Facile synthesis of gold-silver alloy nanoparticles for application in metal enhanced bioluminescence.

In the present study we explored metal enhanced bioluminescence in luciferase enzymes for the first time. For this purpose a simple and reproducible one pot synthesis of gold-silver alloy nanoparticles was developed. By changing the molar ratio of tri-sodium citrate and silver nitrate we could synthesize spherical Au-Ag colloids of sizes ranging from 10 to 50 nm with a wide range of localized surface plasmon resonance (LSPR) peaks (450-550 nm). The optical tunability of the Au-Ag colloids enabled their effective use in enhancement of bioluminescence in a luminescent bacterium Photobacterium leiognathi and in luciferase enzyme systems from fireflies and bacteria. Enhancement of bioluminescence was 250% for bacterial cells, 95% for bacterial luciferase and 52% for firefly luciferase enzyme. The enhancement may be a result of energy transfer or plasmon induced enhancement. Such an increase can lead to higher sensitivity in detection of bioluminescent signals with potential applications in bio-analysis.

ATPase inhibitor based luciferase assay for prolonged and enhanced ATP pool measurement as an efficient fish freshness indicator.

The nucleotide degradation pathway in somatic cells leads to the accumulation of products such as hypoxanthine and inosine, which are commonly used as fish and meat freshness indicators. Assays based on these molecules cannot differentiate the postmortem time over a short period of time (5-10 h). Further, quantification of these degradation products is cumbersome, costly and time-consuming. For the proposed assay, optimal concentrations of 30 and 2 mM, respectively, for the ATPase inhibitors sodium orthovanadate and EDTA were found. Further, it was observed that a firefly luciferase based assay could enhance the sensitivity levels up to 165-fold at 30 °C. In addition, it was observed that the sensitivity for ATP assay was enhanced up to 60-fold even after 12 h. The limit of detection for the ATP assay was 1 pM, unlike other conventional methods, which are sensitive only up to micromolar levels. Moreover, as little as 0.044 g fish fillet was required for the assay, and no time-consuming sample preparation was necessary. Luminescence of prolonged duration was observed in harvested fish kept at -20 °C in comparison with fish kept at 4 and 30 °C, which reflects the shelf life of fish preserved at lower temperatures.

Performance analysis and modelling of circular jets aeration in an open channel using soft computing techniques.

Dissolved oxygen (DO) is an important parameter in assessing water quality. The reduction in DO concentration is the result of eutrophication, which degrades the quality of water. Aeration is the best way to enhance the DO concentration. In the current study, the aeration efficiency (E20) of various numbers of circular jets in an open channel was experimentally investigated for different channel angle of inclination (θ), discharge (Q), number of jets (Jn), Froude number (Fr), and hydraulic radius of each jet (HRJn). The statistical results show that jets from 8 to 64 significantly provide aeration in the open channel. The aeration efficiency and input parameters are modelled into a linear relationship. Additionally, utilizing WEKA software, three soft computing models for predicting aeration efficiency were created with Artificial Neural Network (ANN), M5P, and Random Forest (RF). Performance evaluation results and box plot have shown that ANN is the outperforming model with correlation coefficient (CC) = 0.9823, mean absolute error (MAE) = 0.0098, and root mean square error (RMSE) = 0.0123 during the testing stage. In order to assess the influence of different input factors on the E20 of jets, a sensitivity analysis was conducted using the most effective model, i.e., ANN. The sensitivity analysis results indicate that the angle of inclination is the most influential input variable in predicting E20, followed by discharge and the number of jets.

Aptamer--nanoparticle-based chemiluminescence for p53 protein.

A simple colorimetric biosensing technique based on the interaction of gold nanoparticles (AuNPs) with the aptamer was developed for detection of p53, a tumor suppressor protein, in the current study. Aggregation of AuNPs was induced by desorption of the p53 binding RNA aptamer from the surface of AuNPs as a result of the aptamer target interaction leading to the color change of AuNPs from red to purple. The detection limit of p53 protein by the colorimetric approach was 0.1 ng/ml after successful optimization of the amount of aptamer, AuNPs, salts, and incubation time. Furthermore, the catalytic activity of the aggregated AuNPs was greatly enhanced by chemiluminescence (CL) reaction, where the detection limit was enhanced to 10 pg/ml with a regression coefficient of R2 = 0.9907. Here the sensitivity was increased by 10-fold compared with the AuNP-based colorimetric method. Hence, the sensitivity of detection was increased by employing CL, by using the catalytic activity of aggregated AuNPs, on the luminol-hydrogen peroxide reaction. Thus, the combination of colorimetric and CL-based aptasensor can be of great advantage in increasing the sensitivity of detection for any target analyte.

Biosensors in food processing.

Optical based sensing systems that measure luminescence, fluorescence, reflectance and absorbance, etc., are some of the areas of applications of optical immunosensors. Immunological methods rely on specific binding of an antibody (monoclonal, polyclonal or engineered) to an antigen. Detection of specific microorganisms and microbial toxins requires immobilization of specific antibodies onto a given transducer that can produce signal upon attachment of typical microbe/microbial toxins. Inherent features of immunosensors such as specificity, sensitivity, speed, ease and on-site analysis can be made use for various applications. Safety of food and environment has been a major concern of food technologists and health scientists in recent years. There exists a strong need for rapid and sensitive detection of different components of foods and beverages along with the food borne and water borne pathogens, toxins and pesticide residues with high specificity. Biosensors present attractive, efficient alternative techniques by providing quick and reliable performances. There is a very good potential for application of biosensors for monitoring food quality and safety in food and bioprocessing industries in India.

A review: Aeration efficiency of hydraulic structures in diffusing DO in water.

This paper contemplates the review of aeration efficiency with commonly used different aeration systems such as Venturi flumes, Weirs, Conduits, Stepped channels, In Venturi Aeration, the SAE value grows fast with the number of air holes. In Weir Aeration, it was found that among all the different labyrinth weir structure, triangular notch weirs are known for the optimum results for air entrainment. The ANN model was developed with parameters discharge (Q) and tail water depth (Tw) which showed that Q is more influential parameter than Tw. In conduits structure, it was found that circular high head gated conduits have better aeration performance than other conduits. Aeration efficiency in Stepped channels cascades may range from 30% to 70%. The sensitivity analysis with ANN model showed that discharge (Q) followed by number of steps (N) was the most influential parameter in E20. Bubble size was the important parameter to undertake when using bubble diffuser. The oxygen transfer efficiency (OTE) in jet diffusers was predicted developing an ANN model. It was found in sensitivity analysis that the input of 'velocity' is highly sensitive to OTE. According to literature, jets can provide OTE in the range of 1.91- 21.53kgO2/kW-hr.

Nano RNA aptamer wire for analysis of vitamin B12.

A simple and stable RNA aptamer-based colorimetric sensor for the detection of vitamin B12 using gold nanoparticles (AuNPs) has been proposed. Vitamin B12 belongs to the B vitamin group and prevents pernicious anemia, which is caused by vitamin B12 deficiency. A highly stable RNA aptamer that binds to vitamin B12 was employed by structural modification of 2'-hydroxyl group of ribose to 2'-flouro in all pyrimidines indicated in lowercase in 35-mer aptamer (5' GGA Acc GGu GcG cAu AAc cAc cuc AGu GcG AGc AA 3'). Aggregation of AuNPs was specifically induced by desorption of the vitamin B12 binding RNA aptamer from the surface of AuNPs as a result of the aptamer-target interaction, leading to the color change from red to purple. The level of detection of vitamin B12 was 0.1 µg/ml by successful optimization of the amount of the aptamer, AuNPs, salts, and stability of the aptamer. Analysis of vitamin B12 was carried out, and the observed recovery was 92 to 95.3% with a relative standard deviation in the range of 2.08 to 8.27%. The results obtained were compared with those of the ultraviolet-visible (UV-vis) spectrometry method. This colorimetric aptasensor is advantageous for on-site detection with the naked eye.

Rapid and simple DNA extraction method for the detection of enterotoxigenic Staphylococcus aureus directly from food samples: comparison of PCR and LAMP methods.

AIMS: The study describes the development of simple and rapid DNA extraction method in combination with loop-mediated isothermal amplification (LAMP) to detect enterotoxigenic Staphylococcus aureus in food samples. METHODS AND RESULTS: In this study, isolation of genomic DNA of enterotoxigenic Staph. aureus from spiked milk, milk burfi, khoa, sugarcane juice and boiled rice was carried out by boiling the isolated sample pellets for 10 min with 1% Triton X-100. The isolated DNA was evaluated by polymerase chain reaction (PCR) and LAMP method. The LAMP was found to be 100 times more sensitive than PCR. The LAMP assay was very specific for Staph. aureus, and the presence of other contaminating bacterial DNAs and food matrix did not interfere or inhibit the LAMP assay. CONCLUSIONS: The template DNA extraction method developed in this study for food samples is simple, rapid and cost-effective. LAMP was found to be less sensitive to matrix effect of food, compared to PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is suitable for direct detection of Staph. aureus without any enrichment in contaminated food samples and hence finds its application in food safety analysis, in permutation with LAMP.

An attempt to valorize the only black meat chicken breed of India by delineating superior functional attributes of its meat.

Kadaknath, the only black chicken indigenous to India, faces the threat of extinction due to declining numbers. Its meat is used in tribal medicine for invigorating and health-promoting properties. Expectations of immune-boosting and therapeutic properties in its meat are creating a buzz these days. Thus, Kadaknath meat was explored and further compared with the commercial Cobb 400 broiler (Cobb) for the functional traits that might be contributing towards proclaimed pharmacological benefits. Birds (n = 20/ group) were raised under similar management conditions and the two primal chicken meat cuts (breast and thigh) were collected at the marketing age. Kadaknath meat was found to be an enriched source of functional biomolecules (carnosine, anserine, creatine). Its breast meat carnosine content was more than double of the Cobb broiler, 6.10 ± 0.13 and 2.73 ± 0.1 mg/ g of wet tissue, respectively. Similarly, the thigh meat of Kadaknath was a significantly (P < 0.05) richer source of carnosine. The genetic background was a key determinant for muscle carnosine content as a significant abundance of CARNS1 and SLC36A1 expression was identified in the Kadaknath breast. The superior functional property of Kadaknath meat was established by the antioxidant capacity established by the Oxygen radical absorbance capacity assay and a stronger ability to inhibit the formation of advanced glycation end products (AGEs). The identification of fairly unknown nutritional and functional advantages of Kadaknath meat could potentially change the paradigm with its meat consumption. It will help in developing a brand name for Kadaknath products that will propel an increase in its market share and ultimately conservation of this unique but endangered poultry germplasm.

Competitive immunoassay for analysis of vitamin B(12).

In the current work, direct competitive enzyme-linked immunosorbent assay (ELISA) was developed for derivatized vitamin B(12) by generating chicken egg yolk immunoglobulins (IgY) against derivatized vitamin B(12) and purified using affinity chromatography. Checkerboard assay was performed with vitamin B(12) antibody and vitamin B(12)-alkaline phosphatase conjugate followed by its conjugate characterization using ultraviolet (UV) spectroscopy and high-performance liquid chromatography (HPLC). The limit of detection was 10 ng/ml with a linear working range of 10 to 10,000 ng/ml. The affinity constant (K(a)) of the vitamin B(12) antibody was found to be 4.23×10(8) L/mol. Cross-reactivity with other water-soluble vitamins was found to be less than 0.01% except for analogs of vitamin B(12) that showed 12% to 35%. The intra- and interassay coefficients of variation were found to be in the ranges from 0.0005% to 1.2% and 0.009% to 1.03%, respectively. The assay was validated with the HPLC method in terms of sensitivity, specificity, precision, and recovery of vitamin B(12) with spiked multivitamin injections, tablets, capsules, and chocolates. The HPLC method had a detection limit of 500 ng/ml with a linear working range of 1000 to 10,000 ng/ml. After extraction of vitamin B(12) using Amberlite XAD, the developed ELISA method correlated well with the established HPLC method with a correlation coefficient of 0.90.

Comparative assessment of tea quality by various analytical and sensory methods with emphasis on tea polyphenols.

Attempts were made to evolve an efficient technique for quality assessment of tea (Camellia sinensis) using a tyrosinase based biosensor to detect polyphenols (PP). Tyrosinase catalyzes the polymerization of PP to form theaflavins (Tf) and thearubigins (Tr) contributing to the colour and astringency of tea, which determine tea quality. Variation in biosensor response of tea infusions gave an indication of differential amount of Tf and Tr. A comparative study of quality attributes of 8 varieties of commercially available brands of tea (A-H) was done using biosensor and results were compared with conventional techniques such as spectrophotometry, high performance liquid chromatography (HPLC), Commission Internationale de I'Eclairage (CIE) system and sensory evaluation. Considerable correlation was observed among the biosensor, sensory and spectrophotometric evaluation of tea samples. Sample A showed high Tf content and also showed a relative high biosensor response whereas sample G showed relatively poor response. Application of biosensors would serve as a basis for the evaluation of market value of tea in the near future.

DNA aptamer selection and detection of marine biotoxin 20 Methyl Spirolide G.

This study reports the selection of DNA aptamer for the detection of 20 Methyl Spirolide G (SPXG). After 10 rounds of selection, theenriched pool of aptamers specific to SPXGwas cloned, sequenced and clustered into seven families based onsimilarity. Three sequences SPX1, SPX2 and SPX7, each belonging to different clades were further evaluated for their binding affinity. Surface plasmonresonancestudies determined the highest affinity KDof 0.0345x10-8 M for aptamer SPX7. A label-free microscale thermophoresis-based aptasensing using SPX7 with highest affinity, indicated a linear detection range from 1.9 to 125000 pg/mL (LOD = 0.39 pg/mL; LOQ = 1.17 pg/mL). Spiking studies in simulated contaminated samples of mussel and scallop indicated recoveries in the range of 86 to 108%. Results of this study indicate the successful development of an aptamer for detection of SPXG at picogram levels. It also opens up avenues to develop other sensing platforms for detection of SPXG using the reported aptamer.

Focus on quantum dots as potential fluorescent probes for monitoring food toxicants and foodborne pathogens.

Water-soluble quantum dots (QDs) are fluorescent semiconductor nanoparticles with narrow, very specific, stable emission spectra. Therefore, the bioconjugation of these QDs for biological fluorescent labeling may be of interest due to their unique physical and optical properties as compared to organic fluorescent dyes. These intrinsic properties of QDs have been used for the sensitive detection of target analytes. From the viewpoint of ensuring food safety, there is a need to develop rapid, sensitive and specific detection techniques to monitor food toxicants in food and environmental samples. Even trace levels of these toxicants can inadvertently enter the food chain, creating severe health hazards. The present review emphasizes the application of water-soluble bioconjugated QDs for the detection of food contaminants such as pesticides, pathogenic bacterial toxins such as botulinum toxin, enterotoxins produced by Staphylococcus aureus, Escherichia coli, and for the development of oligonucleotide-based microarrays. This review also emphasizes the application of a possible resonance energy transfer phenomenon resulting from nanobiomolecular interactions obtained through the bioconjugation of QDs with biomolecules. Furthermore, the utilization of significant changes in the spectral behavior of QDs (attributed to resonance energy transfer in the bioconjugate) in future nanobiosensor development is also emphasized.

Transformation of L-tyrosine to L-dopa by a novel fungus, Acremonium rutilum, under submerged fermentation.

The present study deals with the transformation of L-tyrosine to L-dopa by Acremonium rutilum, a fungal tyrosinase producer, isolated from decomposed banana stud. This appears to be the first report on A. rutilum as a polyphenoloxidase producer with both cresolase and catecholase activity. Enriched Czapek-Dox agar was used for plate assay screening. Enriched potato dextrose broth was used for optimization studies, which induced high levels of L-dopa under submerged fermentation. A. rutilum gave the maximum L-dopa production (0.89 mg/ml) and tyrosinase activity (1095 U/mg) under the optimized parameters, that is, a temperature of 25 degrees C, pH 5.5, an inoculum size of 2.5 ml, and an incubation time of 72-120 h, with L-tyrosine (5 mg/ml) as substrate. Five resolved bands, with R(f) values of 0.73, 0.60, 0.54, 0.37, and 0.26, were observed, which confirmed the presence of L-dopa. This study involves the elevated profile of L-dopa production. Such study is needed, as L-dopa has the ability to control Parkinson's disease.

Xanthine scaffold: scope and potential in drug development.

Medicinal plants have been the basis for discovery of various important marketed drugs. Xanthine is one such lead molecule. Xanthines in various forms (caffeine, theophylline, theobromine, etc) are abode in tea, coffee, cocoa, chocolate etc. giving them popular recognition. These compounds are best known for their diverse pharmaceutical applications as cyclic nucleotide phosphodiesterase inhibition, antagonization of adenosine receptor, anti-inflammatory, anti-microbial, anti-oxidant and anti-tumor activities. These properties incentivize to use xanthine as scaffold to develop new derivatives. Chemical synthesis contributes greater diversity in xanthine based derivatisation. With highlighting the existing challenges in chemical synthesis, the present review focuses the probable solution to fill existing lacuna. The review summarizes the available knowledge of xanthine based drugs development along with exploring new xanthine led chemical synthesis path for bringing diversification in xanthine based research. The main objective of this review is to explore the immense potential of xanthine as scaffold in drug development.

Immobilised tyrosinase-based biosensor for the detection of tea polyphenols.

An amperometric principle-based biosensor containing immobilized enzyme tyrosinase has been used for detection of polyphenols in tea. The immobilized tyrosinase-based biosensor could detect tea polyphenols in the concentration range 10-80 mmol L(-1). Immobilization of the enzyme by the crosslinking method gave good stable response to tea polyphenols. The biosensor response reached the steady state within 5 min. The voltage response was found to have a direct linear relationship with the concentration of polyphenols in black tea samples. Enzyme membrane fouling was observed with number of analyses with a single immobilised enzyme membrane. The tyrosinase-based biosensor gave maximum response to tea polyphenols at 30 degrees C. The optimum pH was 7.0. This biosensor system can be applied for analysis of tea polyphenols. Variation in the biosensor response to black tea infusions gave an indication of the different amounts of theaflavins in the samples, which is an important parameter in evaluating tea quality. A comparative study of the quality attributes of a variety of commercially available brands of tea were performed using the biosensor and conventional analytical techniques such as spectrophotometry.

Detection of methyl parathion using immuno-chemiluminescence based image analysis using charge coupled device.

A novel method based on immuno-chemiluminescence and image analysis using charge coupled device (CCD) for the qualitative detection of methyl parathion (MP) with high sensitivity (up to 10 ppt) is described. MP antibodies raised in poultry were used as a biological sensing element for the recognition of MP present in the sample. The immuno-reactor column was prepared by packing in a glass capillary column (150 microl capacity) MP antibodies immobilized on Sepharose CL-4B through periodate oxidation method. Chemiluminescence principle was used for the detection of the pesticide. Light images generated during the chemiluminescence reaction were captured by a CCD camera and further processed for image intensity, which was correlated with pesticide concentrations. K(3)Fe(CN)(6) was used as a light enhancer to obtain detectable light images. Different parameters including concentrations of K(3)Fe(CN)(6), luminol, urea H(2)O(2), antibody, addition sequence of reactants and incubation time to obtain best images were optimized. The results obtained by image analysis method showed very good correlation with that of competitive ELISA for methyl parathion detection. Competitive ELISA method was used as a reference to compare the results obtained by CCD imaging.

Reactivation of immobilized acetyl cholinesterase in an amperometric biosensor for organophosphorus pesticide.

Biosensors based on acetyl cholinesterase (AChE) inhibition have been known for monitoring of pesticides in food and water samples. However, strong inhibition of the enzyme is a major drawback in practical application of the biosensor which can be overcome by reactivation of the enzyme for repeated use. In the present study, enzyme reactivation by oximes was explored for this purpose. Two oximes viz., 1,1'-trimethylene bis 4-formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were compared for the reactivation of the immobilized AChE. TMB-4 was found to be a more efficient reactivator under repeated use, retaining more than 60% of initial activity after 11 reuses, whereas in the case of 2-PAM, the activity retention dropped to less than 50% after only 6 reuses. Investigations also showed that reactivation must be effected within 10 min after each analysis to eliminate the ageing effect, which reduces the efficiency of reactivation.

Biosensor for the determination of phenols based on cross-linked enzyme crystals (CLEC) of laccase.

Cross-linked enzyme crystals (CLECs) are a versatile form of biocatalyst that can also be used for biosensor application. Laccase from Trametes versicolor (E.C.1.10.3.2) was crystallized, cross-linked and lyophilized with beta-cyclodextrin. The CLEC laccase was found to be highly active towards phenols like 2-amino phenol, guaiacol, catechol, pyrogallol, catechin and ABTS (non-phenolic). The CLEC laccase was embedded in 30% polyvinylpropylidone (PVP) gel and mounted into an electrode to make the sensor. The biosensor was used to detect the phenols in 50-1000 micromol concentration level. Phenols with lower molecular weight such as 2-amino phenol, catechol and pyrogallol gave a short response time where as the higher molecular weight substrates like catechin and ABTS had comparatively a long response time. The optimum pH of the analyte was 5.5-6.0 when catechol was used as substrate. The CLEC laccase retained good activity for over 3 months.

Recent advances in nanoparticle based aptasensors for food contaminants.

Food safety and hazard analysis is a prime concern of human life, thus quality assessment of food and water is the need of the day. Recent advances in nano-biotechnology play a significant role in providing possible solutions for developing highly sensitive and affordable detection tools for food analysis. Nanomaterials based aptasensors hold great potential to overcome the drawbacks of conventional analytical techniques. Aptamers comprise a novel class of highly specific bio-recognition elements which are produced by SELEX (systematic evolution of ligands by exponential enrichment) process. They bind to target molecules by folding into 3D structures that can discriminate different chiral compounds. The flexibility in making modifications in aptamers contribute to the design of biosensors, enabling the generation of bio-recognition elements for a wide variety of target molecules. Nanomaterials such as metal nanoparticles, metal nanoclusters, metal oxide nanoparticles, metal and carbon quantum dots, graphene, carbon nanotubes and nanocomposites enable higher sensitivity by signal amplification and introduce several novel transduction principles such as enhanced chemiluminescence, fluorescence, Raman signals, electrochemical signals, enhanced catalytic activity, and super-paramagnetic properties to the biosensor. Although there are a few reviews published recently which deal with the potential of aptamers in various fields, none are devoted exclusively to the potential of aptasensors based on nanomaterials for the analysis of food contaminants. Hence, the current review discusses several transduction systems and their principles used in aptamer based nanosensors which have been developed in the past five years, the challenges faced in their designing, along with their strengths and limitations.