Thangavel Lakshmipriya

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.

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.

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.

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.

Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay

Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors.

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.

Cell-targeting aptamers act as intracellular delivery vehicles

Aptamers are single-stranded nucleic acids or peptides identified from a randomized combinatorial library through specific interaction with the target of interest. Targets can be of any size, from small molecules to whole cells, attesting to the versatility of aptamers for binding a wide range of targets. Aptamers show drug properties that are analogous to antibodies, with high specificity and affinity to their target molecules. Aptamers can penetrate disease-causing microbial and mammalian cells. Generated aptamers that target surface biomarkers act as cell-targeting agents and intracellular delivery vehicles. Within this context, the “cell-internalizing aptamers” are widely investigated via the process of cell uptake with selective binding during in vivo systematic evolution of ligands by exponential enrichment (SELEX) or by cell-internalization SELEX, which targets cell surface antigens to be receptors. These internalizing aptamers are highly preferable for the localization and functional analyses of multiple targets. In this overview, we discuss the ways by which internalizing aptamers are generated and their successful applications. Furthermore, theranostic approaches featuring cell-internalized aptamers are discussed with the purpose of analyzing and diagnosing disease-causing pathogens.

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.

Biotechnological Processes in Microbial Amylase Production

Amylase is an important and indispensable enzyme that plays a pivotal role in the field of biotechnology. It is produced mainly from microbial sources and is used in many industries. Industrial sectors with top-down and bottom-up approaches are currently focusing on improving microbial amylase production levels by implementing bioengineering technologies. The further support of energy consumption studies, such as those on thermodynamics, pinch technology, and environment-friendly technologies, has hastened the large-scale production of the enzyme. Herein, the importance of microbial (bacteria and fungi) amylase is discussed along with its production methods from the laboratory to industrial scales.