Fenugreek hydrogel-agarose composite entrapped gold nanoparticles for acetylcholinesterase based biosensor for carbamates detection.

A biosensor was fabricated to detect pesticides in food samples. Acetylcholinesterase was immobilized in a novel fenugreek hydrogel-agarose matrix with gold nanoparticles. Transparent thin films with superior mechanical strength and stability were obtained with 2% fenugreek hydrogel and 2% agarose. Immobilization of acetylcholinesterase on the membrane resulted in high enzyme retention efficiency (92%) and a significantly prolonged shelf life of the enzyme (half-life, 55 days). Transmission electron microscopy revealed that, gold nanoparticles (10-20 nm in diameter) were uniformly dispersed in the fenugreek hydrogel-agarose-acetylcholinesterase membrane. This immobilized enzyme-gold nanoparticle dip-strip system detected various carbamates, including carbofuran, oxamyl, methomyl, and carbaryl, with limits of detection of 2, 21, 113, and 236 nM (S/N = 3), respectively. Furthermore, the fabricated biosensor exhibited good testing capabilities when used to detect carbamates added to various fruit and vegetable samples.

Invertase-nanogold clusters decorated plant membranes for fluorescence-based sucrose sensor.

In the present study, invertase-mediated nanogold clusters were synthesized on onion membranes, and their application for sucrose biosensor fabrication was investigated. Transmission electron microscopy revealed free nanoparticles of various sizes (diameter ~5 to 50 nm) along with clusters of nanogold (~95 to 200 nm) on the surface of inner epidermal membranes of onions (Allium cepa L.). Most of the polydispersed nanoparticles were spherical, although some were square shaped, triangular, hexagonal or rod-shaped. Ultraviolet-visible spectrophotometric observations showed the characteristic peak for nanoparticles decorated invertase-onion membrane at approximately 301 nm. When excited at 320 nm in the presence of sucrose, the membranes exhibited a photoemission peak at 348 nm. The fluorescence lifetime of this nanogold modified onion membrane was 6.20 ns, compared to 2.47 ns for invertase-onion membrane without nanogold. Therefore, a sucrose detection scheme comprised of an invertase/nanogold decorated onion membrane was successfully developed. This fluorescent nanogold-embedded onion membrane drop-test sensor exhibited wide acidic to neutral working pH range (4.0-7.0) with a response time 30 seconds (<1 min). The fabricated quenching-based probe had a low detection limit (2x10(-9) M) with a linear dynamic range of 2.25x10(-9) to 4.25x10(-8) M for sensing sucrose. A microplate designed with an enzyme-nanomaterial-based sensor platform exhibited a high compliance, with acceptable percentage error for the detection of sucrose in green tea samples in comparison to a traditional method. With some further, modifications, this fabricated enzyme-nanogold onion membrane sensor probe could be used to estimate glucose concentrations for a variety of analytical samples. Graphical abstract Synthesis and characterization of invertase assisted nanogold clusters on onion membranes and their application for fluorescence-based sucrose sensor.

Chitosan-guar gum-silver nanoparticles hybrid matrix with immobilized enzymes for fabrication of beta-glucan and glucose sensing photometric flow injection system.

Simple and fast photometric flow injection analysis system was developed for sensing of ß-1,3-glucan from medicinal mushroom Ganoderma lucidum during fermentation. For this purpose, the chitosan-guar gum-silver nanoparticle-beta glucanase (Ch-GG-AgNPs-ßG) beads and Ch-GG-AgNPs-GOD (glucose oxidase) beads were prepared. The bead packed mini-columns were then used to assemble a flow injection analysis (FIA) system for the detection of ß-(1→3)-d-glucan biomarker or glucose. This colorimetric flow system can detect glucose and glucan with detection limits as low as 50ngmL(-1) and 100ngmL(-1) (S/N=3), respectively. The analysis time of this FIA was approximately 40s, which is faster than the previously reported glucan sensors. The glucose and glucan calibration curves were obtained in the range of 0.25-1.25µgmL(-1) (R(2)=0.988) and 0.2-1.0µgmL(-1)(R(2)=0.979), respectively. The applicability of the nano-bio-composite FIA sensor system for spiked and real ß-(1→3)-d-glucan samples were tested, and the accuracy of the results were greater than 95%. Thus, the designed FIA provides a simple, interference free and rapid tool for monitoring glucose and ß-glucan content, which can be used for various food samples with a little modification.

1,3-ß-Glucanase from Vigna aconitifolia and its possible use in enzyme bioreactor fabrication.

Endo-1,3(4)-ß-glucanase (EC 3.2.1.6) from Vigna aconitifolia sprouts was purified to 14.5 fold by gel filtration and ion-exchange chromatography. The enzyme was found to be a glycoprotein, its activity was Ca(2+) dependent and specific for ß-1,3 linkages in different polysaccharides. The K(m) value of the enzyme was estimated to be 3.0 mg ml(-1) for ß-D-glucan as substrate. Circular dichroism studies revealed 8% α-helix, 48% ß-pleated and 44% random coil in its secondary structure. Purified ß-glucanase was then successfully co-immobilized with glucose oxidase in agarose-chitosan beads, showing better immobilization yield, operational range and stability as compared with the crude ß-glucanase beads. The immobilized ß-glucanase was successfully used for mini-bioreactor fabrication.

Electrochemical ß(1→3)-d-glucan biosensors fabricated by immobilization of enzymes with gold nanoparticles on platinum electrode.

ß(1→3)-d-Glucan sensors were fabricated using bi-enzyme and tri-enzyme immobilized systems with gold nanoparticles (GNPs) to increase sensitivity. The plant ß(1→3)-D-glucanase (ßG), glucose oxidase (GOD) or/and peroxidase (POD) in agarose-corn flour-gelatin (ACG) matrix were coated on platinum disc electrode to detect soluble ß(1→3)-D-glucan. The atomic force microscopy (AFM) revealed that GNPs embedded in ACG formed tiny islands/clusters with enzymes. Both of bi-enzyme sensor (ACG-ßG-GOD-GNPs/Pt) and tri-enzyme sensor (ACG-ßG-GOD-POD-GNPs/Pt) had response time less than 20s for ß(1→3)-D-glucan. A linear calibration plot for bi-enzyme sensor was obtained for ß(1→3)-D-glucan concentration ranged from 100 to 1000 ngmL(-1) (R(2)=0.983). The lower detection limit was 30 ngmL(-1) using applied potential of 200 mV and scan rate of 50 mVs(-1); with signal to noise ratio (S/N) of 3. Fabricated tri-enzyme sensor was also operable under similar conditions with LOD of 50 ngmL(-1) (r(2)=0.989) at -175 mV applied potential and scan rate of 50 mVs(-1). Both sensors were durable and could be repeatedly used for at least 14 times. When the tri-enzyme sensor was employed to analyze ß(1→3)-d-glucan content in alcoholic beverages, the results were comparable to those obtained by standard method.

Fabrication of photometric dip-strip test systems for detection of beta(1-->3)-D-glucan using crude beta(1-->3)-D-glucanase from sprouts of Vigna aconitifolia.

Efforts have been made to fabricate enzyme dip-strip test systems for detecting beta(1-->3)-D-glucan. Beta(1-->3)-D-glucanase from sprouts of Vigna aconitifolia (commonly known as moth bean, 8-day old) with high specific activity (244 U mg(-1)) was co-entrapped with glucose oxidase (GOD) in different combinations of composite polymer matrices of agarose (A), gelatin (G), polyvinyl alcohol (PVA) and corn flour (CF). The enzyme immobilized membranes were checked for immobilization yield, pH and temperature optima, swelling index, thermal, operational and storage stability, and morphology by scanning electron microscopy. The 3% A-2% CF-8% G composite matrix was chosen for fabricating enzyme dip-strip systems for detection of beta-glucan by spectrophotometer using DNSA method (System-I) and AAP method (System-II). Dip-strip System-I and II showed linear dynamic range for detecting glucan concentration ranged from 100 to 500 microg mL(-1) and 10 to 50 microg mL(-1) with contact time 10 and 5 min, respectively. The LOD of System-I and II were found to be 65 microg mL(-1) and 10 microg mL(-1), respectively. Hence System-II was employed for analyzing beta(1-->3)-D-glucan contents in various pharmaceutical samples. It was found that without any sample pre-treatment the percent error of detection was less than 5.

Characterization of alpha-mannosidase from Erythrina indica seeds and influence of endogenous lectin on its activity.

alpha-mannosidase from Erythrina indica seeds is a Zn(2+) dependent glycoprotein with 8.6% carbohydrate. The enzyme has a temperature optimum of 50 degrees C and energy of activation calculated from Arrhenius plot was found to be 23 kJ mol(-1). N-terminal sequence up to five amino acid residues was found to be DTQEN (Asp, Thr, Gln, Glu, and Asn). In chemical modification studies treatment of the enzyme with NBS led to total loss of enzyme activity and modification of a single tryptophan residue led to inactivation. Fluorescence studies over a pH range of 3-8 have shown tryptophan residue to be in highly hydrophobic environment and pH change did not bring about any appreciable change in its environment. Far-UV CD spectrum indicated predominance of alpha-helical structure in the enzyme. alpha-Mannosidase from E indica exhibits immunological identity with alpha-mannosidase from Canavalia ensiformis but not with the same enzyme from Glycine max and Cicer arietinum. Incubation of E. indica seed lectin with alpha-mannosidase resulted in 35% increase in its activity, while no such activation was observed for acid phosphatase from E. indica. Lectin induced activation of alpha-mannosidase could be completely abolished in presence of lactose, a sugar specific for lectin.