Subash C. B. Gopinath

An angular fluidic channel for prism-free surface-plasmon-assisted fluorescence capturing

Surface plasmon excitation provides stronger enhancement of the fluorescence intensity and better sensitivity than other sensing approaches but requires optimal positioning of a prism to ensure optimum output of the incident light. Here we describe a simple, highly sensitive optical sensing system combining surface plasmon excitation and fluorescence to address this limitation. V-shaped fluidic channels are employed to mimic the functions of a prism, sensing plate, and flow channel in a single setup. Superior performance is demonstrated for different biomolecular recognition reactions on a self-assembled monolayer, and the sensitivity reaches 100 fM for biotin-streptavidin interactions. Using an antibody as a probe, we demonstrate the detection of intact influenza viruses at 0.2 HA units ml⁻¹ levels. The convenient sensing system developed here has the advantages of being prism-free and requiring less sample (1-2 μl), making this platform suitable for use in situations requiring low sample volumes.

Strategies to characterize fungal lipases for applications in medicine and dairy industry.

Lipases are water-soluble enzymes that act on insoluble substrates and catalyze the hydrolysis of long-chain triglycerides. Lipases play a vital role in the food, detergent, chemical, and pharmaceutical industries. In the past, fungal lipases gained significant attention in the industries due to their substrate specificity and stability under varied chemical and physical conditions. Fungal enzymes are extracellular in nature, and they can be extracted easily, which significantly reduces the cost and makes this source preferable over bacteria. Soil contaminated with spillage from the products of oil and dairy harbors fungal species, which have the potential to secrete lipases to degrade fats and oils. Herein, the strategies involved in the characterization of fungal lipases, capable of degrading fatty substances, are narrated with a focus on further applications.

Monitoring biomolecular interactions on a digital versatile disk: a BioDVD platform technology.

A spinning-disk biosensor utilizing optical interference of reflected light from a multilayered structure, consisting of dielectric, metal, and optical phase-change thin films, is shown to have the potential to monitor various interactions on its surface. We refer to this platform as a BioDVD, since it utilizes the optical system of a digital versatile disk (DVD) to measure changes in reflected light intensity. Here, we demonstrated that nucleic acid hybridization and RNA-protein interactions can be analyzed efficiently, in a label-free environment, by measuring the reflected light intensity using a DVD-like mechanism. Moreover, our studies revealed that the detection sensitivity for the interactions on the BioDVD can be altered by shifting the state of the phase-change materials, where the amorphous state can be used for analysis and another state (crystalline) can be used both for recording information and selectively masking areas of the disk.

‘Spotted Nanoflowers’: Gold-seeded Zinc Oxide Nanohybrid for Selective Bio-capture

Hybrid gold nanostructures seeded into nanotextured zinc oxide (ZnO) nanoflowers (NFs) were created for novel biosensing applications. The selected ‘spotted NFs’ had a 30-nm-thick gold nanoparticle (AuNP) layer, chosen from a range of AuNP thicknesses, sputtered onto the surface. The generated nanohybrids, characterized by morphological, physical and structural analyses, were uniformly AuNP-seeded onto the ZnO NFs with an average length of 2–3 μm. Selective capture of molecular probes onto the seeded AuNPs was evidence for the specific interaction with DNA from pathogenic Leptospirosis-causing strains via hybridization and mis-match analyses. The attained detection limit was 100 fM as determined via impedance spectroscopy. High levels of stability, reproducibility and regeneration of the sensor were obtained. Selective DNA immobilization and hybridization were confirmed by nitrogen and phosphorus peaks in an X-ray photoelectron spectroscopy analysis. The created nanostructure hybrids illuminate the mechanism of generating multiple-target, high-performance detection on a single NF platform, which opens a new avenue for array-based medical diagnostics.

Generation of anti-influenza aptamers using the systematic evolution of ligands by exponential enrichment for sensing applications.

The systematic evolution of ligands by exponential enrichment (SELEX) is a selection process for identifying high-affinity selective molecules from a randomized combinatorial nucleic acid library against a wide range of target molecules. Using a pool of N25 RNA molecules, the SELEX process was performed against two targets from influenza viruses, namely, intact influenza B/Tokio/53/99 and hemagglutinin of infuluenza B Jilin/20/2003. The selection processes were evaluated by surface plasmon fluorescence spectroscopy (SPFS), and the result was compared to that obtained by a conventional radioisotope method. Clear discrimination among different selection cycles was displayed by SPFS, indicating that this method can be used as an alternative method of radioisotope labeling. The dissociation constant of the selected aptamers against the targets was in the low nanomolar range. The sensitivity of the selected aptamer against intact influenza B/Tokio/53/99 to detect the influenza virus was the low ng/mL level, an approximately 250-fold higher sensitivity than that of the commercially obtained antibody. The target binding sites on the aptamer were predicted by mapping analyses. The selected aptamer could discriminate other influenza strains, and the sensitivity of the selected aptamer was further confirmed by gold-nanoparticle-based sensing on a waveguide-mode sensor. This finding demonstrates that the selected aptamer would be useful for detecting influenza viruses at an early stage of infection and for the purpose of influenza surveillance.

Microbial Enzymes and Their Applications in Industries and Medicine 2014.

Enzymes are considered as a potential biocatalyst for a large number of reactions. Particularly, the microbial enzymes have widespread uses in industries and medicine. The microbial enzymes are also more active and stable than plant and animal enzymes. In addition, the microorganisms represent an alternative source of enzymes because they can be cultured in large quantities in a short time by fermentation and owing to their biochemical diversity and susceptibility to gene manipulation. Industries are looking for new microbial strains in order to produce different enzymes to fulfil the current enzyme requirements. This special issue covers ten articles including three review articles, mainly highlighting the importance and applications of biotechnologically and industrially valuable microbial enzymes. M. Dinarvand et al. in their paper optimized the conditions for overproduction of intraextracellular inulinase and invertase from the fungus Aspergillus niger ATCC 20611. Optimization is one of the most important criteria in developing any new microbial process. Response surface analysis is one of the vital tools to determine the optimal process conditions. This kind of design of a limited set of variables is advantageous compared to the conventional method. The response surface methodology was used for this optimization and achieved the increment until 16 times. This study would be highly useful for the potential application in fermentation industries. In this review, N. Gurung et al. have made an attempt to highlight the importance of different enzymes with a special focus on amylase and lipase. Enzymes generally increase the reaction rates by several million times than normal chemical reactions. Lipases play an important role in the food, detergent, chemical, and pharmaceutical industries. In the past, microbial lipases gained significant attention in the industries due to their substrate specificity and stability under varied conditions. Amylase is an enzyme that catalyses the breakdown of starch into sugars, abundant in the process of animal and human digestion. The major advantage of microbial amylases is being economical and easy to manipulate. Currently, much attention is paid to rapid development of microbial enzyme technology, and these enzymes are relatively more stable than the enzymes derived from plants and animals. P. Mukherjee and P. Roy in their paper have purified and characterized the enzyme hydrocarbon dioxygenase from Stenotrophomonas maltophilia PM102, which has a broad substrate specificity. They found that the presence of copper induces the enzyme activity to be 10.3-fold higher, and NADH induces the increment to be 14.96-fold. Proposed copper enhanced monooxygenase activity and Fourier transform-infrared (FT-IR) characterization of biotransformation products from trichloroethylene satisfy the production of industrially and medically important chemicals and make bioremediation more attractive by improving the development of this technology. C. Huynen et al. in their review paper discuss the importance of protein scaffold to develop hybrid enzymes. The paper discusses the use of class A betalactamase as versatile scaffolds to design hybrid enzymes mentioned as betalactamase hybrid proteins (BHPs), in which an external polypeptide, peptide, protein, or their fragment is inserted at various suitable positions. The paper highlights further how BHPs can be specifically designed to develop as bifunctional proteins to produce and characterize the proteins otherwise difficult to express, to determine the epitope of specific antibodies, to generate antibodies against nonimmunogenic epitopes, and to understand the structure/function relationship of proteins. The hybrid proteins can be applied to produce difficult-to-express peptides/proteins/protein fragments, to map epitopes, to display antigens, and to study protein structure/function relationships. Among other applications, BHPs could be an important player in biosensors and in affinity chromatography, drug screening, and drug targeting. P. Manivasagan et al. in their paper focus on purification and characterization of the protease from Streptomyces sp. MAB18. The authors have optimized the conditions for overproduction of protease using response surface methodology. They have also determined the molecular mass of purified enzyme and great activity and stability of enzyme in different pH and temperatures. Furthermore, the authors confirmed that the protease has an antioxidant ability. In industries, the poultry waste derived protease will be useful as a protein or as an antioxidant. The paper titled “β-Glucosidases from the fungus Trichoderma: an effeicient cellulose machinery in biotechnological applications” is a detailed review on β-glucosidases which are members of the cellulose enzyme complex described by P. Tiwari et al. The authors especially focus on β-glucosidases from the fungus Trichoderma, mostly used for the saccharification of cellulosic biomass for biofuel production. They describe the enzyme family, their classification, structural parameters, properties, and studies at the genomics and proteomics levels. In addition, by bypassing the low enzyme production with hypersecretory strains, they give an insight on using these strains for renewable energy sources like bioethanol production. They imply the importance of fungal β-glucosidases which might be successful for biofuel production in order to meet the need in energy crisis. A. Khoramnia et al. in their paper discuss yeast enzyme application for medium chain fatty acids (MCFAs) modification for industrial purpose and antibacterial applications. The paper focuses on the conceptualization, design, and assay of the enzyme produced from a Malaysian strain of Geotrichum candidum. With the modification on fatty acid processing using a naturally derived enzyme, a free lauric acid rich MCFAs can be obtained which can become a source of antibacterial use for both Gram-positive (Staphylococcus aureus) and Gram-negative (E. coli) bacteria which are difficult microbes due to some of their strains becoming drug resistant. They also describe that the higher lipolysis by the strain specific enzyme is associated with the increased moisture content in the reaction environment on coconut oil hydrolysis. M. A. Hassan et al. in their paper discuss isolation of Bacillus amyloliquefaciencs and B. subtilis from soil and production and characterization of keratinolytic protease. These bacteria were able to degrade the wool completely within 5 days and also produced the highest enzyme activity. The characterization studies confirmed that the enzyme is stable in a broad range of pH and temperatures. Furthermore, they confirmed that the keratinolytic proteases from isolated bacteria are stable in various organic solvents. In this review article, S. C. B. Gopinath et al. put different strategies to characterize fungal lipases for their role in industry and medicine. The advantage of fungal lipases is bestowed with their extracellular nature of production thus reducing the complexities and high operation cost comparing to other bacterial enzymes. The authors provide several illustrations to show how lipolysis can be utilized and put strategies for the characterization of fungal lipases that are capable of degrading fatty substances from different sources, with an effort to highlight further applications. This review would contribute to the isolation and characterization of lipase from various fungal sources and application of lipase for medical and dairy industry and degradation of fatty substance from oil spillages. A. Knob et al. in their paper focus on xylanses and discuss the purification and characterization of a xylanase produced by Penicillium glabrum using brewers spent grain as a substrate in their paper. This study is the first report as the characterization of xylanase was carried out by using such an agroindustrial waste. Furthermore, the researchers also determined the molecular mass of the purified xylanase, the enzyme activity and stability on various pH and temperature ranges, the optimal enzyme production conditions, and the effect of some metal ions and inhibitors on xylanase activity. The authors concluded that the use of substrate brewers spent grain for xylanase production not only decreased the amount of this waste but also reduced the xylanase production cost as desired in biotechnological processes. Periasamy Anbu Subash C. B. Gopinath Arzu Coleri Cihan Bidur Prasad Chaulagain

Biotechnological Aspects and Perspective of Microbial Keratinase Production

Keratinases are proteolytic enzymes predominantly active when keratin substrates are available that attack disulfide bridges in the keratin to convert them from complex to simplified forms. Keratinases are essential in preparation of animal nutrients, protein supplements, leather manufacture, textile processing, detergent formulation, feather meal processing for feed and fertilizer, the pharmaceutical and biomedical industries, and waste management. Accordingly, it is necessary to develop a method for continuous production of keratinase from reliable sources that can be easily managed. Microbial keratinase is less expensive than conventionally produced keratinase and can be obtained from fungi, bacteria, and actinomycetes. In this overview, the expansion of information about microbial keratinases and important considerations in keratinase production are discussed.

Feasibility of graphene in biomedical applications

Nanotechnology is the developing field, bringing the materials in the nanoscale level, has been applied in the interdisciplinary sciences. Different nanomaterials, such as gold, silver, zinc, copper and graphene are shown to have a wide range of applications. Among these, graphene is one of the faster upcoming two-dimensional nanomaterials utilized in various fields due to its positive features including the properties of thermal, electrical, strength and elasticity. Biomedical applications of graphene have been widely attested to be popular among academician and industrial partners for creating next generation medical systems and therapies. In this review, we selectively revealed the current applications of graphene in the interdisciplinary medical sciences.

Biopolymers Regulate Silver Nanoparticle under Microwave Irradiation for Effective Antibacterial and Antibiofilm Activities.

In the current study, facile synthesis of carboxymethyl cellulose (CMC) and sodium alginate capped silver nanoparticles (AgNPs) was examined using microwave radiation and aniline as a reducing agent. The biopolymer matrix embedded nanoparticles were synthesized under various experimental conditions using different concentrations of biopolymer (0.5, 1, 1.5, 2%), volumes of reducing agent (50, 100, 150 μL), and duration of heat treatment (30 s to 240 s). The synthesized nanoparticles were analyzed by scanning electron microscopy, UV-Vis spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy for identification of AgNPs synthesis, crystal nature, shape, size, and type of capping action. In addition, the significant antibacterial efficacy and antibiofilm activity of biopolymer capped AgNPs were demonstrated against different bacterial strains, Staphylococcus aureus MTCC 740 and Escherichia coli MTCC 9492. These results confirmed the potential for production of biopolymer capped AgNPs grown under microwave irradiation, which can be used for industrial and biomedical applications.

Low Temperature Annealed Zinc Oxide Nanostructured Thin Film-Based Transducers: Characterization for Sensing Applications.

The performance of sensing surfaces highly relies on nanostructures to enhance their sensitivity and specificity. Herein, nanostructured zinc oxide (ZnO) thin films of various thicknesses were coated on glass and p-type silicon substrates using a sol-gel spin-coating technique. The deposited films were characterized for morphological, structural, and optoelectronic properties by high-resolution measurements. X-ray diffraction analyses revealed that the deposited films have a c-axis orientation and display peaks that refer to ZnO, which exhibits a hexagonal structure with a preferable plane orientation (002). The thicknesses of ZnO thin films prepared using 1, 3, 5, and 7 cycles were measured to be 40, 60, 100, and 200 nm, respectively. The increment in grain size of the thin film from 21 to 52 nm was noticed, when its thickness was increased from 40 to 200 nm, whereas the band gap value decreased from 3.282 to 3.268 eV. Band gap value of ZnO thin film with thickness of 200 nm at pH ranging from 2 to 10 reduces from 3.263eV to 3.200 eV. Furthermore, to evaluate the transducing capacity of the ZnO nanostructure, the refractive index, optoelectric constant, and bulk modulus were analyzed and correlated. The highest thickness (200 nm) of ZnO film, embedded with an interdigitated electrode that behaves as a pH-sensing electrode, could sense pH variations in the range of 2-10. It showed a highly sensitive response of 444 μAmM-1cm-2 with a linear regression of R2 =0.9304. The measured sensitivity of the developed device for pH per unit is 3.72μA/pH.