Fabrication and Optimization of ChE/ChO/HRP-AuNPs/c-MWCNTs Based Silver Electrode for Determining Total Cholesterol in Serum

The developed method used three enzymes comprised of cholesterol esterase, cholesterol oxidase, and peroxidase for fabrication of amperometric biosensor in order to determine total cholesterol in serum samples. Gold nanoparticles (AuNPs) and carboxylated multiwall carbon nanotubes (cMWCNTs) were used to design core of working electrode, having covalently immobilized ChO, ChE, and HRP. Polyacrylamide layer was finally coated on working electrode in order to prevent enzyme leaching. Chemically synthesised Au nanoparticles were subjected to transmission electron microscopy (TEM) for analysing the shape and size of the particles. Working electrode was subjected to FTIR and XRD. The combined action of AuNP and c-MWCNT showed enhancement in electrocatalytic activity at a very low potential of 0.27 V. The pH 7, temperature 40°C, and response time of 20 seconds, respectively, were observed. The biosensor shows a broad linear range from 0.5 mg/dL to 250 mg/dL (0.01 mM–5.83 mM) with minimum detection limit being 0.5 mg/dL (0.01 mM). The biosensor showed reusability of more than 45 times and was stable for 60 days. The biosensor was successfully tested for determining total cholesterol in serum samples amperometrically with no significant interference by serum components

PURIFICATION AND KINETIC STUDIES OF ORGANOPHOSPHORUS HYDROLASE FROM B. DIMINUTA

Objective: Extraction and purification of Organophosphorus hydrolase (OPH) enzyme from Brevundimonas diminuta and to study kinetic properties of the purified enzyme. Methods: The enzyme was extracted from bacteria and purified by using a combination of gel filtration and ion-exchange chromatography and the purity of an enzyme was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The activity of the purified enzyme was monitored by enzyme assay and total protein content was determined by using Lowry's method. The kinetic properties of the enzyme were also studied. Results: A 72 kDa organophosphorus hydrolase (OPH) enzyme was extracted and purified. The purified enzyme was homodimer and showed a single band on SDS-PAGE. The Michaelis constant (Km) and maximal velocity (Vmax) values of free OPH enzyme for methyl parathion as substrate was 285.71 μM and 50 μM/min respectively. At optimum pH (7.5) and incubation temperature (35°C), free enzyme showed maximum activity with incubation time of 8 min. Conclusion: The bacteria contain OPH enzyme with high potential to detoxify OP pesticides, attractive for bioremediation due to good pH & temperature conditions, were also useful in development of bio analytical techniques such as biosensors for OP pesticide detection

Acetylcholinesterase Biosensors for Electrochemical Detection of Organophosphorus Compounds: A Review

The exponentially growing population, with limited resources, has exerted an intense pressure on the agriculture sector. In order to achieve high productivity the use of pesticide has increased up to many folds. These pesticides contain organophosphorus (OP) toxic compounds which interfere with the proper functioning of enzyme acetylcholinesterase (AChE) and finally affect the central nervous system (CNS). So, there is a need for routine, continuous, on spot detection of OP compounds which are the main limitations associated with conventional analytical methods. AChE based enzymatic biosensors have been reported by researchers as the most promising tool for analysis of pesticide level to control toxicity and for environment conservation. The present review summarises AChE based biosensors by discussing their characteristic features in terms of fabrication, detection limit, linearity range, time of incubation, and storage stability. Use of nanoparticles in recently reported fabrication strategies has improved the efficiency of biosensors to a great extent making them more reliable and robust

NANOMATERIALS BASED ACETYLCHOLINESTERASE BIOSENSORS FOR ORGANOPHOSPHORUS COMPOUNDS DETECTION: REVIEW

Due to intense pressure on agriculture for supporting exponentially growing population pesticides are used on an alarming scale. As these pesticides contain Organophosphorus (OP) compounds which are highly toxic and interfere with functioning of enzyme Acetylcholinestrase (AChE) and finally affecting Central Nervous System (CNS). So, there is an urgent need to monitor OP compounds concentration regularly in the marketed food products and even in the environment (water, soil). Here we focus on the different nanomaterials used for the fabrication of the AChE biosensors for detection of OP compounds which is based on inhibition of AChE. The merits and demerits of the different nanomaterials which are being used as supports are also discussed. The mode of detection, detection limit, linearity range, time of incubation, storage stability of the biosensors is also reviewed. Nanomaterial as an important class of supports used for the AChE biosensors due to their valuable properties. Among all the nanomaterials used Gold nanoparticles (AuNPs) have gained an advantage as they are explored with time.Â

β-sitosterol in different parts of Saraca asoca and herbal drug ashokarista: Quali-quantitative analysis by liquid chromatography-mass spectrometry

β-sitosterol is an important component in food and herbal products and beneficial in hyperlipidemia. Its higher concentrations in serum may lead to coronary artery disease in case of sitosterolemia. Therefore, it is essential to determine the quantity of β-sitosterol in food and herbal drugs. Saraca asoca and its preparations have been widely used by traditional healers are also a source of β-sitosterol. In the present study, quantitative estimation of β-sitosterol present in hot and cold water extracts of bark, regenerated bark, leaves and flowers of the S. asoca and Ashokarista drugs were carried out first time using high performance liquid chromatography coupled (HPLC) with quadrupole time-of-flight mass spectrometry. Different concentrations of β-sitosterol and crude extracts were estimated by HPLC and targeted mass spectrometry. Standard curve for β-sitosterol was prepared from the intensities of transitions (397.50 → 147.0987 m/z) having regression coefficient (r 2) 0.9952. Out of eight extracts and two drugs used in the study bark water, leaves water and leaves hot water extracts were found to have a considerable quantity of β-sitosterol, i.e. 170, 123.5 and 19.3 ng/mL, respectively. The results showed significant differences in the distribution of β-sitosterol among different organs of S. asoca and drugs prepared from its bark. HPLC/electrospray ionizationmass spectroscopy method is accurate, reproducible and requires less specimen, sample preparation and analysis time over HPLC assay. This type of approaches could be helpful for the quality control of herbal medicines and provides necessary information for the rational utilization of plant resources