Immobilization of trypsin on plasma prepared Ag/PPAni nanocomposite film for efficient digestion of protein Dolly Gogoi a , Tapan Barman a , Bula Choudhury b , Mojibur Khan c , Yogesh Chaudhari c , Madhusmita Dehingia c , Arup Ratan Pal a, ⁎, Heremba Bailung a , Joyanti Chutia a a Physical Sciences Division, Institute of Advanced Study in Science and Technology, Guwahati 781035, India b Guwahati Biotech Park, Technology Complex, IIT-Guwahati, Guwahati 781039, India c Life Sciences Division, Institute of Advanced Study in Science and Technology, Guwahati 781035, India abstract article info Article history: Received 23 April 2014 Received in revised form 22 May 2014 Accepted 3 July 2014 Available online 11 July 2014 Keywords: Nanocomposite support matrix Plasma polymerization Sputtering Trypsin immobilization Hydrolyzing capacity This work demonstrates the efficacy of a support matrix prepared by plasma process for trypsin immobilization without any surface activator. Plasma polymerization cum sputtering process is used to prepare the nanocom- posite support matrix. Plasma sputtered silver nanoparticles (AgNPs) are uniformly embedded into plasma poly- merized aniline (PPAni) film. Various characterization tools are employed to study the surface morphology, microstructure and chemical composition of the support matrices. Trypsin is immobilized onto the support ma- trix via the formation of covalent bond between them. Plasma generated free radicals on composite films activate the support matrix and make it efficient for increasing the tertiary enzyme stability via multipoint covalent at- tachment. Trypsin immobilized onto Ag/PPAni matrix has more hydrolyzing capacity of bovine serum albumin (BSA) than free trypsin as well as trypsin immobilized onto PPAni films. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Enzymes play the key role in proteome digestion which facilitates the development of various kinds of enzymatic biosensors [1–9]. In the routinely adopted free enzyme digestion, long (12–20) hours of in- cubation time with free protease (typically trypsin) limit the sample processing throughput and provide incomplete protein proteolysis with complex steric hindrance [6]. Immobilization of enzymes onto polymer surface has significant application in biomaterial and protein research, as it is beneficial to enhance the operational stability of an en- zyme [1–4]. Enzymes immobilized onto supportive matrix are biologi- cally very active and stable in nature towards any bio-environmental changes over free enzymes and such type of matrices can be successfully used for developing bio-sensors, bio-separators and also in food pro- cessing technology [3,4]. Immobilization of an enzyme and its properties are influenced by the method of preparation, choice of support matrix and the physico- chemical characteristics of the matrix [2,5]. Ideal support matrix proper- ties include physical resistance to compression, inertness towards en- zymes, ease of derivatization, biocompatibility, resistance to microbial attack and availability at low cost. Recently, nano-biocomposites have become a thrust area of enzyme immobilization research because the nanostructured materials attain exceptional properties e.g. higher sur- face area to volume ratio, high catalysis rate, high stability, and reusabil- ity [5,10]. Trypsin is one of the important digestive enzymes used for hydroly- sis and proteolysis of high molecular weight proteins into the small pep- tides. During the hydrolysis process, the rapid autolysis of trypsin solution produces unwanted and interfering materials due to least sta- bility of the neutral protease and consequently decreasing the efficiency and rate of catalytic reactions [8,9]. Low stability of trypsin leads to the catalytic reaction to be uncontrollable, slow and expensive. Recently, it is reported that trypsin can be successfully immobilized by forming the co-valent bonding on the surface of polyaniline (PAni) matrix and thereby it can acquire more stability than the free trypsin, exhibiting higher activities at elevated conditions [4]. Immobilization of an enzyme can be carried out in several ways, such as by crosslinking method, encapsulation/inclusion process and at- tachment by co-valent bonding [4,5]. Among these methods, co-valent attachment is more successful in terms of upgraded operational stabili- ty, low performance cost and increased enzyme–polymer ratios [5]. Numbers of studies are made to modify the polymer surface or graft the polymer surface to support co-valent immobilization [5–8]. To the best of our knowledge, immobilization of any enzyme onto plasma pre- pared nanocomposite support matrix and its applicability in protein di- gestion is still to be investigated. Our study explores the immobilization of trypsin onto nano-biocomposite film which is a composite of plasma Materials Science and Engineering C 43 (2014) 237–242 ⁎ Corresponding author at: Physical Sciences Division, Institute of Advanced Study in Science and Technology, Paschim Boragaon, Garchuk, Guwahati 781035, India. Tel.: +91 361 2912073; fax: +91 361 2279909. E-mail address: arpal@iasst.gov.in (A.R. Pal). http://dx.doi.org/10.1016/j.msec.2014.07.025 0928-4931/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec