Metallic nanoparticles: microbial synthesis and unique properties for biotechnological applications,bioavailability and biotransformation

The impact of nanotechnology in all areas of science and technology is evident. The expanding availability of a variety of nanostructures with properties in the nanometer size range has sparked widespread interest in their use in biotechnological systems, including the field of environmental remediation. Nanomaterials can be used as catalysts, adsorbents, membranes, water disinfectants and additives to increase catalytic activity and capability due to their high specific surface areas and nanosize effects. Thus, nanomaterials appear promising for new effective environmental technologies. Definitely, nanotechnology applications for site remediation and wastewater treatment are currently in research and development stages, and new innovations are underway. The synthesis of metallic nanoparticles has been intensively developed not only due to its fundamental scientific interest but also for many technological applications. The use of microorganisms in the synthesis of nanoparticles is a relatively new eco-friendly and promising area of research with considerable potential for expansion. On the other hand, chemical synthesis occurs generally under extreme conditions (e.g. pH, temperature) and also chemicals used may have associated environmental and human health impacts. This review is an overview of current research worldwide on the use of microorganisms during the biosynthesis of metallic nanoparticles and their unique properties that make them good candidates for many applications, including in biotechnology.     

Insights into laccase producing organisms,fermentation states,purification strategies,and biotechnological applications

Laccases are phenol oxidases belonging to the superfamily of multicopper oxidases and are found in bacteria, fungi, lichens, higher plants, and insects. Over the past few decades, laccases and laccase mediator systems (LMS) have found uses in a wide range of technological applications such as textile dye decolorization, industrial wastewater detoxification, pulp bleaching, chemical synthesis, and development of miniaturized biosensors. This has encouraged numerous studies to find and purify laccases with exploitable characteristics. The main aim of the present review is to summarize the rich literature data gained in recent years from the studies on laccases, focusing on the organisms that produce them, the methods used for screening, laccase activity assays, purification strategies, and the application of laccases as eco‐friendly biocatalysts. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1443–1463, 2015     

Biocatalysts: Beautiful creatures

The chemical industry has come under increasing pressure to make chemical production more eco-friendly and independent to fossil resources. The development of industrial processes based on micro-organisms can especially help to eliminate the use or the generation of hazardous substances and can support the transition from dependence on fossil resources towards real sustainable and eco-safety industrial processes. The biocatalysts are the best solution given by nature that can be used to improve some biotechnological applications. In this research review, we report some peculiar properties of biocatalysts, implicated in a range of metabolic pathways and biotechnological tools.     

Polymers in drug delivery

Advances in polymer science have led to the development of several novel drug-delivery systems. A proper consideration of surface and bulk properties can aid in the designing of polymers for various drug-delivery applications. Biodegradable polymers find widespread use in drug delivery as they can be degraded to non-toxic monomers inside the body. Novel supramolecular structures based on polyethylene oxide copolymers and dendrimers are being intensively researched for delivery of genes and macromolecules. Hydrogels that can respond to a variety of physical, chemical and biological stimuli hold enormous potential for design of closed-loop drug-delivery systems. Design and synthesis of novel combinations of polymers will expand the scope of new drug-delivery systems in the future.     

Combinatorial solid phase peptide synthesis and bioassays

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.     

The recent development and applications of fluidic channels by 3D printing

The technology of “Lab-on-a-Chip” allows the synthesis and analysis of chemicals and biological substance within a portable or handheld device. The 3D printed structures enable precise control of various geometries. The combination of these two technologies in recent years makes a significant progress. The current approaches of 3D printing, such as stereolithography, polyjet, and fused deposition modeling, are introduced. Their manufacture specifications, such as surface roughness, resolution, replication fidelity, cost, and fabrication time, are compared with each other. Finally, novel application of 3D printed channel in biology are reviewed, including pathogenic bacteria detection using magnetic nanoparticle clusters in a helical microchannel, cell stimulation by 3D chemical gradients, perfused functional vascular channels, 3D tissue construct, organ-on-a-chip, and miniaturized fluidic “reactionware” devices for chemical syntheses. Overall, the 3D printed fluidic chip is becoming a powerful tool in the both medical and chemical industries.     

A new generation of polymer nanoparticles for drug delivery.

One of the main interests of using polymer nanoparticles as drug carrier systems is to control the delivery of the drugs including their biodistribution. During the last decade, it was clearly demonstrated that surface properties of nanoparticles were the key factor which determined the in vivo fate of such a carrier. Thus, the purpose of this work was to develop a new method which allows the easy fabrication of nanoparticles with versatile surface properties using polysaccharides. This preparation was based on the use of a redox radical polymerization reaction applied for the first time to the emulsion polymerization of alkylcyanoacrylates in aqueous continuous media. The dispersion of nanoparticles was very stable. The nanoparticle surfaces were coated with polysaccharides and their characteristics can be modulated by the type and the molecular weight of the polysaccharides used during the synthesis. Interestingly the biological properties of the polysaccharide immobilized on the nanoparticle surface can be preserved opening very interesting perspectives for such nanoparticles. This method also offers a new strategy for the design of modular biomimetic nanoparticles as drug carrier systems with multiple functions. One of the applications considered in this work was to use these nanoparticles coupled with haemoglobin as an oxygen carrier.     

Synthesis and application of virus-based hybrid nanomaterials

A virus is a nanoscaled biomolecular substance composed of genes, protecting capsid proteins, and envelopes. The nanoscale dimensions and surface functionalities of virions have been exploited to attract and assemble inorganic and organic materials to produce functional nanomaterials with large surface areas. Genetic modifications of virus capsid proteins lead to the selective deposition and controlled growth of inorganic substances producing organized virus-based hybrid materials. Due to these properties, viruses hold promise for development as platforms for the creation of hybrid materials with multiple functionalities. This article reviews the characteristics of commonly used viruses and their fabrication into virus-based hybrid materials that have been applied in engineering applications such as nanowires and catalysts.     

Perspective on the use of humic acids from biomass as natural surfactants for industrial applications

In the context of renewable vs. non-renewable sources of chemical compounds, the development of natural surfactants as a substitute for synthetic surfactants in technological applications is an important issue. In addition, as synthetic surfactants can persist in the environment causing toxic effects, the use of natural products presents a possibility to minimize impact on the environment. Nowadays, a promising new approach in surfactant-based technologies, consists of the use of humic acids (HAs) extracted directly from biomass that exhibit amphiphilic properties, and can be conveniently used as environmentally friendly surfactants. The raw material from which HAs are extracted and their macromolecular composition affect surfactant properties. Therefore fundamental data from more strictly qualitative aspects, needs to be investigated. This review highlights surfactant ability and chemical properties of HA substances coming from renewable sources in comparison to synthetic surfactants, and points out the capacity for HAs to be used effectively in this field of application.     

A miniaturized high-throughput screening assay for fucosyltransferase VII

Fucosyltransferase VII (FucTVII) is a very promising drug target for treatment of inflammatory skin diseases. Its activity is required for synthesis of the sialyl-Lewis X glycoepitopes on the E- and P-selectin ligands, necessary for lymphocyte migration into the skin. High-throughput screening (HTS) of large chemical libraries has become the main source of novel chemical entities for the pharmaceutical industry. The screening of very large compound collections requires the use of specialized assay techniques that minimize time and costs. We describe the development of a miniaturized scintillation proximity assay for human FucTVII based on a oligosaccharide acceptor substrate that is identical to the glycosylation of the physiological substrate. In addition to assay development, the assay performance in a HTS campaign is shown. We screened 798,131 compounds from the Schering AG HTS library and identified 233 IC50 hits; 229 hits were FucTVII specific in so far as they did not inhibit either alpha-fucosidase or galactosyltransferase. In addition to screening a drug-like small-molecule collection, we worked on rational approaches to develop inhibitors or glycosidic decoys based on oligosaccharide-substrate analogues. The structure-activity relationship observed thereby is very narrow and shows strict requirements that are consistent with the described substrate specificity of FucTVII.     

Photo-Attachment of Biomolecules for Miniaturization on Wicking Si-Nanowire Platform

We demonstrated the surface functionalization of a highly three-dimensional, superhydrophilic wicking substrate using light to immobilize functional biomolecules for sensor or microarray applications. We showed here that the three-dimensional substrate was compatible with photo-attachment and the performance of functionalization was greatly improved due to both increased surface capacity and reduced substrate reflectivity. In addition, photo-attachment circumvents the problems induced by wicking effect that was typically encountered on superhydrophilic three-dimensional substrates, thus reducing the difficulty of producing miniaturized sites on such substrate. We have investigated various aspects of photo-attachment process on the nanowire substrate, including the role of different buffers, the effect of wavelength as well as how changing probe structure may affect the functionalization process. We demonstrated that substrate fabrication and functionalization can be achieved with processes compatible with microelectronics processes, hence reducing the cost of array fabrication. Such functionalization method coupled with the high capacity surface makes the substrate an ideal candidate for sensor or microarray for sensitive detection of target analytes.     

Miniaturization of polymerase chain reaction

Polymerase chain reaction (PCR) is one of the most widely used analytical tool and is an important module that would benefit from being miniaturized and integrated onto diagnostic or analytical chips. There are potentially two different approaches for the miniaturization of the PCR module: chamber-type and flow-type micro-PCR. These miniaturized PCRs have distinct characteristics and advantages. In this article, we review the necessity of micro-PCR, the materials for the chip fabrication, the surface modification, and characteristics of the two types of micro-PCR. The motivation underlying the development of micro-PCR, the advantages and disadvantages of the various materials used in fabrication and the surface modification methods will be discussed. And finally, the precise features of the two different types of micro-PCR will be compared.     

Microarrays: The Technology,Analysis and Application

DNA microarray analysis represents one of the major advances leading to the development of functional genomics and proteomics. It involves the fabrication of DNA either by in situ or on‐chip photolithographic synthesis or by inkjet or microjet deposition, as microspots immobilized on the surface of miniaturized substrates like glass or membranes. The immobilized DNA molecules are then allowed to hybridize with labeled complementary DNA. The hybrid DNA so formed is read through scanning devices, such as fluorescence and radioactivity. Further, computational approaches, for example, normalization and clustering allow thousands of genetic parameters in a single experiment to be simultaneously analyzed. This review discusses the fundamental principles and data analysis of the microarray technology, while focusing on its application in gene expression analysis, genotyping for point mutation and diseases diagnostics.     

Fabrication,Densification, and Replica Molding of 3D Carbon Nanotube Microstructures

The introduction of new materials and processes to microfabrication has, in large part, enabled many important advances in microsystems, lab-on-a-chip devices, and their applications. In particular, capabilities for cost-effective fabrication of polymer microstructures were transformed by the advent of soft lithography and other micromolding techniques ^{1, 2}, and this led a revolution in applications of microfabrication to biomedical engineering and biology. Nevertheless, it remains challenging to fabricate microstructures with well-defined nanoscale surface textures, and to fabricate arbitrary 3D shapes at the micro-scale. Robustness of master molds and maintenance of shape integrity is especially important to achieve high fidelity replication of complex structures and preserving their nanoscale surface texture. The combination of hierarchical textures, and heterogeneous shapes, is a profound challenge to existing microfabrication methods that largely rely upon top-down etching using fixed mask templates. On the other hand, the bottom-up synthesis of nanostructures such as nanotubes and nanowires can offer new capabilities to microfabrication, in particular by taking advantage of the collective self-organization of nanostructures, and local control of their growth behavior with respect to microfabricated patterns. Our goal is to introduce vertically aligned carbon nanotubes (CNTs), which we refer to as CNT "forests", as a new microfabrication material. We present details of a suite of related methods recently developed by our group: fabrication of CNT forest microstructures by thermal CVD from lithographically patterned catalyst thin films; self-directed elastocapillary densification of CNT microstructures; and replica molding of polymer microstructures using CNT composite master molds. In particular, our work shows that self-directed capillary densification ("capillary forming"), which is performed by condensation of a solvent onto the substrate with CNT microstructures, significantly increases the packing density of CNTs. This process enables directed transformation of vertical CNT microstructures into straight, inclined, and twisted shapes, which have robust mechanical properties exceeding those of typical microfabrication polymers. This in turn enables formation of nanocomposite CNT master molds by capillary-driven infiltration of polymers. The replica structures exhibit the anisotropic nanoscale texture of the aligned CNTs, and can have walls with sub-micron thickness and aspect ratios exceeding 50:1. Integration of CNT microstructures in fabrication offers further opportunity to exploit the electrical and thermal properties of CNTs, and diverse capabilities for chemical and biochemical functionalization ³.     

Laser microfabricated model surfaces for controlled cell growth

The relatively recent applications of microelectronics technology into the biological sciences arena has drastically revolutionized the field. New foreseeable applications include miniaturized, multiparametric biosensors for high performance multianalyte assays or DNA sequencing, biocomputers, and substrates for controlled cell growth (i.e. tissue engineering). The objectives of this work were to investigate a new method combining microphotolithographical techniques with laser excimer beam technology to create surfaces with well defined 3-D microdomains in order to delineate critical microscopic surface features governing material-cell interaction. Another obvious application of this study pertains to the fabrication of cell-based biosensors. Microfabricated surfaces were obtained with micron resolution, by "microsculpturing" polymer model surfaces using a laser excimer KrF beam coupled with a microlithographic projection technique. The laser beam after exiting a mask was focused onto the polymer target surface via an optical setup allowing for a 10-fold reduction of the mask pattern. Various 3-D micropatterned features were obtained at the micron level. Reproducible submicron features could also be obtained using this method. Subsequently, model osteoblast-like cells were plated onto the laser microfabricated surfaces in order to study the effects of particular surface microtopography on preferential cell deposition and orientation. Preferential cell deposition was observed on surfaces presenting "smooth" microtopographical transitions. This system may provide an interesting model for further insights into correlations between 3-D surface microtopography and cell response with new applications in the field biosensor, biomaterial and pharmaceutical engineering sciences (e.g. new cell based biosensors, controlled synthesis of immobilized cell derived active ingredients).     

Screening for novel biocatalysts

This review attempts to demonstrate the importance of goal-orientated screening for new biocatalysts. Examples of enzymes and microorganisms that have been developed and that have acquired commercial applications are described so as to illustrate the technological potential of biocatalysts. A survey of screening techniques and recently reported examples of screening from food, chemical, pharmaceutical and waste disposal applications etc. are also presented to demonstrate the feasibility of this approach for generating new biocatalysts. An appreciation of some of the difficulties involved, the achievements of Japanese researchers and some examples of the cornucopia of largely unrecognized and potentially valuable microbial activities are also given. An increased effort in screening would have the following benefits: an increased range of biocatalysts with different enzyme activities would be available and more biocatalysts with improved characteristics, suitable for use under industrial conditions, such as resistance to elevated temperatures, extremes of pH and organic solvents would be discovered. Secondly the manpower and other resources required to carry out screening programmes would be reduced, for instance by developing automated techniques. Thirdly, screening procedures would be made much more accessible to non-specialists. Fourthly, improved efforts and expertise in screening would supplement other emerging techniques such as protein engineering. The development of selective, non-random, goal-orientated screening techniques, methods of evaluating biocatalyst performance under operational conditions, and an approach that is more orientated towards commercially desirable goals are essential if these objectives are to be achieved. Screening of naturally occurring microorganisms still appears to be the best way to obtain new strains and/or enzymes for commercial applications. However, two major problems appear to exist. Firstly in identifying applications that are technically feasible and that have sufficient commercial potential to justify the research and development required to generate a new commercially viable biocatalyst and secondly the relatively small number of scientists outside Japan with skill and experience in screening for biocatalysts.     

Free Marine Natural Products Databases for Biotechnology and Bioengineering

Marine organisms and micro‐organisms are a source of natural compounds with unique chemical features. These chemical properties are useful for the discovery of new functions and applications of marine natural products (MNPs). To extensively exploit the potential implementations of MNPs, they are gathered in chemical databases that allow their study and screening for applications of biotechnological interest. However, the classification of MNPs is currently poor in generic chemical databases. The present availability of free‐access‐focused MNP databases is scarce and the molecular diversity of these databases is still very low when compared to the paid‐access ones. In this review paper, the current scenario of free‐access MNP databases is presented as well as the hindrances involved in their development, mainly compound dereplication. Examples and opportunities for using freely accessible MNP databases in several important areas of biotechnology are also assessed. The scope of this paper is, as well, to notify the latent potential of these information sources for the discovery and development of new MNPs in biotechnology, and push future efforts to develop a public domain MNP database freely available for the scientific community.     

The role of proteomics in toxicology: identification of biomarkers of toxicity by protein expression analysis

Proteomics, i.e. the high throughput separation, display and identification of proteins, has the potential to be a powerful tool in drug development. It could increase the predictability of early drug development and identify non-invasive biomarkers of toxicity or efficacy. This review provides an introduction to modern proteomics, with particular reference to applications in toxicology. A literature search was carried out to identify studies in two broad classes: screening/predictive toxicology, and mechanistic toxicology. The strengths and limitations of current methods and the likely impact of techniques in drug development are also considered. Proteomics can increase the speed and sensitivity of toxicological screening by identifying protein markers of toxicity. Proteomics studies have already provided insights into the mechanisms of action of a wide range of substances, from metals to peroxisome proliferators. Current limitations involving speed of throughput are being overcome by increasing automation and the development of new techniques. The isotope-coded affinity tag (ICAT) method appears particularly promising. The application of proteomics to drug development has given rise to the new field of pharmacoproteomics. New associations between proteins and toxicopathological effects are constantly being identified, and major progress is on the horizon as we move into the post-genomic era.     

Improving the ‘tool box’ for robust industrial enzymes

The speed of sequencing of microbial genomes and metagenomes is providing an ever increasing resource for the identification of new robust biocatalysts with industrial applications for many different aspects of industrial biotechnology. Using ‘natures catalysts’ provides a sustainable approach to chemical synthesis of fine chemicals, general chemicals such as surfactants and new consumer-based materials such as biodegradable plastics. This provides a sustainable and ‘green chemistry’ route to chemical synthesis which generates no toxic waste and is environmentally friendly. In addition, enzymes can play important roles in other applications such as carbon dioxide capture, breakdown of food and other waste streams to provide a route to the concept of a ‘circular economy’ where nothing is wasted. The use of improved bioinformatic approaches and the development of new rapid enzyme activity screening methodology can provide an endless resource for new robust industrial biocatalysts.This mini-review will discuss several recent case studies where industrial enzymes of ‘high priority’ have been identified and characterised. It will highlight specific hydrolase enzymes and recent case studies which have been carried out within our group in Exeter.