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Igor L Medintz

Publications and source records attributed to Igor L Medintz.

29 records · Page 2Linked to original sources
PubMed2005

Fluoroimmunoassays using antibody-conjugated quantum dots.

Luminescent colloidal semiconductor nanocrystals (quantum dots) are robust inorganic fluoro phores that have the potential to circumvent some of the functional limitations encountered by organic dyes in sensing and biotechnological applications. Quantum dots exhibit size-dependent tunable, narrow fluorescence emission spectra that span the visible spectrum and have broad absorption spectra. This allows simultaneous excitation of several particle sizes at a single wavelength with emission at multiple wavelengths. Quantum dots also provide a high-resistance threshold to chemical degradation and photodegradation. We have developed a conjugation strategy for the attachment of antibodies to quantum dots based on electrostatic interactions between negatively charged dihydrolipoic acid (DHLA)-capped CdSe-ZnS core-shell quantum dots and positively charged proteins (natural or engineered) that serve to bridge the quantum dot and antibody. This chapter details the materials and methods for synthesis of the DHLA-capped CdSe-ZnS core-shell quantum dots, the construction and preparation of recombinant proteins, the conjugation of antibodies to quantum dots, and the use of antibody-coated quantum dots in a fluoroimmunoassay.

Animals↗
PubMed2004

General strategy for biosensor design and construction employing multifunctional surface-tethered components.

Biosensors function by reversibly linking bioreceptor-target analyte binding with closely integrated signal generation and can either continuously monitor analyte concentrations or be returned to baseline readout values by removal of analyte. We present an approach for producing fully reversible, reagentless, self-assembling biosensors on surfaces. In the prototype biosensor, quencher-dye-labeled biotin-linked E. coli maltose binding protein (MBP) bound in a specific orientation to a NeutrAvidin-coated surface is employed as a bioreceptor. To complete sensor formation, a modular tether arm consisting of a flexible biotinylated DNA oligonucleotide, a fluorescence resonance energy-transfer (FRET) donor dye, and a distal beta-cyclodextrin (beta-CD) analyte analogue is bound in an equimolar amount to the same surface by means of DNA-directed immobilization. After self-assembly, a baseline level of FRET quenching is observed due to specific interaction between the beta-CD of the flexible tether arm and the sugar binding site of MBP, which brings the two dyes into proximity. Addition of the target analyte, the nutrient maltose, displaces the linked beta-CD-dye of the DNA-based tether arm, and a concentration-dependent change in FRET results. Biosensor sensitivity and dynamic range can be controlled by either using MBP variants having different binding constants or by binding of modulator DNA oligonucleotides that are complementary to the flexible DNA tether. The sensor can be regenerated and returned to baseline quenching levels by washing away analyte. A complex set of interactions apparently exists on the sensing surface that may contribute to sensor behavior and range. This approach may represent a general way to assemble a wide range of useful biosensors.

Base Sequence↗
PubMed2004

Surface-immobilized self-assembled protein-based quantum dot nanoassemblies.

Luminescent semiconductor quantum dot (QD)-based optical biosensors have the potential to overcome many of the limitations associated with using conventional organic dyes for biodetection. We have previously demonstrated a hybrid QD-protein-based fluorescence resonance energy transfer (FRET) sensor. Although the QD acted as an energy donor and a protein scaffold in the sensor, recognition and specificity were derived from the proteins. Transitioning this hybrid prototype sensor into flow cells and integrated devices will require a surface-immobilization strategy that allows the QD-based sensor to sample the environment and still maintain a distinct protein-covered QD architecture. We demonstrate a self-assembled strategy designed to accomplish this. Using glass slides coated with a monolayer of neutravidin (NA) as the template, QDs with maltose binding protein (MBP) and avidin coordinated to their surface were attached to the glass slides in discrete patterns using an intermediary bridge of biotinylated MBP or antibody linkers. Control of the surface location and concentration of the QD-protein-based structures is demonstrated. The utility of this self-assembly strategy is further demonstrated by assembling a QD-protein structure that allows the QDs to engage in FRET with a dye located on the surface-covering protein.

Avidin↗
PubMed2004

Multiplexed toxin analysis using four colors of quantum dot fluororeagents.

Quantum dots (QDs) have the potential to simplify the performance of multiplexed analysis. In this work, we prepared bioinorganic conjugates made with highly luminescent semiconductor nanocrystals (CdSe-ZnS core-shell QDs) and antibodies to perform multiplexed fluoroimmunoassays. Sandwich immunoassays for the detection of cholera toxin, ricin, shiga-like toxin 1, and staphylococcal enterotoxin B were performed simultaneously in single wells of a microtiter plate. Initially the assay performance for the detection of each toxin was examined. We then demonstrated the simultaneous detection of the four toxins from a single sample probed with a mixture of all four QD-antibody reagents. Using a simple linear equation-based algorithm, it was possible to deconvolute the signal from mixed toxin samples, which allowed quantitation of all four toxins simultaneously.

Cadmium↗
PubMed2004

Fluorescence resonance energy transfer between quantum dot donors and dye-labeled protein acceptors.

We used luminescent CdSe-ZnS core-shell quantum dots (QDs) as energy donors in fluorescent resonance energy transfer (FRET) assays. Engineered maltose binding protein (MBP) appended with an oligohistidine tail and labeled with an acceptor dye (Cy3) was immobilized on the nanocrystals via a noncovalent self-assembly scheme. This configuration allowed accurate control of the donor-acceptor separation distance to a range smaller than 100 A and provided a good model system to explore FRET phenomena in QD-protein-dye conjugates. This QD-MBP conjugate presents two advantages: (1) it permits one to tune the degree of spectral overlap between donor and acceptor and (2) provides a unique configuration where a single donor can interact with several acceptors simultaneously. The FRET signal was measured for these complexes as a function of both degree of spectral overlap and fraction of dye-labeled proteins in the QD conjugate. Data showed that substantial acceptor signals were measured upon conjugate formation, indicating efficient nonradiative exciton transfer between QD donors and dye-labeled protein acceptors. FRET efficiency can be controlled either by tuning the QD photoemission or by adjusting the number of dye-labeled proteins immobilized on the QD center. Results showed a clear dependence of the efficiency on the spectral overlap between the QD donor and dye acceptor. Apparent donor-acceptor distances were determined from efficiency measurements and corresponding Förster distances, and these results agreed with QD bioconjugate dimensions extracted from structural data and core size variations among QD populations.

Cadmium Compounds↗
PubMed2004

Reversible modulation of quantum dot photoluminescence using a protein- bound photochromic fluorescence resonance energy transfer acceptor.

Multiple copies ( approximately 20) of Escherichia coli maltose binding protein (MBP) were coordinated to luminescent semiconductor quantum dots (QDs) via a C-terminal oligohistidine segment. The MBP was labeled with a sulfo-N-hydroxysuccinimide-activated photochromic BIPS molecule (1',3-dihydro-1'-(2-carboxyethyl)-3,3-dimethyl-6-nitrospiro[2H-1-benzopyran-2,2'-(2H)-indoline]) at two different dye-to-MBP ratios; D/P = 1 and 5. The ability of MBP-BIPS to modulate QD photoluminescence was tested by switching BIPS from the colorless spiropyran (SP) to the colored merocyanine (MC) using white light (>500 nm) or UV light ( approximately 365 nm), respectively. QDs surrounded by MBP-BIPS with D/P = 1 were quenched on average approximately 25% with consecutive repeated switches, while QDs surrounded by MBP-BIPS with D/P = 5 were quenched approximately 60%. This result suggests a possible use of BIPS-labeled proteins in QD-based nanostructures as part of a threshold switch or other biosensing device.

Benzopyrans↗
PubMed2003

Self-assembled nanoscale biosensors based on quantum dot FRET donors.

The potential of luminescent semiconductor quantum dots (QDs) to enable development of hybrid inorganic-bioreceptor sensing materials has remained largely unrealized. We report the design, formation and testing of QD-protein assemblies that function as chemical sensors. In these assemblies, multiple copies of Escherichia coli maltose-binding protein (MBP) coordinate to each QD by a C-terminal oligohistidine segment and function as sugar receptors. Sensors are self-assembled in solution in a controllable manner. In one configuration, a beta-cyclodextrin-QSY9 dark quencher conjugate bound in the MBP saccharide binding site results in fluorescence resonance energy-transfer (FRET) quenching of QD photoluminescence. Added maltose displaces the beta-cyclodextrin-QSY9, and QD photoluminescence increases in a systematic manner. A second maltose sensor assembly consists of QDs coupled with Cy3-labelled MBP bound to beta-cyclodextrin-Cy3.5. In this case, the QD donor drives sensor function through a two-step FRET mechanism that overcomes inherent QD donor-acceptor distance limitations. Quantum dot-biomolecule assemblies constructed using these methods may facilitate development of new hybrid sensing materials.

Biosensing Techniques↗
PubMed2002

Microfabricated 384-lane capillary array electrophoresis bioanalyzer for ultrahigh-throughput genetic analysis.

A microfabricated 384-lane capillary array electrophoresis device is developed and utilized for massively parallel genetic analysis. The 384 capillary lanes, arrayed radially about the center of a 200-mm-diameter glass substrate sandwich, are constructed using scalable microfabrication techniques derived from the semiconductor industry. Samples are loaded into reservoirs on the perimeter of the wafer, separated on the 8-cm-long poly(dimethylacrylamide) gel-filled channels, and detected with a four-color rotary confocal fluorescence scanner. The performance and throughput of this bioanalyzer are demonstrated by simultaneous genotyping 384 individuals for the common hemochromatosis-linked H63D mutation in the human HFE gene in only 325 s. This lab-on-a-chip device thoroughly exploits the power of microfabrication to produce high-density capillary electrophoresis arrays and to use them for high-throughput bioanalysis.

DNA↗
PubMedDate not supplied

A fluorescence resonance energy transfer sensor based on maltose binding protein.

A fluorescence resonance energy-transfer (FRET) sensing system for maltose based on E. coli maltose binding protein (MBP) is demonstrated. The FRET donor portion of the sensing system consists of MBP modified with long wavelength-excitable cyanine dyes (Cy3 or Cy3.5). The novel acceptor portion of the sensor consists of beta-cyclodextrin (beta-CD) modified with either the cyanine dye Cy5 or the dark quencher QSY9. Binding of the modified beta-CD to dye-conjugated MBP results in assembly of the FRET complex. Added maltose displaces the beta-CD-dye adduct and disrupts the FRET complex, resulting in a direct change in fluorescence of the donor moiety. In the use of these FRET pairs, MBP dissociation values for maltose were estimated (0.14-2.90 microM). Maltose limits of detection were in the 50-100 nm range.

Biosensing Techniques↗
PubMedDate not supplied

Self-assembled quantum dot-peptide bioconjugates for selective intracellular delivery.

We demonstrate the use of self-assembled luminescent semiconductor quantum dot (QD)-peptide bioconjugates for the selective intracellular labeling of several eukaryotic cell lines. A bifunctional oligoarginine cell penetrating peptide (based on the HIV-1 Tat protein motif) bearing a terminal polyhistidine tract was synthesized and used to facilitate the transmembrane delivery of the QD bioconjugates. The polyhistidine sequence allows the peptide to self-assemble onto the QD surface via metal-affinity interactions while the oligoarginine sequence allows specific QD delivery across the cellular membrane and intracellular labeling as compared to nonconjugated QDs. This peptide-driven delivery is concentration-dependent and thus can be titrated. Upon internalization, QDs display a punctate-like staining pattern in which some, but not all, of the QD signal is colocalized within endosomes. The effects of constant versus limited exposure to QD-peptide conjugates on cellular viability are evaluated by a metabolic specific assay, and clear differences in cytotoxicity are observed. The efficacy of using peptides for selective intracellular delivery is highlighted by performing a multicolor QD labeling, where we found that the presence or absence of peptide on the QD surface controls cellular uptake.

Animals↗