Matrix For Enzyme Immobilization Research Articles

A facile method for the immobilization of myoglobin on multi-walled carbon nanotubes: Poly(methacrylic acid-co-acrylamide) nanocomposite and its application for direct bio-detection of H2O2

Poly(methacrylic acid-co-acrylamide) (P(MAA-co-AAM)), noncovalently attached on the surface of multiwalled carbon nanotubes (MWCNTs) through hydrophobic interactions, hydrogen bonding or electrostatic attraction, was employed for the first time as a novel matrix for enzyme immobilization to develop highly sensitive amperometric biosensors. The effective interaction between P(MAA-co-AAM) and MWCNTs, and the structural morphology of the as-prepared P(MAA-co-AAM)–MWCNTs nanocomposite were characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Further experimental results show that the solubility and stability of as-prepared P(MAA-co-AAM)–MWCNTs nanocomposites in water are better than that of pristine MWCNTs, and no significant electronic and structural change was observed after functionalization. When myoglobin (Mb) was selected as a model protein, studies by Ultraviolet–visible (UV–vis) and Circular dichroism (CD) spectroscopy indicate that the encapsulated Mb in the as-prepared P(MAA-co-AAM)–MWCNTs nanocomposites, was kept in a near-native state, indicating the P(MAA-co-AAM)–MWCNTs nanocomposites have good biocompatibility for protein/enzyme immobilization. The fabricated electrochemical biosensor based on the immobilized Mb reveals fast response of less than 3 s, wide linear range from 1.47×10−6M to 4.76×10−3M and good detection limit as low as 7.60×10−7M toward the electro-determination of hydrogen peroxide (H2O2) under optimal experimental conditions.

Enzymatic Gas-Diffusion Cathodes with Increased Performance and Stability

The design of a viable enzymatic gas-diffusional cathode has to satisfy the following main requirements: 1) consist of a porous hydrophobic layer that allows the flow of oxygen to feed the catalytic layer; 2) have a porous thin catalytic layer with enzyme-air-electrolyte three-phase interface, and 3) preserve enzymatic activity. Additionally, a direct electron transfer mechanism is desired for enhanced catalytic performance at the electrode surface. Among those requirements, the stability of the enzymatic cathodes is the biggest challenge and the main drawback of the technology due to instability and short lifetime of the enzymatic component.This research introduces the design of gas-diffusional cathode employing bilirubin oxidase (BOx, a multicopper oxidase proven to have a direct electron transfer mechanism [1-8]) immobilized on a complex matrix composed of carbon nanotube(CNT) modified Toray paper (TP) and encapsulated in silica-gel. The developed enzymatic cathode consists of two layers, hydrophobic gas-diffusional layer (GDL) and hydrophilic catalytic layer (CL), pressed together at 1000 psi for five minutes. The GDL (35 wt% teflonized Vulcan carbon powder (XC35)), exposed to air, has hydrophobic and porous properties that allows facilitated oxygen diffusion. The CL (CNTs modified TP) is thin with high surface area providing a 3D CNT/silica-gel matrix for enzyme immobilization and preserved stability. The design of the catalytic layer was improved by growing a CNT “forest”, which was possible by pulse chronoamperometry electrodeposited [11-12] Ni seeds that in turn go through chemical vapor deposition (CVD) mechanism providing highly conductive and high surface area nano-structured layer (Fig. 1). This CNTs-enzyme composite is then utilized as the catalytic layer of the dual layered electrode.The assembly of the bio-cathode on a capillary driven microfluidic system provides a constant flow of electrolyte and facilitates the electrolytic solution transport to the electrode. Therefore this set up design was used to study the performance of the cathode at several different pHs (pH 5 to pH 8) of the electrolytic solution (Fig. 2). All tested cathodes demonstrated an open circuit potential of ~ 0.52V. The performed potentiostatic polarization curves show that the CNT modified TP cathode demonstrates highest performance of approximately 660 μA.cm-2 at 0.0 V and ~350 μA.cm-2at 0.3 V for pH 5.5 (Fig. 2). After this optimal pH, the performance of the cathode decreased demonstrating lowest output at pH 8.To extend the lifetime of the attached enzyme, the advantages of silica encapsulation were explored. Entrapment of BOx is realized using tetramethyl orthosilicate (TMOS) in a CVD process [9-10]. CVD reaction occurs in a chamber of predetermined geometry to ensure sufficient mass transport of TMOS to the electrode surface. TMOS (volatile at room temp.) and water are introduced to the chamber with the modified cathode, having the enzyme deposited on it, causing a reaction with water to take place and a polymerization process to occur. The BOx is entrapped by the bulk silica-gel or silica matrix on the CNT modified TP. This entrapment procedure prolongs the enzymes life up to 6 months (Fig. 3). At day 1 the recorded current density at 0.25 V was 160 μA.cm-2 and at day 180 the current density obtained was 210 μA.cm-2(Fig. 3B). All values of the current densities obtained during the 6 months testing period fall into the confidence limits of the initial output showing preserved enzymatic activity and cathode stability during that time interval. The design and stability demonstrated for the enzymatic electrode can be considered a significant achievement that opens the avenue to new practical applications of biofuel cells. This research demonstrates that increased stability of those systems is a reality.

Functional Interfaces for Biomimetic Energy Harvesting: CNT-DNA Matrix for Enzyme Assembly

The development of 3D structures exploring the properties of nano-materials and biological molecules allows us to mimic natural metabolic processes such as Krebs or Calvin cycle by creating “artificial” metabolic pathways called metabolons. The utilization of metabolons in biomimetic energy-harvesting systems leads to increased fuel oxidation and as a result increased Columbic efficiency. The specific order and minimized distance between the enzymes from those metabolons are the main reasons for subsequent and more effective fuel consumption.In this study a complex carbon nanotubes (CNTs) – DNA matrix for specific enzyme immobilization was created. It composed of “brush” type aligned CNTs covalently attached on electrochemically modified gold surface. This forest like CNTs structure was used as a substrate for the development of a DNA scaffold. This DNA scaffold had a designed sequence that allows strong attachment of DNA to the CNTs surface by wrapping the nanotubes through pi-pi stacking and at the same time has a unique nine base pair motif that was further hybridized. The hybridized part of the DNA was used to specifically attach zinc finger (Znf268) that was genetically fused to small laccase (SLAC).Zinc fingers are small protein structural motifs that can recognize dsDNA and specifically bind to it. We used this property to control the SLAC immobilization by fusing the Znf268 to the enzyme and attaching it to the CNTs-DNA scaffold (Fig. 1). Due to the specific interactions of zinc fingers with DNA we will be able to immobilize enzymes in particular order and distance creating metabolons.Figure 2 shows Atomic Force Microscopy (AFM) images of the gold surface at the different stages of its modification to the creation of the CNTs forest. The modification procedures were also confirmed with x-ray photoelectron spectroscopy (XPS).To prove the concept we used SLAC, which is an enzyme from the family of well-known multi-copper oxidases that are able to reduce oxygen and form water [1]. The enzyme was immobilized by the described approach and characterized by XPS (Fig. 3). The presence of copper in the XPS spectrum unambiguously proofs the success of the immobilization procedure. The developed herein approach for specific enzyme immobilization can be applied for all types of enzymes for the development of bio-cathodes as well as bio-anodes in the design of energy-harvesting systems.The developed herein approach for specific enzyme immobilization can be applied for all types of enzymes for the development of bio-cathodes as well as bio-anodes in the design of energy-harvesting systems.[1] Szilvay, G., S. Brocato, D. Ivnitski, C. Li, P. De La Iglesia, C. Lau, E. Chi, M. Werner-Washburne, S. Banta P. Atanassov Chem. Commun., 2011, 47, 7464–7466.

Direct Electrochemical Tyrosinase Biosensor based on Mesoporous Carbon and Co3O4 Nanorods for the Rapid Detection of Phenolic Pollutants

AbstractA novel tyrosinase (Tyr) biosensor based on a graphitized ordered mesoporous carbon/cobaltosic oxide nanorod (GMC/Co3O4) nanocomposite is developed for the rapid detection of phenolic pollutants. By applying the GMC/Co3O4 nanocomposite as an enzyme immobilization matrix, rapid direct electron transfer between Tyr and the electrode is achieved. The biosensor exhibits a wide linear response for catechol, ranging from 5.0×10−8 to 1.3×10−5 M, with a limit of detection down to 25 nM and a response time of less than 2 s. The sensitivity of the biosensor based on the GMC/Co3O4 nanocomposite (6.4 A M−1 cm−2) is higher than that of the biosensor based on GMC (5.0 A M−1 cm−2) or Co3O4 (3.5 A M−1 cm−2), which can be attributed to the synergistic effect of the GMC/Co3O4 nanocomposite. The biosensor is further used to systematically detect mixed phenolic samples (phenol, catechol, m‐cresol, p‐cresol, and 4‐chlorophenol) and real water samples. The biosensor‐based detection results for river water and tap water samples show outstanding average recovery (92.2–103.3 %) and relative standard deviations (0.9–7.8 %). In comparison with the conventional spectrophotometric method, the biosensor method is more rapid, sensitive, accurate, and convenient. Furthermore, the detection limit of the biosensor method is about 20 times lower than that of the spectrophotometric method. This novel biosensor is proven to be a promising alternative tool for the rapid and on‐site monitoring of environmental phenolic pollutants.

Protein‐coated polymer as a matrix for enzyme immobilization: Immobilization of trypsin on bovine serum albumin‐coated allyl glycidyl ether–ethylene glycol dimethacrylate copolymer

Allyl glycidyl ether (AGE)-ethylene glycol dimethacrylate (EGDM) copolymer with 25% crosslink density (AGE-25) shows excellent bovine serum albumin (BSA) adsorption (up to 16% (w/w)) at pH 8.0 and the adsorbed BSA is strongly bound. This protein-coated polymer provides a novel matrix with naturally existing functional groups such as thiol, amino, and carboxylic acid that are available for covalent immobilization of functional enzymes. Employing appropriate strategies, trypsin as a model protein was covalently bound to BSA-coated matrix both independently, and in a stepwise manner on the same matrix, with less than 5% loss of enzyme activity during immobilization. Glutaraldehyde crosslinking after immobilization provide stable enzyme preparation with activity of 510 units/g recycled up to six times without loss of enzyme activity. AFM studies reveal that the polymer surface has protein peaks and valleys rather than a uniform monolayer distribution of the protein and the immobilized enzyme preparation can best be described as polymer supported cross-linked enzyme aggregates (CLEAs).

Electrochemically induced deposition of poly(benzoxazine) precursors as immobilization matrix for enzymes

Water-soluble benzoxazine oligomers were synthesized by reacting bisphenol A, tetraethylenepentamine and formaldehyde. The pre-formed benzoxazine oligomers can be further electropolymerized from aqueous suspensions under formation of poly(benzoxazine)-based films on electrode surfaces. Integration of glucose oxidase in a poly(benzoxazine) film let to highly reproducible and stable biosensors. Poly(benzoxazines) are proposed as a new family of stable polymers for the design of enzyme electrodes.

Rhoeo discolor leaf extract as a novel immobilizing matrix for the fabrication of an electrochemical glucose and hydrogen peroxide biosensor

This work aims at using non-toxic, easily availableRhoeo discolor(Rd) leaf extract as a matrix for enzyme immobilization in the development of electrochemical biosensors.

A novel conducting polymer based platform for ethanol sensing

A new amperometric ethanol biosensor was fabricated for sensitive and rapid analysis of ethanol. For this purpose, a novel monomer, 9-methyl-9H-carbazole-3-carbohydrazine (MCCH), was designed and electrochemically polymerized on a graphite electrode to achieve a conducting and effective immobilization matrix for enzyme immobilization. Due to the presence of amino groups in the structure of conducting polymer, alcohol oxidase molecules were covalently immobilized onto the functional polymer which brought an effective and long-life analysis of the substrate, ethanol. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) chemistry was used for linking carboxylic acid groups of the enzyme molecules with amino groups of the polymer. After successful immobilization, amperometric biosensor responses were recorded at −0.7V vs. Ag/AgCl in phosphate buffer (pH 7.0). KMapp (8.74mM), Imax (5.94μA), LOD (0.131mM) and sensitivity (4.79μAmM−1cm−2) values were determined. Finally, the prepared biosensor was successfully applied for determination of ethanol content in alcoholic beverages.

Fabrication of Boehmite/Alginate Hybrid Beads for Efficient Enzyme Immobilization

A novel kind of matrix for enzyme immobilization, the boehmite/alginate hybrid beads, was fabricated under mild conditions in this study. The boehmite particles were first prepared via a mild sol–gel technology and were then entrapped into the inherent network of calcium alginate. The characterization results suggest that the hybridization between alginate and boehmite belongs to a physical blending, in which the electrostatic interaction acts an important role. Yeast alcohol dehydrogenase (YADH), which is of high importance for the transformation of CO2, was encapsulated in the boehmite/alginate hybrid beads. The enzyme was loaded as high as 99.4% and uniformly distributed in these hybrid beads according to the fluorescence experiment. It is worthy of noting that the encapsulated YADH can almost reach “zero leaching” after incubation for 67 h and preserve 86.6% activity after 12-recycle using. This kind of hybrid materials may become a promising matrix for the immobilization of industrial enzymes.

Enhanced stability of Kluyveromyces lactis β galactosidase immobilized on glutaraldehyde modified multiwalled carbon nanotubes

Abstract The present study demonstrates covalent immobilization of Kluyveromyces lactis β galactosidase on functionalized multi-walled carbon nanotubes (MWCNTs). Highly efficient surface modification of MWCNTs was achieved by glutaraldehyde for binding greater amount of enzyme. X-ray diffraction analysis and UV visible spectroscopy of MWCNTs showed them to be entirely dispersive in aqueous solution. Transmission electron microscopy showed that MWCNTs were of 20 nm size. Thermogravimetric analysis further revealed the stability of glutaraldehyde modified MWCNT as an ideal matrix for enzyme immobilization. The optimal pH for soluble and immobilized β galactosidase was observed at pH 7.0 while the optimal operating temperatures were observed at 40 °C and 50 °C, respectively. Moreover, our findings demonstrated that β galactosidase immobilized on surface functionalized MWCNTs retained greater biocatalytic activity at higher galactose concentration, and upon repeated uses as compared to enzyme in solution.

Immobilization and catalytic activity of horseradish peroxidase on molybdenum disulfide nanosheets modified electrode

This communication reports the application of layer-structured MoS2 as matrix for enzyme immobilization and biosensor construction. MoS2 nanosheets were prepared using ultrasonic exfoliation in solvent mixture. Data from UV–vis spectra, scanning electron microscopy and transmission electron microscopy demonstrate that the as-prepared MoS2 nanomaterial has ordered layer-structure. The negative charges carried by the MoS2 nanosheets are then used to immobilize horseradish peroxidase (HRP) via the electrostatic attraction, forming HRP–MoS2 hybrid. FTIR spectra indicate that the immobilized HRP retains its native structure. The HRP–MoS2 hybrid modified glassy carbon electrode shows good electrocatalytic activity toward H2O2 reduction. This work suggests that MoS2 nanosheets are a potential candidate for efficient enzyme immobilization and promising in biosensing and biocatalytic applications.

Study on Different Molecular Weights of Chitosan as an Immobilization Matrix for a Glucose Biosensor

Two chitosan samples (medium molecular weight (MMCHI) and low molecular weight (LMCHI)) were investigated as an enzyme immobilization matrix for the fabrication of a glucose biosensor. Chitosan membranes prepared from acetic acid were flexible, transparent, smooth and quick-drying. The FTIR spectra showed the existence of intermolecular interactions between chitosan and glucose oxidase (GOD). Higher catalytic activities were observed on for GOD-MMCHI than GOD-LMCHI and for those crosslinked with glutaraldehyde than using the adsorption technique. Enzyme loading greater than 0.6 mg decreased the activity. Under optimum conditions (pH 6.0, 35°C and applied potential of 0.6 V) response times of 85 s and 65 s were observed for medium molecular weight chitosan glucose biosensor (GOD-MMCHI/PT) and low molecular weight chitosan glucose biosensor (GOD-LMCHI/PT), respectively. The apparent Michaelis-Menten constant () was found to be 12.737 mM for GOD-MMCHI/PT and 17.692 mM for GOD-LMCHI/PT. This indicated that GOD-MMCHI/PT had greater affinity for the enzyme. Moreover, GOD-MMCHI/PT showed higher sensitivity (52.3666 nA/mM glucose) when compared with GOD-LMCHI/PT (9.8579 nA/mM glucose) at S/N>3. Better repeatability and reproducibility were achieved with GOD-MMCHI/PT than GOD-LMCHI/PT regarding glucose measurement. GOD-MMCHI/PT was found to give the highest enzymatic activity among the electrodes under investigation. The extent of interference encountered by GOD-MMCHI/PT and GOD-LMCHI/PT was not significantly different. Although the Nafion coated biosensor significantly reduced the signal due to the interferents under study, it also significantly reduced the response to glucose. The performance of the biosensors in the determination of glucose in rat serum was evaluated. Comparatively better accuracy and recovery results were obtained for GOD-MMCHI/PT. Hence, GOD-MMCHI/PT showed a better performance when compared with GOD-LMCHI/PT. In conclusion, chitosan membranes shave the potential to be a suitable matrix for the development of glucose biosensors.

A sepiolite modified conducting polymer based biosensor

A conducting polymer modified with sepiolite was utilized in the construction of a highly sensitive and fast amperometric cholesterol biosensor. In this study a monomer; (10,13-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)dibenzo[a,c]phenazine (PHED)) was synthesized and then its polymer was coated on a graphite electrode by electropolymerization to obtain a matrix for enzyme immobilization. Cholesterol oxidase was immobilized onto polymer coated electrode by adsorption technique. Sepiolite was introduced for a successful immobilization of the cholesterol oxidase. Immobilized enzyme kinetic parameters (KMapp, Imax) were evaluated by Michaelis-Menten kinetics and calculated as 0.031mM and 6.06μA, respectively. LOD and sensitivity were estimated as 0.36μM and 1.64mA/mMcm2. Characterization of designed biosensor was done to examine the effect of various factors such as enzyme amount, optimum pH and shelf-life. A novel accurate and inexpensive cholesterol biosensor was developed for the determination of total cholesterol in food samples.

Development of bimetal-grown multi-scale carbon micro-nanofibers as an immobilizing matrix for enzymes in biosensor applications

This study describes the development of a novel bimetal (Fe and Cu)-grown hierarchical web of carbon micro-nanofiber-based electrode for biosensor applications, in particular to detect glucose in liquids. Carbon nanofibers (CNFs) are grown on activated carbon microfibers (ACFs) by chemical vapor deposition (CVD) using Cu and Fe as the metal catalysts. The transition metal-fiber composite is used as the working electrode of a biosensor applied to detect glucose in liquids. In such a bi-nanometal-grown multi-scale web of ACF/CNF, Cu nanoparticles adhere to the ACF-surface, whereas Fe nanoparticles used to catalyze the growth of nanofibers attach to the CNF tips. By ultrasonication, Fe nanoparticles are dislodged from the tips of the CNFs. Glucose oxidase (GOx) is subsequently immobilized on the tips by adsorption. The dispersion of Cu nanoparticles at the substrate surface results in increased conductivity, facilitating electron transfer from the glucose solution to the ACF surface during the enzymatic reaction with glucose. The prepared Cu-ACF/CNF/GOx electrode is characterized for various surface and physicochemical properties by different analytical techniques, including scanning electron microscopy (SEM), electron dispersive X-ray analysis (EDX), Fourier-transform infrared spectroscopy (FTIR), BET surface area analysis, and transmission electron microscopy (TEM). The electrochemical tests show that the prepared electrode has fast response current, electrochemical stability, and high electron transfer rate, corroborated by CV and calibration curves. The prepared transition metal-based carbon electrode in this study is cost-effective, simple to develop, and has a stable immobilization matrix for enzymes.

Enzyme-Entrapped Mesoporous Silica for Treatment of Uric Acid Disorders

Gout is an abnormality in the body resulting in the accumulation of uric acid mainly in joints. Dissolution of uric acid crystals into soluble allantoin by the enzyme uricase might provide a better alternative for the treatment of gout. This work aims to investigate the feasibility of a transdermal patch loaded with uricase for better patient compliance. Mesoporous silica (SBA-15) was chosen as the matrix for immobilisation of uricase. Highly oriented mesoporous SBA-15 was synthesized, characterized and uricase was physisorbed in the mesoporous material. The percentage adsorption and release of enzyme in borate buffer was monitored. The release followed linear kinetics and greater than 80% enzyme activity was retained indicating the potential of this system as an effective enzyme immobilization matrix. The enzyme permeability was studied with Wistar rat skin and human cadaver skin. It was found that in case of untreated rat skin 10% of enzyme permeated through skin in 100 h. The permeation increased by adding permeation enhancer (combination of oleic acid in propylene glycol (OA in PG)). The permeation enhancement was studied under two concentrations of OA in PG (1%, 5%) in both rat and human cadaver skin and it was found that 1% OA in PG showed better result in rat skin and 5% OA in PG showed good results in human cadaver skin.

Magnetic composites from minerals: study of the iron phases in clay and diatomite using Mössbauer spectroscopy, magnetic measurements and XRD

Magnetic particles as matrix for enzyme immobilization have been used and due to the enzymatic derivative can be easily removed from the reaction mixture by a magnetic field. This work presents a study about the synthesis and characterization of iron phases into magnetic montmorillonite clay (mMMT) and magnetic diatomaceous earth (mDE) by 57Fe Mossbauer spectroscopy (MS), magnetic measurements and X-ray diffraction (XRD). Also these magnetic materials were assessed as matrices for the immobilization of invertase via covalent binding. Mossbauer spectra of the magnetic composites performed at 4.2 K showed a mixture of magnetite and maghemite about equal proportion in the mMMT, and a pure magnetite phase in the sample mDE. These results were verified using XRD. The residual specific activity of the immobilized invertase on mMMT and mDE were 83 % and 92.5 %, respectively. Thus, both magnetic composites showed to be promising matrices for covalent immobilization of invertase.

Carboxylesterase from Spodoptera Litura: Immobilization and use for the Degradation of Pesticides

The immobilization of carboxylesterase from Spodoptera Litura in mesoporus molecular sieves was studied in this paper. Two different types of mesoporous sieves (MCM-41, SBA-15) were initially used as enzyme support to compare the efficiency of immobilization. The outcome showed the maximum enzyme loadings, twice as many in MCM-41, are 0.0267 L/g in SBA-15 support. The most efficient pH value for immobilization was at 6.5 for both types of the support and the optimal immobilizing time was 2h for SBA-15 and 4h for MCM-41. Hence, the SBA-15 mesoporous support was determined as the enzyme immobilization matrix to continue the investigation because of its superior properties for enzyme immobilization. Then a series of characters of the free and immobilized Carboxylesterase were compared. As a result, the immobilized enzyme was substantiated to be obviously more stable than the free enzyme under the variant conditions. The experiments of pesticides degradation show that, relative to degradation in natural environment, immobilization enzyme had a higher ability to reduce the organic compounds with ester bonds.

Preparation of a Cellulose-based Enzyme Membrane Using Ionic Liquid to Lengthen the Duration of Enzyme Stability

A novel method for preparing enzyme membranes was developed. The enzyme was attached onto the electrode surface by dropping the enzyme solution and allowing it to dry. Glucose oxidase was used for entrapment. Then, the electrode surface was coated with an ionic liquid containing cellulose, and the ionic liquid was removed by immersing the electrode into water. Enzyme activity was retained in the membrane; the enzyme electrode can be used for detecting glucose in the range of 10 µM to 1 mM, and the response time was ~10 s. The stability duration of the electrode was examined: the enzyme electrode could be used for glucose detection for 6 months. The membrane was observed by atomic force microscopy in the force modulation mode; crystalline and amorphous parts were intermingled. In conclusion, the cellulose membrane can be a suitable immobilization matrix for enzymes.

Phyto-inspired Silica Nanowires: Characterization and Application in Lipase Immobilization

Silica nanostructures were phyto-fabricated on different surfaces by using pomegranate (Punica granatum) leaf extracts. On zinc films, nanowires were obtained. On other surfaces such as silica, alumina, zinc oxide, and glass, spherical aggregates, cubic assemblies, microflakes, and acicular structures, respectively, were observed. The nanowires developed on Zn surfaces were characterized by scanning electron microscope-energy dispersive spectrometer (SEM-EDS), X-ray diffraction (XRD), photoluminescence, and Fourier transform infrared (FTIR) spectroscopic analysis. XRD profiles displayed peaks at 2.4, 4.9, and 12.1° indicating the presence of silica nanostructures. When excited at 340 nm, the reaction mixtures displayed a characteristic blue luminescence at 404 nm. FTIR spectra showed the existence of Si-OH and Si-O-Si bonds. The nanowires were functionalized with amine groups and used for the covalent immobilization of Candida rugosa lipase. The immobilized enzyme displayed better pH and temperature stability and retained 80% activity after 20 cycles. This paper highlights a novel route for the phyto-mediated growth of silica nanowires on Zn surfaces, their characterization and effective use as a matrix for enzyme immobilization.

Immobilization of barley oxalate oxidase onto gold–nanoparticle-porous CaCO3 microsphere hybrid for amperometric determination of oxalate in biological materials

The use of Au nanoparticles (AuNPs)-CaCO(3) porous microsphere hybrid as a matrix for enzyme immobilization to increase the performance of an amperometric biosensor. A method is described for construction of an amperometric oxalate biosensor by immobilizing a barley root oxalate oxidase (OxOx) onto AuNPs-CaCO(3) porous microsphere hybrid encapsulated in silica sol and deposited on Au electrode. The biosensor showed optimum response within 4s at pH 5.0 and 30°C, when polarized at +0.4V vs. Ag/agCl. The biosensor possesses high sensitivity and measures oxalate concentrations as low as 1.0 μmol/L. The working linear range was from 1.0 to 1000 μmol/L of oxalate. The biosensor measured urinary and plasma oxalate of normal persons and stone formers. The use of AuNPs-CaCO(3) porous microsphere hybrid as a support for immobilization of OxOx has resulted into an improved amperometric oxalate biosensor.