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

Printed Graphene Electrochemical Biosensors Fabricated by Inkjet Maskless Lithography for Rapid and Sensitive Detection of Organophosphates

Solution phase printing of graphene-based electrodes has recently become an attractive low-cost, scalable manufacturing technique to create in-field electrochemical biosensors. Here, we report a graphene-based electrode developed via inkjet maskless lithography (IML) for the direct and rapid monitoring of triple-O linked phosphonate organophosphates (OPs); these constitute the active compounds found in chemical warfare agents and pesticides that exhibit acute toxicity as well as long-term pollution to soils and waterways. The IML-printed graphene electrode is nano/microstructured with a 1000 mW benchtop laser engraver and electrochemically deposited platinum nanoparticles (dia. ∼25 nm) to improve its electrical conductivity (sheet resistance decreased from ∼10 000 to 100 Ω/sq), surface area, and electroactive nature for subsequent enzyme functionalization and biosensing. The enzyme phosphotriesterase (PTE) was conjugated to the electrode surface via glutaraldehyde cross-linking. The resulting biosensor was able to rapidly measure (5 s response time) the insecticide paraoxon (a model OP) with a low detection limit (3 nM), and high sensitivity (370 nA/μM) with negligible interference from similar nerve agents. Moreover, the biosensor exhibited high reusability (average of 0.3% decrease in sensitivity per sensing event), stability (90% anodic current signal retention over 1000 s), longevity (70% retained sensitivity after 8 weeks), and the ability to selectively sense OP in actual soil and water samples. Hence, this work presents a scalable printed graphene manufacturing technique that can be used to create OP biosensors that are suitable for in-field applications as well as, more generally, for low-cost biosensor test strips that could be incorporated into wearable or disposable sensing paradigms

A triangular three-dye DNA switch capable of reconfigurable molecular logic

Structural DNA nanotechnology has developed profoundly in the last several years allowing for the creation of increasingly sophisticated devices capable of discrete sensing, locomotion, and molecular logic. The latter research field is particularly attractive as it provides information processing capabilities that may eventually be applied in situ, for example in cells, with potential for even further coupling to an active response such as drug delivery. Rather than design a new DNA assembly for each intended logic application, it would be useful to have one generalized design that could provide multiple different logic gates or states for a targeted use. In pursuit of this goal, we demonstrate a switchable, triangular dye-labeled three-arm DNA scaffold where the individual arms can be assembled in different combinations and the linkage between each arm can be physically removed using toehold-mediated strand displacement and then replaced by a rapid anneal. Rearranging this core structure alters the rates of Förster resonance energy transfer (FRET) between each of the two or three pendant dyes giving rise to a rich library of unique spectral signatures that ultimately form the basis for molecular photonic logic gates. The DNA scaffold is designed such that different linker lengths joining each arm, and which are used as the inputs here, can also be used independently of one another thus enhancing the range of molecular gates. The functionality of this platform structure is highlighted by easily configuring it into a series of one-, two- and three-input photonic Boolean logic gates such as OR, AND, INHIBIT, etc., along with a photonic keypad lock. Different gates can be realized in the same structure by altering which dyes are interrogated and implementation of toehold-mediated strand displacement and/or annealing allows reconfigurable switching between input states within a single logic gate as well as between two different gating devices

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

Enhanced enzymatic activity from phosphotriesterase trimer gold nanoparticle bioconjugates for pesticide detection

The rapid detection of organophosphates (OPs), a class of strong neurotoxins, is critically important for monitoring acute insecticide exposure and potential chemical warfare agent use. Herein, we improve the enzymatic activity of a phosphotriesterase trimer (PTE3), an enzyme that selectively recognizes OPs directly, by conjugation with distinctly sized (i.e., 5, 10, and 20 nm diameter) gold nanoparticles (AuNPs). The number of enzymes immobilized on the AuNP was controlled by conjugating increasing molar ratios of PTE3 onto the AuNP surface via metal affinity coordination. This occurs between the PTE3-His6 termini and the AuNP-displayed Ni2+-nitrilotriacetic acid end groups and was confirmed with gel electrophoresis. The enzymatic efficiency of the resultant PTE3–AuNP bioconjugates was analyzed via enzyme progress curves acquired from two distinct assay formats that compared free unbound PTE3 with the following PTE3–AuNP bioconjugates: (1) fixed concentration of AuNPs while increasing the bioconjugate molar ratio of PTE3 displayed around the AuNP and (2) fixed concentration of PTE3 while increasing the bioconjugate molar ratio of PTE3–AuNP by decreasing the AuNP concentration. Both assay formats monitored the absorbance of p-nitrophenol that was produced as PTE3 hydrolyzed the substrate paraoxon, a commercial insecticide and OP nerve agent simulant. Results demonstrate a general equivalent trend between the two formats. For all experiments, a maximum enzymatic velocity (Vmax) increased by 17-fold over free enzyme for the lowest PTE3–AuNP ratio and the largest AuNP (i.e., ratio of 1[thin space (1/6-em)]:[thin space (1/6-em)]1, 20 nm dia. AuNP). This work provides a route to improve enzymatic OP detection strategies with enzyme–NP bioconjugates

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

Platinum-paper micromotors: An urchin-like nanohybrid catalyst for green monopropellant bubble-thrusters

Platinum nanourchins supported on microfibrilated cellulose films (MFC) were fabricated and evaluated as hydrogen peroxide catalysts for small-scale, autonomous underwater vehicle (AUV) propulsion systems. The catalytic substrate was synthesized through the reduction of chloroplatinic acid to create a thick film of Pt coral-like microstructures coated with Pt urchin-like nanowires that are arrayed in three dimensions on a two-dimensional MFC film. This organic/inorganic nanohybrid displays high catalytic ability (reduced activation energy of 50-63% over conventional materials and 13-19% for similar Pt nanoparticle-based structures) during hydrogen peroxide (H2O2) decomposition as well as sufficient propulsive thrust (\u3e0.5 N) from reagent grade H2O2 (30% w/w) fuel within a small underwater reaction vessel. The results demonstrate that these layered nanohybrid sheets are robust and catalytically effective for green, H2O2-based micro-AUV propulsion where the storage and handling of highly explosive, toxic fuels are prohibitive due to size-requirements, cost limitations, and close person-to-machine contact

High Aspect Ratio Carbon Nanotube Membranes Decorated with Pt Nanoparticle Urchins for Micro Underwater Vehicle Propulsion via H2O2 Decomposition

The utility of unmanned micro underwater vehicles (MUVs) is paramount for exploring confined spaces, but their spatial agility is often impaired when maneuvers require burst-propulsion. Herein we develop high-aspect ratio (150:1), multiwalled carbon nanotube microarray membranes (CNT-MMs) for propulsive, MUV thrust generation by the decomposition of hydrogen peroxide (H2O2). The CNT-MMs are grown via chemical vapor deposition with diamond shaped pores (nominal diagonal dimensions of 4.5 × 9.0 μm) and subsequently decorated with urchin-like, platinum (Pt) nanoparticles via a facile, electroless, chemical deposition process. The Pt-CNT-MMs display robust, high catalytic ability with an effective activation energy of 26.96 kJ mol–1 capable of producing a thrust of 0.209 ± 0.049 N from 50% [w/w] H2O2 decomposition within a compact reaction chamber of eight Pt-CNT-MMs in series