Vinita Hooda

Immobilization and Kinetics of Catalase on Calcium Carbonate Nanoparticles Attached Epoxy Support

A novel hybrid epoxy/nano CaCO3 composite matrix for catalase immobilization was prepared by polymerizing epoxy resin in the presence of CaCO3 nanoparticles. The hybrid support was characterized using scanning electron microscopy and Fourier transform infrared spectroscopy. Catalase was successfully immobilized onto epoxy/nano CaCO3 support with a conjugation yield of 0.67u2009±u20090.01xa0mg/cm2 and 92.63u2009±u20090.80xa0% retention of activity. Optimum pH and optimum temperature of free and immobilized catalases were found to be 7.0 and 35xa0°C. The value of Km for H2O2 was higher for immobilized enzyme (31.42xa0mM) than native enzyme (27.73xa0mM). A decrease in Vmax value from 1,500 to 421.10xa0μmol (minxa0mg protein)−1 was observed after immobilization. Thermal and storage stabilities of catalase improved immensely after immobilization. Immobilized enzyme retained three times than the activity of free enzyme when kept at 75xa0°C for 1xa0h and the half-life of enzyme increased five times when stored in phosphate buffer (0.01xa0M, pH 7.0) at 5xa0°C. The enzyme could be reused 30 times without any significant loss of its initial activity. Desorption of catalase from the hybrid support was minimum at pH 7.0.

A new immobilization and sensing platform for nitrate quantification.

Nitrate reductase (NR) from Aspergillus niger was covalently coupled to the epoxy affixed gold nanoparticles (epoxy/AuNPs) with a conjugation yield of 35.40±0.01 μg/cm(2) and 93.90±0.85% retention of specific activity. The bare and NR bound epoxy/AuNPs support was characterized using scanning electron microscopy and Fourier Transform Infrared Spectroscopy. The immobilized enzyme system was optimized with respect to pH, temperature and substrate concentrations and successfully employed for determination of nitrate contents in ground water. The minimum detection limit of the method was 0.05 mM with linearity from 0.1 to 10.0 mM. The % recoveries of added nitrates (0.1 and 0.2 mM) were >95.0% and within-day and between-day coefficients of variations were 1.012% and 3.125% respectively. The method showed good correlation (R(2)=0.998) with the popular Griess reaction method. Epoxy/AuNPs bound NR showed good thermal and storage stabilities and retained 50% activity after 16 reuses.

Immobilization of nitrate reductase onto epoxy affixed silver nanoparticles for determination of soil nitrates

Epoxy glued silver nanoparticles were used as immobilization support for nitrate reductase (NR). The resulting epoxy/AgNPs/NR conjugates were characterized at successive stages of fabrication by scanning electron microscopy and fourier transform infrared spectroscopy. The immobilized enzyme system exhibited reasonably high conjugation yield (37.6±0.01 μg/cm(2)), with 93.54±0.88% retention of specific activity. Most favorable working conditions of pH, temperature and substrate concentration were ascertained to optimize the performance of epoxy/AgNPs/NR conjugates for soil nitrate quantification. The analytical results for soil nitrate determination were consistent, reliable and reproducible. Minimum detection limit of the method was 0.05 mM with linearity from 0.1 to 11.0 mM. The % recoveries of added nitrates (0.1 and 0.2 mM) were<95.0% and within-day and between-day coefficients of variations were 0.556% and 1.63% respectively. The method showed good correlation (R(2)=0.998) with the popular Griess reaction method. Epoxy/AgNPs bound NR had a half-life of 18 days at 4 °C and retained 50% activity after 15 reuses.

Effect of changing the nanoscale environment on activity and stability of nitrate reductase

Nitrate reductase (NR) is employed for fabrication of nitrate sensing devices in which the enzyme in immobilized form is used to catalyze the conversion of nitrate to nitrite in the presence of a suitable cofactor. So far, instability of immobilized NR due to the use of inappropriate immobilization matrices has limited the practical applications of these devices. Present study is an attempt to improve the kinetic properties and stability of NR using nanoscale iron oxide (nFe3O4) and zinc oxide (nZnO) particles. The desired nanoparticles were synthesized, surface functionalized, characterized and affixed onto the epoxy resin to yield two nanocomposite supports (epoxy/nFe3O4 and epoxy/nZnO) for immobilizing NR. Epoxy/nFe3O4 and epoxy/nZnO support could load as much as 35.8±0.01 and 33.20±0.01μg/cm(2) of NR with retention of about 93.72±0.50 and 84.81±0.80% of its initial activity respectively. Changes in surface morphology and chemical bonding structure of both the nanocomposite supports after addition of NR were confirmed by scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FTIR). Optimum working conditions of pH, temperature and substrate concentration were ascertained for free as well as immobilized NR preparations. Further, storage stability at 4°C and thermal stability between 25-50°C were determined for all the NR preparations. Analytical applications of immobilized NR for determination of soil and water nitrates along with reusability data has been included to make sure the usefulness of the procedure.

In vitro effects of metal oxide nanoparticles on barley oxalate oxidase

Barley oxalate oxidase (OxO), a manganese-containing protein, is largely employed for determination of oxalate in various biologic materials. The present report describes in vitro effects of nanoparticles (NPs) of three metal oxides, i.e., zinc oxide (ZnO), copper oxide (CuO), and manganese oxide (MnO2), on the activity and stability of OxO purified from barley roots. The transmission electron microscopy and X-ray diffraction studies of these NPs revealed their very fine crystalline structure with the dimeter in the range 30–70, 50–60, and 20–60xa0nm for ZnO NPs, CuO NPs, and MnO2 NPs, respectively. The addition of suspension of these three NPs into assay mixture of enzyme individually, led to the adsorption of OxO over their surface, as confirmed by Fourier transform infrared spectra and UV–Vis spectroscopic studies. Compared to free enzyme, MnO2 NPs-bound enzyme showed improved activity (35xa0% stimulation at 2.5xa0mg/ml concentration), while ZnO NPs- and CuO NPs-bound enzyme had no substantial improvement. The kinetic properties of individually NPs-bound enzyme were studied and compared with those of free enzyme. The MnO2 NPs-bound enzyme also showed marked improvement in its storage and thermal stability compared to free enzyme.

Increasing the efficiency of immobilization and chitin determination using copper oxide nanoparticles

A novel polyurethane (PU) support with and without copper oxide nanoparticles (nCuO) was employed for immobilization of two chitinolytic enzymes, chitinase and N-acetyl β glucosaminidase (NAGase) to yield PU/nCuO/chitinase/NAGase and PU/chitinase/NAGase conjugates respectively. The surface morphologies, topologies and bonded interactions between different components of the immobilized enzyme conjugates were characterized using Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and Fourier Transform Infrared Spectroscopy (FTIR) respectively. High conjugation yield of 0.811±0.10mg/cm2 with 97.66±0.50% retention of specific activity of enzymes on PU/nCuO support vis-a-vis a conjugation yield of 0.531±0.50mg/cm2 with 75.23±0.60% retention activity on PU support was achieved. Additionally, increased pH and temperature tolerance, better kinetic parameters and enhanced stabilities of PU/nCuO/chitinase/NAGase conjugates established their superiority over the PU/chitinase/NAGase conjugates. However, both the conjugates were successfully employed for the determination of chitin contents in the apparently healthy stored rice grains. The methods were tested on analytical parameters such as linearity, limit of detection, the coefficient of variation and reproducibility of the externally added chitin, which were again found to be superior for PU/nCuO/chitinase/NAGase conjugates.

Enzymes loaded chitosan/coconut fibre/zinc oxide nanoparticles strip for polyamine determination

Most often, the immobilized enzyme based quantification is an attractive alternative to other chromatographic, electrochemical and mass spectrometry based methods due to its specificity and simplicity. In the present study, polyamine oxidase specific for spermine and spermidine and diamine oxidase specific for putrescine, were co-immobilized onto a novel chitosan/coconut fibre/zinc oxide nanoparticles (CS/CF/nZnO) hybrid support to yield a polyamine sensing strip. The strip worked optimally at pH 7.0, temperature 25°C and 6min of incubation time. Pretty good values for kinetic constants Kmapp (6.60mM), Vmax (17.69μmol/min mg protein) and kcatapp (1987.64s-1) as well as for thermal (<50 % activity retained at 40°C), storage (half life-40days) and operational stabilities (<90 % activity retained after 20 reuses) were obtained. The strip was employed for polyamine determination in some of the locally grown fruit and vegetables and the results were found to be comparable, reliable and reproducible.

Nitrate quantification: recent insights into enzyme-based methods

Abstract Nitrate monitoring of environmental samples is essential to safeguard human and environmental health. Various non-enzymatic methods such as Griess reaction-based chemical method; Fourier transform infrared spectroscopy; chromatographic, electrochemical and optical sensors yield reproducible results but suffer from drawbacks such as use of hazardous chemicals, interference from coexistent anions, and bulky and expensive instrumentation and hence are not favored for routine analysis. On the other hand, nitrate reductase (NR)-based methods are simple, sensitive, specific, environment friendly, easy to carry out, and, therefore, suitable for routine analysis. NR in these methods is employed in both free (in commercially available kits) and immobilized form. In comparison to the native NR, immobilized NR shows better activity and stability accompanied by overall reduction in the cost of the method. The review gives a brief account of non-enzymatic nitrate quantification, whereas recent advances in enzyme-based determination have been explored in more detail.

Alcohol quantification: recent insights into amperometric enzyme biosensors

Abstract Biosensors are the switching channels that make sense. The biosensors have found an empirical role in health applications (e.g. clinical diagnostics) as they represent the technological counterpart of living senses. On a global scale, alcohol analysis is indispensable for criminal justice systems, monitoring medical conditions of HIV patients & pregnant women as well as public safety issues regarding pilots, metro drivers, doctors etc. For addressing the clinical and toxicological problems, much advancement in the improvement of biosensors have been witnessed in recent decades. Currently, electrochemical biosensors dominate the field which harnesses the synergistic action of enzymes and nanomaterials for the analysis of ethanol. The enzymatic biosensors are the most explored biosensing devices among all the types of biosensors, and employment of nanomaterials has paved a way to the further improvements in this gem of a discovery. The relative comparison to precise the alcohol biosensors has been aptly discussed in the review on the basis of several analytical properties including fabrication, linearity, sensitivity, response time, detection limit as well as storage stability. Finally, the recent trends and emerging future prospects of alcohol biosensors have been reviewed.

Bilirubin enzyme biosensor: potentiality and recent advances towards clinical bioanalysis

Bilirubin detection plays a major role in healthcare. Its high concentration in human serum is lethal and must be determined accurately. Clinically, it is vital for assessing patients with deleterious health conditions such as jaundice or icterus, hepatitis, mental disorders, cerebral palsy and brain damage especially in the case of neonates. In evaluating the drawbacks regarding the conventional methodology of bilirubin detection, there is need for a superior analytical tool. Bilirubin oxidase (BOx)-based sensors have been designed for the ultrasensitive analysis of bilirubin and quality deliverance of treatment and this review highlights the different mechanisms of bilirubin detection using different modified electrodes. Further, it also addresses the exploitation of highly attractive electrocatalytic properties of elite nanoparticles such as gold and zirconia- coated silica nanoparticles in enhancing the reproducibility and specificity of bilirubin biosensors.