Browsing by Author "Mahe Talat"

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A Facile and Simple Strategy for the Synthesis of Label Free Carbon Quantum Dots from the latex of Euphorbia milii and Its Peroxidase-Mimic Activity for the Naked Eye Detection of Glutathione in a Human Blood Serum
(American Chemical Society, 2019) Daraksha Bano; Vijay Kumar; Vikas Kumar Singh; Subhash Chandra; Devendra Kumar Singh; Pradeep Kumar Yadav; Mahe Talat; Syed Hadi Hasan
Herein a green synthetic route has been developed for the synthesis of water-soluble CQDs by facile single-step hydrothermal treatment of latexes of E. milii plant for the first time. This methodology is zero-cost; uses ultrapure water as a green solvent; does not use strong concentrated acid; and avoids the use of post surface passivating agents. The as-prepared CQDs exhibited excellent optical properties, including high QY up to 39.2%, resistance to high salt strength, and long time photostability. Furthermore, the as-prepared CQDs served as an intrinsic peroxidase-mimic activity to catalyze the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) associated with H 2 O 2 , which resulted into a blue-colored reaction with a characteristic absorbance peak at 652 nm. Afterward, the proposed TMB-based oxidation system act as a probe for the detection of GSH and offers the high selectivity relative to the different amino acids and various other interfering agents which can be easily seen with naked eye. The limit of detection (LOD) was found to be 5.3 nM in a linear range 0.02 to 0.1 μM of GSH concentration which showed to be superior under the optimal condition as compared with another probe. To demonstrate the practical feasibility for the GSH detection, the present system was successfully applied on human blood serums with good recovery. © 2018 American Chemical Society.
Amine functionalized graphene sheet conjugated with amphotericin B for the treatment of visceral leishmaniasis
(Advanced Engineering Solutions, 2014) Mahe Talat; Shyam M. Lal; Madhukar Rai; Shyam Sundar; Onkar Nath Srivastava
The treatment options for visceral leishmaniasis (VL), potentially caused by Leishmania donovani, are limited and unsatisfactory. Conventional AmB exhibits low solubility and instability at gastric pH and is unable to penetrate the brush border membrane of the small intestine and have have serious toxicity. Different nano vectors have been explored for the transportation of these drugs to the target. We evaluated a novel formulation of Amphotericin B (AmB) conjugated to amine-functionalized graphene (f-Gr) for safety and efficacy over conventional AmB. Graphene sheet have been synthesized by thermal exfoliation of chemically prepared graphite oxide by rapid heating at 1050°C in an Ar atmosphere. As synthesized graphene sheet was amine functionalized non-covalently with L-cysteine at room temperature. As functionalized graphene sheet was further used to conjugate AmB. The conjugate (f-Gr-AmB) was characterized by FTIR, SEM and TEM that established successful attachment of AmB to f-Gr. The f-Gr-AmB was found to exhibit lesser cytotoxity as compared to AmB also it showed significantly enhanced efficacy of f-Gr-AmB over AmB. However the results of in vivo and in vitro studies are not described here. © 2014, Advanced Engineering Solutions (AES.COM) Ottawa, Canada.
Application of response surface methodology for the determination of optimum reaction conditions (Temperature and pH) for starch hydrolysis by α-amylase
(2011) Nivedita Jaiswal; Om Prakash; Mahe Talat; S.H. Hasan; Rajesh Kumar Pandey
α-amylase from soybean seeds was purified to apparent homogeneity by affinity precipitation via entrapment in alginate with 84% recovery and about 20-fold purification. The α-amylase activity and stability was characterized at various pH and temperature. Response Surface Methodology (RSM) using two-level-two-factor full factorial Central Composite Design (CCD) model was employed to optimize process parameters like pH and temperature which affects the kinetics of α-amylase catalyzed hydrolysis of starch. The results predicted by the design were found in good agreement (R2 = 97.85%) 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 unto 5.5 and temperature 75°C. Thus, the RSM was successful in determining the optimum reaction conditions for starch hydrolysis by α-amylase. © 2011 Academic Journals Inc.
Biosynthesis of gold and silver nanoparticles by natural precursor clove and their functionalization with amine group
(2010) Ashwani Kumar Singh; Mahe Talat; D.P. Singh; O.N. Srivastava
We report a simple and cost effective way for synthesis of metallic nanoparticles (Au and Ag) using natural precursor clove. Au and Ag nanoparticles have been synthesized by reducing the aqueous solution of AuCl4 and AgNO3 with clove extract. One interesting aspect here is that reduction time is quite small (few minutes instead of hours as compared to other natural precursors). We synthesized gold and silver nanoparticles of different shape and size by varying the ratio of AuCl4 and AgNO3 with respect to clove extract, where the dominant component is eugenol. The evolution of Au and Ag nanoparticles from the reduction of different ratios of AuCl4 and AgNO3 with optimised concentration of the clove extract has been evaluated through monitoring of surface plasmon behaviour as a function of time. The reduction of AuCl4 and AgNO3 by eugenol is because of the inductive effect of methoxy and allyl groups which are present at ortho and para positions of proton releasing -OH group as two electrons are released from one molecule of eugenol. This is followed by the formation of resonating structure of the anionic form of eugenol. The presence of methoxy and allyl groups has been confirmed by FTIR. To the best of our knowledge, use of clove as reducing agent, the consequent very short time (minutes instead of hours and without any scavenger) and the elucidation of mechanism of reduction based on FTIR analysis has not been attempted earlier.
Bright-blue-emission nitrogen and phosphorus-doped carbon quantum dots as a promising nanoprobe for detection of Cr(vi) and ascorbic acid in pure aqueous solution and in living cells
(Royal Society of Chemistry, 2018) Vikas Kumar Singh; Virendra Singh; Pradeep Kumar Yadav; Subhash Chandra; Daraksha Bano; Vijay Kumar; Biplob Koch; Mahe Talat; Syed Hadi Hasan
Highly fluorescent nitrogen and phosphorus-doped carbon quantum dots (N,P-CQDs) were synthesized via a one-step hydrothermal method and fully characterized via various techniques such as TEM, DLS, FT-IR, P-XRD and XPS analysis. The as-synthesized N,P-CQDs showed excellent optical properties and exhibited bright blue colour under UV-light with CIE coordinate (0.20, 0.22) along with a high quantum yield of 73%, due to which they could act as on-off fluorescent nanoprobes for the selective and sensitive detection of highly toxic Cr(vi) below its permissible limit via the inner filter effect (IFE) and static quenching mechanism. In addition, Cr(vi) could be reduced to lower valent chromium species. Therefore, the N,P-CQDs + Cr(vi) system further acted as a selective off-on sensor for reductant ascorbic acid (AA) because it reduced Cr(vi) to Cr(iii) species, resulting in the elimination of IFE and recovery of fluorescence of N,P-CQDs. Notably, this system possesses excellent biocompatibility and negligible cytotoxicity; therefore, it can be potentially applied for fluorescence imaging of intracellular Cr(vi) and ascorbic acid (AA) in living cells and complex biological systems. © 2018 The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.
Characterization and evaluation of amine-modified graphene amphotericin B for the treatment of visceral leishmaniasis: In vivo and in vitro studies
(Dove Medical Press Ltd., 2014) Shyam Lal Mudavath; Mahe Talat; Madhukar Rai; Onkar Nath Srivastava; Shyam Sundar
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. © 2014 Mudavath et al.
Cicer a-galactosidase immobilization onto functionalized graphene nanosheets using response surface method and its applications
(Elsevier Ltd, 2014) Neelesh Singh; Garima Srivastava; Mahe Talat; Himanshu Raghubanshi; Onkar Nath Srivastava; Arvind M. Kayastha
Cicer a-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 a-galactosidase application in food processing industries. © 2013 Elsevier Ltd.
Covalent immobilization of peanut β-amylase for producing industrial nano-biocatalysts: A comparative study of kinetics, stability and reusability of the immobilized enzyme
(Elsevier Ltd, 2018) Ranjana Das; Mahe Talat; O.N. Srivastava; Arvind M. Kayastha
Stability of enzymes is an important parameter for their industrial applicability. Here, we report successful immobilization of β-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. β-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. © 2017 Elsevier Ltd
Deployment of new carbon nanostructure: Graphene for drug delivery and biomedical applications
(Springer Verlag, 2016) Mahe Talat; O.N. Srivastava
Graphene, the well awarded and popularly known as 2D carbon allotrope, is a versatile material. Its unique physico-chemical properties finds its application in a wide range of areas ranging from quantum physics, nanoelectronics, energy research, catalysis and engineering of nanocomposites, biomaterials and drug delivery. In principle, it is possible to produce graphene from high purity graphite sheet. Since graphite is stacked layers of many graphene sheets, bonded together by week van der Waals force and if these forces are disrupted individual graphene sheets can be separated out. In the present chapter we will discuss different methods of the synthesis of graphene like LPCVD, CVD (Chemical Vapor Deposition), thermal exfoliation, arc discharge, electrochemical exfoliation and chemical reduction. Since graphene having an innate property of hydrophobicity have the tendency to agglomeration and insolubility. To overcome the aforementioned insolubility some functionality needs to be attached to the graphene sheet. Therefore, different methods of functionalization like covalent and non-covalent approaches are also discussed. The last part of the chapter will deal with the application of graphene particularly in the drug delivery and biomedical field. © Springer India 2016.
Detection of copper (II) in aqueous solution by immobilized urease obtained from agro-waste: Optimization of process variables
(2009) Mahe Talat
Urease was used for estimation of Cu2+ in aqueous solution. Process variables were optimized by Central Composite Design using MINITAB® 15 software. Results predicted by the design were close to experimental value indicating suitability of the model. 3D surface plot and isoresponse contour plot were helpful in predicting results by performing limited set of experiments. Urease was extracted from discarded seeds of pumpkin to apparent homogeneity by heat fractionation and gel filtration. Homogeneous enzyme preparation was further immobilized in 3.5% alginate. Effect of Cu2+ on the activity of soluble and immobilized enzyme was investigated. Enzyme inhibition was biphasic, irreversible, and non-competitive (Ki = 1.06 μM). © The Korean Society for Biotechnology and Bioengineering and Springer Berlin Heidelberg 2009.
Effective removal of fluoride from water by coconut husk activated carbon in fixed bed column: Experimental and breakthrough curves analysis
(Elsevier B.V., 2018) Mahe Talat; Sweta Mohan; Viney Dixit; Devendra Kumar Singh; Syed Hadi Hasan; Onkar Nath Srivastava
The bio-waste coconut husk was converted into activated carbon through merely treating with KOH and thus obtained activated carbon was characterized by XRD, TGA, SEM and TEM analysis. The prepared activated carbon having very high surface area (1448 m2/g) was utilized as an adsorbent for the removal of fluoride (F-) from water. Fluoride adsorption experiments were performed on the laboratory-scale column at different bed height, flow rates, and F- concentrations to explore the potential of prepared adsorbent and it was found to be very efficient adsorbent as it showed high adsorption capacity 6.5 mg/g at pH 5, F- concentration 10 mg/L and adsorbent dose 1.4 g/L. Various breakthrough models i.e. Bed Depth Service Time (BDST), Thomas and Yoon–Nelson were applied on breakthrough data to analyze the breakthrough curves. The high R2 values obtained for the BDST model revealed its validity for this adsorption system. Breakthrough curves were successfully analyzed and described by both Thomas and Yoon–Nelson models. The exhausted adsorbent was efficiently regenerated with the 10% NaOH solution and regenerated adsorbent showed remarkable uptake capacity with a slight reduction in adsorption performance up to the 3 cycles. Life factor calculation indicated that adsorbent bed would have sufficient bed capacity up to 8.3 cycles to avoid breakthrough at time t = 0 and the bed would be completely exhausted after 9.0 cycles. © 2018
Enzymatic detection of As(III) in aqueous solution using alginate immobilized pumpkin urease: Optimization of process variables by response surface methodology
(2009) Mahe Talat; Om Prakash; S.H. Hasan
Urease immobilized on alginate was utilized to detect and quantify As3+ 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 As3+. The process variables effecting the quantitation of As3+ was investigated using central composite design with Minitab® 15 software. The predicted results were found in good agreement (R2 = 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 As3+ concentration and interaction time. 3D plot and contour plot between As3+ concentration and interaction time was helpful to predict residual activity of enzyme for a particular As3+ at a particular time. © 2009 Elsevier Ltd. All rights reserved.
Enzymatic detection of heavy metal ions in aqueous solution from vegetable wastes by immobilizing pumpkin (Cucumis melo) urease in calcium alginate beads
(2008) Om Prakash; Mahe Talat; S.H. Hasan; Rajesh K. Pandey
Enzyme urease is extracted from the discarded seeds of pumpkin. Urease was purified to apparent homogeneity (5.2 fold) by heat treatment at 48 ± 1°C and gel filtration through Sephadex G-200. Effect of model metal ions on the activity of the homogeneous enzyme preparation (sp. activity 353 U/mg protein, A280/A260 = 1.12) of soluble as well as immobilized enzyme was investigated. The soluble and immobilized urease has been used for the quantitative estimation of general water pollution with heavy metal ions like Hg2+, Cu2+, Cd2+, and Co2+. The measurements of the urease residual activity have been carried out in tris-acetate buffer after pre-incubation of model metal salt. The inhibition was found to be biphasic with an initial rapid loss of activity and remainder in slow phase of 10-15 min. The immobilization was done in 3.5% alginate beads leading to 86% of entrapment. There was no leaching of the enzyme over a period of 15 days at 4°C. The beads were fairly stable up to 50°C and exhibited activity even at -10°C. The inhibition by these ions was non-competitive and irreversible, hence could not be restored by dialysis. Based on the values of inhibition constant Ki the heavy-metal ions were found to inhibit urease in the following order Hg2+ > Cu2+ > Cd2+ > Co2+. © KSBB.
Enzymatic detection of mercuric ions in ground-water from vegetable wastes by immobilizing pumpkin (Cucumis melo) urease in calcium alginate beads
(2008) Om Prakash; Mahe Talat; Syed Hadi Hasan; Rajesh K. Pandey
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 °C and gel filtration through Sephadex G-200. The homogeneous enzyme preparation (Sp activity 353 U/mg protein, A280/A260 = 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. Hg2+ 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 °C. The inhibition was non-competitive and the Ki was found to be 1.26 × 10-1 μM. Time-dependent interaction of urease with Hg2+ 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 Hg2+ with the enzyme was largely irreversible, as the activity could not be restored by dialysis. The significance of the observations is discussed. © 2007 Elsevier Ltd. All rights reserved.
Facile synthesis of large surface area graphene and its applications
(Springer Verlag, 2017) Mahe Talat; Prashant Tripathi; Onkar Nath Srivastava
In 2008, one of the most costly materials on earth was graphene which was obtained by exfoliation method, having a model area of a human hair cross section costing more than $1000 as of April 2008 [2]. Since then, hunger for searching low-cost, non-hazardous alternative methods for scalable production of graphene continued. Graphene’s flexible chemistry with an atomic thickness makes it superlatives in material science, an ideal candidate for countless applications. Incredibly light weight and flexible, yet 200 times stronger than steel. Highly conductive but at the same time transparent, first 2D material existing but is one million times smaller than the diameter of a single human hair. Having all these amazing properties, graphene is creating an impact in wide range of industries including in fields of electronics, EMI shielding, composites, sensing devices as well as in energy storage, biomedical devices, and many more. Application of this wonder material is restricted to our imagination only. These engineering applications necessitate accessibility of graphene on the large scale, and methods used to synthesize this are facile, cost-effective, simple, quick, and single-step process, and thus appropriate processes are essential for synthesizing it down to a single-sheet level. The quality and quantity of graphene also plays an essential role, as the occurrence of defects, structural disorders, impurities, multiple domains, grain boundaries, and wrinkles in the graphene sheet can leave an unwanted effect on its electronic and optical properties. The present chapter will be aimed at the preparation of high quality few-layer graphene on a bulk scale from graphite in an affordable, nontoxic, and easy method. The methods which will be discussed in this chapter will be mostly on microwave-assisted synthesis of graphene and electrochemical exfoliation method used in our lab as well as reported by other workers, and how these methods are advantageous over conventional exfoliation methods. Last segment of this chapter will deal with applications of graphene particularly in EMI shielding. © 2017, Springer Nature Singapore Pte Ltd.
Factorial design for the optimization of enzymatic detection of cadmium in aqueous solution using immobilized urease from vegetable waste
(2008) Om Prakash; Mahe Talat; S.H. Hasan; Rajesh K. Pandey
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 °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 °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 × 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 (22), 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. © 2008 Elsevier Ltd. All rights reserved.
Fluoride quantitation in aqueous solution by agarose immobilized pumpkin urease
(2011) Om Prakash; Rajesh K. Pandey; Mahe Talat; Nivedita Jaiswal
Soluble as well as agarose immobilized pumpkin urease was utilized for easy and rapid detection and quantitation of fluoride in aqueous solution. Pumpkin urease was immobilized in 1.5 % agarose leading to 74.2 % immobilization. Inhibition by both soluble and agarose immobilized enzyme revealed a clear dependence on concentration and time. In order to inhibit the activity completely in soluble enzyme, 45 mM fluoride was required while agarose immobilized enzyme required almost double the concentration of the inhibitor i.e., 80 mM. The inhibition caused by fluoride was noncompetitive with K i value of 3.9 and 2.3 mM. The significance of the observations is discussed. © 2011 Bentham Science Publishers.
Functionalized graphene sheets as immobilization matrix for fenugreek β-Amylase: Enzyme kinetics and stability studies
(Public Library of Science, 2014) Garima Srivastava; Kritika Singh; Mahe Talat; Onkar Nath Srivastava; Arvind M. Kayastha
β-Amylase finds application in food and pharmaceutical industries. Functionalized graphene sheets were customised as a matrix for covalent immobilization of Fenugreek β-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. © 2014 Srivastava et al.
Green Synthesis of Fluorescent Carbon Quantum Dots from Azadirachta indica Leaves and Their Peroxidase-Mimetic Activity for the Detection of H 2 O 2 and Ascorbic Acid in Common Fresh Fruits
(American Chemical Society, 2019) Pradeep Kumar Yadav; Vikas Kumar Singh; Subhash Chandra; Daraksha Bano; Vijay Kumar; Mahe Talat; Syed Hadi Hasan
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 (H 2 O 2 ). Further, the kinetics of peroxidase-like catalytic activity follows the Michaelis-Menten and ping-pong pathway. In addition, the H 2 O 2 sensitive TMB oxidation motivated the colorimetric detection of H 2 O 2 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. Copyright © 2018 American Chemical Society.
Immobilization of β-galactosidase onto functionalized graphene nano-sheets using response surface methodology and its analytical applications
(2012) Devesh Kishore; Mahe Talat; Onkar Nath Srivastava; Arvind M. Kayastha
Background: β-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 (Ea) remains unchanged whereas optimum pH and Km were changed after immobilization. Increased thermal stability of enzyme was observed after conjugating the enzyme with functionalized graphene. Significance: Immobilized β-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. © 2012 Kishore et al.