Diagnosis of pleural tuberculosis by multi-targeted loop-mediated isothermal amplification assay based on SYBR Green I reaction: comparison with GeneXpert® MTB/RIF assay.

BACKGROUND: Diagnosis of pleural tuberculosis (TB) is tedious owing to its close resemblance with malignant pleural effusion and sparse bacterial load in clinical specimens. There is an immediate need to design a rapid and dependable diagnostic test to prevent unnecessary morbidity/mortality. RESEARCH DESIGN AND METHODS: A multi-targeted loop-mediated isothermal amplification (MT-LAMP) was deliberated using mpt64 and IS6110 to diagnose pleural TB within pleural fluids/biopsies. MT-LAMP products were analyzed by gel-based and visual detection methods, viz. SYBR Green I, SYBR Green I+deoxyuridine triphosphate uracil-N-glycosylase (dUTP-UNG), and dry methyl green reactions. RESULTS: In a pilot study, while assessing pleural TB/non-TB control subjects (n = 40), both SYBR Green I+dUTP-UNG/gel-based MT-LAMP assays exhibited better sensitivity/specificity than SYBR Green I and dry methyl green MT-LAMP. Since it is facile to work with SYBR Green I+dUTP-UNG than gel-based MT-LAMP, we validated the performance of SYBR Green I+dUTP-UNG in a higher number of specimens (n = 97), which revealed somewhat higher sensitivity (85.2 vs. 81.5%) and specificity (97.7 vs. 90.7%) than SYBR Green I MT-LAMP. Furthermore, the sensitivity attained by SYBR Green I+dUTP-UNG MT-LAMP was significantly higher (p < 0.001) than GeneXpert. CONCLUSIONS: Our SYBR Green I+dUTP-UNG MT-LAMP is a simple and reliable method to diagnose pleural TB, which may translate into a point-of-care test.

Recent advances in development of electrochemical biosensors for the detection of biogenic amines.

Biogenic amines (BAs) are widely found in food as a consequence of diverse factors including free amino acid availability, microbial production of decarboxylases, and variations in processing and storage conditions. Hence, BAs are considered as an important marker for determining the freshness and quality of food. Owing to the documentation of BAs in different dietary products, their numerous negative impacts on human health have reported to be a serious concern in past few decades. Therefore, the quantification of these chemical species in food becomes crucial as it can immensely contributes toward control of new episodes on food intoxication in humans. In this line, various chromatographic and colorimetric methods have been developed to detect BAs. However, these methods are in use from a longer time, still are limited by high cost, lengthy procedures, huge infrastructure and skilled personnel requirements that hinder their on-field application. In pursuit of a reliable method offering accurate detection of BAs, this review presents the state-of-the-art of electrochemical strategies for BAs sensing in food. The core of the review discusses about the widely employed electrochemical transducers, such as amperometric, potentiometric, impedimetric and conductometric-based BAs biosensors with significant findings of research work conducted previously. The application of electrochemical sensors to analyze BAs in different fields including food systems (fermented and non-fermented types) and smart packaging systems has been reviewed. Moreover, existing challenges and further available prospects for the development of rapid, facile, and sensitive electrochemical strategies for on-site determination of BAs have also been discussed.

Cell-free arsenic biosensors with applied nanomaterials: critical analysis.

Arsenic is a ubiquitously found metalloid in our ecosystem because of natural and anthropogenic activities. People exposed to a higher level of arsenic become susceptible to several disorders, including cancer. According to current statistics, the population chronically exposed to arsenic has surpassed 200 million. Therefore, its detection in our environment is of great importance. There are many analytical techniques for the assessment of arsenic in different kinds of environmental samples. Among these techniques, the biosensor is considered a convenient platform and a widely applied analytical device for rapid qualitative and quantitative analysis in the field of environmental monitoring, food safety, and disease diagnosis. Today, there is a trend of including nanomaterials in sensors and biosensors because it empowers researchers to explore new arsenic detection methods and to enhance their analytical capabilities. In this review article, we summarized the latest developments in arsenic biosensors in particular with emphasis on the works based on cell-free approaches that are protein/enzyme-based, DNA-based, and aptamer-based utilizing various transduction platforms. In the meantime, we compared the capabilities that were related to these cell-free arsenic biosensors. This review article also highlights the development and application of novel nanomaterials for arsenic detection.

A novel amperometric biosensor for rapid detection of ethanol utilizing gold nanoparticles and enzyme coupled PVC reaction cell.

This research was aimed at the fabrication of an improved enzyme-based amperometric biosensor for rapid quantification of ethanol. Alcohol oxidase (AOX) from Pichia pastoris was covalently immobilized on chemically treated polyvinylchloride (PVC) beaker and subsequently horseradish peroxidase (HRP), nafion (Nf), carboxylated multi-walled carbon nanotubes (c-MWCNTs), chitosan (CHIT) and gold nanoparticles (AuNPs) were immobilized onto Au electrode to fabricate a working electrode. The enzyme-coated PVC surface was analysed morphologically via scanning electron microscopy (SEM). At different stages of construction, the electrochemical properties of working electrode were deciphered by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor displayed optimal response in a short time span of 12 s at pH 7.5 and 35°C temperature. The working range exhibited by the proposed biosensor was 0.01-42 mM with a limit of detection (LOD) of 0.0001 µM and storage stability of 180 days at 4°C. When level of alcohol was evaluated in commercial samples via standard assay kit and existing biosensor, a good correlation (R2 = 0.98) was observed which authenticates its reliability.

Reusable Enzymatic Strip for Detection of Arsenic

HIGHLIGHTS Arsenic is considered as one of the highly hazardous elements in the environment and a serious carcinogen for the human health. An enzymatic method has been described by using arsenite oxidase for arsenic detection. Residual activity of the immobilized enzyme was 43% of the initial activity after being recycled 10 times.

Abstract Arsenic is considered as one of the highly hazardous elements in the environment and a serious carcinogen for the human health. More attention has taken towards the arsenic due to its presence in ground water in India, China, Bangladesh, Inner Mongolia and several other regions of the world. It's been a challenge to remove arsenic due to the lack of its efficient detection approach in the complicated environmental matrix. The proposed method describes an enzymatic method for arsenic determination using arsenite oxidase, which catalyzes the oxidation of arsenite to arsenate. Hence, a colorimetric PVC strip with immobilized arsenite oxidase has been developed to detect the arsenic concentration and also having potential for the field-testing. The influence of the optimal conditions i.e. pH, temperature, storage stability, and reusability of free and immobilized enzyme were evaluated and compared. The results have shown that the stabilities were significantly enhanced compared with free counterpart. Residual activity of the immobilized enzyme was 43% of the initial activity after being recycled 10 times. We approve that this novel low cost immobilized carrier presents a new approach in large scale applications and expected to act as a model for establishment of indigenous arsenic sensor in miniature form.

Biosensors for penicillin quantification: a comprehensive review.

Penicillins represent most commonly prescribed and salient part of antibacterial armamentarium. Due to lack of proper guidance and regulations, especially in developing countries they are indiscriminately applied in agricultural sectors (livestock farming, aquaculture and plants). Major concerns related to remaining residues of penicillins in food or environment are allergy, development of antibacterial resistance, environmental pollution, economical losses to food industries, consumers and food safety. Researchers of different disciplines are developing reliable detection methods for penicillins in various samples. Among them biosensors are attracting considerable attention primarily for their instant detection, convenience in application, on-site monitoring and portability. Nowadays, they are becoming more sophisticated with the help of modern approaches such as nanotechnology. This review article summarizes the research literature on advancements and recent trends in the field of biosensors for penicillins quantification till date. Different domains of biosensors (electrochemical, optical, mass-sensitive and thermal) are discussed for penicillins along with their applicability.

Biosynthesis, characterization, bactericidal and sporicidal activity of silver nanoparticles using the leaves extract of Litchi chinensis.

Biosynthesis of silver nanoparticles (AgNPs) using plant extracts has become a promising alternative to the conventional chemical synthesis approach. In this study, cost-effective synthesis of AgNPs was attempted using leaves extract of Litchi chinensis. Bio-reduction reaction for the synthesis of NPs was checked by confirming the presence of AgNPs in solution by UV-vis spectrophotometry and with further characterization by fourier-transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). Surface plasmon resonance (SPR) band showed absorption peak at 422 nm indicating the formation of AgNPs, and FTIR spectra confirmed the presence of biological molecules involved in AgNPs synthesis. TEM analysis revealed the spherical shape of AgNPs with particle size distribution in a range of 5-15 nm. Further, the biosynthesized AgNPs showed significant bactericidal and sporicidal activity against model spore former Bacillus subtilis. AgNPs at concentrations ranging from 25 to 100 µg/mL showed bactericidal activity with inhibition zone ranging from 4-19 mm and sporicidal activity at 100-200 µg/mL in a range of 4.46-61.6% with an exposure time of 2-8 h. These findings exhibit distinctive potential of biogenic AgNPs for their efficient use in developing novel bactericidal and sporicidal agent against spore forming bacilli.

Novel approach using activated cellulose film for efficient immobilization of purified diamine oxidase to enhance enzyme performance and stability.

The presence of various contaminants in foodstuffs has led to serious public health concerns. Diamine oxidase (DAO) has attracted tremendous attention for guarding food safety as well as clinical and environmental industries. In this study, DAO from Pisum sativum (Pea) seedlings was extracted and purified by dialysis and gel filtration. Purified DAO was covalently immobilized onto the surface of nitrocellulose membrane using glutaraldehyde. The obtained bioaffinity support has efficiently shown high yield immobilization of DAO from pea seedlings. The optimal conditions of free and immobilized DAO activity were evaluated against the substrate, Putrescine dihydrochloride. The influence of pH, temperature, storage stability, and reusability of immobilized enzyme with comparison to the free enzyme was studied and the results showed that the stabilities were significantly enhanced compared with free counterpart. Residual activity of the immobilized enzyme was 59% of the initial activity after being recycled 10 times. We approve that this novel low cost immobilized DAO carrier presents a new approach in large scale applications.

Enzymatic biosensors for the quantification of biogenic amines: a literature update.

The present review emphasizes on the quantification of biogenic amines (BAs) which are regarded as a quality indicator of food freshness or spoilage and for evaluating microbial action while food processing. BAs have various potential adverse effects on human health and they are widely found in varying concentrations in different food stuffs. In the quest for a reliable method for their precise detection, BA biosensors have emerged as an efficient tool which enables rapid and accurate assessment in miniature form. Various combinations of amine oxidase enzymes have been used for the fabrication of biosensors in order to enhance specific biorecognition and signal transduction. This article also summarizes the widely employed components used in the construction of a pertinent biosensor and the research results conducted previously. The meticulous description regarding the choice of transducers and the significant role of mediators in a high response biosensor has been reviewed. Moreover, it also encompasses the utilization of highly attractive electrolytic characteristics of nanoparticles to enhance the specificity and accuracy of BA biosensors.

Enzyme-Based Ultrasensitive Electrochemical Biosensors for Rapid Assessment of Nitrite Toxicity: Recent Advances and Perspectives.

In the present era of rapid international globalization and industrialization, intensive use of nitrite as a fertilizing agent in agriculture, preservative, dyeing agent, food additive and as corrosion inhibitor in industrial sectors is adversely effecting environment, natural habitats and human health. The issue of toxicity and carcinogenicity due to excessive ingestion of nitrites via the dietary intake has led to an imminent need for its efficient real-time monitoring in situ. Nitrite detection employing electrochemical biosensors has been gaining high credibility in the field of clinical research. Nitrite biosensors have emerged as an outstanding choice for portable point of care testing of nitrite quantification owing to the excellent properties, such as rapidity, miniaturization, ultra-low limits of detection, multiplexing and enhanced detection sensitivity. The article is enclosed with an interesting outlook on latest emerging trends in the development of nitrite biosensors utilizing nanomaterials, such as metal nanoparticles, carbon nanotubes, metal oxide nanoparticles, nanocomposites, polymers and biomaterials. The present review embarks on the highlights relevant to the nitrite quantification in real samples, then proceeds with a meticulous description of the most pertinent electrochemical nitrite biosensors, which have been proposed by adopting diverse materials and strategies of fabrication and finally end with the achievements and future outlook signifying the application of these nanoengineered biosensors for environmental surveillance and human safety.

A novel amperometric bienzymatic biosensor based on alcohol oxidase coupled PVC reaction cell and nanomaterials modified working electrode for rapid quantification of alcohol.

A new amperometric sensor has been fabricated for sensitive and rapid quantification of ethanol. The biosensor assembly was prepared by covalently immobilizing alcohol oxidase (AOX) from Pichia pastoris onto chemically modified surface of polyvinylchloride (PVC) beaker with glutaraldehyde as a coupling agent followed by immobilization of horseradish peroxidase (HRP), silver nanoparticles (AgNPs), chitosan (CHIT), carboxylated multi-walled carbon nanotubes (c-MWCNTs) and nafion (Nf) nanocomposite onto the surface of Au electrode (working electrode). Owing to properties such as chemical inertness, light weight, weather resistance, corrosion resistance, toughness and cost-effectiveness, PVC membrane has attracted a growing interest as a support for enzyme immobilization in the development of biosensors. The amperometric biosensor displayed optimum response within 8 s at pH 7.5 and 35°C temperature. A linear response to alcohol in the range of 0.01mM-50 mM and 0.0001 µM as a minimum limit of detection was displayed by the proposed biosensor with excellent storage stability (190 days) at 4°C. The sensitivity of the sensor was found to be 155 µA mM-1 cm-2. A good correlation (R2 = 0.99) was found between alcohol level in commercial samples as evaluated by standard ethanol assay kit and the current biosensor which validates its performance.

Recent trends and perspectives in enzyme based biosensor development for the screening of triglycerides: a comprehensive review.

Clinical manifestations of the elevated plasma triacylglycerol (TG) include a greater prevalence of atherosclerotic heart disease, acute pancreatitis, diabetes mellitus, hypertension, and ischemic vascular disease. Hence, these significant health troubles have attracted scientific attention for the precise detection of TG in biological samples. Numerous techniques have been employed to quantify TG over many decades, but biosensors hold the leading position owing to their superior traits such as highly specific recognition for target molecules, accuracy, minituarization, small sample requirement and rapid response. Enzyme-based electrochemical biosensors represent an instantaneous resolution for the foremost bottlenecks constraining laboratory prototypes to reach real time bedside applications. We highlight the choice of transducers and constructive strategies to design high-performance biosensor for the quantification of triglycerides in sera and early diagnosis of health problems related to it. In the present review, a small effort has been made to emphasize the significant role of enzymes, nanostructured metal oxides, graphene, conducting polypyrrole, nanoparticles, porous silicon, EISCAP and ENFET in enabling TG biosensors more proficient and taking a revolutionary step forward.

Alcohol quantification: recent insights into amperometric enzyme biosensors.

Biosensors are the switching channels that make sense. The biosensors have found an empirical role in health applications (e.g. clinical diagnostics) as they represent the technological counterpart of living senses. On a global scale, alcohol analysis is indispensable for criminal justice systems, monitoring medical conditions of HIV patients & pregnant women as well as public safety issues regarding pilots, metro drivers, doctors etc. For addressing the clinical and toxicological problems, much advancement in the improvement of biosensors have been witnessed in recent decades. Currently, electrochemical biosensors dominate the field which harnesses the synergistic action of enzymes and nanomaterials for the analysis of ethanol. The enzymatic biosensors are the most explored biosensing devices among all the types of biosensors, and employment of nanomaterials has paved a way to the further improvements in this gem of a discovery. The relative comparison to precise the alcohol biosensors has been aptly discussed in the review on the basis of several analytical properties including fabrication, linearity, sensitivity, response time, detection limit as well as storage stability. Finally, the recent trends and emerging future prospects of alcohol biosensors have been reviewed.

Recent approaches to ameliorate selectivity and sensitivity of enzyme based cholesterol biosensors: a review.

The healthcare area is often reluctant to execute new technology unless they are proven to be safe, constructive and secure. Eventually, an aspiration stands for providing point-of-care testing service to allow a better estimation of the biochemical levels of a patient that entails an insistent remedial action. With increasing mortality rate due to cardiovascular diseases (CVDs) in present scenario, it has become the need of hour to develop more advance methods for their diagnosis, so that it can be determined at sensitive levels and can be prevented from being fatal. Elevated level of cholesterol in blood stream is one of the utmost risk factors which lead to CVDs. Discernible from the vast research in this field, worth of cholesterol biosensors is already recognized and flourished in the clinical analysis of brain and cardiac vascular diseases. It necessitates unremitting progress in the development of biosensing technology towards fabrication, miniaturization and multiplexing ability of cholesterol quantification devices so that they can endow with lab-on-chip-analysis systems to the medical field. Different strategies have been meticulously explored for the engineering of cholesterol biosensors utilizing nanocomposites, conducting polymers, nanotubes and nanoparticles. Foremost, this article reviews the contemporary evolution in cholesterol biosensors, which encompass various strategies for immobilization of enzymes and roles of various matrices and artificial mediators used for the biosensor fabrication. Still there remains an enormous challenge to congregate the demands of performance and yield in a cost effective manner for its application in successful treatments of CVDs.

Bilirubin enzyme biosensor: potentiality and recent advances towards clinical bioanalysis.

Bilirubin detection plays a major role in healthcare. Its high concentration in human serum is lethal and must be determined accurately. Clinically, it is vital for assessing patients with deleterious health conditions such as jaundice or icterus, hepatitis, mental disorders, cerebral palsy and brain damage especially in the case of neonates. In evaluating the drawbacks regarding the conventional methodology of bilirubin detection, there is need for a superior analytical tool. Bilirubin oxidase (BOx)-based sensors have been designed for the ultrasensitive analysis of bilirubin and quality deliverance of treatment and this review highlights the different mechanisms of bilirubin detection using different modified electrodes. Further, it also addresses the exploitation of highly attractive electrocatalytic properties of elite nanoparticles such as gold and zirconia- coated silica nanoparticles in enhancing the reproducibility and specificity of bilirubin biosensors.

Fabrication and Optimization of ChE/ChO/HRP-AuNPs/c-MWCNTs Based Silver Electrode for Determining Total Cholesterol in Serum.

The developed method used three enzymes comprised of cholesterol esterase, cholesterol oxidase, and peroxidase for fabrication of amperometric biosensor in order to determine total cholesterol in serum samples. Gold nanoparticles (AuNPs) and carboxylated multiwall carbon nanotubes (cMWCNTs) were used to design core of working electrode, having covalently immobilized ChO, ChE, and HRP. Polyacrylamide layer was finally coated on working electrode in order to prevent enzyme leaching. Chemically synthesised Au nanoparticles were subjected to transmission electron microscopy (TEM) for analysing the shape and size of the particles. Working electrode was subjected to FTIR and XRD. The combined action of AuNP and c-MWCNT showed enhancement in electrocatalytic activity at a very low potential of 0.27 V. The pH 7, temperature 40°C, and response time of 20 seconds, respectively, were observed. The biosensor shows a broad linear range from 0.5 mg/dL to 250 mg/dL (0.01 mM-5.83 mM) with minimum detection limit being 0.5 mg/dL (0.01 mM). The biosensor showed reusability of more than 45 times and was stable for 60 days. The biosensor was successfully tested for determining total cholesterol in serum samples amperometrically with no significant interference by serum components.

Preparation of electrochemical biosensor for detection of organophosphorus pesticides.

Polyvinyl chloride (PVC) can be used to develop reaction beaker which acts as electrochemical cell for the measurement of OP pesticides. Being chemically inert, corrosion resistant, and easy in molding to various shapes and size, PVC can be used for the immobilization of enzyme. Organophosphorus hydrolase was immobilized covalently onto the chemically activated inner surface of PVC beaker by using glutaraldehyde as a coupling agent. The carbon nanotubes paste working electrode was constructed for amperometric measurement at a potential of +0.8 V. The biosensor showed optimum response at pH 8.0 with incubation temperature of 40°C. K m and I max for substrate (methyl parathion) were 322.58 µM and 1.1 µA, respectively. Evaluation study showed a correlation of 0.985, which was in agreement with the standard method. The OPH biosensor lost 50% of its initial activity after its regular use for 25 times over a period of 50 days when stored in 0.1 M sodium phosphate buffer, pH 8.0 at 4°C. No interference was observed by interfering species.

ß-sitosterol in different parts of Saraca asoca and herbal drug ashokarista: Quali-quantitative analysis by liquid chromatography-mass spectrometry.

ß-sitosterol is an important component in food and herbal products and beneficial in hyperlipidemia. Its higher concentrations in serum may lead to coronary artery disease in case of sitosterolemia. Therefore, it is essential to determine the quantity of ß-sitosterol in food and herbal drugs. Saraca asoca and its preparations have been widely used by traditional healers are also a source of ß-sitosterol. In the present study, quantitative estimation of ß-sitosterol present in hot and cold water extracts of bark, regenerated bark, leaves and flowers of the S. asoca and Ashokarista drugs were carried out first time using high performance liquid chromatography coupled (HPLC) with quadrupole time-of-flight mass spectrometry. Different concentrations of ß-sitosterol and crude extracts were estimated by HPLC and targeted mass spectrometry. Standard curve for ß-sitosterol was prepared from the intensities of transitions (397.50 → 147.0987 m/z) having regression coefficient (r (2)) 0.9952. Out of eight extracts and two drugs used in the study bark water, leaves water and leaves hot water extracts were found to have a considerable quantity of ß-sitosterol, i.e. 170, 123.5 and 19.3 ng/mL, respectively. The results showed significant differences in the distribution of ß-sitosterol among different organs of S. asoca and drugs prepared from its bark. HPLC/electrospray ionizationmass spectroscopy method is accurate, reproducible and requires less specimen, sample preparation and analysis time over HPLC assay. This type of approaches could be helpful for the quality control of herbal medicines and provides necessary information for the rational utilization of plant resources.

Acetylcholinesterase biosensors for electrochemical detection of organophosphorus compounds: a review.

The exponentially growing population, with limited resources, has exerted an intense pressure on the agriculture sector. In order to achieve high productivity the use of pesticide has increased up to many folds. These pesticides contain organophosphorus (OP) toxic compounds which interfere with the proper functioning of enzyme acetylcholinesterase (AChE) and finally affect the central nervous system (CNS). So, there is a need for routine, continuous, on spot detection of OP compounds which are the main limitations associated with conventional analytical methods. AChE based enzymatic biosensors have been reported by researchers as the most promising tool for analysis of pesticide level to control toxicity and for environment conservation. The present review summarises AChE based biosensors by discussing their characteristic features in terms of fabrication, detection limit, linearity range, time of incubation, and storage stability. Use of nanoparticles in recently reported fabrication strategies has improved the efficiency of biosensors to a great extent making them more reliable and robust.

Antibodies raised against hemolymph of Anopheles culicifacies reduce the fecundity and malaria parasite development.

BACKGROUND & OBJECTIVES: Several studies have been made to study the effect of antisera raised against different tissues (hemolymh, ovary, midgut and salivary glands) on the fecundity and malaria parasite development in the different species of mosquitoes but there are no reports on the antisera raised against the hemolymph of Anopheles culicifacies, the principal malaria vector in India accounting for 65% of malaria cases. Hence, an attempt was made to study the same and evaluate its impact on malaria parasite development. METHODS: Polyclonal and multifactorial antibodies were produced in rabbits against heterogenous mixture of hemolymph proteins. Antibodies against hemolymph proteins were screened for their potential to influence reproductive performance of mosquitoes. Antibody titer in rabbit serum was determined by ELISA and putative candidate antigens were identified in the hemolymph of An. culicifacies by western blotting. Cross reactivity amongst various tissues vis-a-vis hemolymph protein was also identified. In addition, a significant reduction in oocyst development was also observed in An. culicifacies mosquitoes that ingested antihemolymph antibodies along with Plasmodium vivax. RESULTS: The maximum reduction in fecundity (57%) was observed during fourth week, after the last booster and number of oocyts per infected mosquito reduced by 73.35% in the group of mosquitoes that ingested antihemolymph antibodies along with the infected blood meal respectively. However, the ingestion of antibodies against hemolymph proteins did not have significant influence on hatchability. Antisera raised against hemolymph proteins of An. culicifacies recognized 11 polypeptides by western blotting. INTERPRETATION & CONCLUSION: During the present study, 11 putative candidate antigens were identified in the hemolymph of An. culicifacies, against which antibodies produced significantly reduced the fecundity by 57%. In addition, a significant reduction in oocyst development was also observed in An. culicifacies that ingested antihemolymph antibodies along with P.vivax.