BackgroundThere is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein.MethodologySolid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours.ConclusionsThe Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.

The cellulosic part of rice straw was modified to develop N-halamine 11 derivatives for disinfection. The process involved cross-linking of the cellulosic material 12 with amino/amide/imide containing compounds; cyclic and acyclic. The structures of the 13 prepared materials were identified using FTIR and solid state 13 CNMR. The modified 14 materials were halogenated to form N-halamines and the antimicrobial activity of each 15 evaluated against examples of Gram-positive (Staphylococcus aureus) and Gram-16 negative bacteria (Escherichia coli) using a variety of methods; agar plate, blended agar, 17 stirred flask and in columns. One of the N-halamines achieved a 9 log reduction against 18 both E. coli and S. aureus in 4 hours. In addition, no S. aureus growth was recorded on 19 agar plates blended with 0.5g of this same material. 20

The mechanism of reaction between 1-ethyl-3-methylimidazolium acetate and the difunctional diglycidyl ether of bisphenol A (DGEBA) is explored using thermal and spectroscopic methods. Investigation of the 1,3-dialkylimidazolium based ionic liquids comprising the common cation (1-ethyl-3-methylimidazolium) and different anions (acetate, diethyl phosphate, dicyanamide or thiocyanate) via thermogravimetric analysis revealed 1-ethyl-3-methylimidazolium acetate to be the least thermally stable, both in air and nitrogen, and 1-ethyl-3-methylimidazolium dicyanamide to be the most thermally stable. Dynamic differential scanning calorimetry reveals the formulations comprising DGEBA and ionic liquid where it was revealed that the lowest and highest temperature for the onset of reaction were observed for formulations with 1-ethyl-3-methylimidazolium acetate and 1-ethyl-3-methylimidazolium dicyanamide respectively. 1-Ethyl-3-methylimidazolium acetate was shown, via nuclear magnetic resonance (NMR) spectroscopy and residual gas analysis, to degrade at 150 C to yield dealkylated products including methyl acetate and ethyl acetate as well as 1-methylimidazole and 1-ethylimidazole. The dealkylated imidazole ring is proposed as a route for initiation of the epoxy ring. Adduct formation between 1-ethyl-3-methylimidazoloium acetate and benzaldehyde at room temperature was observed leading to the proposal of the generation of a carbene species as a route for initiation of the epoxy ring in formulations with the acetate anion. NMR analysis of formulations comprising 1-ethyl-3-methylimidazolium thiocyanate and epoxy are believed, at room temperature, to initiate via reaction of the thiocyanate anion with the epoxy ring. 2 At elevated temperatures, it is proposed that a second, competing reaction, involving deprotonation of the imidazolium ring, also becomes active. The three proposed reaction pathways, namely the carbene route, the imidazole route and the counter-ion route, are all proposed to occur when an ionic liquid is used to initiate an epoxy resin.

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Microparticles incorporating micrometer-sized diffractive bar codes have been modified with oligonucleotides and immunoglobulin Gs to enable DNA hybridization and immunoassays. The bar codes are manufactured using photolithography of a chemically functional commercial epoxy photoresist (SU-8). When attached by suitable linkers, immobilized probe molecules exhibit high affinity for analytes and fast reaction kinetics, allowing detection of single nucleotide differences in DNA sequences and multiplexed immunoassays in <45 min. Analysis of raw data from assays carried out on the diffractive microparticles indicates that the reproducibility and sensitivity approach those of commercial encoding platforms. Micrometer-sized particles, imprinted with several superimposed diffraction gratings, can encode many million unique codes. The high encoding capacity of this technology along with the applicability of the manufactured bar codes to multiplexed assays will allow accurate measurement of a wide variety of molecular interactions, leading to new opportunities in diverse areas of biotechnology such as genomics, proteomics, high-throughput screening, and medical diagnostics.

An N-Halamine biocidal polymer was prepared by co-polymerizing toluene-2,6-diisocyanate with a new heterocyclic uramil-based azo-monomer, followed by halogenation. The mode of action of N-halamine polymers on bacteria was investigated and halogenation conditions (temperature, halogenation time, halogen concentration) were optimized for bactericidal action against E. coli and S. aureus. It was found that the mode of action of this type of polymer is a combination of different factors; contact, release, and through interaction of the polymer with the bacterial medium. The most effective halogenation conditions were stirring 1 g polymer in 10 mL sodium hypochlorite (10 %) for 1 h at ambient temperature (22 C).

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