Immobilized antibodies are useful for the detection of antigens in highly sensitive microarray diagnostic applications. Arrays with the antibodies are attached regioselectively in a uniform orientation are typically more sensitive than those with random orientations. Direct regioselective immobilization of antibodies on a solid support typically requires a modified form of the protein. We now report a general approach for the regioselective attachment of antibodies to a surface using truncated forms of antibody binding proteins A, G, and L that retain the structural motifs required for antibody binding. The recombinant proteins have a C-terminal CVIX protein farnesyltransferase recognition motif that allows us to append a bioorthogonal azide or alkyne moiety and use the Cu(I)-catalyzed Huisgen cycloaddition to attach the binding proteins to a suitably modified glass surface. This approach offers several advantages. The recombinant antibody binding proteins are produced in E. coli, chemoselectively modified posttranslationally in the cell-free homogenate, and directly attached to the glass surface without the need for purification at any stage of the process. Complexes between immobilized recombinant proteins A, G, and L and their respective strongly bound antibodies were stable to repeated washing with PBST buffer at pH 7.2. However, the antibodies could be stripped from the slides by treatment with 0.1 M glycine·HCl buffer, pH 2.6, for 30 min and regenerated by shaking with PBS buffer, pH 7.2, at 4 ° C overnight. The recombinant forms of proteins A, G, and L can be used separately or in combination to give glass surfaces capable of binding a wide variety of antibodies.

prepared by adding the PAN to N,N-dimethylformamide and sonicating (80 W) for 20 min. 1±4 wt.-% graphite nanoplatelets (relative to PAN) were added to the PAN solutions, each of which was then sonicated for 30 min to ensure an even dispersion. A 30 kV high-voltage power supply (Gamma High Voltage Research, Ormond Beach, FL) was used as the voltage source. The electrospun fibers were collected on a 75 mm 75 mm aluminum-foil-covered copper plate.For scanning electron microscopy analysis, the nanofiber mats were affixed to graphite disks with carbon tape, coated with gold (20 thickness), and imaged with a Cambridge Stereoscan 250. For transmission electron microscopy analysis, the samples were dispersed on lacey carbon grids (Ted Pella, Redding, CA) using ethanol and imaged with a JEOL-2000 FX operated at 200 kV. Thermal gravimetric analysis (TGA) was performed using a TA Instruments Hi-Res TGA 2950. Experiments were carried out from room temperature to 1000 C, ramped at 5 C min ±1 in air. Atomic force microscopy analysis was performed using a Nanoscope IIIa (Digital Instruments/Veeco) with a 5 nm radius silicon-carbide tip. In preparation for imaging, the electrospun composite fibrils were stabilized in an oven at 200 C for 20 min in air. The composite-fibril specimens were then fixed on a 5 mm 5 mm mica sheet with a drop of DMF solution. The specimens were left overnight to allow the solvent to evaporate. One of the most promising approaches to overcome the fundamental limitation of conventional membranes has been the addition of microporous molecular-sieving materials (e.g., zeolites, silica and carbon molecular sieves) to organic polymers in the hope of combining the processability of the polymers with the strong size selectivity of spatially well-defined inorganic pores. Since zeolites and carbon molecular sieves have highly attractive permeation properties, with permeabilities and selectivities significantly higher than polymeric materials, the size and shape selectivity of these nanoporous materials would be expected to generate precise molecular-sieving discrimination by permitting smaller-sized gas penetrants to diffuse at much higher rates than larger-sized penetrants. Composite materials exploiting the mass-transport properties of polymer matrices, and nanoporous complexes, may lead to membranes with enhanced gas separation properties. In this study, highly selective organic molecular sieve composite membranes have been prepared using molecular dispersions of three-dimensional (3D)-network nanoporous transitionmetal complexes, {[Cu 2 (PF 6 )(NO 3 )(4,4¢-bpy) 4 ] . 2PF 6 . 2H 2 O} n , confined within amorphous glassy polysulfone (PSf). The organic molecular sieves exhibited enhanced selectivities for hydrogen over methane (ca. 200 at 5 wt.-% loading), which is the highest ideal selectivity reported for molecular sieve composite membranes. COMMUNICATIONS 80

The systematic oxidation reactions of a wide range of alcohols have been carried out by using an iron porphyrin complex in order to understand their relation to cytochrome P-450 enzymes and to have a practical application to organic synthesis. The iron porphyrin complex catalyzed efficiently alcohol oxidation to the respective carbonyl compound via a high-valent iron-oxo porphyrin intermediate ((Porp)Fe=O+). Several mechanistic studies such as isotope 18O labeling, deuterium isotope effect, linear free energy relationship, and ring-opening of radical clock substrate, have suggested that the alcohol is oxidized by a sequence of reactions involving an alpha-hydroxyalkyl radical intermediate and oxygen rebound to form the gem-diol, dehydration of which yields the carbonyl compounds. Moreover, it has been proposed that a two-state reactivity mechanism can also be adopted for alcohol oxidation reactions in iron porphyrin model systems as exhibited by P-450 enzymes.

Protein chips are powerful tools as analytical and diagnostic devices for detection of biomolecular interactions, where the proteins are covalently or noncovalently attached to biosensing surfaces to capture and detect target molecules or biomarkers. Thus, fabrication of biosensing surfaces for regio- and chemoselective immobilization of biomolecules is a crucial step for better biosensor performance. In our previous studies, a regio- and chemoselective immobilization strategy was demonstrated on glass surfaces. This strategy is now used to regioselectively attach proteins to self-assembled monolayers (SAMs) on gold surfaces. Recombinant green fluorescent protein (GFP), glutathione S-transferase (GST), and antibody-binding protein G, bearing a C-terminal CVIA motif, were prepared and a farnesyl analogue with an ω-alkyne moiety was attached to the sulfhydryl moiety in the cysteine side chain by protein farnesyltransferase. The proteins, modified with the bioorthogonal alkyne functional group, were covalently and regioselectively immobilized on thiol or dithiocarbamate (DTC) SAMs on a gold surface by a Huigsen [3 + 2] cycloaddition reaction with minimal nonspecific binding. A concentration-dependent increase of fluorescence intensity was observed in wells treated with GFP on both thiol- and DTC-SAMs. The highly ordered, densely packed layer allowed for a high loading of immobilized protein, with a concomitant increase in substrate binding capacity. The DTC-SAMs were substantially more resistant to displacement of the immobilized proteins from the gold surface by β-mercaptoethanol than alkane-thiol SAMs.

Antibody arrays are a useful for detecting antigens and other antibodies. This technique typically requires a uniform and well-defined orientation of antibodies attached to a surface for optimal performance. A uniform orientation can be achieved by modification of antibodies to include a single site for attachment. Thus, uniformly oriented antibody arrays require a bioengineered modification for the antibodies directly immobilization on the solid surface. In this study, we describe a “sandwich-type” antibody array where unmodified antibodies are oriented through binding with regioselectively immobilized recombinant antibody-binding protein L. Recombinant proL-CVIA bearing C-terminal CVIA motif is post-translationally modified with an alkyne group by protein farnesyltransferase (PFTase) at the cysteine residue in the CVIA sequence to give proL-CVIApf, which is covalently attached to an azido-modified glass slide by a Huisgen [3 + 2] cycloaddition reaction. Slides bearing antibodies bound to slides coated with regioselectively immobilized proL-CVIApf gave stronger fluorescence outputs and those where the antibody-binding protein was immobilized in random orientations on an epoxy-modified slide. Properly selected capture and detection antibodies did not cross-react with immobilized proL-CVIApf in sandwich arrays, and the proL-CVIApf slides can be used for multiple cycles of detected over a period of several months.