Ultra-rapid laser protein micropatterning: screening for directed polarization of single neurons

Protein micropatterning is a powerful tool for studying the effects of extracellular signals on cell development and regeneration. Laser micropatterning of proteins is the most flexible method for patterning many different geometries, protein densities, and concentration gradients. Despite these advantages, laser micropatterning remains prohibitively slow for most applications. Here, we take advantage of the rapid multi-photon induced photobleaching of fluorophores to generate sub-micron resolution patterns of full-length proteins on polymer monolayers, with sub-microsecond exposure times, i.e. one to five orders of magnitude faster than all previous laser micropatterning methods. We screened a range of different PEG monolayer coupling chemistries, chain-lengths and functional caps, and found that long-chain acrylated PEG monolayers are effective at resisting non-specific protein adhesion, while permitting efficient cross-linking of biotin-4-fluorescein to the PEG monolayers upon exposure to femtosecond laser pulses. We find evidence that the dominant photopatterning chemistry switches from a two-photon process to three- and four-photon absorption processes as the laser intensity increases, generating increasingly volatile excited triplet-state fluorophores, leading to faster patterning. Using this technology, we were able to generate over a hundred thousand protein patterns with varying geometries and protein densities to direct the polarization of hippocampal neurons with single-cell precision. We found that certain arrays of patterned triangles as small as neurite growth cones can direct polarization by impeding the elongation of reverse-projecting neurites, while permitting elongation of forward-projecting neurites. The ability to rapidly generate and screen such protein micropatterns can enable discovery of conditions necessary to create in vitro neural networks with single-neuron precision for basic discovery, drug screening, as well as for tissue scaffolding in therapeutics.     

A simple approach to micropatterning and surface modification of poly(dimethylsiloxane)

Ozone treatment is an efficient economical, alternative method for surface activation of poly(dimethylsiloxane) (PDMS). This is illustrated by the derivatization of a PDMS surface with (3-aminopropyl)triethoxysilane (APTES). The apparent surface concentration of amino groups was found to be ca. 10(-8) mol/cm2 using UV/visible spectroscopy of the product from the reaction of the amino groups and fluorescamine. Potential application for micropatterning of biologically active interfaces was demonstrated by the covalent immobilization of oligonucleotides. A simple process for photolithographic patterning on PDMS surfaces has been developed.     

Photocatalytic patterning and modification of graphene

TiO(2)-based photocatalysis has been widely used to decompose various organic pollutants for the purpose of environmental protection. Such a "green" photochemical process can ultimately degrade organic compounds into CO(2) and H(2)O under ambient conditions. We demonstrate here its extended application on the engineering of single- or few-layer graphene. Using a patterned TiO(2) photomask, we have achieved various photochemical tailorings of graphene, including ribbon cutting, arbitrary patterning on any substrate, layer-by-layer thinning, and localized graphene to graphene oxide conversion. UV-visible spectroscopic studies indicate that the photogenerated, highly reactive ·OH radicals work as sharp chemical scissors. Being a solution-free, cost-effective, scalable, and easy handling technique, the presented photocatalytic patterning and modification approach allows for the versatile design and fabrication of graphene-based devices and circuits, compatible with current microelectronic technology, as demonstrated by this fabricated all-carbon field effect transistor (FET) array.     

One-step synthesis of labeled sugar nucleotides for protein O-GlcNAc modification studies by chemical function analysis of an archaeal protein

Herein we present the chemical function analysis of a recombinant sugar nucleotidyltransferase from the hyperthermophile Pyrococcus furiosus and its use in the one-pot synthesis of chloroacetyl- and alkyne-tagged analogues of uridinediphospho-N-acetylglucosamine (UDP-GlcNAc). The gene was originally annotated as a glucose-1-phosphate deoxythymidylyltransferase; however, kinetic analysis of a panel of sugar-1-phosphates with the protein shows that it is better described as a bifunctional protein that synthesizes UDP-GlcNAc from glucosamine-1-phosphate and acetyl coenzyme A (CoA). A new mass-spectrometry-based assay for the rapid analysis of the acyltransferase activity demonstrates that the enzyme can also accept cheaper truncated N-acetylcysteamine thioester substrates in place of the natural acetyl CoA. The enzyme can tolerate alkyne or chloride substitutions in the acyl moiety, thereby allowing the facile synthesis of tagged sugar nucleotides for future use in protein O-GlcNAc modification studies.     

Targeted analysis of protein citrullination using chemical modification and tandem mass spectrometry

Protein citrullination originates from enzymatic deimination of polypeptide‐bound arginine and is involved in various biological processes during health and disease. However, tools required for a detailed and targeted proteomic analysis of citrullinated proteins in situ, including their citrullination sites, are limited. A widely used technique for detection of citrullinated proteins relies on antibody staining after specific derivatization of citrulline residues by 2,3‐butanedione and antipyrine. We have recently reported on the details of this reaction. Here, we show that this chemical modification can be utilized to specifically detect and identify citrullinated peptides and their citrullination sites by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Using model compounds, we demonstrate that in collision‐induced dissociation (CID) a specific, modification‐derived fragment ion appears as the dominating signal at m/z 201.1 in the MS/MS spectra. When applying electron transfer dissociation (ETD), however, the chemical modification of citrulline remained intact and extensive sequence coverage allowed identification of peptides and their citrullination sites. Therefore, LC/MS/MS analysis with alternating CID and ETD has been performed, using CID for specific, signature ion‐based detection of derivatized citrullinated peptides and ETD for sequence determination. The usefulness of this targeted analysis was demonstrated by identifying citrullination sites in myelin basic protein deiminated in vitro. Combining antibody‐based enrichment of chemically modified citrulline‐containing peptides with specific mass spectrometric detection will increase the potential of such a targeted analysis of protein citrullination in the future. Copyright © 2009 John Wiley & Sons, Ltd.     

Suzuki coupling for protein modification

Suzuki-coupling, a representative cross-coupling reaction between small molecules, can proceed on a protein surface in aqueous solution under mild conditions. The modified protein produced by the coupling reaction maintained its native like structure and function. In addition, fluorescent dye appended-protein acts as a fluorescent biosensor.     

Steam etched porous graphene oxide network for chemical sensing

Oxidative etching of graphene flakes was observed to initiate from edges and the occasional defect sites in the basal plane, leading to reduced lateral size and a small number of etch pits. In contrast, etching of highly defective graphene oxide and its reduced form resulted in rapid homogeneous fracturing of the sheets into smaller pieces. On the basis of these observations, a slow and more controllable etching route was designed to produce nanoporous reduced graphene oxide sheets by hydrothermal steaming at 200 °C. The degree of etching and the concomitant porosity can be conveniently tuned by etching time. In contrast to nonporous reduced graphene oxide annealed at the same temperature, the steamed nanoporous graphene oxide exhibited nearly 2 orders of magnitude increase in the sensitivity and improved recovery time when used as chemiresistor sensor platform for NO(2) detection. The results underscore the efficacy of the highly distributed nanoporous network in the low temperature steam etched GO.     

Use of fullerene additions for modification of chemical absorbers

The characteristics of lime chemical absorber, both conventional and modified with fullerenes, were examined under dynamic conditions (kinetic characteristics of the CO₂ uptake, water retention capacity, and pore structure).     

Simulating the conditions for the formation of graphene and graphene nanowalls by semiempirical quantum chemical methods

A “substrate-surface graphene island” is simulated by semiempirical quantum chemical methods for different substrates. It is established that such systems with various island sizes correspond to graphene growth on corresponding substrates when CVD is used. Carbon atoms can be incorporated into the island from either the side of the substrate or the side of the CVD reactor exposed to it. It is shown for a wide range of island sizes that the best configuration with respect to the substrate is the structure of a carbon nanowall oriented perpendicular to the substrate. It is emphasized that a transition to this configuration is possible in reality only if CVD is plasma-stimulated when there is a strong near-electrode field near the surface, and the preliminary scission of the carbon carrier material occurs simultaneously in the CVD reactor volume.     

Visualizing chemical reactions confined under graphene

An undercover agent: graphene has been used as an imaging agent to visualize interfacial reactions under its cover, and exhibits a strong confinement effect on the chemistry of molecules underneath. In a CO atmosphere, CO penetrates into the graphene/Pt(111) interface and reacts with O(2) therein, whereas intercalated CO desorbs from the Pt surface.     

From nanographene and graphene nanoribbons to graphene sheets: chemical synthesis

Graphene, an individual two-dimensional, atomically thick sheet of graphite composed of a hexagonal network of sp(2) carbon atoms, has been intensively investigated since its first isolation in 2004, which was based on repeated peeling of highly oriented pyrolyzed graphite (HOPG). The extraordinary electronic, thermal, and mechanical properties of graphene make it a promising candidate for practical applications in electronics, sensing, catalysis, energy storage, conversion, etc. Both the theoretical and experimental studies proved that the properties of graphene are mainly dependent on their geometric structures. Precise control over graphene synthesis is therefore crucial for probing their fundamental physical properties and introduction in promising applications. In this Minireview, we highlight the recent progress that has led to the successful chemical synthesis of graphene with a range of different sizes and chemical compositions based on both top-down and bottom-up strategies.     

The necessity of structural irregularities for the chemical applications of graphene

Graphene is a fashionable material in the realm of nanotechnology, but its potentials are somehow misunderstood. Although a defect-free flat graphene looks perfect with exceptional mechanical properties, this perfectly ordered hexagonal structure is chemically less active, and not of practical interest in the applications involving chemical reactions. In most applications (e.g., in sensors, energy storage, electrochemical systems, catalysis, etc.) of graphene, the superior properties are usually because of the reactivity of intrinsic defects and dangling bonds. Unfortunately, the ordered structure of graphene mistakenly gets all the credits, and then, all attentions and even market supplies are shifted towards ideal graphene (i.e., chemically inert). There is a gradual shift to using graphene nanoribbons and quantum dots instead of ideal seamless graphene because the results are better. Understanding the roles of possible irregularities can pave the path for subtly designing graphene for the practical applications. Reducing the reactive sites may make graphene more well-defined structure-wise but practically less useful. While briefly describing common approaches for manipulating the classic structure of graphene, we summarize their successful utilization for various applications, e.g., in electrochemistry, luminescence, and catalysis.     

A rapid room temperature chemical route for the synthesis of graphene: metal-mediated reduction of graphene oxide

A rapid and facile route for the synthesis of reduced graphene oxide sheets (rGOs) at room temperature by the chemical reduction of graphene oxide using Zn/acid in aqueous solution is demonstrated.     

On-chip graphene oxide aptasensor for multiple protein detection

The versatility of an on-chip graphene oxide (GO) aptasensor was successfully confirmed by the detection of three different proteins, namely, thrombin (TB), prostate specific antigen (PSA), and hemagglutinin (HA), simply by changing the aptamers but with the sensor composition remaining the same. The results indicate that both DNA and RNA aptamers immobilized on the GO surface are sufficiently active to realize an on-chip aptasensor. Molecular selectivity and concentration dependence were investigated in relation to TB and PSA detection by using a dual, triple, and quintuple microchannel configuration. The multiple target detection of TB and PSA on a single chip was also demonstrated by using a 2 × 3 linear-array GO aptasensor. This work enables us to apply this sensor to the development of a multicomponent analysis system for a wide variety of targets by choosing appropriate aptamers.     

Challenges in polypropylene by chemical modification

Radical reactions of i-PP are a well known technical process for the chemical degradation to increase the flowability of the i-PP melt. By decreasing the temperature and increasing the life time of the PP-radicals, a process to synthesize long chain branched i-PP, was developed. The long chain branched-i-PP allows to introduce the i-PP in processing technologies as blow moulding film technology or foaming technology. The mechanism will be discussed. The radical grafting of Polypropylene (PP) becomes more important for the developing of PP-alloys with extended properties. Methylmethacrylate and styrene were polymerized and grafted in PP at low temperatures in solid state. Grafting and polymerizing in solid state means solving the monomers in the PP-powder directly from the reactor without contacting with oxygen (air). The reaction is started by the thermal decomposition of a peroxide. The reactivity of the primary radicals from the peroxide and the transfer reactions of the polymer radical of the PMMA or PS influences the amount of the grafted polymer. The solubility of the monomers and the peroxide in the amorphous i-PP-phase was measured. The grafting yield and the dispersity of the second polymer depends on the solubility and dispersity of peroxide and monomers in the PP-powder particles.     

High-performance polyolefin composites by chemical modification

The paper will give a short introducing survey on the present state of polyolefin and copolyolefin cross linking. The combination of high-density polyethylene (PEHD) and polypropylene (PP) with elastomers, particularly with ethylene-propylene terpolymer (EPDM), results in high-impact strength PP (HI-PP) and in thermoplastic elastomers (TPE) considered as high-performance polyolefin composites (HP-POC). By filling of PEHD and PP, HP-POCs with increased stiffness, modulus, and abrasion resistance can be produced. They are cost - advantageous substitutes for construction materials as, for example, ABS. Possibilities and limits are demonstrated by own investigations in this field. Best mechanical properties are obtained by reinforcement with fibres, particularly with glass fibres. Requirement for superior parameters is a chemical coupling between glass-fibre surface or glass-fibre size, respectively, and the PP matrix. A new adhesive agent on the basis of siloxane allows to reach high strength and toughness of the composites. The effect seems to be due to a better mutual penetration of the adhesive agent, the size, and the PP matrix. Outlooks will be given on the further development of HP-POC in the field of reinforcement and reactive combination with other polymers.     

Ribozyme 的结构及其化学修饰

本文对四种类型Ribozyme的结构进行了综述, 并着重讨论了锤头型Ribozyme的化学修饰及对其稳定性和催化活性的影响。