Investigation of the Glucose-Induced Inactivation of Maltose Permease in Saccharomyces

The addition of glucose to maltose fermenting Saccharomyces cerevisiae cells results in the rapid loss of maltose transport activity. This results both from the repression of the maltose permease gene transcription and from the post-translational inactivation of maltose permease through a process termed glucose-induced inactivation of maltose permease. The inactivation consists of two separable processes, a rapid inhibition of maltose transport activity and by a slower degradation of maltose permease protein. Degradation is dependent on endocytosis, vesicular sorting and vacuolar proteolysis, and is independent of the proteasome. Furthermore, maltose permease exists in differentially phosphorylated forms. Ubiquitin and the ubiquitin-conjugating system are necessary for glucose-induced proteolysis of maltose permease. The rate of glucose-induced proteolysis of maltose permease is slowed by deletion of DOA4 encoding a ubiquitin recycling enzyme and this is suppressed by overexpression of ubiquitin. Moreover, maltose permease exists in a ubiquitinated state whose abundance increases upon the addition of glucose. A mutation that reduces the expression of NPI1/RSP5, encoding a putative ubiquitin-protein ligase, also dramatically decreases the rate of glucose-induced proteolysis of maltose permease. Double null mutations in ubc1 ubc4 and ubc4 ubc5 had no significant impact on the rate of glucose-induced proteolysis of maltose permease. Mutational analysis was carried out on the predicted cytoplasmic N- and C-terminal domains of maltose permease. The N-terminal cytoplasmic domain of maltose permease contains a putative PEST sequence and a dileucine sequence. The C-terminal cytoplasmic domain contains a putative endocytosis signal, NPF, and numerous lysine, serine, and threonine residues. The PEST sequence is required for rapid glucose-induced inhibition and degradation of maltose permease while alterations in the C-terminal domain cause only a modest decrease. In summary, our results indicate that glucose stimulates the ubiquitination of maltose permease and that this marks this protein for endocytosis and vacuolar proteolysis. The PEST sequence found in residues 49–78 of the N-terminal cytoplasmic domain plays an essential role in this process and appears to be required for recognition by the ubiquitin conjugating enzymes

Potential clinical applications of quantum dots

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

Design of Biotin-Functionalized Luminescent Quantum Dots

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 ∼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

Utilizing the Organizational Power of DNA Scaffolds for New Nanophotonic Applications

AbstractRapid development of DNA technology has provided a feasible route to creating nanoscale materials. DNA acts as a self‐assembled nanoscaffold capable of assuming any three‐dimensional shape. The ability to integrate dyes and new optical materials such as quantum dots and plasmonic nanoparticles precisely onto these architectures provides new ways to exploit their near‐ and far‐field interactions. A fundamental understanding of these optical processes will help drive development of next‐generation photonic nanomaterials. This review is focused on latest progress in DNA‐based photonic materials and highlights DNA scaffolds for rapidly assembling and prototyping nanoscale optical devices. Three areas are discussed including intrinsically active DNA structures displaying chiral properties, DNA scaffolds hosting plasmonic nanomaterials, and fluorophore‐labeled DNAs that engage in Förster resonance energy transfer and give rise to complex molecular photonic wires. An explanation of what is desired from these optical processes when harnessed sets the tone for what DNA scaffolds are providing toward each focus. Examples from the literature illustrate current progress along with a discussion of challenges to overcome for further improvements. Opportunities to integrate diverse classes of optically active molecules including light‐generating enzymes, fluorescent proteins, nanoclusters, and metal–chelates in new structural combinations on DNA scaffolds are also highlighted

Optimizing Two-Color Semiconductor Nanocrystal Immunoassays in Single Well Microtiter Plate Formats

The simultaneous detection of two analytes, chicken IgY (IgG) and Staphylococcal enterotoxin B (SEB), in the single well of a 96-well plate is demonstrated using luminescent semiconductor quantum dot nanocrystal (NC) tracers. The NC-labeled antibodies were prepared via sulfhydryl-reactive chemistry using a facile protocol that took <3 h. Dose response curves for each target were evaluated in a single immunoassay format and compared to Cy5, a fluorophore commonly used in fluorescent immunoassays, and found to be equivalent. Immunoassays were then performed in a duplex format, demonstrating multiplex detection in a single well with limits of detection equivalent to the single assay format: 9.8 ng/mL chicken IgG and 7.8 ng/mL SEB

Artificial Multienzyme Scaffolds: Pursuing in Vitro Substrate Channeling with an Overview of Current Progress

Artificial multienzyme scaffolds are being developed for in vitro cascaded biocatalytic activity and, in particular, accessing substrate channeling. This review covers progress in this field over t..

Pursuing Excitonic Energy Transfer with Programmable DNA-Based Optical Breadboards

DNA nanotechnology has now enabled the self-assembly of almost any prescribed 3-dimensional nanoscale structure in large numbers and with high fidelity. These structures are also amenable to site-specific modification with a variety of small molecules ranging from drugs to reporter dyes. Beyond obvious application in biotechnology, such DNA structures are being pursued as programmable nanoscale optical breadboards where multiple different/identical fluorophores can be positioned with sub-nanometer resolution in a manner designed to allow them to engage in multistep excitonic energy-transfer (ET) via Förster resonance energy transfer (FRET) or other related processes. Not only is the ability to create such complex optical structures unique, more importantly, the ability to rapidly redesign and prototype almost all structural and optical analogues in a massively parallel format allows for deep insight into the underlying photophysical processes. Dynamic DNA structures further provide the unparalleled capability to reconfigure a DNA scaffold on the fly in situ and thus switch between ET pathways within a given assembly, actively change its properties, and even repeatedly toggle between two states such as on/off. Here, we review progress in developing these composite materials for potential applications that include artificial light harvesting, smart sensors, nanoactuators, optical barcoding, bioprobes, cryptography, computing, charge conversion, and theranostics to even new forms of optical data storage. Along with an introduction into the DNA scaffolding itself, the diverse fluorophores utilized in these structures, their incorporation chemistry, and the photophysical processes they are designed to exploit, we highlight the evolution of DNA architectures implemented in the pursuit of increased transfer efficiency and the key lessons about ET learned from each iteration. We also focus on recent and growing efforts to exploit DNA as a scaffold for assembling molecular dye aggregates that host delocalized excitons as a test bed for creating excitonic circuits and accessing other quantum-like optical phenomena. We conclude with an outlook on what is still required to transition these materials from a research pursuit to application specific prototypes and beyond

Terbium to Quantum Dot FRET Bioconjugates for Clinical Diagnostics: Influence of Human Plasma on Optical and Assembly Properties

Förster resonance energy transfer (FRET) from luminescent terbium complexes (LTC) as donors to semiconductor quantum dots (QDs) as acceptors allows extraordinary large FRET efficiencies due to the long Förster distances afforded. Moreover, time-gated detection permits an efficient suppression of autofluorescent background leading to sub-picomolar detection limits even within multiplexed detection formats. These characteristics make FRET-systems with LTC and QDs excellent candidates for clinical diagnostics. So far, such proofs of principle for highly sensitive multiplexed biosensing have only been performed under optimized buffer conditions and interactions between real-life clinical media such as human serum or plasma and LTC-QD-FRET-systems have not yet been taken into account. Here we present an extensive spectroscopic analysis of absorption, excitation and emission spectra along with the luminescence decay times of both the single components as well as the assembled FRET-systems in TRIS-buffer, TRIS-buffer with 2% bovine serum albumin, and fresh human plasma. Moreover, we evaluated homogeneous LTC-QD FRET assays in QD conjugates assembled with either the well-known, specific biotin-streptavidin biological interaction or, alternatively, the metal-affinity coordination of histidine to zinc. In the case of conjugates assembled with biotin-streptavidin no significant interference with the optical and binding properties occurs whereas the histidine-zinc system appears to be affected by human plasma

A Nanobody‐on‐Quantum Dot Displacement Assay for Rapid and Sensitive Quantification of the Epidermal Growth Factor Receptor (EGFR)

Biosensing approaches that combine small, engineered antibodies (nanobodies) with nanoparticles are often complicated. Here, we show that nanobodies with different C-terminal tags can be efficiently attached to a range of the most widely used biocompatible semiconductor quantum dots (QDs). Direct implementation into simplified assay formats was demonstrated by designing a rapid and wash-free mix-and-measure immunoassay for the epidermal growth factor receptor (EGFR). Terbium complex (Tb)-labeled hexahistidine-tagged nanobodies were specifically displaced from QD surfaces via EGFR-nanobody binding, leading to an EGFR concentration-dependent decrease of the Tb-to-QD Förster resonance energy transfer (FRET) signal. The detection limit of 80±20 pM (16±4 ng mL−1) was 3-fold lower than the clinical cut-off concentration for soluble EGFR and up to 10-fold lower compared to conventional sandwich FRET assays that required a pair of different nanobodies

Platinum Nanoparticle Decorated SiO 2 Microfibers as Catalysts for Micro Unmanned Underwater Vehicle Propulsion

Micro unmanned underwater vehicles (UUVs) need to house propulsion mechanisms that are small in size but sufficiently powerful to deliver on-demand acceleration for tight radius turns, burst-driven docking maneuvers, and low-speed course corrections. Recently, small-scale hydrogen peroxide (H2O2) propulsion mechanisms have shown great promise in delivering pulsatile thrust for such acceleration needs. However, the need for robust, high surface area nanocatalysts that can be manufactured on a large scale for integration into micro UUV reaction chambers is still needed. In this report a thermal/electrical insulator, silicon oxide (SiO2) microfibers, are used as a support for platinum nanoparticle (PtNP) catalysts. The mercapto-silanization of the SiO2 microfibers enables strong covalent attachment with PtNPs and the resultant PtNP-SiO2 fibers act as a robust, high surface area catalyst for H2O2 decomposition. The PtNP-SiO2 catalysts are fitted inside a micro UUV reaction chamber for vehicular propulsion; the catalysts can propel a micro UUV for 5.9 meters at a velocity of 1.18 m/s with 50 mL of 50% (w/w) H2O2.The concomitance of facile fabrication, economic and scalable processing, and high performance —including a reduction in H2O2 decomposition activation energy of 40-50% over conventional material catalysts—paves the way for using these nanostructured microfibers in modern, small-scale underwater vehicle propulsion systems