Novel approach using activated cellulose film for efficient immobilization of purified diamine oxidase to enhance enzyme performance and stability.

The presence of various contaminants in foodstuffs has led to serious public health concerns. Diamine oxidase (DAO) has attracted tremendous attention for guarding food safety as well as clinical and environmental industries. In this study, DAO from Pisum sativum (Pea) seedlings was extracted and purified by dialysis and gel filtration. Purified DAO was covalently immobilized onto the surface of nitrocellulose membrane using glutaraldehyde. The obtained bioaffinity support has efficiently shown high yield immobilization of DAO from pea seedlings. The optimal conditions of free and immobilized DAO activity were evaluated against the substrate, Putrescine dihydrochloride. The influence of pH, temperature, storage stability, and reusability of immobilized enzyme with comparison to the free enzyme was studied and the results showed that the stabilities were significantly enhanced compared with free counterpart. Residual activity of the immobilized enzyme was 59% of the initial activity after being recycled 10 times. We approve that this novel low cost immobilized DAO carrier presents a new approach in large scale applications.

A novel amperometric bienzymatic biosensor based on alcohol oxidase coupled PVC reaction cell and nanomaterials modified working electrode for rapid quantification of alcohol.

A new amperometric sensor has been fabricated for sensitive and rapid quantification of ethanol. The biosensor assembly was prepared by covalently immobilizing alcohol oxidase (AOX) from Pichia pastoris onto chemically modified surface of polyvinylchloride (PVC) beaker with glutaraldehyde as a coupling agent followed by immobilization of horseradish peroxidase (HRP), silver nanoparticles (AgNPs), chitosan (CHIT), carboxylated multi-walled carbon nanotubes (c-MWCNTs) and nafion (Nf) nanocomposite onto the surface of Au electrode (working electrode). Owing to properties such as chemical inertness, light weight, weather resistance, corrosion resistance, toughness and cost-effectiveness, PVC membrane has attracted a growing interest as a support for enzyme immobilization in the development of biosensors. The amperometric biosensor displayed optimum response within 8 s at pH 7.5 and 35°C temperature. A linear response to alcohol in the range of 0.01mM-50 mM and 0.0001 µM as a minimum limit of detection was displayed by the proposed biosensor with excellent storage stability (190 days) at 4°C. The sensitivity of the sensor was found to be 155 µA mM-1 cm-2. A good correlation (R2 = 0.99) was found between alcohol level in commercial samples as evaluated by standard ethanol assay kit and the current biosensor which validates its performance.

A novel amperometric biosensor for rapid detection of ethanol utilizing gold nanoparticles and enzyme coupled PVC reaction cell.

This research was aimed at the fabrication of an improved enzyme-based amperometric biosensor for rapid quantification of ethanol. Alcohol oxidase (AOX) from Pichia pastoris was covalently immobilized on chemically treated polyvinylchloride (PVC) beaker and subsequently horseradish peroxidase (HRP), nafion (Nf), carboxylated multi-walled carbon nanotubes (c-MWCNTs), chitosan (CHIT) and gold nanoparticles (AuNPs) were immobilized onto Au electrode to fabricate a working electrode. The enzyme-coated PVC surface was analysed morphologically via scanning electron microscopy (SEM). At different stages of construction, the electrochemical properties of working electrode were deciphered by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor displayed optimal response in a short time span of 12 s at pH 7.5 and 35°C temperature. The working range exhibited by the proposed biosensor was 0.01-42 mM with a limit of detection (LOD) of 0.0001 µM and storage stability of 180 days at 4°C. When level of alcohol was evaluated in commercial samples via standard assay kit and existing biosensor, a good correlation (R2 = 0.98) was observed which authenticates its reliability.

A novel polyurethane/nano ZnO matrix for immobilization of chitinolytic enzymes and optical sensing of chitin.

Purified chitinase from Vigna mungo and N-acetyl ß glucosaminidase (NAGase) from Canavalia ensiformis were immobilized on to the novel polyurethane (PU)/zinc oxide nanoparticles (nano ZnO) composite matrix with a conjugation yield of 0.785±0.01mg/cm2 and 96.19±0.85% retention of specific activity. Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR) also confirmed the presence of nano ZnO and enzymes on the PU support. Thus synthesized PU/nano ZnO/chitinase/NAGase conjugates were optimized with respect to pH, temperature and substrate concentration and successfully employed for development of an absorbance based optical biosensor for chitin determination in stored wheat grains. The limit of detection was 0.01mM with linearity from 0.1 to 10.0mM. The% recoveries of added chitin (0.1 and 0.2mM) were >95.0% and >96.5% respectively and within-day and between-day coefficients of variations were 1.03% and 1.78% respectively. The method showed good correlation (R2=0.996) with the popular 3,5-dinitrosalicylic acid method. PU/nano ZnO bound chitinase/NAGase showed good thermal and storage stabilities and could be reused 10 times without any appreciable loss of activity.

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.

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 0.67 ± 0.01 mg/cm(2) and 92.63 ± 0.80 % retention of activity. Optimum pH and optimum temperature of free and immobilized catalases were found to be 7.0 and 35 °C. The value of Km for H2O2 was higher for immobilized enzyme (31.42 mM) than native enzyme (27.73 mM). A decrease in Vmax value from 1,500 to 421.10 µmol (min mg 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 75 °C for 1 h and the half-life of enzyme increased five times when stored in phosphate buffer (0.01 M, pH 7.0) at 5 °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.