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Quantum dots (QDs) are loaded with a series of peptides and proteins of increasing size, including a <20 residue peptide, myoglobin, mCherry, and maltose binding protein, which together cover a range of masses from <2.2 to approximately 44 kDa. Conjugation to the surface of dihydrolipoic acid-functionalized QDs is facilitated by polyhistidine metal affinity coordination. Increasing ratios of dye-labeled peptides and proteins are self-assembled to the QDs and then the bioconjugates are separated and analyzed using agarose gel electrophoresis. Fluorescent visualization of both conjugated and unbound species allows determination of an experimentally derived maximum loading number. Molecular modeling utilizing crystallographic coordinates or space-filling structures of the peptides and proteins also allow the predicted maximum loadings to the QDs to be estimated. Comparison of the two sets of results provides insight into the nature of the QD surface and reflects the important role played by the nanoparticle's hydrophilic solubilizing surface ligands. It is found that for the larger protein molecules steric hindrance is the major packing constraint. In contrast, for the smaller peptides, the number of available QD binding sites is the principal determinant. These results can contribute towards an overall understanding of how to engineer designer bioconjugates for both QDs and other nanoparticle materials.

A simple bifunctional colorimetric/fluorescent sensing assay is demonstrated for the detection of HIV-1 specific antibodies. This assay makes use of a short peptide sequence coupled to an environmentally sensitive dye that absorbs and emits in the visible portion of the spectrum. The core peptide sequence is derived from the highly antigenic six-residue epitope of the HIV-1 p17 protein and is situated adjacent to a terminal cysteine residue which enables site-specific fluorescent labeling with Cy3 cyanine dye. Interaction of the Cy3-labeled p17 peptide with monoclonal anti-p17 antibody resulted in an up to 4-fold increase in dye absorption and greater than 5-fold increase in fluorescent emission, yielding a limit of detection as low as 73 pM for the target antibody. This initial study demonstrates both proof-of-concept for this approach and suggests that the resulting sensor could potentially be used as a rapid screening method for HIV-1 infection while requiring minimal equipment and reagents. The potential for utilizing this assay in simple field-portable point-of-care and diagnostic devices is discussed.

One of the principle hurdles to wider incorporation of semiconductor quantum dots (QDs) in biology is the lack of facile linkage chemistries to create different types of functional QD--bioconjugates. A two-step modular strategy for the presentation of biomolecules on CdSe/ZnS core/shell QDs is described here which utilizes a chemoselective, aniline-catalyzed hydrazone coupling chemistry to append hexahistidine sequences onto peptides and DNA. This specifically provides them the ability to ratiometrically self-assemble to hydrophilic QDs. The versatility of this labeling approach was highlighted by ligating proteolytic substrate peptides, an oligoarginine cell-penetrating peptide, or a DNA-probe to cognate hexahistidine peptidyl sequences. The modularity allowed subsequently self-assembled QD constructs to engage in different types of targeted bioassays. The self-assembly and photophysical properties of individual QD conjugates were first confirmed by gel electrophoresis and Forster resonance energy transfer analysis. QD-dye-labeled peptide conjugates were then used as biosensors to quantitatively monitor the proteolytic activity of caspase-3 or elastase enzymes from different species. These sensors allowed the determination of the corresponding kinetic parameters, including the Michaelis constant (K(M)) and the maximum proteolytic activity (V(max)). QDs decorated with cell-penetrating peptides were shown to be successfully internalized by HEK 293T/17 cells, while nanocrystals displaying peptide--DNA conjugates were utilized as fluorescent probes in hybridization microarray assays. This modular approach for displaying peptides or DNA on QDs may be extended to other more complex biomolecules such as proteins or utilized with different types of nanoparticle materials.

We characterized the resonance energy transfer interactions for conjugates consisting of QD donors self-assembled with three distinct fluorescent protein acceptors: two monomeric fluorescent proteins, the dsRed derivative mCherry or yellow fluorescent protein and the multi-chromophore b-phycoerythrin light harvesting complex. Using steady-state and time-resolved fluorescence, we showed that nonradiative transfer of excitation energy in these conjugates can be described within the Förster dipole-dipole formalism, with transfer efficiencies that vary with the degree of spectral overlap, donor-acceptor separation distance and the number of acceptors per QD. Comparison between the quenching data and simulation of the conjugate structures indicated that while energy transfer to monomeric proteins was identical to what was measured for QD-dye pairs, interactions with b-phycoerythrin were more complex. For the latter, the overall transfer efficiency results from the cumulative contribution of individual channels between the central QD and the chromophores distributed throughout the protein structure. Due to the biocompatible nature of fluorescent proteins, these QD-assemblies may have great potential for use in intracellular imaging and sensing.

Nanoparticle-mediated drug delivery (NMDD) is an emerging research area that seeks to address many of the pharmacokinetic issues encountered with traditional systemically administered drug therapies. Although the field is still in its infancy, recent research has already highlighted the potential for improved drug delivery and targeted therapeutics; however, the real promise lies in combining drug therapy with diagnostic imaging, nucleic acid delivery/gene therapy and/or biosensing applications all in one engineered nanoparticle vector. In this review, the authors discuss the unique contributions that luminescent semiconductor nanocrystals or quantum dots (QDs) offer for NMDD, how they can function as a powerful nanoscale platform to understand this process at its most basic level, and even provide drug-related properties in certain circumstances. Selected examples from the current literature are utilized to describe both their potential and the contributions they have already made towards the design and implementation of NMDD vectors. Important related issues such as QD biofunctionalization and toxicity are also discussed. The paper concludes with a perspective of how this field can be expected to develop in the future.

Simultaneous detection of multiple independent fluorescent signals or signal multiplexing has the potential to significantly improve bioassay throughput and to allow visualization of concurrent cellular events. Applications based on signal multiplexing, however, remain hard to achieve in practice due to difficulties in both implementing hardware and the photophysical liabilities associated with available organic dye and protein fluorophores. Here, we used charge-transfer interactions between luminescent semiconductor quantum dots (QDs) and proximal redox complexes to demonstrate controlled quenching of QD photoemission in a multiplexed format. In particular, we show that, because of the ability of the Ru complex to effectively interact with CdSe-ZnS QDs emitting over a broad window of the optical spectrum, higher orders of multiplexed quenching can be achieved in a relatively facile manner. Polyhistidine-appended peptides were site-specifically labeled with a redox-active ruthenium (Ru) phenanthroline complex and self-assembled onto QDs, resulting in controlled quenching of the QD emission. Different QD colors either alone or coupled to Ru-phen-peptide were then mixed together and optically interrogated. Composite spectra collected from mixtures ranging from four up to eight distinct QD colors were deconvoluted, and the individual QD photoluminescence (PL) loss due to charge transfer was quantified. The current multiplexing modality provides a simpler format for exploiting the narrow, size-tunable QD emissions than that offered by resonance energy transfer; for the latter, higher orders of multiplexing are limited by spectral overlap requirements.

Functional DNA scaffolds can be defined as DNA-based structures comprising chemical moieties facilitating and guiding the immobilization of additional nanocomponents. Due to the limited reactivity of DNA there is currently a need to develop rapid routes to expand its chemical repertoire and increase its versatility as a nanostructuring scaffold. We report a simple synthetic strategy for generating linear and stable double-stranded DNA scaffolds functionalized with multiple sites reactive towards free thiols, and the utility of this approach is demonstrated by immobilizing a model protein containing an accessible free thiol. This procedure is very versatile and could be easily expanded to other types of chemistries. This approach could also potentially be employed for the specific, oriented immobilization of various biomolecules and nanoparticles on predefined DNA architectures.

We demonstrate the use of a hybrid fluorescent protein semiconductor quantum dot (QD) sensor capable of specifically monitoring caspase 3 proteolytic activity. mCherry monomeric red fluorescent protein engineered to express an N-terminal caspase 3 cleavage site was ratiometrically self-assembled to the surface of QDs using metal-affinity coordination. The proximity of the fluorescent protein to the QD allows it to function as an efficient fluorescence resonance energy transfer acceptor. Addition of caspase 3 enzyme to the QD-mCherry conjugates specifically cleaved the engineered mCherry linker sequence, altering the energy transfer with the QD and allowing quantitative monitoring of proteolytic activity. Inherent advantages of this sensing approach include bacterial expression of the protease substrate in a fluorescently appended form, facile self-assembly to QDs, and the ability to recombinantly modify the substrate to target other proteases of interest.

The use of semiconductor quantum dots (QDs) in biological sensing and labeling continues to grow with each year. Current and projected applications include use as fluorescent labels for cellular labeling, intracellular sensors, deep-tissue and tumor imaging agents, sensitizers for photodynamic therapy, and more recently interest has been sparked in using them as vectors for studying nanoparticle-mediated drug delivery. Many of these applications will ultimately require the QDs to undergo targeted intracellular delivery, not only to specific cells, but also to a variety of subcellular compartments and organelles. It is apparent that this issue will be critical in determining the efficacy of using QDs, and indeed a variety of other nanoparticles, for these types of applications. In this review, we provide an overview of the current methods for delivering QDs into cells. Methods that are covered include facilitated techniques such as those that utilize specific peptide sequences or polymer delivery reagents and active methods such as electroporation and microinjection. We critically examine the benefits and liabilities of each strategy and illustrate them with selected examples from the literature. Several important related issues such as QD size and surface coating, methods for QD biofunctionalization, cellular physiology and toxicity are also discussed. Finally, we conclude by providing a perspective of how this field can be expected to develop in the future.

We provide an overview of the progress made in the past few years in investigating fluorescence resonance energy transfer (FRET) using semiconductor quantum dots (QDs) and the application of QD-based FRET to probe specific biological processes. We start by providing some of the pertinent conceptual elements involved in resonance energy transfer, and then discuss why the Förster dipole-dipole mechanism applies to QD fluorophores. We then describe the unique QD photophysical properties of direct relevance to FRET and summarize the main advantages offered, along with some of the limitations encountered by QDs as exciton donors and/or acceptors. Next we describe the overall progress made and discuss a few representative examples where QD-based FRET sensing of specific biological processes has been demonstrated. We also detail some of the advances of single molecule FRET using QD-conjugates and highlight the unique information that can be extracted. We conclude by providing an assessment of where QD-based FRET investigations may be evolving in the near future.

We describe a simple and versatile scheme to prepare a series of poly(ethylene glycol)-based bidentate ligands that permit strong interactions with colloidal semiconductor nanocrystals (quantum dots, QDs) and gold nanoparticles (AuNPs) alike and promote their dispersion in aqueous solutions. These ligands are synthesized by coupling poly(ethylene glycol)s of various chain length to thioctic acid, followed by ring opening of the 1,2-dithiolane moiety to create a bidentate thiol anchoring group with enhanced affinity for CdSe-ZnS core-shell QDs. These ligands provide a straightforward means of preparing QDs and AuNPs that exhibit greater resistance to environmental changes, facilitating their effective use in bioassays and live cell imaging.

Colloidal quantum dots (QDs) have a large fraction of their atoms arrayed on their surfaces and are capped with bifunctional ligands, which make their photoluminescence highly sensitive to potential charge transfer to or from the surrounding environment. In this report, we used peptides as bridges between CdSe-ZnS QDs and metal complexes to promote charge transfer between the metal complexes and QDs. We found that quenching of the QD emission is highly dependent on the relative position of the oxidation levels of QDs and metal complex used; it also traces the number of metal complexes brought in close proximity of the nanocrystal surface. In addition, partial bleaching of the absorption was measured for the QD-metal complex assemblies. These proximity driven interactions were further used to construct sensing assemblies to detect proteolytic enzyme activity.

We utilize cell penetrating peptide functionalized QDs as specific vectors for the intracellular delivery of model fluorescent protein cargos. Multiple copies of two structurally diverse fluorescent proteins, the 27 kDa monomeric yellow fluorescent protein and the 240 kDa multichromophore b-phycoerythrin complex, were attached to QDs using either metal-affinity driven self-assembly or biotin-Streptavidin binding, respectively. Cellular uptake of these complexes was found to depend on the additional presence of cell-penetrating peptides within the QD-protein conjugates. Once inside the cells, the QD conjugates were mostly distributed within endolysosomal compartments, indicating that intracellular delivery of both QD assemblies was primarily driven by endocytotic uptake. Cellular microinjection of QD-fluorescent protein assemblies was also utilized as an alternate delivery strategy that could bypass the endocytic pathway. Simultaneous signals from both the QDs and the fluorescent proteins allowed verification of their colocalization and conjugate integrity upon delivery inside live cells. Due to their intrinsic fluorescence properties, this class of proteins provides a unique tool to test the ability of QDs functionalized with cell penetrating peptides to mediate the intracellular delivery of both small and large size protein cargos. Use of QD-peptide/fluorescent protein vectors may make powerful tools for understanding the mechanisms of nanoparticle-mediated drug delivery.

Background: Microfabricated genetic-analysis devices have great potential for delivering complex clinical diagnostic technology to the point of care. As a demonstration of the potential of these devices, we used a microfabricated capillary array electrophoresis (microCAE) instrument to rapidly characterize the familial and genotypic relationship of twins who had been assigned fraternal (dizygous) status at birth., Methods: We extracted the genomic DNA from buccal samples collected from the twin sons, the parents, another sibling, and an unrelated control individual. We then carried out multiplex PCR amplification of sequences at 16 short tandem repeat loci commonly used in forensic identity testing. We simultaneously separated the amplicons from all of the individuals on a microCAE device and fluorescently detected the amplicons with single-base resolution in <30 min., Results: The genotypic analysis confirmed the identical status of the twins and revealed, in conjunction with the medical data, that their twin status arose from the rarer dichorionic, diamniotic process., Conclusions: The ability to rapidly analyze complex genetic samples with microCAE devices demonstrates that this approach can help meet the growing need for rapid genetics-based diagnostics.

The use of luminescent colloidal quantum dots in biological investigations has increased dramatically over the past several years due to their unique size-dependent optical properties and recent advances in biofunctionalization. In this review, we describe the methods for generating high-quality nanocrystals and report on current and potential uses of these versatile materials. Numerous examples are provided in several key areas including cell labeling, biosensing, in vivo imaging, bimodal magnetic-luminescent imaging, and diagnostics. We also explore toxicity issues surrounding these materials and speculate about the future uses of quantum dots in a clinical setting.

We have designed and synthesized a series of modular ligands based on poly(ethylene glycol) (PEG) coupled with functional terminal groups to promote water-solubility and biocompatibility of quantum dots (QDs). Each ligand is comprised of three modules: a PEG single chain to promote hydrophilicity, a dihydrolipoic acid (DHLA) unit connected to one end of the PEG chain for strong anchoring onto the QD surface, and a potential biological functional group (biotin, carboxyl, and amine) at the other end of the PEG. Water-soluble QDs capped with these functional ligands were prepared via cap exchange with the native hydrophobic caps. Homogeneous QD solutions that are stable over extended periods of time and over a broad pH range were prepared. Surface binding assays and cellular internalization and imaging showed that QDs capped with DHLA-PEG-biotin strongly interacted with either NeutrAvidin immobilized on surfaces or streptavidin coupled to proteins which were subsequently taken up by live cells. EDC coupling in aqueous buffer solutions was also demonstrated using resonance energy transfer between DHLA-PEG-COOH-functionalized QDs and an amine-terminated dye. The new functional surface ligands described here provide not only stable and highly water-soluble QDs but also simple and easy access to various biological entities.

Luminescent quantum dots (QDs) were proven to be very effective fluorescence resonance energy transfer donors with an array of organic dye acceptors, and several fluorescence resonance energy transfer based biosensing assemblies utilizing QDs have been demonstrated in the past few years. Conversely, gold nanoparticles (Au-NPs) are known for their capacity to induce strong fluorescence quenching of conventional dye donors. Using a rigid variable-length polypeptide as a bifunctional biological linker, we monitor the photoluminescence quenching of CdSe-ZnS QDs by Au-NP acceptors arrayed around the QD surface, where the center-to-center separation distance was varied over a broad range of values (approximately 50-200 Angstrom). We measure the Au-NP-induced quenching rates for such QD conjugates using steady-state and time-resolved fluorescence measurements and examine the results within the context of theoretical treatments based on the Förster dipole-dipole resonance energy transfer, dipole-metal particle energy transfer, and nanosurface energy transfer. Our results indicate that nonradiative quenching of the QD emission by proximal Au-NPs is due to long-distance dipole-metal interactions that extend significantly beyond the classical Förster range, in agreement with previous studies using organic dye-Au-NP donor-acceptor pairs.

A microfluidic separation system is developed to perform two-dimensional differential gel electrophoretic (DIGE) separations of complex, cellular protein mixtures produced by induced protein expression in E. coli. The micro-DIGE analyzer is a two-layer borosilicate glass microdevice consisting of a single 3.75 cm long channel for isoelectric focusing, which is sampled in parallel by 20 channels effecting a second-dimension separation by native electrophoresis. The connection between the orthogonal separation systems is accomplished by smaller channels comprising a microfluidic interface (MFI) that prevents media leakage between the two dimensions and enables facile loading of discontinuous gel systems in each dimension. Proteins are covalently labeled with Cy2 and Cy3 DIGE and detected simultaneously with a rotary confocal fluorescence scanner. Reproducible two-dimensional separations of both purified proteins and complex protein mixtures are performed with minimal run-to-run variation by including 7 M urea in the second-dimension separation matrix. The capabilities of the micro-DIGE analyzer are demonstrated by following the induced expression of maltose binding protein in E. coli. Although the absence of sodium dodecyl sulfate (SDS) in the second-dimension sizing separation limits the orthogonality and peak capacity of the separation, this analyzer is a significant first step toward the reproducible two-dimensional analysis of complex protein samples in microfabricated devices. Furthermore, the microchannel interface structures developed here will facilitate other multidimensional separations in microdevices.

In response to limited nitrogen and abundant carbon sources, diploid Saccharomyces cerevisiae strains undergo a filamentous transition in cell growth as part of pseudohyphal differentiation. Use of the disaccharide maltose as the principal carbon source, in contrast to the preferred nutrient monosaccharide glucose, has been shown to induce a hyper-filamentous growth phenotype in a strain deficient for GPA2 which codes for a Galpha protein component that interacts with the glucose-sensing receptor Gpr1p to regulate filamentous growth. In this report, we compare the global transcript and proteomic profiles of wild-type and Gpa2p deficient diploid yeast strains grown on both rich and nitrogen starved maltose media. We find that deletion of GPA2 results in significantly different transcript and protein profiles when switching from rich to nitrogen starvation media. The results are discussed with a focus on the genes associated with carbon utilization, or regulation thereof, and a model for the contribution of carbon sensing/metabolism-based signal transduction to pseudohyphal differentiation is proposed.

Self-assembly of proteins, peptides, DNA, and other biomolecules to semiconductor quantum dots (QD) is an attractive bioconjugation route that can circumvent many of the problems associated with covalent chemistry and subsequent purification. Polyhistidine sequences have been shown to facilitate self-assembly of proteins and peptides to ZnS-overcoated CdSe QDs via complexation to unoccupied coordination metal sites on the nanocrystal surface. We describe the synthesis and characterization of a thiol-reactive hexahistidine peptidic linker that can be chemically attached to thiolated-DNA oligomers and mediate their self-assembly to CdSe-ZnS core-shell QDs. The self-assembly of hexahistidine-appended DNA to QDs is probed with gel electrophoresis and fluorescence resonance energy transfer techniques, and the results confirm high-affinity conjugate formation with control over the average molar ratio of DNA assembled per QD. To demonstrate the potential of this reactive peptide linker strategy, a prototype QD-DNA-dye molecular beacon is self-assembled and tested against both specific and nonspecific target DNAs. This conjugation route is potentially versatile, as altering the reactivity of the peptide linker may allow targeting of different functional groups such as amines and facilitate self-assembly of other nanoparticle-biomolecule structures.

We report the design and synthesis of a tetraethylene glycol- (TEG-) based bidentate ligand functionalized with dihydrolipoic acid (DHLA) and biotin (DHLA-TEG-biotin) to promote biocompatibility of luminescent quantum dots (QD's). This new ligand readily binds to CdSe-ZnS core-shell QDs via surface ligand exchange. QDs capped with a mixture of DHLA and DHLA-TEG-biotin or polyethylene glycol- (PEG-) (molecular weight average approximately 600) modified DHLA (DHLA-PEG600) and DHLA-TEG-biotin are easily dispersed in aqueous buffer solutions. In particular, homogeneous buffer solutions of QDs capped with a mixture of DHLA-PEG600 and DHLA-TEG-biotin that are stable over broad pH range have been prepared. QDs coated with mixtures of DHLA/DHLA-TEG-biotin and with DHLA-PEG600/DHLA-TEG-biotin were tested in surface binding assays and the results indicate that biotin groups on the QD surface interact specifically with NeutrAvidin-functionalized microtiter well plates.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The first generation of luminescent semiconductor quantum dot (QD)-based hybrid inorganic biomaterials and sensors is now being developed. It is crucial to understand how bioreceptors, especially proteins, interact with these inorganic nanomaterials. As a model system for study, we use Rhodamine red-labeled engineered variants of Escherichia coli maltose-binding protein (MBP) coordinated to the surface of 555-nm emitting CdSe-ZnS core-shell QDs. Fluorescence resonance energy transfer studies were performed to determine the distance from each of six unique MBP-Rhodamine red dye-acceptor locations to the center of the energy-donating QD. In a strategy analogous to a nanoscale global positioning system determination, we use the intraassembly distances determined from the fluorescence resonance energy transfer measurements, the MBP crystallographic coordinates, and a least-squares approach to determine the orientation of the MBP relative to the QD surface. Results indicate that MBP has a preferred orientation on the QD surface. The refined model is in agreement with other evidence, which indicates coordination of the protein to the QD occurs by means of its C-terminal pentahistidine tail, and the size of the QD estimated from the model is in good agreement with physical measurements of QD size. The approach detailed here may be useful in determining the orientation of proteins in other hybrid protein-nanoparticle materials. To our knowledge, this is the first structural model of a hybrid luminescent QD-protein receptor assembly elucidated by using spectroscopic measurements in conjunction with crystallographic and other data.

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.

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.

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.

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.

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.

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.