Protein Kinase-Actuated Resonance Energy Transfer in Quantum Dot−Peptide Conjugates

Bioconjugates of quantum dot nanocrystals possess unique optical properties that allow them to serve as exceptional biological imaging and sensing reagents. Protein kinases are an important family of enzymes that phosphorylate serine, threonine, or tyrosine side chains and are critical in cell signaling and cancer biology, but despite their biomedical and pharmaceutical significance, their activity has been little explored using quantum dot technology. We demonstrate that self-assembled peptide−quantum dot conjugates can serve as surrogate substrates in a simple homogeneous assay for protein kinase activity. Enzymatic phosphorylation of the peptide-conjugates is detected by means of a complementary FRET-acceptor labeled antiphosphotyrosine antibody, with formation of the immunocomplex resulting in energy transfer between the quantum dot and FRET acceptor molecules. This approach should facilitate the development of new assays for protein kinases and other enzymes based on quantum dot FRET donors

Caged Quantum Dots

Caged Quantum Dot

Multiplex Sensing of Protease and Kinase Enzyme Activity <i>via</i> Orthogonal Coupling of Quantum Dot–Peptide Conjugates

Nanoparticle-based labels are emerging as simpler and more sensitive alternatives to traditional fluorescent small molecules and radioactive reporters in biomarker assays. The determination of biomarker levels is a recommended clinical practice for the assessment of many diseases, and detection of multiple analytes in a single assay, known as multiplexing, can increase predictive accuracy. While multiplexed detection can also simplify assay procedures and reduce systematic variability, combining multiple assays into a single procedure can lead to complications such as substrate cross-reactivity, signal overlap, and loss of sensitivity. By combining the specificity of biomolecular interactions with the tunability of quantum dot optical properties, we have developed a detection system capable of simultaneous evaluation of the activity of two critical enzyme classes, proteases and kinases. We avoid cross-reactivity and signal overlap by synthesizing enzyme-specific peptide sequences with orthogonal terminal functionalization for attachment to quantum dots with distinct emission spectra. Enzyme activity is reported <i>via</i> binding of either gold nanoparticle–peptide conjugates or FRET acceptor dye-labeled antibodies, which mediate changes in quantum dot emission spectra. To the best of our knowledge, this is the first demonstration of the multiplexed sensing of the activity of two different classes of enzymes <i>via</i> a nanoparticle-based activity assay. Using the quantum dot-based assay described herein, we were able to detect the protease activity of urokinase-type plasminogen activator at concentrations ≥ 50 ng/mL and the kinase activity of human epidermal growth factor receptor 2 at concentrations ≥ 7.5 nM, levels that are clinically relevant for determination of breast cancer prognosis. The modular nature of this assay design allows for the detection of different classes of enzymes simultaneously and represents a generic platform for high-throughput enzyme screening in rapid disease diagnosis and drug discovery

Measurement of Solvation Responses at Multiple Sites in a Globular Protein

Proteins respond to electrostatic perturbations through complex reorganizations of their charged and polar groups, as well as those of the surrounding media. These solvation responses occur both in the protein interior and on its surface, though the exact mechanisms of solvation are not well understood, in part because of limited data on the solvation responses for any given protein. Here, we characterize the solvation kinetics at sites throughout the sequence of a small globular protein, the B1 domain of streptococcal protein G (GB1), using the synthetic fluorescent amino acid Aladan. Aladan was incorporated into seven different GB1 sites, and the time-dependent Stokes shift was measured over the femtosecond to nanosecond time scales by fluorescence upconversion and time-correlated single photon counting. The seven sites range from buried within the protein core to fully solvent-exposed on the protein surface, and are located on different protein secondary structures including β-sheets, helices, and loops. The dynamics in the protein sites were compared against the free fluorophore in buffer. All protein sites exhibited an initial, ultrafast Stokes shift on the subpicosecond time scale similar to that observed for the free fluorophore, but smaller in magnitude. As the probe is moved from the surface to more buried sites, the dynamics of the solvation response become slower, while no clear correlation between dynamics and secondary structure is observed. We suggest that restricted movements of the surrounding protein residues give rise to the observed long time dynamics and that such movements comprise a large portion of the protein's solvation response. The proper treatment of dynamic Stokes shift data when the time scale for solvation is comparable to the fluorescence lifetime is discussed

Azide–Alkyne Click Conjugation on Quantum Dots by Selective Copper Coordination

Functionalization of nanocrystals is essential for their practical application, but synthesis on nanocrystal surfaces is limited by incompatibilities with certain key reagents. The copper-catalyzed azide–alkyne cycloaddition is among the most useful methods for ligating molecules to surfaces, but has been largely useless for semiconductor quantum dots (QDs) because Cu⁺ ions quickly and irreversibly quench QD fluorescence. To discover nonquenching synthetic conditions for Cu-catalyzed click reactions on QD surfaces, we developed a combinatorial fluorescence assay to screen >2000 reaction conditions to maximize cycloaddition efficiency while minimizing QD quenching. We identify conditions for complete coupling without significant quenching, which are compatible with common QD polymer surfaces and various azide/alkyne pairs. Based on insight from the combinatorial screen and mechanistic studies of Cu coordination and quenching, we find that superstoichiometric concentrations of Cu can promote full coupling if accompanied by ligands that selectively compete with the Cu from the QD surface but allow it to remain catalytically active. Applied to the conjugation of a K⁺ channel-specific peptidyl toxin to CdSe/ZnS QDs, we synthesize unquenched QD conjugates and image their specific and voltage-dependent affinity for K⁺ channels in live cells

Rapid Cytosolic Delivery of Luminescent Nanocrystals in Live Cells with Endosome-Disrupting Polymer Colloids

Luminescent nanocrystals hold great potential for bioimaging because of their exceptional optical properties, but their use in live cells has been limited. When nanocrystals enter live cells, they are taken up in vesicles. This vesicular sequestration is persistent and precludes nanocrystals from reaching intracellular targets. Here, we describe a unique, cationic core−shell polymer colloid that translocates nanocrystals to the cytosol by disrupting endosomal membranes via a low-pH triggered mechanism. Confocal fluorescence microscopy and flow cytometry indicate that picomolar concentrations of quantum dots are sufficient for cytosolic labeling, with the process occurring within a few hours of incubation. We anticipate a host of advanced applications arising from efficient cytosolic delivery of nanocrystal imaging probes: from single particle tracking experiments to monitoring protein−protein interactions in live cells for extended periods

Dual-Emitting Quantum Dot/Quantum Rod-Based Nanothermometers with Enhanced Response and Sensitivity in Live Cells

Temperature is a key parameter in physiological processes, and probes able to detect small changes in local temperature are necessary for accurate and quantitative physical descriptions of cellular events. Several have recently emerged that offer excellent temperature sensitivity, spatial resolution, or cellular compatibility, but it has been challenging to realize all of these properties in a single construct. Here, we introduce a luminescent nanocrystal-based sensor that achieves this with a 2.4% change/°C ratiometric response over physiological temperatures in aqueous buffers, with a precision of at least 0.2 °C. Thermoresponsive dual emission is conferred by a Förster resonant energy transfer (FRET) process between CdSe–CdS quantum dot–quantum rods (QD–QRs) as donors and cyanine dyes as acceptors, which are conjugated to QD–QRs using an amphiphilic polymer coating. The nanothermometers were delivered to live cells using a pH-responsive cationic polymer colloid, which served to both improve uptake and release nanocrystals from endosomal confinement. Within cells, they showed an unexpected enhancement in their temperature response and sensitivity, highlighting the need to calibrate these and similar probes within the cell

Reproducible, High-Throughput Synthesis of Colloidal Nanocrystals for Optimization in Multidimensional Parameter Space

While colloidal nanocrystals hold tremendous potential for both enhancing fundamental understanding of materials scaling and enabling advanced technologies, progress in both realms can be inhibited by the limited reproducibility of traditional synthetic methods and by the difficulty of optimizing syntheses over a large number of synthetic parameters. Here, we describe an automated platform for the reproducible synthesis of colloidal nanocrystals and for the high-throughput optimization of physical properties relevant to emerging applications of nanomaterials. This robotic platform enables precise control over reaction conditions while performing workflows analogous to those of traditional flask syntheses. We demonstrate control over the size, size distribution, kinetics, and concentration of reactions by synthesizing CdSe nanocrystals with 0.2% coefficient of variation in the mean diameters across an array of batch reactors and over multiple runs. Leveraging this precise control along with high-throughput optical and diffraction characterization, we effectively map multidimensional parameter space to tune the size and polydispersity of CdSe nanocrystals, to maximize the photoluminescence efficiency of CdTe nanocrystals, and to control the crystal phase and maximize the upconverted luminescence of lanthanide-doped NaYF4 nanocrystals. On the basis of these demonstrative examples, we conclude that this automated synthesis approach will be of great utility for the development of diverse colloidal nanomaterials for electronic assemblies, luminescent biological labels, electroluminescent devices, and other emerging applications

sj-pdf-1-fao-10.1177_24730114231216985 – Supplemental material for Prospective Clinical and Computed Tomography Evaluation of Calcaneus Fractures Treated Through Sinus Tarsi Approach

Supplemental material, sj-pdf-1-fao-10.1177_24730114231216985 for Prospective Clinical and Computed Tomography Evaluation of Calcaneus Fractures Treated Through Sinus Tarsi Approach by Julia C. Mastracci, Alexander R. Dombrowsky, Bruce E. Cohen, J. Kent Ellington, Samuel E. Ford, Scott B. Shawen, Todd A. Irwin and Carroll P. Jones in Foot & Ankle Orthopaedics</p

Intrinsic Optical Bistability of Photon Avalanching Nanocrystals

Optically bistable materials respond to a single input with two possible optical outputs, contingent upon excitation history. Such materials would be ideal for optical switching and memory, yet limited understanding of intrinsic optical bistability (IOB) prevents development of nanoscale IOB materials suitable for devices. Here, we demonstrate IOB in Nd3+-doped KPb2Cl5 avalanching nanoparticles (ANPs), which switch with high contrast between luminescent and non-luminescent states, with hysteresis characteristic of bistability. We elucidate a nonthermal mechanism in which IOB originates from suppressed nonradiative relaxation in Nd3+ ions and from the positive feedback of photon avalanching, resulting in extreme, >200th-order optical nonlinearities. Modulation of laser pulsing tunes hysteresis widths, and dual-laser excitation enables transistor-like optical switching. This control over nanoscale IOB establishes ANPs for photonic devices in which light is used to manipulate light