Methylene Blue Assisted Electrochemical Detection of Bacterial Biofilm.

This paper presents a novel electrochemical transduction method for the rapid and straightforward detection of bacterial biofilm. Briefly, fifteen isolates from various sources were collected and evaluated for their ability to generate biofilm. The Congo red-based agar method and the tube test were used for preliminary screening. A microtiter experiment was also performed to quantitatively examine the screening results and validate the outcomes of the proposed methylene blue-based electrochemical detection method. Electrochemical sensing was performed on the two selected isolates using methylene blue as a redox indicator. For optimization goals, several methylene blue concentrations were studied. Methylene blue at a concentration of 0.4 mM was used for the analysis conclusion. The developed electrochemical method displayed a linear R2 value of 0.9747. The new electrochemical approach demonstrated great sensitivity and rapid response compared to conventional microtiter test methods.

Utilization of non-pathogenic bacteria to obtain optimum biofilm production for beneficial applications.

Depending on the bacteria embedded in the extracellular polymeric layer, biofilms can be advantageous or harmful. The isolated strains used in this investigation are already established to be beneficial biofilm-producing bacteria. In order to use them effectively in various domains, it is necessary to characterize them and understand their ideal physiological characteristics for maximum biofilm growth. This study used genome sequence analysis to identify and characterize strains isolated from water samples in Raipur, Chhattisgarh, India. The nucleotide sequences were submitted to NCBI GenBank under the accession numbers Bacillus tequilensis (MN889418) and Pseudomonas beteli (MN889419) and the strains were further characterized using some advanced techniques (phase contrast microscopy, Raman spectroscopy, Fourier-transform infrared spectroscopy, and scanning electron microscope). For maximum biofilm formation by isolated bacterial strains, many physiochemical factors including incubation duration, temperature, pH, carbon source concentration, and nitrogen source concentration were further examined and optimized. The fact that these non-pathogenic strains were found in public water supplies is another important part of this research because there is a chance that they could change into pathogenic state in future and cause disease in humans.

Microplastics: An overview on separation, identification and characterization of microplastics.

At present plastic residues has become grave threat to the environment. Microplastics are plastic residues with a size <5 mm, due to their small size it is very difficult to remove them from water bodies, sediments and air with available techniques. Nanoplastics are different in size range as nanoplastics are smaller than 1 µm in size. This review is an attempt to gather an insight towards microplastic and its associated point of concerns. The review will highlight some of the methods appropriate for microplastics sampling and techniques for its identification in environmental samples. Some of the sampling methods include sieving, filtration, visual sorting, digestion, density separation. While, identification techniques in practice are SEM-EDS, FTIR, NIR, Raman, NMR spectroscopy, etc. Still there is a need and scope for development of more economical and portable techniques in this direction.

Microbial exopolysaccharides: Synthesis pathways, types and their commercial applications.

Polysaccharides are essential natural metabolites found in all life forms such as microorganisms, animals and plants with various biochemical structures and biological functions. Among all the life forms microbial exopolysaccharides are produced in shorter time duration as they responsible for the microbial cell adhesion and protection during unfavorable growth conditions. Microbial exopolysaccharides are composed of repeated sugar units of same or different types and form a complex by associating with proteins, lipids, metal ions, extracellular DNA (eDNA), organic and inorganic compounds to form a protective layer around the microbial colonies collectively known as biofilm. Specific functions of exopolysaccharides depend on structural composition and habitat of a host microorganism. There are various techniques to study the composition and structure of exopolysaccharides such as High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection, Size Exclusion Chromatography coupled with multi-laser light scattering (SEC-MALLS),X-Ray diffraction (XRD), Differential Scanning Calorimetry (DSC), Fourier Transform Infrared (FTIR) and Thermal Gravimetric Analysis (TGA), etc. In the current article, we reviewed microbial exopolysaccharides physiochemical properties, composition, analyzing techniques through which possible commercial applications in dairy products, cosmetics, research, agriculture and petroleum industry can be performed.

Enzyme Immobilization over Polystyrene Surface Using Cysteine Functionalized Copper Nanoparticle as a Linker Molecule.

The work focus on the development of a simple and efficient method of enzyme immobilization over a polystyrene surface using cysteine functionalized copper nanoparticle as linker molecule. The polystyrene surface is activated by generating -NO2 groups by the process of nitration reaction. The nitrated polystyrene plate then is silanized with (3-mercaptopropyl) trimethoxysilane (MPTS) followed with the coupling of cysteine-capped copper nanoparticles on the silanized surface through thiol moiety. A nanoparticle layer is thus created over the polystyrene surface which is efficiently used for covalent immobilization of urease via an amino group of cysteine through glutaraldehyde treatment. The technique resulted in an enhancement in the enzymatic activity by 72.37% over the soluble counterpart. The immobilized enzyme also exhibited appreciable reusability of about 10 times with activity retention of 82% of its initial activity. Immobilization also offered an increased thermal and pH stability to the immobilized enzyme over the soluble enzyme.

An efficient protocol to use iron oxide nanoparticles in microfluidic paper device for arsenic detection.

This method describes a rapid ecofriendly and affordable method for detecting arsenic in the water sample. The system designed works on the principle that involves generation of arsine due to reduction of arsenic by bare and cysteine capped iron oxide nanoparticles and its further reaction with silver nitrate present on the microfluidic paper analytical device (µPAD). Change in the color of µPAD from colorless to reddish brown is a result of reaction between arsine gas and silver nitrate, and is the detection criteria. The sample solution of arsenic was prepared in lemon juice to provide the required acidic environment for hydride generation. This proposed method has detection limit of 0.01 ppm (10 ppb) and 1 ppm for cysteine capped and bare iron oxide nanoparticles respectively. This is for the first time that iron oxide nanoparticles are being used for detection and reduction arsenic species in environmental sample. The same device can be used for on-site detection in an ecofriendly manner.

A three step approach for the purification of alkaline phosphatase from non-pasteurized milk.

In this study, a three step purification of alkaline phosphatase from non-pasteurized milk has been described. It included cream extraction, n-butanol treatment and acetone precipitation. Different parameters such as buffer concentration, temperature, pH, substrate concentration, acetone and n-butanol treatment were optimized to maximize the enzyme activity. The enzyme was fruitfully purified up to homogeneity from the milk, with percentage recovery and fold purification of 56.17 and 17.67 respectively. The kinetic parameters were determined to be 0.927 mM (Km) and 55.86 µM/min (Vmax), with specific activity of 11.31 U/mg. Other optimized parameters were estimated as a buffer concentration of 0.5 M with pH 9.0, temperature optima at 37 °C, with n-butanol and acetone concentration of 20 % (v/v) and 50 % (v/v) respectively. This approach provides a simple and effective method for the purification of alkaline phosphatase from non-pasteurized milk.

Recent advances in phosphate biosensors.

A number of biosensors have been developed for phosphate analysis particularly, concerning its negative impact within the environmental and biological systems. Enzymatic biosensors comprising either a single or multiple enzymatic system have been extensively used for the direct and indirect analysis of phosphate ions. Furthermore, some non-enzymatic biosensors, such as affinity-based biosensors, provide an alternative analytical approach with a higher selectivity. This article reviews the recent advances in the field of biosensor developed for phosphate estimation in clinical and environmental samples, concerning the techniques involved, and the sensitivity toward phosphate ions. The biosensors have been classified and discussed on the basis of the number of enzymes used to develop the analytical system, and a comparative analysis has been performed.

Alkaline phosphatase inhibition based conductometric biosensor for phosphate estimation in biological fluids.

Determination of phosphate ions concentration is very important from both, environmental and clinical point of view. In this study, a simple and novel conductometric biosensor for indirect determination of the phosphate ions in aqueous solution has been developed. The developed biosensor is based on the inhibition of immobilized alkaline phosphatase activity, in the presence of the phosphate ions. This is the first time we developed a mono-enzymatic biosensor for indirect estimation of phosphate ions. The developed biosensor showed a broad linear response (as compared to other reported biosensors) for phosphate ions in the range of 0.5-5.0 mM (correlation coefficient=0.995), with a detection limit of 50 µM. Different optimized parameters were obtained as the buffer concentration of 30 mM, substrate concentration of 1.0mM, and a pH of 9.0. All the optimized parameters were analyzed by analysis of variance, and were found to be statistically significant at a level of α=0.05. The developed biosensor is also suitable to determine the serum phosphate concentration, with a recovery of 86-104%, while a recovery of 102% was obtained from the water samples that were spiked with 500 µM phosphate. A relative standard deviation in the conductance response for five successive measurements (n=5) did not exceed 7%, with a shelf life of 30 days. With a lower detection limit and a higher recovery, the biosensor provides a facile approach for phosphate estimation in biological fluids.

Highly efficient production of inverted syrup in an analytical column with immobilized invertase.

This paper presents a procedure by which a simple and economical analytical column containing immobilized invertase was developed. This column has high efficiency of converting sucrose into inverted syrup rapidly. Gelatine beads were used for the immobilization of invertase. The enzyme was entrapped efficiently and was found to be stable and retained its activity over a period of 3 months. Immobilization parameters for maximum enzyme activity were estimated as temperature optima at 60 °C, pH optima 7.0 and 30 mg/mL enzyme concentration was found to give maximum immobilization (72 %). The reusability of the gelatine immobilized invertase was found to be seven times with a time interval of 24 h. The immobilized invertase presented a KM of 51.28 mM and Vmax of 0.334 mM/min. The time required to hydrolyse 50 % sucrose solution by a column of length 10 cm and diameter of 1.5 cm was found to be 15 min at room temperature. The column was found effective for inversion of biological samples like sugar cane juice.

Synthesis and characterization of cysteine functionalized silver nanoparticles for biomolecule immobilization.

A facile method for the aqueous phase synthesis of cysteine-functionalized silver nanoparticles by potato extract has been reported in the present work. These functionalized nanoparticles were then used for the covalent immobilization of a biomolecule, alkaline phosphatase, on its surface through carbodiimide coupling. Different reaction parameters such as cysteine concentration, reducing agent concentration, temperature, pH and reaction time were varied during the nanoparticles' formation, and their effects on plasmon resonance were studied using Ultraviolet-visible spectroscopy. Fourier transform infrared spectroscopy was used to confirm the surface modification of silver nanoparticles by cysteine and the particle size analysis was done using particle size analyzer, which showed the average nanoparticles' size of 61 nm for bare silver nanoparticles and 201 nm for the enzyme-immobilized nanoparticles. The synthesized nanoparticles were found to be highly efficient for the covalent immobilization of alkaline phosphatase on its surface and retained 67% of its initial enzyme activity (9.44 U/mg), with 75% binding efficiency. The shelf life of the enzyme-nanoparticle bioconjugates was found to be 60 days, with a 12% loss in the initial enzyme activity. With a simple synthesis strategy, high immobilization efficiency and enhanced stability, these enzyme-coated nanoparticles have the potential for further integration into the biosensor technology.

Acetohydroxamate inhibition of the activity of urease from dehusked seeds of water melon (Citrullus vulgaris).

Urease from the seeds of watermelon (Citrullus vulgaris) was purified to apparent homogeneity, using two acetone fractionation steps, heat treatment at 48 degrees C and gel filtration through Sephadex G-200. Effect of acetohydroxamic acid (AHA) on the activity of the homogeneous enzyme preparation (sp. act. 3000 +/- 550U/mg protein) was investigated. AHA exhibited a concentration-dependent inhibition both in the presence and absence of the substrate. The inhibition was uncompetitive and the Ki was 2.5 mM. Binding of AHA with the enzyme was reversible, as 63% activity could be restored by dialysis. Time-dependent inhibition revealed a monophasic inhibition of the activity. Addition of beta-mercaptoethanol (ME) gradually abolished the inhibition. Pre-treatment of native enzyme with 8.0 mM ME for 5 min at 30 degrees C exhibited protection against AHA-induced inhibition. The significance of these observations is discussed.