On the biocatalytic synthesis of silicone polymers.

Polysiloxanes, with poly(dimethyl)siloxane (PDMS) being the most common example, are widely used in various industrial and consumer applications due to the physicochemical properties imparted by their Si-O-Si backbone structure. The conventional synthesis of PDMS involves the hydrolysis of dichlorodimethylsilane, which raises environmental concerns due to the usage of chlorinated compounds. Herein, a biocatalytic approach for PDMS synthesis is demonstrated using silicatein-α (Silα), an enzyme from marine sponges that is known to catalyse the hydrolysis and condensation of Si-O bonds. Using dialkoxysilane precursors, it was found that Silα catalyses the formation of PDMS in non-aqueous media, yielding polymers with higher molecular weights (approximately 1000-2000 Da). However, on prolonged exposure, the gradual degradation of the polymers was also observed. Overall these observations indicate that Silα catalyses the formation polysiloxanes, demonstrating the potential of biocatalysis for more sustainable polysiloxane production.

Recombinant silicateins as model biocatalysts in organosiloxane chemistry.

The family of silicatein enzymes from marine sponges (phylum Porifera) is unique in nature for catalyzing the formation of inorganic silica structures, which the organisms incorporate into their skeleton. However, the synthesis of organosiloxanes catalyzed by these enzymes has thus far remained largely unexplored. To investigate the reactivity of these enzymes in relation to this important class of compounds, their catalysis of Si-O bond hydrolysis and condensation was investigated with a range of model organosilanols and silyl ethers. The enzymes' kinetic parameters were obtained by a high-throughput colorimetric assay based on the hydrolysis of 4-nitrophenyl silyl ethers. These assays showed unambiguous catalysis with kcat/Km values on the order of 2-50 min-1 µM-1 Condensation reactions were also demonstrated by the generation of silyl ethers from their corresponding silanols and alcohols. Notably, when presented with a substrate bearing both aliphatic and aromatic hydroxy groups the enzyme preferentially silylates the latter group, in clear contrast to nonenzymatic silylations. Furthermore, the silicateins are able to catalyze transetherifications, where the silyl group from one silyl ether may be transferred to a recipient alcohol. Despite close sequence homology to the protease cathepsin L, the silicateins seem to exhibit no significant protease or esterase activity when tested against analogous substrates. Overall, these results suggest the silicateins are promising candidates for future elaboration into efficient and selective biocatalysts for organosiloxane chemistry.

A Method for Metal/Protein Stoichiometry Determination Using Thin-Film Energy Dispersive X-ray Fluorescence Spectroscopy.

A convenient approach is proposed for the quantitation of elemental cofactors in proteins and the determination of metal/protein stoichiometry, on the basis of energy dispersive X-ray fluorescence spectroscopy (EDXRF). The analysis of proteins containing the metals Cu, Fe, Zn, and Ca and also the nonmetallic element P is shown as a demonstration of the generality of the method. In general, the reported method gives limit of detection (LOD) and limit of quantification (LOQ) values in the low ppm range and requires only a few microliters of protein sample at micromolar concentrations. Moreover, sample preparation does not require any digestion steps before the analysis. The expected metal/protein stoichiometry was observed for each protein analyzed, highlighting the precision and accuracy of the method in all the tested cases. Furthermore, it is shown that the method is compatible with multimeric proteins and those with post-translational modifications such as glycosylation.

Photoresponsive Hydrogels with Photoswitchable Stiffness: Emerging Platforms to Study Temporal Aspects of Mesenchymal Stem Cell Responses to Extracellular Stiffness Regulation.

An extensive number of cell-matrix interaction studies have identified matrix stiffness as a potent regulator of cellular properties and behaviours. Perhaps most notably, matrix stiffness has been demonstrated to regulate mesenchymal stem cell (MSC) phenotype and lineage commitment. Given the therapeutic potential for MSCs in regenerative medicine, significant efforts have been made to understand the molecular mechanisms involved in stiffness regulation. These efforts have predominantly focused on using stiffness-defined polyacrylamide (PA) hydrogels to culture cells in 2D and have enabled elucidation of a number of mechano-sensitive signalling pathways. However, despite proving to be a valuable tool, these stiffness-defined hydrogels do not reflect the dynamic nature of living tissues, which are subject to continuous remodelling during processes such as development, ageing, disease and regeneration. Therefore, in order to study temporal aspects of stiffness regulation, researchers have developed and exploited novel hydrogel substrates with in situ tuneable stiffness. In particular, photoresponsive hydrogels with photoswitchable stiffness are emerging as exciting platforms to study MSC stiffness regulation. This chapter provides an introduction to the use of PA hydrogel substrates, the molecular mechanisms of mechanotransduction currently under investigation and the development of these emerging photoresponsive hydrogel platforms.

An Enzyme Cascade for Selective Modification of Tyrosine Residues in Structurally Diverse Peptides and Proteins.

Bioorthogonal chemistry enables a specific moiety in a complex biomolecule to be selectively modified in the presence of many reactive functional groups and other cellular entities. Such selectivity has become indispensable in biology, enabling biomolecules to be derivatized, conjugated, labeled, or immobilized for imaging, biochemical assays, or therapeutic applications. Methyltransferase enzymes (MTase) that accept analogues of the cofactor S-adenosyl methionine have been widely deployed for alkyl-diversification and bioorthogonal labeling. However, MTases typically possess tight substrate specificity. Here we introduce a more flexible methodology for selective derivatization of phenolic moieties in complex biomolecules. Our approach relies on the tandem enzymatic reaction of a fungal tyrosinase and the mammalian catechol-O-methyltransferase (COMT), which can effect the sequential hydroxylation of the phenolic group to give an intermediate catechol moiety that is subsequently O-alkylated. When used in this combination, the alkoxylation is highly selective for tyrosine residues in peptides and proteins, yet remarkably tolerant to changes in the peptide sequence. Tyrosinase-COMT are shown to provide highly versatile and regioselective modification of a diverse range of substrates including peptide antitumor agents, hormones, cyclic peptide antibiotics, and model proteins.

A high-throughput assay for arylamine halogenation based on a peroxidase-mediated quinone-amine coupling with applications in the screening of enzymatic halogenations.

Arylhalides are important building blocks in many fine chemicals, pharmaceuticals and agrochemicals, and there has been increasing interest in the development of more "green" halogenation methods based on enzyme catalysis. However, the screening and development of new enzymes for biohalogenation has been hampered by a lack of high-throughput screening methods. Described herein is the development of a colorimetric assay for detecting both chemical and enzymatic arylamine halogenation reactions in an aqueous environment. The assay is based on the unique UV/Vis spectrum created by the formation of an ortho-benzoquinone-amine adduct, which is produced by the peroxidase-catalysed benzoquinone generation, followed by Michael addition of either a halogenated or non-halogenated arylamine. This assay is sensitive, rapid and amenable to high-throughput screening platforms. We have also shown this assay to be easily coupled to a flavin-dependent halogenase, which currently lacks any convenient colorimetric assay, in a "one-pot" workflow.

Single-molecule protein arrays enabled by scanning probe block copolymer lithography.

The ability to control the placement of individual protein molecules on surfaces could enable advances in a wide range of areas, from the development of nanoscale biomolecular devices to fundamental studies in cell biology. Such control, however, remains a challenge in nanobiotechnology due to the limitations of current lithographic techniques. Herein we report an approach that combines scanning probe block copolymer lithography with site-selective immobilization strategies to create arrays of proteins down to the single-molecule level with arbitrary pattern control. Scanning probe block copolymer lithography was used to synthesize individual sub-10-nm single crystal gold nanoparticles that can act as scaffolds for the adsorption of functionalized alkylthiol monolayers, which facilitate the immobilization of specific proteins. The number of protein molecules that adsorb onto the nanoparticles is dependent upon particle size; when the particle size approaches the dimensions of a protein molecule, each particle can support a single protein. This was demonstrated with both gold nanoparticle and quantum dot labeling coupled with transmission electron microscopy imaging experiments. The immobilized proteins remain bioactive, as evidenced by enzymatic assays and antigen-antibody binding experiments. Importantly, this approach to generate single-biomolecule arrays is, in principle, applicable to many parallelized cantilever and cantilever-free scanning probe molecular printing methods.

Development of Improved Spectrophotometric Assays for Biocatalytic Silyl Ether Hydrolysis.

Reported herein is the development of assays for the spectrophotometric quantification of biocatalytic silicon-oxygen bond hydrolysis. Central to these assays are a series of chromogenic substrates that release highly absorbing phenoxy anions upon cleavage of the sessile bond. These substrates were tested with silicatein, an enzyme from a marine sponge that is known to catalyse the hydrolysis and condensation of silyl ethers. It was found that, of the substrates tested, tert-butyldimethyl(2-methyl-4-nitrophenoxy)silane provided the best assay performance, as evidenced by the highest ratio of enzyme catalysed reaction rate compared with the background (uncatalysed) reaction. These substrates were also found to be suitable for detailed enzyme kinetics measurements, as demonstrated by their use to determine the Michaelis-Menten kinetic parameters for silicatein.

Evaluation of Fluorescence-Based Screening Assays for the Detection and Quantification of Silyl Hydrolase Activity.

This study reports the development of fluorometric assays for the detection and quantification of silyl hydrolase activity using silicatein as a model enzyme. These assays employed a series of organosilane substrates containing either mycophenolate or umbelliferone moieties, which become fluorescent upon hydrolysis of a scissile Si-O bond. Among these substrates, the mycophenolate-derived molecule MycoF, emerged as the most promising candidate due to its relative stability in aqueous media, which resulted in good differentiation between the enzyme-catalyzed and uncatalyzed background hydrolysis. The utility of MycoF was also demonstrated in the detection of enzyme activity in cell lysates and was found to be capable of qualitative identification of positive "hit" candidates in a high-throughput format. These fluorogenic substrates were also suitable for use in quantitative kinetic assays, as demonstrated by the acquisition of their Michaelis-Menten parameters.

Biocatalytic Generation of o-Quinone Imines in the Synthesis of 1,4-Benzoxazines and Its Comparative Green Chemistry Metrics.

1,4-Benzoxazines are important motifs in many pharmaceuticals and can be formed by a reaction sequence involving the oxidation of o-aminophenols to their corresponding quinone imine followed by an in situ inverse electron demand Diels-Alder (IEDDA) cycloaddition with a suitable dienophile. Reported herein is the development of a reaction sequence that employs horseradish peroxidase to catalyze the oxidation of the aminophenols prior to the IEDDA as a more sustainable alternative to the use of conventional stoichiometric oxidants. The synthesis of 10 example benzoxazines is demonstrated in this "one-pot, two-step" procedure with yields between 42% and 92%. The green chemistry metrics, including the E-factor and generalized reaction mass efficiency, for this biocatalytic reaction were compared against the conventional chemical approach. It was found that the reported biocatalytic route was approximately twice as green by these measures.

S-adenosyl-methionine-dependent methyltransferases: highly versatile enzymes in biocatalysis, biosynthesis and other biotechnological applications.

S-adenosyl methionine (SAM) is a universal biological cofactor that is found in all branches of life where it plays a critical role in the transfer of methyl groups to various biomolecules, including DNA, proteins and small-molecule secondary metabolites. The methylation process thus has important implications in various disease processes and applications in industrial chemical processing. This methyl transfer is catalysed by SAM-dependent methyltransferases (MTases), which are by far the largest groups of SAM-dependent enzymes. A significant amount is now known regarding the structural biology and enzymology of these enzymes, and, consequently, there is now significant scope for the development of new MTases and SAM analogues for applications from biomolecular imaging to biocatalytic industrial processes. This review will focus on current efforts in the manipulation of class I and V SAM-dependent MTases and the use of synthetic SAM analogues, which together offer the best prospects for rational redesign towards biotechnological applications. Firstly, metabolic engineering of organisms incorporating small-molecule MTases is discussed; this can be applied in a variety of areas from the industrial bioprocessing of flavourants and antibiotics to frontier research in biofuel production and bioremediation. Secondly, the application of MTases in combination with SAM analogues is reviewed; this allows the tagging of proteins and oligonucleotides with moieties other than the methyl group. Such tagging allows the isolation of the tagged biomolecule and aids its visualisation by a range of analytical methods. The review then summarises the potential advantages of MTase-mediated chemistry and offers some future perspectives on downstream applications.

Regulation of Mesenchymal Stem Cell Morphology Using Hydrogel Substrates with Tunable Topography and Photoswitchable Stiffness.

Cell function can be directly influenced by the mechanical and structural properties of the extracellular environment. In particular, cell morphology and phenotype can be regulated via the modulation of both the stiffness and surface topography of cell culture substrates. Previous studies have highlighted the ability to design cell culture substrates to optimise cell function. Many such examples, however, employ photo-crosslinkable polymers with a terminal stiffness or surface profile. This study presents a system of polyacrylamide hydrogels, where the surface topography can be tailored and the matrix stiffness can be altered in situ with photoirradiation. The process allows for the temporal regulation of the extracellular environment. Specifically, the surface topography can be tailored via reticulation parameters to include creased features with control over the periodicity, length and branching. The matrix stiffness can also be dynamically tuned via exposure to an appropriate dosage and wavelength of light, thus, allowing for the temporal regulation of the extracellular environment. When cultured on the surface of the hydrogels, the morphology and alignment of immortalised human mesenchymal stem cells can be directly influenced through the tailoring of surface creases, while cell size can be altered via changes in matrix stiffness. This system offers a new platform to study cellular mechanosensing and the influence of extracellular cues on cell phenotype and function.

Protein micro- and nanopatterning using aminosilanes with protein-resistant photolabile protecting groups.

An approach to the integration of nanolithography with synthetic chemical methodology is described, in which near-field optical techniques are used to selectively deprotect films formed by the adsorption of aminosilanes protected by modified 2-nitrophenylethoxycarbonyl (NPEOC) groups. The NPEOC groups are functionalized at the m- or p-position with either a tetraethyleneglycol or a heptaethylene glycol adduct. We describe the synthesis of these bioresistant aminosilanes and the characterization of the resulting photoreactive films. Photodeprotection by exposure to UV light (λ = 325 nm) yielded the amine with high efficiency, at a similar rate for all four adsorbates, and was complete after an exposure of 2.24 J cm(-2). Following photodeprotection, derivatization by trifluoroacetic anhydride was carried out with high efficiency. Micropatterned samples, formed using a mask, were derivatized with aldehyde-functionalized polymer nanoparticles and, following derivatization with biotin, were used to form patterns of avidin-coated polymer particles. Fluorescence microscopy and atomic force microscopy data demonstrated that the intact protecting groups conferred excellent resistance to nonspecific adsorption. Nanometer-scale patterns were created using scanning near-field photolithography and were derivatized with biotin. Subsequent conjugation with avidin-functionalized polymer nanoparticles yielded clear fluorescence images that indicated dense attachment to the nanostructures and excellent protein resistance on the surrounding surface. These simple photocleavable protecting group strategies, combined with the use of near-field exposure, offer excellent prospects for the control of surface reactivity at nanometer resolution in biological systems and offer promise for integrating the top-down and bottom-up molecular fabrication paradigms.

Parallel scanning near-field photolithography: the snomipede.

The "Millipede", developed by Binnig and co-workers (Bining, G. K.; et al. IBM J. Res. Devel. 2000, 44, 323.), elegantly solves the problem of the serial nature of scanning probe lithography processes, by deploying massive parallelism. Here we fuse the "Millipede" concept with scanning near-field photolithography to yield a "Snomipede" that is capable of executing parallel chemical transformations at high resolution over macroscopic areas. Our prototype has sixteen probes that are separately controllable using a methodology that is, in principle, scalable to much larger arrays. Light beams generated by a spatial modulator or a zone plate array are coupled to arrays of cantilever probes with hollow, pyramidal tips. We demonstrate selective photodeprotection of nitrophenylpropyloxycarbonyl-protected aminosiloxane monolayers on silicon dioxide and subsequent growth of nanostructured polymer brushes by atom-transfer radical polymerization, and the fabrication of 70 nm structures in photoresist by a Snomipede probe array immersed under water. Such approaches offer a powerful means of integrating the top-down and bottom-up fabrication paradigms, facilitating the reactive processing of materials at nanometer resolution over macroscopic areas.

Lithographic Processes for the Scalable Fabrication of Micro- and Nanostructures for Biochips and Biosensors.

Since the early 2000s, extensive research has been performed to address numerous challenges in biochip and biosensor fabrication in order to use them for various biomedical applications. These biochips and biosensor devices either integrate biological elements (e.g., DNA, proteins or cells) in the fabrication processes or experience post fabrication of biofunctionalization for different downstream applications, including sensing, diagnostics, drug screening, and therapy. Scalable lithographic techniques that are well established in the semiconductor industry are now being harnessed for large-scale production of such devices, with additional development to meet the demand of precise deposition of various biological elements on device substrates with retained biological activities and precisely specified topography. In this review, the lithographic methods that are capable of large-scale and mass fabrication of biochips and biosensors will be discussed. In particular, those allowing patterning of large areas from 10 cm2 to m2, maintaining cost effectiveness, high throughput (>100 cm2 h-1), high resolution (from micrometer down to nanometer scale), accuracy, and reproducibility. This review will compare various fabrication technologies and comment on their resolution limit and throughput, and how they can be related to the device performance, including sensitivity, detection limit, reproducibility, and robustness.

Nanoscale biomolecular structures on self-assembled monolayers generated from modular pegylated disulfides.

A solid-phase synthetic strategy was developed that uses modular building blocks to prepare symmetric oligo(ethylene glycol)-terminated disulfides with a variety of lengths and terminal functionalities. The modular disulfides, composed of alkyl amino groups linked by an amide group to oligoethylene chains were used to generate self-assembled monolayers (SAMs), which were characterised to determine their applicability for biomolecular applications. X-ray photoelectron spectroscopy (XPS) of the SAMs obtained from these molecules demonstrated improved stability towards displacement by 16-hexadecanethiol, while surface plasmon resonance (SPR) analyses of SAMs prepared with the hydroxy-terminated oligoethylene disulfide showed equal resistance to non-specific protein adsorption in comparison to 11-mercaptoundecyl tri(ethylene glycol). SAMs made from these adsorbates were amenable to nanoscale patterning by scanning near-field photolithography (SNP), facilitating the fabrication of nanopatterned, protein-functionalised surfaces. Such SAMs may be further developed for bionanotechnology applications such as the fabrication of nanoscale biological arrays and sensor devices.

Site-selective immobilisation of functional enzymes on to polystyrene nanoparticles.

The immobilisation of proteins on to nanoparticles has a number of applications ranging from biocatalysis through to cellular delivery of biopharmaceuticals. Here we describe a phosphopantetheinyl transferase (Sfp)-catalysed method for immobilising proteins bearing a small 12-mer "ybbR" tag on to nanoparticles functionalised with coenzyme A. The Sfp-catalysed immobilisation of proteins on to nanoparticles is a highly efficient, single step reaction that proceeds under mild conditions and results in a homogeneous population of proteins that are covalently and site-specifically attached to the surface of the nanoparticles. Several enzymes of interest for biocatalysis, including an arylmalonate decarboxylase (AMDase) and a glutamate racemase (GluR), were immobilised on to nanoparticles using this approach. These enzymes retained their activity and showed high operational stability upon immobilisation.

Improved Production and Biophysical Analysis of Recombinant Silicatein-α.

Silicatein-α is a hydrolase found in siliceous sea sponges with a unique ability to condense and hydrolyse silicon-oxygen bonds. The enzyme is thus of interest from the perspective of its unusual enzymology, and for potential applications in the sustainable synthesis of siloxane-containing compounds. However, research into this enzyme has previously been hindered by the tendency of silicatein-α towards aggregation and insolubility. Herein, we report the development of an improved method for the production of a trigger factor-silicatein fusion protein by switching the previous hexahistidine tag for a Strep-II tag, resulting in 244-fold improvement in protein yield compared to previous methods. Light scattering and thermal denaturation analyses show that under the best storage conditions, although oligomerisation is never entirely abolished, these nanoscale aggregates of the Strep-tagged protein exhibit improved colloidal stability and solubility. Enzymatic assays show that the Strep-tagged protein retains catalytic competency, but exhibits lower activity compared to the His6-tagged protein. These results suggest that the hexahistidine tag is capable of non-specific catalysis through their imidazole side chains, highlighting the importance of careful consideration when selecting a purification tag. Overall, the Strep-tagged fusion protein reported here can be produced to a higher yield, exhibits greater stability, and allows the native catalytic properties of this protein to be assessed.