An Efficient Probe for Rapid Detection of Cyanide in Water at Parts per Billion Levels and Naked-Eye Detection of Endogenous Cyanide

A new molecular probe based on an oxidized bis-indolyl skeleton has been developed for rapid and sensitive visual detection of cyanide ions in water and also for the detection of endogenously bound cyanide. The probe allows the naked-eye detection of cyanide ions in water with a visual color change from red to yellow ((max)=80nm) with the immediate addition of the probe. It shows high selectivity towards the cyanide ion without any interference from other anions. The detection of cyanide by the probe is ratiometric, thus making the detection quantitative. A Michael-type addition reaction of the probe with the cyanide ion takes place during this chemodosimetric process. In water, the detection limit was found to be at the parts per million level, which improved drastically when a neutral micellar medium was employed, and it showed a parts-per-billion-level detection, which is even 25-fold lower than the permitted limits of cyanide in water. The probe could also efficiently detect the endogenously bound cyanide in cassava (a staple food) with a clear visual color change without requiring any sample pretreatment and/or any special reaction conditions such as pH or temperature. Thus the probe could serve as a practical naked-eye probe for in-field experiments without requiring any sophisticated instruments.

Highly sensitive and rapid detection of acetylcholine using an ITO plate modified with platinum-graphene nanoparticles

Determining the concentrations of acetylcholine (ACh) and choline (Ch) is clinically important. ACh is a neurotransmitter that acts as a key link in the communication between neurons in the spinal cord and in nerve skeletal junctions in vertebrates, and plays an important role in transmitting signals in the brain. A bienzymatic sensor for the detection of ACh was prepared by co-immobilizing choline oxidase (ChO) and acetylcholinesterase (AChE) on graphene matrix/platinum nanoparticles, and then electrodepositing them on an ITO-coated glass plate. Graphene nanoparticles were decorated with platinum nanoparticles and were electrodeposited on a modified ITO-coated glass plate to form a modified electrode. The modified electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) studies. The optimum response of the enzyme electrode was obtained at pH 7.0 and 35 degrees C. The response time of this ACh-sensing system was shown to be 4 s. The linear range of responses to ACh was 0.005-700 mu M. This biosensor exhibits excellent anti-interferential abilities and good stability, retaining 50% of its original current even after 4 months. It has been applied for the detection of ACh levels in human serum samples.

An enzyme-linked immuno focus assay for rapid detection and enumeration, and a newborn mouse model for human non-polio enteroviruses associated with acute diarrhea

We have recently reported significant association of non-polio enteroviruses (NPEVs) with acute and persistent diarrhea (18-21% of total diarrheal cases), and non-diarrheal Increased Frequency of Bowel Movements (IFoBM-ND) (about 29% of the NPEV infections) in children and that the NPEV-associated diarrhea was as significant as rotavirus diarrhea. However, their diarrhea-causing potential is yet to be demonstrated in an animal model system. Since the determination of virus titers by the traditional plaque assay takes 4-7 days, there is a need for development of a rapid method for virus titer determination to facilitate active clinical research on enterovirus-associated diarrhea. The goal of this study is to develop a cell-based rapid detection and enumeration method and to demonstrate the diarrhea-inducing potential of purified and characterized non-polio enteroviruses, which were isolated from diarrheic children. Here we describe generation of monoclonal and polyclonal antibodies against purified strains belonging to different serotypes, and development of an enzyme-linked immuno focus assay (ELIFA) for detection and enumeration of live NPEV particles in clinical and purified virus samples, and a newborn mouse model for NPEV diarrhea. Plaque-purified NPVEs, belonging to different serotypes, isolated from children with diarrhea, were grown in cell culture and purified by isopycnic CsCl density gradient centrifugation. By ELIFA, NPEVs could be detected and enumerated within 12 h post-infection. Our results demonstrated that Coxsackievirus B1 (CVB1) and CVB5 strains, isolated from diarrheic children, induced severe diarrhea in orally-inoculated 9-12 day-old mouse pups, fulfilling Koch's postulates. The methods described here would facilitate studies on NPEV-associated gastrointestinal disease. (C) 2015 Elsevier B.V. All rights reserved.

Development of a probe-based blotting technique for the detection of Tobacco streak virus

Digoxigenin (DIG)-labeled DNA probe was developed for a sensitive and rapid detection of the Tobacco streak virus (TSV) isolates in India by dot-blot and tissue print hybridization techniques. DIG-labeled DNA probe complementary to the coat protein (CP) region of TSV sunflower isolate was designed and used to detect the TSV presence at field levels. Dot-blot hybridization was used to check a large number of TSV isolates with a single probe. In addition, a sensitivity of the technique was examined with the different sample extraction methods. Another technique, the tissue blot hybridization offered a simple, reliable procedure and did not require a sample processing. Thus, both non-radioactively labeled probe techniques could facilitate the sample screening during TSV outbreaks and offer an advantage in quarantine services.

Employing denaturation for rapid electrochemical detection of myoglobin using TiO2 nanotubes

An alternative antibody-free strategy for the rapid electrochemical detection of cardiac myoglobin has been demonstrated here using hydrothermally synthesized TiO2 nanotubes (Ti-NT). The denaturant induced unfolding of myoglobin led to easy access of the deeply buried electroactive heme center and thus the efficient reversible electron transfer from protein to electrode surface. The sensing performance of the Ti-NT modified electrodes were compared vis a vis commercially available titania and GCEs. The tubular morphology of the Ti-NT led to facile transfer of electrons to the electrode surface, which eventually provided a linear current response (obtained from cyclic voltammetry) over a wide range of Mb concentration. The sensitivity of the Ti-NT based sensor was remarkable and was equal to 18 mu A mg(-1) ml (detection limit = 50 nM). This coupled with the rapid analysis time of a few tens of minutes (compared to a few days for ELISA) demonstrates its potential usefulness for the early detection of acute myocardial infarction (AMI).

ELISA for the detection of venoms from four medically important snakes of India

A double antibody sandwich enzyme linked immunosorbent assay (ELISA) was developed to detect Echis carinatus venom in various organs (brain, heart, lungs, liver, spleen and kidneys) as well as tissue at the site of injection of mice, at various time intervals (1, 6, 12, 18, 24 h and 12 h intervals up to 72 h) after death. The assay could detect E. carinatus venom levels up to 2.5 ng/ml of tissue homogenate and the venom was detected up to 72 h after death. A highly sensitive and species-specific avidin-biotin microtitre ELISA was also developed to detect venoms of four medically important Indian snakes (Bungarus caeruleus, Naja naja, E. carinatus and Daboia russelli russelli) in autopsy specimens of human victims of snake bite. The assay could detect venom levels as low as 100 pg/ml of tissue homogenate. Venoms were detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area and postmortem blood. In all 12 human victim cadavers tested the culprit species were identified. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart and lungs. Development of a simple, rapid and species-specific diagnostic kit based on this ELISA technique useful to clinicians is discussed.

Detection of alpha(2u)-globulin and its bound putative pheromones in the preputial gland of the Indian commensal rat (Rattus rattus) using mass spectrometry

The role of pheromones and pheromone-binding proteins in the laboratory rat has been extensively investigated. However, we have previously reported that the preputial gland of the Indian commensal rat produces a variety of pheromonal molecules and preputial glands would seem to be the predominant source for pheromonal communication. The presence of pheromone-binding proteins has not yet been identified in the preputial gland of the Indian commensal rat; therefore, the experiments were designed to unravel the alpha(2u)-globulin (alpha 2u) and its bound volatiles in the commensal rat. Total preputial glandular proteins were first fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently analyzed by mass spectrometry. Further, we purified alpha 2u and screened for the presence of bound pheromonal molecules with the aid of gas chromatography/mass spectrometry (GC/MS). A novel alpha 2u was identified with a high score and this protein has not been previously described as present in the preputial gland of Indian commensal rats.This novel alpha 2u was then characterized by tandem mass spectrometry (MS/MS). Peptides with m/z values of 969, 1192, 1303 and 1876 were further fragmented with the aid of MS/MS and generated de novo sequences which provided additional evidence for the presence of alpha 2u in the preputial gland. Finally, we identified the presence of farnesol 1 and 2 bound to alpha 2u. The present investigation confirms the presence of alpha 2u (18.54 kDa) in the preputial gland of the Indian commensal rat and identifies farnesol 1 and 2 as probably involved in chemo-communication by the Indian commensal rat.Copyright (C) 2010 John Wiley & Sons, Ltd.

Rapid localization of damage using a circular sensor array and Lamb wave based triangulation

A damage detection and imaging methodology based on symmetry of neighborhood sensor path and similarity of signal patterns with respect to radial paths in a circular array of sensors has been developed It uses information regarding Limb wave propagation along with a triangulation scheme to rapidly locate and quantify the severity of damage without using all of the sensor data. In a plate like structure, such a scheme can be effectively employed besides full field imaging of wave scattering pattern from the damage, if present in the plate. This new scheme is validated experimentally. Hole and corrosion type damages have been detected and quantified using the proposed scheme successfully. A wavelet based cumulative damage index has been studied which shows monotonic sensitivity against the severity of the damage. which is most desired in a Structural Health Monitoring system. (C) 2010 Elsevier Ltd. All rights reserved.

A Rapid Bioassay for Measuring Inhibin Activity

A sensitive and rapid bioassay of inhibin is described. Swiss albino female mice, 27 days old, weighing 25-30 g, were primed with 20 IU hCG, given in 2 equal doses, administered at 0900 h and 1800 h. An increment of 200% in uterine weight could be observed when the animals were autopsied the next day at 1000 h. The validity of the endpoint chosen, as a function dependent on FSH, was shown by the fact that neutralization of endogenous FSH by FSH antiserum led to an inhibition of uterine weight increase. Further injection of the inhibin material caused an actual reduction in the heightened levels of FSH (ng/ml) brought about by hCG: Control, 207 ± 13.6; hCG, 365 ± 28.8 and hCG inhibin, below the detection level of radioimmunoassay. Inhibin preparation from ovine testicular extract when tested at 3 dose levels showed a dose dependent and significant suppression in uterine weight increase. The assay was statistically validε = 2.5; slope = 23.9; λ = 0.104 and intra- and interassay variation = 8.3% and 11.7%, respectively. Since the mouse uterine weight assay is specific, has greater sensitivity and is uniformly reproducible, it is suggested that this assay procedure is ideally suited to assess the activity of different inhibin test preparations as well as to follow the purification of inhibin activity from crude material.

Development and characterization of monoclonal antibodies against WbSXP-1 for the detection of circulating filarial antigens

The importance of developing effective assays to diagnose, monitor and evaluate human lymphatic filariasis has been emphasized by the World Health Organization. Presently, few immunodiagnostics are available for filarial monitoring programmes. The Wuchereria bancrofti (Wb) SXP-1 parasite protein, with 84% homology to Brugia malayi (Bm) SXP-1, was found to be highly immunogenic. WbSXP-1 is one among the diagnostic candidate molecules that were used for developing a rapid-antibody-flow-through diagnostic kit for filariasis. Studies were initiated with the aim of developing monoclonal antibodies against recombinant WbSXP-1 and prospective applications for the detection of both circulating Wb and Bm antigens in serum samples from infected individuals. The monoclones 1A6C2 of subclass IgG1k, and 2A12F8 of class IgM, specifically detected Wb and Bm microfilaria isolated from patients and did not show cross-reactivity with other filarial recombinant antigens. We anticipate that this work will address the problems faced in the rapid diagnosis of human lymphatic filariasis in endemic areas in developing countries.

A transient detection algorithm for audio using iterative analysis of STFT

We propose an iterative algorithm to detect transient segments in audio signals. Short time Fourier transform(STFT) is used to detect rapid local changes in the audio signal. The algorithm has two steps that iteratively - (a) calculate a function of the STFT and (b) build a transient signal. A dynamic thresholding scheme is used to locate the potential positions of transients in the signal. The iterative procedure ensures that genuine transients are built up while the localised spectral noise are suppressed by using an energy criterion. The extracted transient signal is later compared to a ground truth dataset. The algorithm performed well on two databases. On the EBU-SQAM database of monophonic sounds, the algorithm achieved an F-measure of 90% while on our database of polyphonic audio an F-measure of 91% was achieved. This technique is being used as a preprocessing step for a tempo analysis algorithm and a TSR (Transients + Sines + Residue) decomposition scheme.

Remarkable role of positional isomers in the design of sensors for the ratiometric detection of copper and mercury ions in water

Cation sensing properties of the three positional isomers of rhodamine based sensors (1-3) are studied in water. The sensors differ only in the position of pyridine's nitrogen. The chemosensor 1, with pyridine nitrogen at ortho-position, showed a selective colorimetric detection of Cu(II) ions in water, at physiological pH 7.4 and also in medium containing BSA (bovine serum albumin) and blood serum. Notably the compound 2 and 3, with pyridine end located at meta-and para-positions did not show any color change with Cu(II) ions, although both the compounds showed turn-on change both in color and fluorescence with Hg(II) ions specifically. All the probes showed ratiometric changes with the specific metal ions. The changing position of nitrogen also changed the complexation pattern of the sensors with the metal ions. Probe 1 showed 2 : 1 complexation with Cu(II), whereas 2 and 3 showed 1 : 1 complexation with Hg(II) ions. The mechanism investigation showed that the change in color upon addition of metal ions is due to the ring-opening of the spirolactam ring of the probes. Cu(II) interacted with ligand 1 through a three-point interaction mode comprising carbonyl oxygen, amido nitrogen and pyridine nitrogen end. But in case of 2 and 3, Hg2+ only interacted through pyridine nitrogen ends. Quantitative estimation of Cu2+ and Hg2+ in complex biological media such as bovine albumin protein (BSA) and human blood serum were performed using these sensors. Rapid on-site detection as well as discrimination of these toxic ions was demonstrated using easily prepared portable test-strips.

Selective and Efficient Detection of Nitro-Aromatic Explosives in Multiple Media including Water, Micelles, Organogel, and Solid Support

Selective detection of nitro-aromatic compounds (NACs) at nanomolar concentration is achieved for the first time in multiple media including water, micelles or in organogels as well as using test strips. Mechanism of interaction of NACs with highly fluorescent p-phenylenevinylene-based molecules has been described as the electron transfer phenomenon from the electron-rich chromophoric probe to the electron deficient NACs. The selectivity in sensing is guided by the pK(a) of the probes as well as the NACs under consideration. TNP-induced selective gel-to-sol transition in THF medium is also observed through the reorganization of molecular self-assembly and the portable test trips are made successfully for rapid on-site detection purpose.

A rapid, efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library

With the development of deep sequencing methodologies, it has become important to construct site saturation mutant (SSM) libraries in which every nucleotide/codon in a gene is individually randomized. We describe methodologies for the rapid, efficient, and economical construction of such libraries using inverse polymerase chain reaction (PCR). We show that if the degenerate codon is in the middle of the mutagenic primer, there is an inherent PCR bias due to the thermodynamic mismatch penalty, which decreases the proportion of unique mutants. Introducing a nucleotide bias in the primer can alleviate the problem. Alternatively, if the degenerate codon is placed at the 5' end, there is no PCR bias, which results in a higher proportion of unique mutants. This also facilitates detection of deletion mutants resulting from errors during primer synthesis. This method can be used to rapidly generate SSM libraries for any gene or nucleotide sequence, which can subsequently be screened and analyzed by deep sequencing. (C) 2013 Elsevier Inc. All rights reserved.

Titania nanotube-modified screen printed carbon electrodes enhance the sensitivity in the electrochemical detection of proteins

The use of titania nanotubes (TiO2-NT) as the working electrode provides a substantial improvement in the electrochemical detection of proteins. A biosensor designed using this strategy provided a robust method to detect protein samples at very low concentrations (C-protein ca 1 ng/mu l). Reproducible measurements on protein samples at this concentration (I-p,I-a of 80 +/- 1.2 mu A) could be achieved using a sample volume of ca 30 mu l. We demonstrate the feasibility of this strategy for the accurate detection of penicillin binding protein, PBP2a, a marker for methicillin resistant Staphylococcus aureus (MRSA). The selectivity and efficiency of this sensor were also validated using other diverse protein preparations such as a recombinant protein tyrosine phosphatase (PTP10D) and bovine serum albumin (BSA). This electrochemical method also presents a substantial improvement in the time taken (few minutes) when compared to conventional enzyme-linked immunosorbent assay (ELISA) protocols. It is envisaged that this sensor could substantially aid in the rapid diagnosis of bacterial infections in resource strapped environments. (C) 2014 Elsevier B.V. All rights reserved.