Protein-sheathed SWNT as a versatile scaffold for nanoparticle assembly and superstructured nanowires

One dimensional (1D) nanostructures have many possible applications in electronic, optical, and sensing devices associated with their nanosized lateral dimensions. In this regard, a general and bottom-up strategy to synthesize 1D nanoparticle arrays and conductive nanowires with a facile structural/compositional control is highly desired. We herein report a protein-sheathed single walled carbon nanotube (SWNT) that satisfies the criteria for an ideal template to assemble micron-long gold nanoparticle (AuNP) linear arrays with high structural rigidity. The resulting AuNP array has minimized inter-particle gaps, which is especially useful to template the overgrowth of Ag, Pd, and Pd/Ag metals into continuous nanowires (Au@M, M=Ag, Pd, Ag/ Pd). Our method successfully overcomes the incompatibility between carbon and metal materials, and the resulting superstructured metal nanowires have a tunable diameter below 100 nm and a shape closely replicating a SWNT. The Ag nanowires are composed of coalesced Au@Ag coreshell nanoparticles, while the Pd and Pd/Ag nanowires are made of very fine Pd nanocrystallites around the AuNP cores. These unique structural features are pivotal to various applications including surface enhanced Raman scattering (SERS), electrocatalysis, and gas sensors.     

Construction of a graphene oxide based noncovalent multiple nanosupramolecular assembly as a scaffold for drug delivery

A multiple supramolecular assembly, in which a folic acid-modified β-cyclodextrin (1) acted as a target unit, an adamantanyl porphyrin (2) acted as a linker unit, and graphene oxide acted as a carrier unit, was successfully fabricated through non-covalent interactions and comprehensively investigated by means of UV/Vis, fluorescence, and X-ray photoelectron spectroscopies, and electron microscopy. Significantly, the graphene oxide unit could associate with the anticancer drug doxorubicin through π-π interactions, and the folic acid-modified β-cyclodextrin unit could recognize the folic acid receptors in cancer cells. Owing to the cooperative contribution of these three units, the resulting multiple supramolecular assembly, after association with doxorubicin, exhibited better drug activity and much lower toxicity than free doxorubicin in vivo.     

Artificial polypeptide scaffold for protein immobilization

An artificial polypeptide scaffold composed of surface anchor and protein capture domains was designed and expressed in vivo. By using a mutant E. coli phenylalanyl-tRNA synthetase, the photoreactive amino acid para-azidophenylalanine was incorporated into the surface anchor domain. Octyltrichlorosilane-treated surfaces were functionalized with this polypeptide by spin coating and photocrosslinking. The resulting protein films were shown to immobilize recombinant proteins through association of coiled coil heterodimer.     

Elastin-calmodulin scaffold for protein microarray fabrication

In this work, we report a new method to reversibly immobilize proteins to a surface in a functionally active orientation directly from cell lysate by employing a fusion protein consisting of a thermal-responsive elastin (ELP) domain as the surface anchor and a calcium-responsive calmodulin (CalM) domain for protein capturing. Incorporation of an M13 tag into recombinant proteins enables not only easy surface immobilization but also direct purification from cell lysates. The feasibility of concept was demonstrated using the M13-tagged yellow fluorescent protein (M13-YFP). The ELP-CalM functionalized surfaces were shown to capture M13-YFP directly from cell lysate through the specific calmodulin-M13 association in a calcium-dependent manner. We also demonstrated that immobilization is reversible; the bound proteins were released from the surface in the presence of EDTA.     

A polymer scaffold for protein oligomerization

We report the design and synthesis of well-defined polymers for the noncovalent oligomerization of proteins. The reported scaffolds, which were generated by atom-transfer radical polymerization (ATRP), take advantage of the well-characterized interaction between a Ni2+ complex and an oligohistidine sequence (His tag). Thus, our polymers are designed to facilitate the oligomerization of any protein possessing a His tag. We demonstrate that they can oligomerize fibroblast growth factor-8b (FGF-8b) and promote FGF-8b-mediated cell proliferation in the absence of heparin.     

Single-label kinase and phosphatase assays for tyrosine phosphorylation using nanosecond time-resolved fluorescence detection

The collision-induced fluorescence quenching of a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) by hydrogen atom abstraction from the tyrosine residue in peptide substrates was introduced as a single-labeling strategy to assay the activity of tyrosine kinases and phosphatases. The assays were tested for 12 different combinations of Dbo-labeled substrates and with the enzymes p60c-Src Src kinase, EGFR kinase, YOP protein tyrosine phosphatase, as well as acid and alkaline phosphatases, thereby demonstrating a broad application potential. The steady-state fluorescence changed by a factor of up to 7 in the course of the enzymatic reaction, which allowed for a sufficient sensitivity of continuous monitoring in steady-state experiments. The fluorescence lifetimes (and intensities) were found to be rather constant for the phosphotyrosine peptides (ca. 300 ns in aerated water), while those of the unphosphorylated peptides were as short as 40 ns (at pH 7) and 7 ns (at pH 13) as a result of intramolecular quenching. Owing to the exceptionally long fluorescence lifetime of Dbo, the assays were alternatively performed by using nanosecond time-resolved fluorescence (Nano-TRF) detection, which leads to an improved discrimination of background fluorescence and an increased sensitivity. The potential for inhibitor screening was demonstrated through the inhibition of acid and alkaline phosphatases by molybdate.     

Prototype for integrated two-dimensional gel electrophoresis for protein separation

Two-dimensional gel electrophoresis practitioners have long waited for a fully automated system. This article presents an integrated platform that is capable of complete automation from sample introduction to spots detection. The strip gel for the first dimensional separation is fixed on the edge of a discrete planar stage before separation. A pair of platinum pin electrodes for isoelectric focusing (IEF) makes contact from underneath the stage. IEF is performed directly after rehydration and protein loading. After the first dimensional separation, sodium dodecyl sulfate (SDS) equilibration is done on the same stage without moving the gel. The IEF stage is then moved horizontally to couple with a precast second dimensional gel. The <0.5 mm gap between the two gels is filled with poly (ethylene oxide) solution. After SDS-polyacrylamide gel electrohporesis separation, a charge-coupled device camera is used to detect spots via protein native fluorescence excited by a Hg (Xe) lamp with the gel inside the running cell. Potential for full automation is demonstrated with 0.5 microg of Escherichia coli proteins on this miniaturized platform. More than 240 spots are detected in a total experiment time of <2.5 h.     

Indole as a scaffold for anion recognition

Indole has an acidic N-H that can be used to form hydrogen bonds to anions and in this paper the synthesis of three new suitably functionalised indole based anion receptors is presented along with their evaluation using (1)H NMR titration techniques.     

Submicron streptavidin patterns for protein assembly

Micron and submicron-scale features of aldehyde functionality were fabricated in polymer films by photolithography to develop a platform for protein immobilization and assembly at a biologically relevant scale. Films containing the pH-reactive polymer poly(3,3'-diethoxypropyl methacrylate) and a photoacid generator (PAG) were patterned from 500 nm to 40 mum by exposure to 365 nm (i-line) light. Upon PAG activation and hydrolysis of acetals, aldehyde groups formed. After the films were incubated with a biotinylated aldehyde reactive probe, the X-ray photoelectron spectroscopy results were consistent with biotin being attached to the surface. The background was subsequently passivated by flood exposure and incubation with an aminooxy-terminated poly(ethylene glycol), resulting in a 98% reduction in nonspecific protein adsorption. Protein patterning and assembly was demonstrated using streptavidin, biotinylated anthrax toxin receptor-1, and the protective antigen moiety of anthrax toxin and confirmed by fluorescence microscopy and atomic force microscopy (AFM). AFM demonstrated that 500 nm protein features were achieved. Because of the abundance of biotinylated proteins, this methodology provides a platform for protein immobilization and assembly for various applications in biotechnology.     

Fluorescence assays for high-throughput screening of protein kinases

Protein kinases comprise one of the most important group of targets for drug discovery research today. Methods to identify novel kinase inhibitors by high-throughput screening have evolved rapidly in recent years. An important aspect is the availability of fluorescent probes that can be applied in a homogeneous, or mix-and-measure, assay format. Here, we illustrate the application of fluorescence read-out technologies for kinase targets in light of our own experiences in assay development and high-throughput screening.     

Cuttlebone-derived organic matrix as a scaffold for assembly of silver nanoparticles and application of the composite films in surface-enhanced Raman scattering

Biologically derived materials provide a rich variety of approaches toward new functional materials because of their fascinating structures and environment-friendly features, which is currently a topic of research interest. In this paper, we show that the cuttlebone-derived organic matrix (CDOM) is an excellent scaffold for the one-step synthesis and assembly of silver nanoparticles (AgNPs), which can be further used as substrate for surface-enhanced Raman scattering (SERS). Formation of AgNPs–CDOM composite was accomplished by the reaction of CDOM with AgNO₃ and NH₃·H₂O solution at 80 °C without using any other stabilizer and reducing agents. UV–vis spectra and TEM were utilized to characterize the AgNPs and investigate their formation process. Results demonstrate that the size and distribution of AgNPs can be partly regulated by changing incubation time; the concentration of NH₃·H₂O is critical to the formation rate of AgNPs. As a proof of principle, we show that the AgNPs–CDOM composite can be employed in trace analysis using SERS.     

Dendrimers as a scaffold for nitric oxide release

The preparation and characterization of nitric oxide (NO)-releasing dendrimer conjugates are reported. Generation 3 and 5 polypropylenimine dendrimers (DAB-Am-16 and DAB-Am-64) were modified at the exterior to impart different amine functionalities. The ability to store NO on a dendritic scaffold using N-diazeniumdiolate NO donors was examined via the reaction of primary amine, secondary amine, and amide functionalities with high pressures of NO (5 atm). The secondary amine dendrimer conjugates exhibited a high storage capacity for NO (up to 5.6 micromol NO/mg), greatly increasing the "payload" of released NO over existing macromolecular NO donors. The mechanism of diazeniumdiolate decomposition was proton initiated, generating NO spontaneously under physiological conditions (pH 7.4, 37 degrees C). The NO release durations (>16 h) observed for the secondary amine dendrimers were significantly longer compared to small molecule alkyl secondary amine diazeniumdiolates, thus illustrating a dendritic effect on NO release kinetics. The multivalent exterior of dendrimers allows for the future combination of NO donors and other functionalities on a single molecular scaffold, enabling diverse utility as NO storage/delivery systems.     

Tetronic-oligolactide-heparin hydrogel as a multi-functional scaffold for tissue regeneration

A novel cell-supporting scaffold, Tetronic-oligolactide-heparin (TLH) hydrogel, was prepared by coupling heparin to polymerized Tetronic-oligolactide for use in improving tissue regeneration. Aqueous TLH solutions showed thermosensitive behavior, demonstrating potential for use as injectable hydrogels. The content and activity of conjugated heparin were determined to be 61 wt.-% of total polymer and 67.2% of intact heparin activity, respectively. The basic fibroblast growth factor (bFGF) binding assay showed TLH hydrogel had a relatively high bFGF affinity, which indicates applicability for growth factor delivery. Chondrocyte culture on hydrogels revealed that the cell viability and the amount of synthesized glycosaminoglycan for TLH hydrogel were higher than those for alginate gel.     

2-Aminopyrimidine as a novel scaffold for biofilm modulation

An efficient synthetic route to a series of substituted 2-aminopyrimidine (2-AP) derivatives has been developed. Subsequent biofilm screening has allowed comparison between the biological activity of these new derivatives and that of the 2-aminoimidazole class of anti-biofilm compounds. Several derivatives displayed the ability to modulate bacterial biofilm formation, exhibiting greater activity against Gram-positive strains than Gram-negative strains. Additionally some 2-aminopyrmidines were able to suppress MRSA resistance to conventional antibiotics.     

l-Menthol as new scaffold for designing chiral phase-transfer catalysts

We herein report, a new class of chiral phase-transfer catalysts derived from l-menthol. The moderately good enantioselectivity exhibited by these catalysts have been explained based on single crystal X-ray data and molecular modelling studies.     

New HIV-protease assays applying self-quenching peptide substrates in combination with time-resolved fluorescence single-molecule spectroscopy

This work describes the optimization and adoption of an assay system for the Human Immunodeficiency Virus (HIV)-protease, whose inhibition plays a central role in HIV therapy. The HIV-protease, which is an essential enzyme during viral maturation, has a specific cleavage site of eight amino acid residues (SQNY*PIV). Adding two amino acid residues at the N-terminus and enclosing the resulting sequence by a dye-labelled lysine residue and a tryptophan residue leads to the substrate (K(dye)CGSQNY*PIVW) in which the fluorescence of the fluorophore is efficiently quenched by the intrinsic tryptophan due to a photoinduced electron transfer reaction. After cleavage of the substrate by the target enzyme, the dye and the tryptophan residue are separated, effecting a significant increase in fluorescence intensity. Measuring the fluorescence versus time enables an online-monitoring of the enzyme activity. With this method, a HIV-PR concentration of 10^?9?M is detectable within minutes, which is comparable with commercially available assays using doubly labelled substrates based on a fluorescence resonance energy transfer. We were able to further increase the sensitivity to the subnanomolar range by using confocal single-molecule spectroscopy.     

Supported lipopolymer membranes as nanoscale filters: simultaneous protein recognition and size-selection assays

A technique for size-selective discrimination of protein analytes was developed by incorporating poly(ethylene glycol) (PEG) lipopolymers into supported lipid bilayers. The membranes also contained biotinylated lipids, which recognized both streptavidin and anti-biotin IgG. By employing various PEG lipopolymer concentrations, clear discrimination against anti-biotin (Mw = 150 000 Da) binding could be observed, which became more pronounced at higher polymer densities. On the other hand, streptavidin (Mw = 52 800) binding to the membrane remained unaffected even at PEG concentrations that were well into the mushroom-to-brush phase transition. These observations were exploited to create an on-chip ligand-receptor binding assay that favored streptavidin binding over anti-biotin by several orders of magnitude in the presence of the lipopolymer. Control experiments revealed that the two proteins are bound to similar extents from a multi-protein analyte solution in the absence of PEG.     

Chitosan biotinylation and electrodeposition for selective protein assembly

An alternative route to protein assembly at surfaces based on using the unique capabilities of biological materials for the spatially selective assembly of proteins is described. Specifically, the stimuli-responsive properties of aminopolysaccharide chitosan are combined with the molecular-recognition capabilities of biotin-streptavidin binding. Biotinylated chitosan retains its stimuli-responsive properties and is capable of electrodepositing at specific electrode addresses. Once deposited, it is capable of binding streptavidin, which can mediate the subsequent assembly of biotinylated proteins. Spatially selective protein assembly using biotinylated Protein A and fluorescently-labeled antibodies is demonstrated.     

Engineering of a redox protein for DNA-directed assembly

Engineered enzyme conjugate of the small laccase enzyme from Streptomyces coelicolor and zinc finger DNA binding domain from Zif268 is demonstrated to bind double stranded DNA in a site specific manner while retaining enzymatic activity.