A Facile Synthesis of Alkaline Electrolyte Based Graphene Sheets, Their Functionalization and Attachment of Some Drugs.

High-quality graphene is highly enviable material due to its seminal role amongst several areas in modern technology including its role as nanocarrier for site selective drug grafting and delivery applications. Here, we report a facile, cost-effective and single-step method to produce high-quality graphene through customised electrochemical exfoliation of graphite anode in alkaline electrolyte medium. The quality of graphene sheets (GS) were investigated by Raman, TEM/HRTEM, AFM, and FTIR techniques. The high quality as well as excellent Π-Π stacking nature of the honeycomb lattice of graphene was confirmed by measuring the quenching capability through photo-luminescence spectroscopy using organic dyes. A plausible mechanism for the graphite exfoliation has been given where evolution of high density of oxygen molecules exerts large force on the graphitic layers leads to exfoliation and consequent synthesis of graphene. Furthermore, to explore the application of the graphene sheets so synthesized, we carried out studies which may make them as suitable carriers for drug delivery. For this, graphene sheets were functionalized with L-cysteine and attached with the drugs Amphotericin-B (AmB) and Tamoxifen citrate (TMX). The conjugation of drugs with L-cysteine functionalized graphene has been confirmed through FTIR and Raman spectroscopic techniques. The drug loading efficiency of FGS for AmB and TMX was 75.00% and 94.31%, respectively. The present formulation of drugs (AmB and TMX) conjugated with graphene is suitable for the targeted drug delivery as it will enhance the efficacy and reduce cytotoxicity associated with drug.

Optimization of process variables by central composite design for the immobilization of urease enzyme on functionalized gold nanoparticles for various applications.

In the present study, enzyme urease has been immobilized on amine-functionalized gold nanoparticles (AuNPs). AuNPs were synthesized using natural precursor, i.e., clove extract and amine functionalized through 0.004 M L: -cysteine. Enzyme (urease) was extracted and purified from the vegetable waste, i.e., seeds of pumpkin to apparent homogeneity (sp. activity 353 U/mg protein). FTIR spectroscopy and transmission electron microscopy was used to characterize the immobilized enzyme. The immobilized enzyme exhibited enhanced activity as compared with the enzyme in the solution, especially, at lower enzyme concentration. Based on the evaluation of activity assay of the immobilized enzyme, it was found that the immobilized enzyme was quite stable for about a month and could successfully be used even after eight cycles having enzyme activity of about 47%. In addition to this central composite design (CCD) with the help of MINITAB version 15 Software was utilized to optimize the process variables viz., pH and temperature affecting the enzyme activity upon immobilization on AuNPs. The results predicted by the design were found in good agreement (R2 = 96.38%) with the experimental results indicating the applicability of proposed model. The multiple regression analysis and ANOVA showed the individual and cumulative effect of pH and temperature on enzyme activity indicating that the activity increased with the increase of pH up to 7.5 and temperature 75 °C. The effects of each variables represented by main effect plot, 3D surface plot, isoresponse contour plot and optimized plot were helpful in predicting results by performing a limited set of experiments.

Green Synthesis of Fluorescent Carbon Quantum Dots from Azadirachta indica Leaves and Their Peroxidase-Mimetic Activity for the Detection of H2O2 and Ascorbic Acid in Common Fresh Fruits.

In the present study, an ecofriendly and zero-cost approach has been demonstrated for the preparation of carbon quantum dots by one-pot hydrothermal treatment of leaf extracts of neem (Azadirachta indica). The as-synthesized neem carbon quantum dots (N-CQDs) exhibited high fluorescent quantum yields (QYs) up to 27.2%. Moreover, N-CQDs also act with a peroxidase-like-mimetic activity toward the oxidation of peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) in association with hydrogen peroxide (H2O2). Further, the kinetics of peroxidase-like catalytic activity follows the Michaelis-Menten and ping-pong pathway. In addition, the H2O2 sensitive TMB oxidation motivated the colorimetric detection of H2O2 which showed linearity from 0.1 to 0.5 mmol/L with a detection limit (LOD) of 0.035 mmol/L. Furthermore, the blue colors of oxidized TMB (ox-TMB) were selectively reduced in native TMB with ascorbic acid (AA) without any interference of other reducing agents. The linear range of AA detection was lying between 5 and 40 µM with a LOD up to 1.773 µM. The practicability assay of the proposed sensing system toward the detection of AA was also investigated in real sample analysis such as common fruits which showed good sensitivity to the presence of AA. Therefore, this convenient, ecofriendly, and cost-effective peroxidase-based sensing system opens a new platform for analysis of AA in real samples and in complex biological systems.

Mustard seeds derived fluorescent carbon quantum dots and their peroxidase-like activity for colorimetric detection of H2O2 and ascorbic acid in a real sample.

Herein, we were synthesized fluorescent carbon quantum dots via facile one-step hydrothermal treatment of mustard seeds (M-CQDs). It showed excellent optical property with fluorescent quantum yield 4.6%. The as-prepared M-CQDs exhibited peroxidase-like mimetic activity and catalyzed the oxidation of chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2 to produce a blue color reaction mixture with the prominent peak at 652 nm. Furthermore, the peroxidase-like catalytic performance of M-CQDs followed the steady-state kinetics behavior and exhibited similar catalytic activity as that of natural enzyme Horseradish peroxidase (HRP). In addition to this, the double reciprocal plot showed a parallel line which suggested the occurrence of Ping-Pong type of mechanism. The H2O2 dependent oxidation of TMB was helpful for the colorimetric detection of H2O2 in the linear range of 0.02-0.20 mM with the limit of detection (LOD) of 0.015 mM. Interestingly, the oxidized TMB (ox-TMB) was further reduced to native TMB by the reducing agent ascorbic acid. Hence M-CQDs showed its potential towards the selective and sensitive detection of ascorbic acid in the linear range of 10-70 µM having a correlation coefficient of 0.998 with LOD of 3.26 µM. The practical feasibility of the proposed detection method of AA was also investigated in common fresh fruits.

Employment of pumpkin (Cucumis melo) urease entrapped in alginate beads for quantitation of cadmium in aqueous media.

The potential of employment of free as well as alginate-immobilized urease for the quantitation of cadmium (Cd(2+)) was explored. Urease from the seeds of pumpkin (Cucumis melo) was purified to apparent homogeneity by heat treatment at 48 +/- 0.1 degrees C and gel filtration through Sephadex G-200. The purified enzyme exhibited a single band on native PAGE under coomassie brilliant blue and silver staining. The enzyme entrapped in 3.5% alginate beads (with 86% immobilization) exhibited no leaching over a period of 15 days at 4 degrees C. Urease-catalyzed urea hydrolysis by both soluble and immobilized enzyme revealed a dependence on the inhibitor concentration. The inhibition caused by Cd(2+) was non-competitive and the interaction of Cd(2+) with the enzyme was irreversible as the activity could not be restored by dialysis. The time-dependent inhibition both in the presence and in absence of substrate revealed a biphasic inhibition of the activity. The significance of the results is discussed.

Enzymatic detection of mercuric ions in ground-water from vegetable wastes by immobilizing pumpkin (Cucumis melo) urease in calcium alginate beads.

Present report describes a quick and simple test based on enzyme inhibition for the detection of mercury in aqueous medium by urease immobilized in alginate beads. Urease was extracted from the discarded seeds of pumpkin (Cucumis melo) and was purified to apparent homogeneity (5.2-fold) by heat treatment at 48+/-0.1 degrees C and gel filtration through Sephadex G-200. The homogeneous enzyme preparation (Sp activity 353 U/mg protein, A(280)/A(260)=1.12) was immobilized in 3.5% alginate leading to 86% immobilization. Effect of mercuric ion on the activity of soluble as well as immobilized enzyme was investigated. Hg(2+) exhibited a concentration-dependent inhibition both in the presence and absence of the substrate. The alginate immobilized enzyme showed less inhibition. There was no leaching of the enzyme over a period of 15 days at 4 degrees C. The inhibition was non-competitive and the K(i) was found to be 1.26x10(-1)microM. Time-dependent interaction of urease with Hg(2+) exhibited a biphasic inhibition behavior in which approximately half of the initial activity was lost rapidly (within 10 min) and reminder in a slow phase. Binding of Hg(2+) with the enzyme was largely irreversible, as the activity could not be restored by dialysis. The significance of the observations is discussed.

Factorial design for the optimization of enzymatic detection of cadmium in aqueous solution using immobilized urease from vegetable waste.

Free as well as alginate immobilized urease was utilized for detection and quantitation of cadmium (Cd2+) in aqueous samples. Urease from the seeds of pumpkin (Cucumis melo), being a vegetable waste, was extracted and purified to apparent homogeneity (Sp. Activity 353 U/mg protein; A280/A260=1.12) by heat treatment at 48+/-0.1 degrees C and gel filtration through Sephadex G-200. The homogeneous enzyme preparation was immobilized in 3.5% alginate leading to 86% immobilization and no leaching of the enzyme was found over a period of 15 days at 4 degrees C. Urease catalyzed urea hydrolysis by both soluble and immobilized enzyme revealed a clear dependence on the concentration of Cd2+. The inhibition caused by Cd2+ was non-competitive (Ki=1.41 x 10(-5) M). The time dependent inhibition both in the presence and in absence of Cd2+ ion revealed a biphasic inhibition in the activity. A Response Surface Methodology (RSM) for the parametric optimization of this process was performed using two-level-two-full factorial (2(2)), central composite design (CCD). The regression coefficient, regression equation and analysis of variance (ANOVA) was obtained using MINITAB 15 software. The predicted values thus obtained were closed to the experimental value indicating suitability of the model. In addition to this 3D response surface plot and isoresponse contour plot were helpful to predict the results by performing only limited set of experiments.

Covalent immobilization of peanut ß-amylase for producing industrial nano-biocatalysts: A comparative study of kinetics, stability and reusability of the immobilized enzyme.

Stability of enzymes is an important parameter for their industrial applicability. Here, we report successful immobilization of ß-amylase (bamyl) from peanut (Arachis hypogaea) onto Graphene oxide-carbon nanotube composite (GO-CNT), Graphene oxide nanosheets (GO) and Iron oxide nanoparticles (Fe3O4). The Box-Behnken Design of Response Surface Methodology (RSM) was used which optimized parameters affecting immobilization and gave 90%, 88% and 71% immobilization efficiency, respectively, for the above matrices. ß-Amylase immobilization onto GO-CNT (bamyl@GO-CNT) and Fe3O4 (bamyl@Fe3O4), resulted into approximately 70% retention of activity at 65 °C after 100 min of exposure. We used atomic force microscopy (AFM), scanning and transmission electron microscopy (SEM and TEM), Fourier transformed infrared (FT-IR) spectroscopy and fluorescence microscopy for characterization of free and enzyme bound nanostructures (NS). Due to the non-toxic nature of immobilization matrices and simple but elegant immobilization procedure, these may have potential utility as industrial biocatalysts for production of maltose.

Peroxidase mimetic activity of fluorescent NS-carbon quantum dots and their application in colorimetric detection of H2O2 and glutathione in human blood serum.

Interest is growing in the development of artificial enzymes to overcome the drawbacks of natural enzymes. Herein, we have synthesized nitrogen-sulphur dual-doped carbon quantum dots (NS-CQDs) via a one-step hydrothermal method; the NS-CQDs possess excellent optical properties and a high fluorescent quantum yield (46%). Significantly, the NS-CQDs exhibited peroxidase mimetic enzyme activity without support from metals or polymeric materials and efficiently catalyzed the oxidation of peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H2O2 to produce a blue solution with an absorption maximum at 652 nm. Mechanistic studies suggest that the small size and high electron density of NS-CQDs play vital roles and accelerate the reduction of H2O2 to generate ˙OH radical, which facilitates the oxidation of TMB. The catalytic activity is based on Michaelis-Menten kinetic behavior, and steady state kinetic analysis suggests that the NS-CQDs exhibit a higher affinity for H2O2 than TMB, similar to the natural enzyme horseradish peroxidase (HRP). Moreover, the catalytic pathway follows a ping-pong mechanism. Therefore, these findings offer a worthy platform for colorimetric detection of H2O2 in a linear range of 0.02 mM to 0.1 mM with a limit of detection of 0.004 mM. Interestingly, the blue colour of oxidized TMB showed excellent selectivity over non-thiolate biological molecules, especially amino acids, and glutathione can be detected up to 0.07 µM via colorimetric and fluorimetric assays. Additionally, this system showed excellent recovery (96.0-108.3%) of GSH from human blood serum. Thus, the proposed sensing system is simple, convenient, and straightforward and can be potentially applied for real time monitoring of H2O2 and glutathione in biological samples.

α-Amylase immobilization onto functionalized graphene nanosheets as scaffolds: Its characterization, kinetics and potential applications in starch based industries.

α-Amylase is imperative for starch and its deriviatized industries. Functionalized graphene sheets were tailored and optimized as scaffold for α-amylase immobilization using Response Surface Methodology based on Box-Behnken design, with an overall immobilization efficiency of 85.16%. Analysis of variance provided adequacy to the mathematical model for further studies. Native and immobilized functionalized graphene were characterized using transmission and scanning electron microscopy, followed by Fourier transform infrared (FTIR) spectroscopy. Wheat α-amylase conjugated with functionalized graphene sheets were visually evident on transmission and scanning micrographs while the FTIR spectra showed interplay of various chemical interactions and bonding, during and after immobilization. Optimum pH and optimum temperature for immobilized enzyme though remained unchanged but showed broader range whereas Km showed a slight decrease (1.32 mg/mL). It also showed enhanced thermal and storage stability and retained 73% residual activity after 10 uses. These ensemble of properties and non-toxic nature of functionalized graphene, makes it viable to be absorbed commercially in starch processing industries.

Functionalized graphene sheets as immobilization matrix for Fenugreek ß-amylase: enzyme kinetics and stability studies.

ß-Amylase finds application in food and pharmaceutical industries. Functionalized graphene sheets were customised as a matrix for covalent immobilization of Fenugreek ß-amylase using glutaraldehyde as a cross-linker. The factors affecting the process were optimized using Response Surface Methodology based Box-Behnken design of experiment which resulted in 84% immobilization efficiency. Scanning and Transmission Electron Microscopy (SEM, TEM) and Fourier Tansform Infrared (FTIR) spectroscopy were employed for the purpose of characterization of attachment of enzyme on the graphene. The enzyme kinetic studies were carried out for obtaining best catalytic performance and enhanced reusability. Optimum temperature remained unchanged, whereas optimum pH showed shift towards acidic range for immobilized enzyme. Increase in thermal stability of immobilized enzyme and non-toxic nature of functionalized graphene can be exploited for production of maltose in food and pharmaceutical industries.

Cicer α-galactosidase immobilization onto functionalized graphene nanosheets using response surface method and its applications.

Cicer α-galactosidase was immobilized onto functionalized graphene with immobilization efficiency of 84% using response surface methodology (Box-Behnken design). The immobilized enzyme had higher thermal stability than the soluble one, attractive for industrial applications. Immobilization of the enzyme lowered the Km to 1/3rd compared to the soluble enzyme. Raffinose family oligosaccharides (RFOs) are mainly responsible for flatulence by taking soybean derived food products. The immobilized enzyme can be used effectively for the hydrolysis of RFOs. After ten successive runs, the immobilized enzyme still retained approximately 60% activity, with soybean RFOs. The easy availability of enzyme source, ease of its immobilization on matrices, non-toxicity, increased stability of immobilized enzyme and effective hydrolysis of RFOs increase the Cicer α-galactosidase application in food processing industries.

Characterization and evaluation of amine-modified graphene amphotericin B for the treatment of visceral leishmaniasis: in vivo and in vitro studies.

Amphotericin B (AmB) has been the first-line treatment for visceral leishmaniasis (VL), a neglected protozoan disease, especially in regions like Bihar, India, where resistance to antimonials is widespread. However, adverse drug reactions are a major limiting factor. We evaluated a novel formulation of AmB conjugated to amine-modified graphene (f-Gr) for safety and efficacy over conventional AmB. The f-Gr was prepared in a gentle one-step process of noncovalent (amine) functionalization with the help of amino acid L-cysteine. This f-Gr was further conjugated to AmB by peptide bond. The conjugate (f-Gr-AmB) was characterized by Raman spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, and transmission electron microscopy. f-Gr-AmB was found to exhibit lesser cytotoxicity toward J774A.1 cells than AmB, and did not induce any hepatic or renal toxicity in Swiss albino mice. In vitro antileishmanial assay in J774A.1 cells showed significantly enhanced efficacy of f-Gr-AmB over AmB. Furthermore, percentage inhibition of amastigote replication in a hamster model of VL was significantly higher in the f-Gr-AmB treated group (87.8%) compared to the AmB group (70.4%). These results suggest that f-Gr-AmB could be a safe and effective alternative to conventional AmB in the treatment of VL.

Immobilization of ß-galactosidase onto functionalized graphene nano-sheets using response surface methodology and its analytical applications.

BACKGROUND: ß-Galactosidase is a vital enzyme with diverse application in molecular biology and industries. It was covalently attached onto functionalized graphene nano-sheets for various analytical applications based on lactose reduction. METHODOLOGY/PRINCIPAL FINDINGS: Response surface methodology based on Box-Behnken design of experiment was used for determination of optimal immobilization conditions, which resulted in 84.2% immobilization efficiency. Native and immobilized functionalized graphene was characterized with the help of transmission and scanning electron microscopy, followed by Fourier transform infrared (FTIR) spectroscopy. Functionalized graphene sheets decorated with islands of immobilized enzyme were evidently visualized under both transmission and scanning electron microscopy after immobilization. FTIR spectra provided insight on various chemical interactions and bonding, involved during and after immobilization. Optimum temperature and energy of activation (E(a)) remains unchanged whereas optimum pH and K(m) were changed after immobilization. Increased thermal stability of enzyme was observed after conjugating the enzyme with functionalized graphene. SIGNIFICANCE: Immobilized ß-galactosidase showed excellent reusability with a retention of more than 92% enzymatic activity after 10 reuses and an ideal performance at broad ranges of industrial environment.

Enzymatic detection of As(III) in aqueous solution using alginate immobilized pumpkin urease: optimization of process variables by response surface methodology.

Urease immobilized on alginate was utilized to detect and quantify As(3+) in aqueous solution. Urease from the seeds of pumpkin (vegetable waste) was purified to apparent homogeneity by heat treatment and gel filtration (Sephadex G-200). Further enzyme was entrapped in 3.5% alginate beads. Urea hydrolysis by enzyme revealed a clear dependence on the concentration and interaction time of As(3+). The process variables effecting the quantitation of As(3+) was investigated using central composite design with Minitab 15 software. The predicted results were found in good agreement (R(2)=96.71%) with experimental results indicating the applicability of proposed model. The multiple regression analysis and ANOVA showed that enzyme activity decreased with increase of As(3+) concentration and interaction time. 3D plot and contour plot between As(3+) concentration and interaction time was helpful to predict residual activity of enzyme for a particular As(3+) at a particular time.