M. Talat, R. Kumar Pandey, J.V. Medicherla, O. Nath Srivastava
Banaras Hindu University,
Keywords: asparginase, expanded, graphite, leukemia, TEM
Summary:The current study presents the development of a novel, diagnostic and cost-effective L-Asparaginase based biosensor conjugated to graphene for monitoring asparagines in leukemic patients. L-Asparaginase (E.C. 126.96.36.199) acts as an anti-leukemic agent by inhibiting the protein synthesis via depletion of asparagine in leukemic cells. It is accessible as a drug under different trade name in the market . It is also used in food industry to reduce the formation of acrylamide, from the baked product which is highly carcinogenic [3,4]. Considering the importance of L-asparaginase, this antileukimic enzyme was purified and further attached to graphene for the biosensing purpose. High quality graphene oxide was synthesized using expanded graphite following a modified Hummer’s method. This method is facile, inexpensive and avoids use of strong acids. Graphite powder was treated with potassium permanganate and H2SO4 in an ice water bath under continuous stirring until a uniform liquid paste was formed with a large volumetric expansion. The suspension was then filtered and was subjected to repeated washing and centrifugation to remove unwanted impurities. To fully exfoliate, the Graphene Oxide (GO) sheets were thermally exfoliated by rapid heating at 10500C for 30 seconds under an Argon atmosphere. The product was isolated by filtration and washed copiously with water and vacuum dried. Further the antileukemic enzyme Asparaginase was isolated from a bacterial source, Bacillus cereus strain BHU-MBU BC1 (ACCESSION No. KJ472206). The isolated enzyme was further purified in a single step through DEAE-Sepharose column at 4 °C. Kinetic parameters of the as purified enzyme were thoroughly studied. As purified enzyme was further attached to amine functionalized graphene . It was observed that graphene conjugated enzyme showed the enhanced activity and the reusability of the enzyme was found to be 12 times retaining activity upto 80%. The temperature optima of the immobilized enzyme was also improved from 45 º to 55º C as compared to soluble enzyme.Scanning and Transmission Electron Microscopy (SEM, TEM) and Fourier Transform Infrared (FTIR) spectroscopy were employed for the purpose of characterization. Thus, as conjugated asparginase to amine functionalized graphene will further be used for the detection of asparagines level present in the blood of leukemia patients. References: 1. Bhat M R , T Marar. Purification and characterization of L-asperginase from Salinicoccus sp. M KJ997975. International Journal of Recent Trends in Science And Technology,Vol.14 ( 1), 2015 p.42-48. 2. Richa Jain, K.U. Zaidi, Yogita Verma, Pooja Saxena.People’s Journal of Scientific Research Vol. 5(1), 2012 3. Pedreschi, F; Mery, D. and Marique, T., Grading of potatoes. In Computer Vision Technology for Food Quality Evaluation. ed. D.-W. Sun. pp.305–318. 2008 4. Kuilman, M. and Wilms, L., Toxicol. Lett. Vol.172, pp. S196-S197. 2007 5. Mudavath, S.L., Talat, M., Rai, M., Srivastava, O.N., Sundar, S. Characterization and evaluation of amine-modified graphene amphotericin B for the treatment of visceral leishmaniasis: In vivo and in vitro studies (2014) Drug Design, Development and Therapy,Vol. 8, pp. 1235-1247.