QDs make them appealing as in vivo and in vitro fluorophores in a variety of biological investigations, in which traditional fluorescent labels based on organic molecules fall short of providing long-term stability and simultaneous detection of multiple signals. The ability to make QDs water soluble and target them to specific biomolecules has led to promising applications in cellular labelling, deep-tissue imaging, assay labelling and as efficient fluorescence resonance energy transfer donors. Despite recent progress, much work still needs to be done to achieve reproducible and robust surface functionalization and develop flexible bioconjugation techniques. In this review, we look at current methods for preparing QD bioconjugates as well as presenting an overview of applications. The potential of QDs in biology has just begun to be realized and new avenues will arise as our ability to manipulate these materials improves.

Biosensing Techniques↗