Detection of HIV-1 Specific Monoclonal Antibodies Using Enhancement of Dye-Labeled Antigenic Peptides

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

Toward Single Molecule Detection of Staphylococcal Enterotoxin B: Mobile Sandwich Immunoassay on Gliding Microtubules

An immunoassay based on gliding microtubules (MTs) is described for the detection of staphylococcal enterotoxin B. Detection is performed in a sandwich immunoassay format. Gliding microtubules carry the antigen-specific “capture” antibody, and bound analyte is detected using a fluorescent viral scaffold as the tracer. A detailed modification scheme for the MTs postpolymerization is described along with corresponding quantification by fluorescence spectroscopy. The resultant antibody−MTs maintain their morphology and gliding capabilities. We report a limit of detection down to 0.5 ng/mL during active transport in a 30 min assay time and down to 1 ng/mL on static surfaces. This study demonstrates the kinesin/MT-mediated capture, transport, and detection of the biowarfare agent SEB in a microfluidic format

Kinetics of Metal-Affinity Driven Self-Assembly between Proteins or Peptides and CdSe−ZnS Quantum Dots

We present a molecular characterization of metal-affinity driven self-assembly between CdSe−ZnS core−shell quantum dots (QDs) and a series of proteins and peptides appended with various length polyhistidine tags. In particular, we investigated the kinetics of self-assembly between surface-immobilized QDs and proteins/peptides under flow conditions, as well as between freely diffusing QDs and proteins/peptides (solution phase). In the first configuration, QDs were immobilized onto functionalized substrates and then exposed to dye-labeled peptides/proteins. Using evanescent wave excitation, we assessed self-assembly by monitoring the time-dependent changes in the dye fluorescence. In solution, the kinetics of self-assembly was monitored via energy transfer between QDs and dye-labeled proteins/peptides. These measurements allowed determination of the kinetic parameters, including the association and dissociation rates (kon and koff) and the apparent binding constant (Kd). We find that self-assembly is rapid with an equilibrium constant Kd-1 ≈ 1 nM for solution self-assembly, confirming that metal-affinity interactions provide QD bioconjugates that are functional and stable

On the Quenching of Semiconductor Quantum Dot Photoluminescence by Proximal Gold Nanoparticles

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 (∼50−200 Å). 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

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 β-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 β-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 β-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

Monitoring Botulinum Neurotoxin A Activity with Peptide-Functionalized Quantum Dot Resonance Energy Transfer Sensors

Botulinum neurotoxins (BoNTs) are extremely potent bacterial toxins that contaminate food supplies along with having a high potential for exploitation as bioterrorism agents. There is a continuing need to rapidly and sensitively detect exposure to these toxins and to verify their active state, as the latter directly affects diagnosis and helps provide effective treatments. We investigate the use of semiconductor quantum dot (QD)−peptide Förster resonance energy transfer (FRET) assemblies to monitor the activity of the BoNT serotype A light chain protease (LcA). A modular LcA peptide substrate was designed and optimized to contain a central LcA recognition/cleavage region, a unique residue to allow labeling with a Cy3 acceptor dye, an extended linker-spacer sequence, and a terminal oligohistidine that allows for final ratiometric peptide−QD-self-assembly. A number of different QD materials displaying charged or PEGylated surface-coatings were evaluated for their ability to self-assemble dye-labeled LcA peptide substrates by monitoring FRET interactions. Proteolytic assays were performed utilizing either a direct peptide-on-QD format or alternatively an indirect pre-exposure of peptide to LcA prior to QD assembly. Variable activities were obtained depending on QD materials and formats used with the most sensitive pre-exposure assay result demonstrating a 350 pM LcA limit of detection. Modeling the various QD−peptide sensor constructs provided insight into how the resulting assembly architecture influenced LcA recognition interactions and subsequent activity. These results also highlight the unique roles that both peptide design and QD features, especially surface-capping agents, contribute to overall sensor activity

Functionalizing Nanoparticles with Biological Molecules: Developing Chemistries that Facilitate Nanotechnology

Functionalizing Nanoparticles with Biological Molecules: Developing Chemistries that Facilitate Nanotechnolog

Cellular Uptake and Fate of PEGylated Gold Nanoparticles Is Dependent on Both Cell-Penetration Peptides and Particle Size

Numerous studies have examined how the cellular delivery of gold nanoparticles (AuNPs) is influenced by different physical and chemical characteristics; however, the complex relationship between AuNP size, uptake efficiency and intracellular localization remains only partially understood. Here we examine the cellular uptake of a series of AuNPs ranging in diameter from 2.4 to 89 nm that are synthesized and made soluble with poly(ethylene glycol)-functionalized dithiolane ligands terminating in either carboxyl or methoxy groups and covalently conjugated to cell penetrating peptides. Following synthesis, extensive physical characterization of the AuNPs was performed with UV–vis absorption, gel electrophoresis, zeta potential, dynamic light scattering, and high resolution transmission electron microscopy. Uptake efficiency and intracellular localization of the AuNP–peptide conjugates in a model COS-1 cell line were probed with a combination of silver staining, fluorescent counterstaining, and dual mode fluorescence coupled to nonfluorescent scattering. Our findings show that AuNP cellular uptake is directly dependent on the surface display of the cell-penetrating peptide and that the ultimate intracellular destination is further determined by AuNP diameter. The smallest 2.4 nm AuNPs were found to localize in the nucleus, while intermediate 5.5 and 8.2 nm particles were partially delivered into the cytoplasm, showing a primarily perinuclear fate along with a portion of the nanoparticles appearing to remain at the membrane. The 16 nm and larger AuNPs did not enter the cells and were located at the cellular periphery. A preliminary assessment of cytotoxicity demonstrated minimal effects on cellular viability following peptide-mediated uptake

Reactive Semiconductor Nanocrystals for Chemoselective Biolabeling and Multiplexed Analysis

Effective biological application of nanocrystalline semiconductor quantum dots continues to be hampered by the lack of easily implemented and widely applicable labeling chemistries. Here, we introduce two new orthogonal nanocrystal bioconjugation chemistries that overcome many of the labeling issues associated with currently utilized approaches. These chemistries specifically target either (1) the ubiquitous amines found on proteins or (2) thiols present in either antibody hinge regions or recombinantly introduced into other proteins to facilitate site-specific labeling. The amine chemistry incorporates aniline-catalyzed hydrazone bond formation, while the sulfhydryl chemistry utilizes nanocrystals displaying surface activated maleimide groups. Both reactive chemistries are rapidly implemented, yielding purified nanocrystal–protein bioconjugates in as little as 3 h. Following initial characterization of the nanocrystal materials, the wide applicability and strong multiplexing potential of these chemistries are demonstrated in an array of applications including immunoassays, immunolabeling in both cellular and tissue samples, in vivo cellular uptake, and flow cytometry. Side-by-side comparison of the immunolabeled cells suggested a functional equivalence between results generated with the amine and thiol-labeled antibody–nanocrystal bioconjugates in that format. Three-color labeling was achieved in the cellular uptake format, with no significant toxicity observed while simultaneous five-color labeling of different epitopes was demonstrated for the immunolabeled tissue sample. Novel labeling applications are also facilitated by these chemistries, as highlighted by the ability to directly label cellular membranes in adherent cell cultures with the thiol-reactive chemistry