Profile diversity of galacto-oligosaccharides from disaccharides to hexasaccharides by porous graphitic carbon liquid chromatography-orbitrap tandem mass spectrometry.

Galacto-oligosaccharides (GOS) are important prebiotic supplements for commercial nutraceutical food. The prebiotic efficacy of functional GOS is dependent on their chemical profile. Screening potential markers aids specifications and quality control of GOS materials. However, profiling analysis of GOS with a degree of polymerization (DP) ≥ 4 is still challenging. This study presents a porous graphitic carbon liquid chromatography-orbitrap tandem mass spectrometry-based method that characterized 58 linear and 10 branched GOS and detected 59 non-reducing GOS from DP2 to DP6. The results indicated that 15 major group components with DP2-DP5 accounted for more than 65% of total GOS content in GOS samples, while non-reducing GOS components accounted for only 2.8-7.6%. Substantial variations in components occurred in samples from different batches and sources. Structural and constitutive diversity were dominated by DP3-DP5. This method can help control the quality of GOS products and be used to investigate the structural and prebiotic-efficacy relationships.

Tamarindus indica seed-shell nanoparticles­silver nanoparticles-Ceratonia silique bean gum composite for copper-micro mesh grid electrode fabrication and its application for glucose detection in artificial salivary samples.

This study used a new approach to fabricate a glucose detection system based on nano-engineered biomaterials. The fabrication steps included strategic synthesis, integration and stabilization of biological and metal nanoparticles in superabsorbent hydrogel gum matrix. The design of the high-performance electrochemical biosensor platform includes copper-micro mesh grid electrode modified with polymer phase comprising of silver nanoparticles surface coroneted with Ceratonia silique locust bean gum (LBG), Tamarindus indica seed-shell nanoparticles and glucose oxidase (GOx). Fundamental assessment of catalytic properties of the nanobiocomposite films on copper grid probe were performed by cyclic voltammetry, amperometry, differential pulse voltammetry. Probes showed good repeatability, reproducibility, selectivity, and long-term stability. The GOx was well-immobilized and stabilized by C. siliqua nano-matrix, with 85% and 98% activity retention when stored at different condiions for 6 month and 3 months, respectively. The fabricated grid-platform exhibited linear response in a wide range of glucose concentration, with detection limit of 1.0 nM (S/N = 3) and sensitivity 38.7 mA nM-1 cm-2. The bionanomaterial-based sensor was successfully applied for ultra-low glucose detection in artificial salivary samples. The designed sensor, perhaps with further modifications, has potential for the next generation of sensing platform in various biological fluids especially for non-invasive glucose detection for diabetic patients.

Exploration of Chitinous Scaffold-Based Interfaces for Glucose Sensing Assemblies.

: The nanomaterial-integrated chitinous polymers have promoted the technological advancements in personal health care apparatus, particularly for enzyme-based devices like the glucometer. Chitin and chitosan, being natural biopolymers, have attracted great attention in the field of biocatalysts engineering. Their remarkable tunable properties have been explored for enhancing enzyme performance and biosensor advancements. Currently, incorporation of nanomaterials in chitin and chitosan-based biosensors are also widely exploited for enzyme stability and interference-free detection. Therefore, in this review, we focus on various innovative multi-faceted strategies used for the fabrication of biological assemblies using chitinous biomaterial interface. We aim to summarize the current development on chitin/chitosan and their nano-architecture scaffolds for interdisciplinary biosensor research, especially for analytes like glucose. This review article will be useful for understanding the overall multifunctional aspects and progress of chitin and chitosan-based polysaccharides in the food, biomedical, pharmaceutical, environmental, and other diverse applications.

Electrically nanowired-enzymes for probe modification and sensor fabrication.

Enzymes are highly specific and selective due to their precise, intricate three-dimensional catalytic- structure. Electron transfer in enzymes normally occurs through an active-metal centers or tunneling events that are highly insulated by the surrounding globular protein structure. In case of electrochemically active enzymes/proteins, the distance between the redox-active cofactor and the electrode surface plays key role during direct communication. Therefore, the long electron-tunneling distance can be overcome by introducing mobile redox mediators such as nanostructures specially nanowires which can diffuse into and out of the enzyme active site, ferrying reducing or oxidizing equivalents with them. Therefore, nanowire-conjugated enzymes have gained great interest in the development of biosensor devices and other electrocatalytic-biological applications. Herein we present a comprehensive review about the electrochemical enzyme-based sensor using nanowires. Over the past decade, nanowires were investigated as a versatile platform for various applications including sensors and biosensors because of their high aspect ratio and a high surface-to-volume ratio. This review aimed to summarize some of the recent developments in the enzyme based sensor research that have been achieved with various metallic and non-metallic one-dimensional nanostructure i.e. nanowires. Due to low or no toxicity and biocompatibility, enzymes conjugated with nanowires are still highly specific, sensitive and biologically active. This review demonstrates the potential usability of nanowired-enzymes for the bioanalytical applications. The review includes various types of nanowires, mode of the enzyme integration or immobilization methodologies, probe modification, biosensor fabrication and real or spiked sample testing. Biosensor parameters such as linear range and sensitivity, selectivity and detection limit of reported sensors were also considered herein. We also introduce some of the new nanowire materials which have not yet been used for biosensing or biosensor application. The limitations, challenges and prospects for the use of nanowired-enzymes in electrochemical and other real-time sensing systems as well as fabrication technologies are also discussed in this review.

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.

Analysis and enhancement of nutritional and antioxidant properties of Vigna aconitifolia sprouts.

Vigna aconitifolia sprouts (Moth bean sprouts, MBS) were analyzed for their nutritional and antioxidant properties during sprouting. Sprouting for six days led to a 7.0 fold increase in fresh weight, 2.4 fold increase in soluble proteins, 3.0 fold increase in carbohydrates, and a 5.5 fold increase in mineral content. Phenolic content also increased by 28% during germination. Caffeic acid, ferulic acid, cinnamic acid and kaempferol were the predominant phenolic compounds detected in the ethanolic extracts of MBS by HPLC. Following supplementation with metal ions (200 µg ml⁻¹), the sprouts demonstrated a considerable increase in metal ion uptake, with improved phenolic content. MBS ethanolic extracts also reduced intracellular oxidative stress in HepG2 cells.

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.

Invertase inhibition based electrochemical sensor for the detection of heavy metal ions in aqueous system: Application of ultra-microelectrode to enhance sucrose biosensor's sensitivity.

We are reporting fabrication and characterization of electrochemical sucrose biosensor using ultra-microelectrode (UME) for the detection of heavy metal ions (Hg(II), Ag(I), Pb(II) and Cd(II)). The working UME, with 25 microm diameter, was modified with invertase (INV, EC: 3.2.1.26) and glucose oxidase (GOD, EC: 1.1.3.4) entrapped in agarose-guar gum. The hydrophilic character of the agarose-guar gum composite matrix was checked by water contact angle measurement. The atomic force microscopy (AFM) images of the membranes showed proper confinement of both the enzymes during co-immobilization. The dynamic range for sucrose biosensor was achieved in the range of 1 x 10(-10) to 1 x 10(-7)M with lower detection limit 1 x 10(-10)M at pH 5.5 with 9 cycles of reuse. The spectrophotometric and electrochemical studies showed linear relationship between concentration of heavy metal ions and degree of inhibition of invertase. The toxicity sequence for invertase using both methods was observed as Hg(2+)>Pb(2+)>Ag(+)>Cd(2+). The dynamic linear range for mercury using electrochemical biosensor was observed in the range of 5 x 10(-10) to 12.5 x 10(-10)M for sucrose. The lower detection limit for the fabricated biosensor was found to be 5 x 10(-10)M. The reliability of the electrochemical biosensor was conformed by testing the spike samples and the results were comparable with the conventional photometric DNSA method.