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Biomedical subjects

M S Thakur

Publications and source records attributed to M S Thakur.

16 recordsLinked to original sources
PubMed2005

Detection of methyl parathion using immuno-chemiluminescence based image analysis using charge coupled device.

A novel method based on immuno-chemiluminescence and image analysis using charge coupled device (CCD) for the qualitative detection of methyl parathion (MP) with high sensitivity (up to 10 ppt) is described. MP antibodies raised in poultry were used as a biological sensing element for the recognition of MP present in the sample. The immuno-reactor column was prepared by packing in a glass capillary column (150 microl capacity) MP antibodies immobilized on Sepharose CL-4B through periodate oxidation method. Chemiluminescence principle was used for the detection of the pesticide. Light images generated during the chemiluminescence reaction were captured by a CCD camera and further processed for image intensity, which was correlated with pesticide concentrations. K(3)Fe(CN)(6) was used as a light enhancer to obtain detectable light images. Different parameters including concentrations of K(3)Fe(CN)(6), luminol, urea H(2)O(2), antibody, addition sequence of reactants and incubation time to obtain best images were optimized. The results obtained by image analysis method showed very good correlation with that of competitive ELISA for methyl parathion detection. Competitive ELISA method was used as a reference to compare the results obtained by CCD imaging.

Equipment Design↗
PubMed2005

Biosensor for the determination of phenols based on cross-linked enzyme crystals (CLEC) of laccase.

Cross-linked enzyme crystals (CLECs) are a versatile form of biocatalyst that can also be used for biosensor application. Laccase from Trametes versicolor (E.C.1.10.3.2) was crystallized, cross-linked and lyophilized with beta-cyclodextrin. The CLEC laccase was found to be highly active towards phenols like 2-amino phenol, guaiacol, catechol, pyrogallol, catechin and ABTS (non-phenolic). The CLEC laccase was embedded in 30% polyvinylpropylidone (PVP) gel and mounted into an electrode to make the sensor. The biosensor was used to detect the phenols in 50-1000 micromol concentration level. Phenols with lower molecular weight such as 2-amino phenol, catechol and pyrogallol gave a short response time where as the higher molecular weight substrates like catechin and ABTS had comparatively a long response time. The optimum pH of the analyte was 5.5-6.0 when catechol was used as substrate. The CLEC laccase retained good activity for over 3 months.

Biosensing Techniques↗
PubMed2004

Regeneration of ethyl parathion antibodies for repeated use in immunosensor: a study on dissociation of antigens from antibodies.

Reliable analysis using an immunosensor strongly depends on the specificity, activity, and sensitivity of the antibody. Immobilization of antibody on the solid matrix enables its repeated use, for which it is required to dissociate the antigens and antigen-enzyme conjugate from the immobilized antibody matrix after each use and while doing so, a maximum retention of activity and specificity are crucial requirements. In the present investigation, on the development of an immunosensor for the organophosphorus pesticide ethyl parathion (EP) using EP antibodies, different dissociating agents such as organic solvents, detergents and acidic buffers, that is, dimethyl sulphoxide (DMSO), Tween-20, cetyl trimethylammonium bromide (CTAB), methanol, chloroform, guanidium chloride (GdmCl), glycine-HCl (Gly-HCl) buffer in the pH range of 1.5-3.0, pierce buffer and combination of DMSO and methanol in phosphate buffer and Gly-HCl buffer and salts like NaCl and MgCl2 were used. Generally about 50-60% dissociation was obtained with some degree of denaturation of the antibody immobilized on the sepharose matrix. However, 1% DMSO in combination with 0.2 M Gly-HCl buffer at a pH of 2.3 showed 97% dissociation and the immobilized antibody retained sufficient activity to carry out 14 reproducible assays for EP.

Antigen-Antibody Complex↗
PubMed2004

Stabilization of immobilized glucose oxidase against thermal inactivation by silanization for biosensor applications.

An important requirement of immobilized enzyme based biosensors is the thermal stability of the enzyme. Studies were carried out to increase thermal stability of glucose oxidase (GOD) for biosensor applications. Immobilization of the enzyme was carried out using glass beads as support and the effect of silane concentration (in the range 1-10%) during the silanization step on the thermal stability of GOD has been investigated. Upon incubation at 70 degrees C for 3h, the activity retention with 1% silane was only 23%, which increased with silane concentration to reach a maximum up to 250% of the initial activity with 4% silane. Above this concentration the activity decreased. The increased stability of the enzyme in the presence of high silane concentrations may be attributed to the increase in the surface hydrophobicity of the support. The decrease in the enzyme stability for silane concentrations above 4% was apparently due to the uneven deposition of the silane layer on the glass bead support. Further work on thermal stability above 70 degrees C was carried out by using 4% silane and it was found that the enzyme was stable up to 75 degrees C with an increased activity of 180% after 3-h incubation. Although silanization has been used for the modification of the supports for immobilization of enzymes, the use of higher concentrations to stabilize immobilized enzymes is being reported for the first time.

Biosensing Techniques↗
PubMed2004

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.

Biosensing Techniques↗
PubMed2002

Reactivation of immobilized acetyl cholinesterase in an amperometric biosensor for organophosphorus pesticide.

Biosensors based on acetyl cholinesterase (AChE) inhibition have been known for monitoring of pesticides in food and water samples. However, strong inhibition of the enzyme is a major drawback in practical application of the biosensor which can be overcome by reactivation of the enzyme for repeated use. In the present study, enzyme reactivation by oximes was explored for this purpose. Two oximes viz., 1,1'-trimethylene bis 4-formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were compared for the reactivation of the immobilized AChE. TMB-4 was found to be a more efficient reactivator under repeated use, retaining more than 60% of initial activity after 11 reuses, whereas in the case of 2-PAM, the activity retention dropped to less than 50% after only 6 reuses. Investigations also showed that reactivation must be effected within 10 min after each analysis to eliminate the ageing effect, which reduces the efficiency of reactivation.

Acetylcholinesterase↗
PubMed2002

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.

Animals↗
PubMed1993

Downstream processing of microbial rennet from solid state fermented moldy bran.

In recent years due to acute shortage of calf-rennet, microbial rennets seem to be an effective alternative and are commercially produced. Mucor miehei was cultivated under the solid state fermentation conditions, and the moldy bran was extracted using a semicontinuous multiple contact forced percolation method. The treated extract was then filtered through 5% R16 clay which enabled easy and efficient removal of impurities such as lipase and protease without involving costly chemical treatments. The ethyl alcohol precipitated enzyme was dried and made into powder form having activity of 1.5 x 10(5)Soxhlet units/gm.

Journal Article↗
PubMed1990

Use of dexamethasone for induction of parturition in goats.

Parturition was induced in 5 goats of Beetal and Beetal x Black Bengal cross in advanced stages of pregnancy, using intramuscular injection of 10 mg of dexamethasone. Goats required 47.7 +/- 1.26 hours on average after injection for parturition. There were no deleterious effects of induction on placental expulsion, kid weight, kid survival, and postpartum fertility of does. Plasma concentrations of progesterone and oestradiol 17 beta followed a similar trend in controls and treated animals. While plasma progesterone concentrations declined before parturition, the oestradiol concentration rose to its peak on the day of parturition.

Animals↗
PubMed1988

Cultural stability of Streptomyces fradiae in the production of xylose isomerase: studies in shake flasks.

Comprehensive studies of pure colonies of Streptomyces fradiae in the production of xylose isomerase by submerged fermentation at shake flask level revealed poor culture stability with respect to enzyme production, biomass formation, degree of pigmentation, quantity of glucose and xylose utilization, level of enzyme in cell-free culture broth and final pH of the fermentation medium. The results serve to stress obligatory evaluation of culture stability of Streptomyces strains in determining their suitability for use in developing fermentation processes for commercial exploitation.

Aldose-Ketose Isomerases↗
PubMed1983

Production of plant growth regulators by some Fusarium species.

About 10 species of Fusarium were screened and tested for their growth regulatory activity on oat coleoptile straight growth test. Culture filtrates of four species contained growth-stimulating factors while others showed growth-inhibition responses on oat sections. On the whole, F. moniliforme was found to be produce higher stimulatory effects. Chromatographic analysis revealed the presence of several indole auxin and gibberellin-like plant-growth regulators in their culture filtrates. The quantity and biological activity of each indole spot was also measured.

Edible Grain↗
PubMedDate not supplied

Reversible denaturation behavior of immobilized glucose oxidase.

Glucose oxidase (GOD) was immobilized by using glutaraldehyde crosslinking and various stabilizing agents such as BSA, gelatin, lysozyme, and polyethylenimine (PEI). Studies on the denaturation of the soluble as well as immobilized GOD were carried out for 1 h at various concentrations of guanidine hydrochloride (GdmCl) in 50 mM phosphate buffer, pH 6.0 at 25 +/- 1 degrees C. The soluble enzyme required a GdmCl concentration of 5 M for total activity loss, whereas for GOD immobilized with BSA, gelatin, lysozyme, and heat-inactivated lysozyme, the corresponding GdmCl concentration required was 8 M. GOD immobilized with PEI, however, was more stable and retained 25% activity when denatured for 1 h using 8 M GdmCl. However, after undergoing denaturation for 1 h, GOD immobilized with lysozyme regained 72% original activity within 20 min of renaturation, while GOD immobilized with BSA, PEI, gelatin, and heat-inactivated lysozyme regained only 39, 21, 20, and 25% of activity, respectively. After five cycles of repeated denaturation and renaturation with 8 M GdmCl, GOD immobilized with lysozyme retained 70% of the original activity. Refolding ability of lysozyme, glutaraldehyde crosslinkages between lysozyme and GOD, together with ionic interactions between them, appear to play an important role in the denaturation-renaturation behavior of the immobilized enzyme.

Buffers↗