M.S. Thakur

Enhancement of operational stability of an enzyme biosensor for glucose and sucrose using protein based stabilizing agents

With the incorporation of lysozyme during the immobilization step, considerable enhancement of the operational stability of a biosensor has been demonstrated in the case of an immobilized single enzyme (glucose oxidase) system for glucose and multienzyme (invertase, mutarotase and glucose oxidase) system for sucrose. Thus an increased number of repeated analyses of 750 samples during 230 days for glucose and 400 samples during 40 days of operation for sucrose have been achieved. The increased operational stability of immobilized single and multienzyme system, will improve the operating cost effectiveness of the biosensor.

Ascorbate oxidase based amperometric biosensor for organophosphorous pesticide monitoring

An amperometric principle based biosensor containing tissues of cucumber, rich in ascorbic acid oxidase, was used for the detection of organophosphorous (OP) pesticide ethyl paraoxon, which inhibits the activity of ascorbic acid oxidase. The optimal concentration of ascorbic acid used as substrate was found to be 5.67 mM. The biosensor response was found to reach steady state within 2 min. A measurable inhibition (> 10%) was obtained with 10 min incubation of the enzyme electrode with different concentrations of the pesticide. There was a linear relationship between the percentage of inhibition of the enzyme substrate reaction and the pesticide (ethyl paraoxon) concentration in the range 1-10 ppm with a regression value 0.9942.

Photoabsorption and Resonance Energy Transfer Phenomenon in CdTe−Protein Bioconjugates: An Insight into QD−Biomolecular Interactions

Luminescent quantum dots (QDs) possess unique photophysical properties, which are advantageous in the development of new generation robust fluorescent probes based on Forster resonance energy transfer (FRET) phenomena. Bioconjugation of these QDs with biomolecules create hybrid materials having unique photophysical properties along with biological activity. The present study is aimed at characterizing QD bioconjugates in terms of optical behavior. Colloidal CdTe QDs capped with 3-mercaptopropionic acid (MPA) were conjugated to different proteins by the carbodiimide protocol using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and a coupling reagent like N-hydroxysuccinimide (NHS). The photoabsorption of these QD-protein bioconjugates demonstrated an effective coupling of electronic orbitals of constituents. A linear variation in absorbance of bioconjugates at 330 nm proportionate to conjugation suggests a covalent attachment as confirmed by gel electrophoresis. A red shift in the fluorescence of bovine serum albumin (BSA) due to conjugation inferred a decrease in Stokes shift and solvent polarization effects on protein. A proportionate quenching in BSA fluorescence followed by an enhancement of QD fluorescence point toward nonradiative dipolar interactions. Further, reduction in photobleaching of BSA suggests QD-biomolecular interactions. Bioconjugation has significantly influenced the photoabsorption spectrum of QD bioconjugates suggesting the formation of a possible protein shell on the surface of QD. The experimental result suggests that these bioconjugates can be considered nanoparticle (NP) superstructures for the development of a new generation of robust nanoprobes.

Enhancement of chemiluminescence for vitamin B12 analysis.

In the current article, chemiluminescence (CL) from the vitamin B(12) and luminol reaction was studied under alkaline conditions to develop a sensitive analytical method for vitamin B(12) using the carbonate enhancement effect. The method was successfully applied to the determination of vitamin B(12) in vitamin B(12) tablets, multivitamin capsules, and vitamin B(12) injections. Experimental parameters were optimized, including luminol concentration, urea-hydrogen peroxide (urea-H(2)O(2)) concentration, effect of pH, and sequence of addition of reactants for obtaining maximum CL, which was not explored previously. The limit of detection was 5 pg/ml, and the linear range was 10 pg/ml to 1 microg/ml with a regression coefficient of R(2)=0.9998. The importance of these experimental parameters and the carbonate enhancement effect is discussed based on the knowledge of the mechanism of oxidation of luminol and decomposition of urea-H2O2 in the presence of vitamin B(12). Extraction of vitamin B(12) was carried out, and the observed recovery was 97-99.2% with a relative standard deviation in the range of 0.30-1.09%. The results obtained were compared with those of the flame atomic absorption spectrometry method.

Enhancement of stability of immobilized glucose oxidase by modification of free thiols generated by reducing disulfide bonds and using additives

Stability of glucose oxidase (GOD) immobilized with lysozyme has been considerably enhanced by modification of free thiols generated by reducing disulfide bonds using beta-mercaptoethanol and N-ethylmaleimide in conjunction with additives like antibiotics and salts. Thermal stability of immobilized GOD was quantified by means of the transition temperature, Tm and the operational stability by half-life t1/2 at 70 degrees C. Modification of the free thiols in the enzyme coupled with the presence of kanamycin, NaCl, and K2SO4, led to increase in Tm, to 80, 82 and 84 degrees C (compared to 75 degrees C in control) and t1/2 by 7.7-, 11- and 22-fold, respectively, indicating that this method can be effectively used for enhancing the stability of enzymes.

Optimization of the multienzyme system for sucrose biosensor by response surface methodology

An immobilized multienzyme- and cathodic amperometry-based biosensor for sucrose was constructed for the analysis of food and fermentation samples. The multienzyme system, comprising invertase, mutarotase and glucose oxidase (GOD), was immobilized by using glutaraldehyde as cross-linking agent. Operating parameters of the biosensor for the estimation of sucrose in the range 1–10% were standardized. Response surface methodology (RSM) based on three-factor, three-variable design was used to evaluate the effect of important variables (concentration of enzymes, (varied in the range invertase (10–50 IU), mutarotase (5–105 IU) and GOD (1–9 IU)) on the response of biosensor. In the range of parameters studied, response time decreased with decrease in the invertase and with increase in mutarotase and GOD. Mutarotase concentration above 75 IU was found to result in an increased response time due to inhibition of mutarotase by its product β-D-glucose. The optimal conditions achieved for the analysis of sucrose were: invertase 10 IU, mutarotase 40 IU, and GOD 9 IU. With these conditions, the predicted and actual experimental response time values were 2.26 and 2.35 min respectively, showing good agreement.

An automated flow injection analysis system for on-line monitoring of glucose and L-lactate during lactic acid fermentation in a recycle bioreactor

The study concerns on-line sequential analysis of glucose and L-lactate during lactic acid fermentation using a flow injection analysis (FIA) system. Enzyme electrodes containing immobilized glucose oxidase and L-lactate oxidase were used with an amperometric detection system. A 12-bit data acquisition card with 16 analog input channels and 8 digital output channels was used. The software for data acquisition was developed using Visual C++, and was devised for sampling every hour for sequential analyses of lactate and glucose. The detection range was found to be 2–100 g l−1 for glucose and 1–60 g l−1 for L-lactate using the biosensors. This FIA system was used for monitoring glucose utilization and L-lactate production by immobilized cells of Lactobacillus casei subsp. rhamnosus during a lactic acid fermentation process in a recycle batch reactor. After 13 h of fermentation, complete sugar utilization and maximal L-lactate production was observed. A good agreement was observed between analysis data obtained using the biosensors and data from standard analyses of reducing sugar and L-lactate. The biosensors exhibited excellent stability during continuous operation for at least 45 days.

Aptamer--nanoparticle-based chemiluminescence for p53 protein.

A simple colorimetric biosensing technique based on the interaction of gold nanoparticles (AuNPs) with the aptamer was developed for detection of p53, a tumor suppressor protein, in the current study. Aggregation of AuNPs was induced by desorption of the p53 binding RNA aptamer from the surface of AuNPs as a result of the aptamer target interaction leading to the color change of AuNPs from red to purple. The detection limit of p53 protein by the colorimetric approach was 0.1 ng/ml after successful optimization of the amount of aptamer, AuNPs, salts, and incubation time. Furthermore, the catalytic activity of the aggregated AuNPs was greatly enhanced by chemiluminescence (CL) reaction, where the detection limit was enhanced to 10 pg/ml with a regression coefficient of R2 = 0.9907. Here the sensitivity was increased by 10-fold compared with the AuNP-based colorimetric method. Hence, the sensitivity of detection was increased by employing CL, by using the catalytic activity of aggregated AuNPs, on the luminol-hydrogen peroxide reaction. Thus, the combination of colorimetric and CL-based aptasensor can be of great advantage in increasing the sensitivity of detection for any target analyte.

Purification, Identification, and Characterization of Methylcobalamin from Spirulina platensis

The present study reports methylcobalamin in Spirulina platensis using high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), microbiological assay, chemiluminescence assay, liquid chromatography-mass spectrometry (LC-MS), and tandem mass spectrometry (MS/MS). Extraction of vitamin B12 from S. platensis was carried out without using cyanide. Partial purification was achieved using Amberlite XAD-2 followed by elution with 80% (v/v) methanol. Activated charcoal facilitated removal of impurities in S. platensis extract and in further purification of vitamin B12. The purified fraction was identified to contain methylcobalamin as analyzed by HPLC and TLC. Authenticity of methylcobalamin was further confirmed by LC-MS and MS/MS. Quantitation of methylcobalamin in a test sample of S. platensis biomass was performed using microbiological assay and chemiluminescence assay and was found to be 38.5±2 and 35.7±2 μg/100 g of dry biomass, respectively.

Monitoring of biomass in solid state fermentation using light reflectance

Abstract The nature of growth of filamentous fungi on solid substrates renders the estimation of biomass during the course of solid state fermentation difficult. The changing colour of the substrate during the growth of the microorganism can be quantified by light reflectance measurements. The experiments reported in the present paper indicate the potential of using this method for monitoring biomass in the presence of solid substrates.