Marimuthu Citartan

Assays for aptamer-based platforms

Aptamers are single stranded DNA or RNA oligonucleotides that have high affinity and specificity towards a wide range of target molecules. Aptamers have low molecular weight, amenable to chemical modifications and exhibit stability undeterred by repetitive denaturation and renaturation. Owing to these indispensable advantages, aptamers have been implemented as molecular recognition element as alternative to antibodies in various assays for diagnostics. By amalgamating with a number of methods that can provide information on the aptamer-target complex formation, aptamers have become the elemental tool for numerous biosensor developments. In this review, administration of aptamers in applications involving assays of fluorescence, electrochemistry, nano-label and nano-constructs are discussed. Although detection strategies are different for various aptamer-based assays, the core of the design strategies is similar towards reporting the presence of specific target binding to the corresponding aptamers. It is prognosticated that aptamers will find even broader applications with the development of new methods of transducing aptamer target binding.

Aptamers as a replacement for antibodies in enzyme-linked immunosorbent assay

The application of antibodies in enzyme-linked immunosorbent assay (ELISA) is the basis of this diagnostic technique which is designed to detect a potpourri of complex target molecules such as cell surface antigens, allergens, and food contaminants. However, development of the systematic evolution of Ligands by Exponential Enrichment (SELEX) method, which can generate a nucleic acid-based probe (aptamer) that possess numerous advantages compared to antibodies, offers the possibility of using aptamers as an alternative molecular recognition element in ELISA. Compared to antibodies, aptamers are smaller in size, can be easily modified, are cheaper to produce, and can be generated against a wide array of target molecules. The application of aptamers in ELISA gives rise to an ELISA-derived assay called enzyme-linked apta-sorbent assay (ELASA). As with the ELISA method, ELASA can be used in several different configurations, including direct, indirect, and sandwich assays. This review provides an overview of the strategies involved in aptamer-based ELASA.

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

Sensing strategies for influenza surveillance

Influenza viruses, which are RNA viruses belonging to the family Orthomyxoviridae, cause respiratory diseases in birds and mammals. With seasonal epidemics, influenza spreads all over the world, resulting in pandemics that cause millions of deaths. Emergence of various types and subtypes of influenza, such as H1N1 and H7N9, requires effective surveillance to prevent their spread and to develop appropriate anti-influenza vaccines. Diagnostic probes such as glycans, aptamers, and antibodies now allow discrimination among the influenza strains, including new subtypes. Several sensors have been developed based on these probes, efforts made to augment influenza detection. Herein, we review the currently available sensing strategies to detect influenza viruses.

Lipase-Secreting Bacillus Species in an Oil-Contaminated Habitat: Promising Strains to Alleviate Oil Pollution

Lipases are of great interest for different industrial applications due to their diversity and versatility. Among different lipases, microbial lipases are preferable due to their broad substrate specificity, and higher stability with lower production costs compared to the lipases from plants and animals. In the past, a vast number of bacterial species have been reported as potential lipases producers. In this study, the lipases-producing bacterial species were isolated from an oil spillage area in the conventional night market. Isolated species were identified as Bacillus species by biochemical tests which indicate their predominant establishment, and further screened on the agar solid surfaces using lipid and gelatin as the substrates. Out of the ten strains tested, four potential strains were subjected to comparison analysis of the lipolytic versus proteolytic activities. Strain 10 exhibited the highest lipolytic and proteolytic activity. In all the strains, the proteolytic activity is higher than the lipolytic activity except for strain 8, suggesting the possibility for substrate-based extracellular gene induction. The simultaneous secretion of both the lipase and protease is a mean of survival. The isolated bacterial species which harbour both lipase and protease enzymes could render potential industrial-based applications and solve environmental issues.

Aptahistochemistry in diagnostic pathology: technical scrutiny and feasibility

Antibodies have been the workhorse for diagnostic immunohistochemistry to specifically interrogate the expression of certain protein to aid in histopathological diagnosis. This review introduces another dimension of histochemistry that employs aptamers as the core tool, the so-called aptahistochemistry. Aptamers are an emerging class of molecular recognition elements that could recapitulate the roles of antibodies. The many advantageous properties of aptamers suited for this diagnostic platform are scrutinized. An in-depth discussion on the technical aspects of aptahistochemistry is provided with close step-by-step comparison to the more familiarized immunohistochemical procedures, namely functionalization of the aptamer as a probe, antigen retrieval, optimization with emphasis on incubation parameters and visualization methods. This review offers rationales to overcome the anticipated challenges in transition from immunohistochemistry to aptahistochemistry, which is deemed feasible for an average diagnostic pathology laboratory.

Use of UV-vis-NIR Spectroscopy to monitor label-free Interaction between Molecular Recognition Elements and Erythropoietin on a Gold-coated Polycarbonate Platform

Label-free-based detection is pivotal for real-time monitoring of biomolecular interactions and to eliminate the need for labeling with tags that can occupy important binding sites of biomolecules. One simplest form of label-free-based detection is ultraviolet-visible-near-infrared (UV-vis-NIR) spectroscopy, which measure changes in reflectivity as a means to monitor immobilization and interaction of biomolecules with their corresponding partners. In biosensor development, the platform used for the biomolecular interaction should be suitable for different molecular recognition elements. In this study, gold (Au)-coated polycarbonate was used as a platform and as a proof-of-concept, erythropoietin (EPO), a doping substance widely abused by the athletes was used as the target. The interaction of EPO with its corresponding molecular recognition elements (anti-EPO monoclonal antibody and anti-EPO DNA aptamer) is monitored by UV-vis-NIR spectroscopy. Prior to this, to show that UV-vis-NIR spectroscopy is a suitable method for measuring biomolecular interaction, the interaction between biotin and streptavidin was demonstrated via this strategy and reflectivity of this interaction decreased by 25%. Subsequent to this, interaction of the EPO with anti-EPO monoclonal antibody and anti-EPO DNA aptamer resulted in the decrease of reflectivity by 5% and 10%, respectively. The results indicated that Au-coated polycarbonate could be an ideal biosensor platform for monitoring biomolecular interactions using UV-vis-NIR spectroscopy. A smaller version of the Au-coated polycarbonate substrates can be derived from the recent set-up, to be applied towards detecting EPO abuse among atheletes.

Monitoring recombinant human erythropoietin abuse among athletes

The illegal administration of recombinant human erythropoietin (rHuEPO) among athletes is largely preferred over blood doping to enhance stamina. The advent of recombinant DNA technology allowed the expression of EPO-encoding genes in several eukaryotic hosts to produce rHuEPO, and today these performance-enhancing drugs are readily available. As a mimetic of endogenous EPO (eEPO), rHuEPO augments the oxygen carrying capacity of blood. Thus, monitoring the illicit use of rHuEPO among athletes is crucial in ensuring an even playing field and maintaining the welfare of athletes. A number of rHuEPO detection methods currently exist, including measurement of hematologic parameters, gene-based detection methods, glycomics, use of peptide markers, electrophoresis, isoelectric focusing (IEF)-double immunoblotting, aptamer/antibody-based methods, and lateral flow tests. This review gleans these different strategies and highlights the leading molecular recognition elements that have potential roles in rHuEPO doping detection.

Non-Protein Coding RNA Genes as the Novel Diagnostic Markers for the Discrimination of Salmonella Species Using PCR

Salmonellosis, a communicable disease caused by members of the Salmonella species, transmitted to humans through contaminated food or water. It is of paramount importance, to generate accurate detection methods for discriminating the various Salmonella species that cause severe infection in humans, including S. Typhi and S. Paratyphi A. Here, we formulated a strategy of detection and differentiation of salmonellosis by a multiplex polymerase chain reaction assay using S. Typhi non-protein coding RNA (sRNA) genes. With the designed sequences that specifically detect sRNA genes from S. Typhi and S. Paratyphi A, a detection limit of up to 10 pg was achieved. Moreover, in a stool-seeding experiment with S. Typhi and S. Paratyphi A, we have attained a respective detection limit of 15 and 1.5 CFU/mL. The designed strategy using sRNA genes shown here is comparatively sensitive and specific, suitable for clinical diagnosis and disease surveillance, and sRNAs represent an excellent molecular target for infectious disease.

Current and Potential Developments of Cortisol Aptasensing towards Point-of-Care Diagnostics (POTC)

Anxiety is a psychological problem that often emerges during the normal course of human life. The detection of anxiety often involves a physical exam and a self-reporting questionnaire. However, these approaches have limitations, as the data might lack reliability and consistency upon application to the same population over time. Furthermore, there might be varying understanding and interpretations of the particular question by the participant, which necessitating the approach of using biomarker-based measurement for stress diagnosis. The most prominent biomarker related to stress, hormone cortisol, plays a key role in the fight-or-flight situation, alters the immune response, and suppresses the digestive and the reproductive systems. We have taken the endeavour to review the available aptamer-based biosensor (aptasensor) for cortisol detection. The potential point-of-care diagnostic strategies that could be harnessed for the aptasensing of cortisol were also envisaged.