Materials Science and Engineering C 43 (2014) 237–242

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Materials Science and Engineering C

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Immobilization of trypsin on plasma prepared Ag/PPAni nanocompositefilm 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, Indiab Guwahati Biotech Park, Technology Complex, IIT-Guwahati, Guwahati 781039, Indiac Life Sciences Division, Institute of Advanced Study in Science and Technology, Guwahati 781035, India

⁎ Corresponding author at: Physical Sciences Division,Science and Technology, Paschim Boragaon, Garchuk, Guw361 2912073; fax: +91 361 2279909.

E-mail address: arpal@iasst.gov.in (A.R. Pal).

http://dx.doi.org/10.1016/j.msec.2014.07.0250928-4931/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 23 April 2014Received in revised form 22 May 2014Accepted 3 July 2014Available online 11 July 2014

Keywords:Nanocomposite support matrixPlasma polymerizationSputteringTrypsin immobilizationHydrolyzing capacity

This work demonstrates the efficacy of a support matrix prepared by plasma process for trypsin immobilizationwithout any surface activator. Plasma polymerization cum sputtering process is used to prepare the nanocom-posite supportmatrix. 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 activatethe 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.

Institute of Advanced Study inahati 781035, India. Tel.: +91

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Enzymes play the key role in proteome digestion which facilitatesthe development of various kinds of enzymatic biosensors [1–9]. Inthe routinely adopted free enzyme digestion, long (12–20) hours of in-cubation time with free protease (typically trypsin) limit the sampleprocessing throughput and provide incomplete protein proteolysiswith complex steric hindrance [6]. Immobilization of enzymes ontopolymer surface has significant application in biomaterial and proteinresearch, 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-environmentalchanges over free enzymes and such type ofmatrices can be successfullyused for developing bio-sensors, bio-separators and also in food pro-cessing technology [3,4].

Immobilization of an enzyme and its properties are influenced bythe method of preparation, choice of support matrix and the physico-chemical characteristics of thematrix [2,5]. Ideal supportmatrix proper-ties include physical resistance to compression, inertness towards en-zymes, ease of derivatization, biocompatibility, resistance to microbialattack and availability at low cost. Recently, nano-biocomposites have

become a thrust area of enzyme immobilization research because thenanostructured 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 highmolecularweight proteins into the small pep-tides. During the hydrolysis process, the rapid autolysis of trypsinsolution produces unwanted and interfering materials due to least sta-bility of the neutral protease and consequently decreasing the efficiencyand rate of catalytic reactions [8,9]. Low stability of trypsin leads to thecatalytic reaction to be uncontrollable, slow and expensive. Recently, itis reported that trypsin can be successfully immobilized by formingthe co-valent bonding on the surface of polyaniline (PAni) matrix andthereby it can acquire more stability than the free trypsin, exhibitinghigher activities at elevated conditions [4].

Immobilization of an enzyme can be carried out in several ways,such as by crosslinkingmethod, encapsulation/inclusion process and at-tachment by co-valent bonding [4,5]. Among these methods, co-valentattachment 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 graftthe polymer surface to support co-valent immobilization [5–8]. To thebest 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 immobilizationof trypsin onto nano-biocomposite film which is a composite of plasma

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polymerized aniline (PPAni) and silver nanoparticles (AgNPs), termedas Ag/PPAni.

Plasma polymerization is a versatile technology that has enormousadvantages over wet chemical processes, such as wide range of mono-mers can polymerized; synthesis does not require the use of solventsand oxidants and thus gives a product with less contamination and su-perior stability [11–14]. Polymers prepared from this technique arehighly crosslinked, pin-hole free and prone to rapid generation of activesites and free radicals [12,13]. The most important advantage of PPAnisupport matrix is the direct enzyme immobilization and matrix co-valent linking without any surface activator or binders e.g. glutaralde-hyde [4,5]. PPAni is an environmentally stable polymer [9,10]. Highsynthetic yield and stabilitywith temperature and pH are the character-istics of PPAni. Biocompatibility and high resistance tomicrobial activityare the key factor to choose PPAni matrix for immobilization purpose[9]. This is possible because PPAnifilm has large numbers of free radicalson its surface, which permit the active site for immobilized enzyme tobe readily attached by the matrix substrate. In this case the time in-volvement for immobilization process is reduced along with matrixpreparation cost. AgNPS are embedded into PPAni film to increase thesurface to volume ratio for co-valent attachment of enzymes [15,16].AgNPs are cost-effective and safe biocidal material which enhancesthe facile combination with biomolecules along with linkage stability[18,19]. Hence, immobilization of trypsin on Ag/PPAni helps the proteinfor favorable orientation or to make conduction between prostheticgroups because AgNPs permit adsorption of protein molecules withretained enzymatic activity [15–19].

Fourier transform infrared spectroscopy (FTIR), X-ray photoelectronspectroscopy (XPS), Transmission electron microscopy (TEM) andField-emission scanning electron microscopy (FESEM) are used tocharacterize the nanocomposite matrices before and after the trypsinimmobilization. The hydrolytic capacity of the immobilized trypsin tohydrolyze BSA protein has been evaluated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis and comparedwith that of native trypsin.

2. Materials and method

2.1. Reagents

Aniline, trypsin and bovine serum albumin (BSA) are purchasedfrom Sigma-Aldrich. All the necessary reagents are of analytical gradeand required solutions are prepared with distilled deionized water.

2.2. Matrix preparation

The nano-composite matrix (Ag/PPAni) for trypsin immobilization isprepared by plasma polymerization cum sputtering combined processand is carried out in a vertically placed stainless steel cylindrical chamberof 30 cm in diameter and 40 cm in height. The schematic diagram of theexperimental unit used for this work is similar to that of a previous re-port with some minor modifications [19]. The experimental set-up isequipped with a water cooled planar magnetron with silver (Ag) targetof 99.99% purity (ACI alloys, USA) and a stainless steel water cooled sub-strate RF electrode placed 12.5 cm apart from each other in a cylindricalvacuum vessel. Before the introduction of the Argon (Ar) gas into thechamber, it is evacuated to a base pressure of 1 × 10−5 Torr using aturbo pump backed by a dry roughing pump. Aniline monomer is fedinto the chamber in vapor form at a flow rate of 8 sccm in such a waythat the chamber pressure can be maintained at 5 × 10−2 Torr. Flow ofargon (12 sccm) is controlled using mass flow controllers (AALBORG,USA) whereas the flow of aniline is controlled using a vapor phasemass flow controller (MKS Instruments, USA, Model 1150). A pulsedDC power supply (Advanced energy, USA) is used for the generation ofplasma for sputtering of Ag. Themagnetron sputtering process is carriedout at applied power of 40Wand simultaneously plasma polymerization

is done at radiofrequency (RF) power of 7 W with self bias of −68 V.Thus, combined plasma polymerization of aniline monomer andsputtering of Ag metal target develops the composite film by a processenergized by both pulsed DC and RF power in 20 min. For pure PPAnifilmdeposition, themagnetron remains grounded, other than that all pa-rameters are same.

2.3. Immobilization method

All immobilization tests are performed at least in triplicates. Bovinepancreas trypsin solution is prepared in 0.1 M phosphate buffer, pH7.6 (1 g/l). Prepared samples (PPAni and Ag/PPAni) are soaked in 4 mlof the prepared trypsin solution at 4 °C with shaking condition forovernight. After that the samples are rinsed in phosphate buffer(0.1 M; pH 7.6) for 30 min and again for 60 min. Finally the samplesare washed with combined solution of phosphate buffer and tween20 (0.1 ml of tween 20 in 100 ml of 0.1 phosphate buffer) and dried inambient condition. The amount of trypsin immobilized is quantifiedusing Lowry's method [20].

2.4. Characterization techniques

For structural information of the nanocomposite, Fourier TransformInfrared (FTIR) Spectrometer (Vector 22 FT-IR Spectrometer, Bruker,Germany) is used. The spectra are obtained in the transmittancemode, within the spectral range of 4000–400 cm−1. All FTIR measure-ments are performed with 32 scans and a resolution of 4 cm−1.

The surface chemistry of composite matrix and immobilized sam-ples is further investigated by X-ray photoelectron spectroscopy(XPS). XPS spectra are recorded with an ESCA 3000 spectrometer (VGMicrotech, UK), equipped with an Mg Kα X-ray source (1253.6 eV)and a hemispherical electron analyzer. The X-ray source is operated at150 W. The residual pressure in the ion-pumped analysis chamber ismaintained at 1.0 × 10−7 Pa during data acquisition. The XPS curvefitting is performed using “XPSPEAK-4.1” software.

Field emission scanning electron microscope (FESEM, SIGMA VP,Carl Zeiss) is used to observe the surface morphologies of the samples.The samples are coated with gold–palladium alloy in an ion-sputtercoater (SC7620, Emitech) in a low vacuum prior to examination. Thecrystal structure of the embedded AgNPs is investigated using a (JEOLJEM 2100) transmission electron microscope (TEM) having resolutionof 1.9 Å to 1.4 Å.

2.5. Determination of hydrolyzing capacity

In order to determine hydrolytic capacity of native and immobilizedtrypsin, an assay was performed using BSA protein as a substrate at aconcentration of 1%. BSA (66.4 kDa) is the most widely used substrateprotein for evaluating the digestion performance of an enzyme. For hy-drolytic performance, free trypsin and immobilized trypsin are incubat-edwith BSA (1%) at 37 °C for 50min. Hydrolyzed products are subject toSodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis. Same amount of trypsin (native and immobilized) isused in electrophoresis for 2 h at 120 V and gel is stained withCoomassie Brilliant Blue.

3. Results and discussion

3.1. FTIR analysis

Fig. 1 represents the FTIR spectra for PPAni and Ag/PPAni film beforeand after the immobilization of trypsin. All the characteristic bands ofPPAni, Ag/PPAni and Ag/PPAni/Trypsin are presented in Table S1 (sup-plementary data). For PPAni sample (Fig. 1(a)) appearance of secondaryamine peak in the range of 3378–3369 cm−1 is due to the N\Hstretching. Retention of aromaticity is confirmed by the appearance of

Fig. 1. FT-IR spectra of (a) PPAni, (b) Ag/PPAni, (c) PPAni/trypsin and (d) Ag/PPAni/trypsin collected in the spectral region (4000–400 cm−1).

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intense bands at 1601 cm−1 and 1496 cm−1 due to C_C stretch of ben-zenoid and quinoid units, respectively [12,13]. The evolution of the bandat 1307 cm−1 assigned to C\N stretch suggests aromatic linkages. Thebands at 690 cm−1 are due to 1, 3 di-substituted aromatic ring (meta)and at 753 cm−1 are due to 1, 2 di-substituted aromatic ring (ortho)which indicates the cross-linked PAni. The weak intensity band at835 cm−1 due to 1, 4 di-substituted aromatic ring (para) comparedwith the bands 690 cm−1 and 753 cm−1 is an indication of branchedand crosslinked structure during plasma polymerization process.

For Ag/PPAni sample (Fig. 1(b)), due to the incorporation of Agnanostructure some of the characteristic bands of PPAni are found tobe shifted towards the higher wavenumber [9]. C\N stretching peakfor nitrile group at 2212 cm−1 is found to be more intense and thepeak at 1383 cm−1 has shifted towards 1396 cm−1 for C\N Stretching.Similarly, the peak at 1307 cm−1 for PPAni is moved to 1313 cm−1 pos-sibly due to the increase in the order of the amide C\N bond in the Ag/PPAni composite film.

Fig. 1(c) and (d) is the representative spectra for trypsinimmobilized PPAni and Ag/PPAni samples. Appearance of new bandsat 3524 cm−1 and 3348 cm−1 corresponds to N\H symmetric andN\H asymmetric vibrations [7]. Characteristic bands for pure trypsin(not shown in Fig. 1) such as methyl asymmetric at 2933 cm−1 andmethylene symmetric at 2900 cm−1 are found to be shifted towardslower wavenumbers of 2929 cm−1 and 2877 cm−1 respectively [7,8].Co-valent bond attachment of trypsin onto PPAni and Ag/PPAni film isconfirmed by the appearance of 1655 cm−1 band for Amide I and1511 cm−1 for Amide II. Hence, it is indicated that trypsin is successfullyadsorbed on the surface and its secondary structure is unperturbed dur-ing the immobilization process [7,8]. It is possible because in plasma en-vironment electrons acquire sufficiently high energies to dissociatesignificant numbers of chemical bonds involved in the organic structureand during this process the formation of unsaturated bonds also occursalongwith the generation ofmultiple free radicals,whichhave potentialto strongly support the immobilization process.

3.2. SEM and EDX analyses

The surface morphology of the PPAni, Ag/PPAni, PPAni/trypsin andAg/PPAni/trypsin is presented in Fig. 2 along with EDX analysis. Insetsare the SEM images of the same samples taken at higher magnification(×300,000). Fig. 2(a) is the morphological image of PPAni matrix pre-pared by plasma polymerization process; correspondingly the EDX

confirms that the film is composed of C, N and O. However, inFig. 2(b) Ag is detected along with C, N and O for Ag/PPAni matrix.Magnified image confirms that Ag particles deposited by plasmasputtering process have formed some nanostructures. The topographyof the Ag/PPAni nanocomposite film has uniform surface as revealedin Fig. 2(b). The topography of immobilization samples PPAni/trypsinand Ag/PPAni film/trypsin as shown in Fig. 2(c) and (d) has unique sur-face morphology. In both samples, the formation of porous layer withmicro-flower and rod like patterns possibly demonstrates the trypsinimmobilization. Formation of free radicals in plasma polymerized com-posite matrix provides helpful support for enzyme immobilization. EDXresult of Ag/PPAni/trypsin shows the presence of Ag even after the im-mobilization process. It is important to mention that Si peak appearsin EDX spectrums; this is because siliconwafer is used as base substrate.

3.3. TEM and SAED analyses

For detailed understanding of the form of AgNPs in Ag/PPAni/trypsinsample, TEM and selected-area electron diffraction (SAED) analyses areemployed. Representative TEM image and corresponding SAED ringpatterns for Ag/PPAni/trypsin are shown in Fig. 2(e). It is obvious fromthe figure that the AgNPs are spherical in shape having a diameter with-in the range of 10–15 nm. It is apparent from Fig. 2(e) that these silveroxide (AgxO) NPs are apparently embedded in a matrix of amorphousPPAni. SAED ring pattern confirms that the existingmaterial is polycrys-talline in nature [21,22]. Crystal planes are evaluated from the SAED ringpatterns by calculating the value of d-spacing. The d-spacing analysis re-veals that the existence of AgxO is in the form of Ag2O (cubic structure)and AgO. This finding is well correlated with the XPS results. The mea-sured d-values are presented in the inset of Fig. 2(e) along with the in-dication of crystal plane. In the HRTEM images it is confirmed thatamorphous zones exist between discrete crystalline oxide particles,suggesting that Ag/PPAni/trypsin is actually a composite of crystallinesilver oxide particles and an amorphous-like polymer phase.

3.4. XPS analysis

XPS study is employed to analyze the chemical state of the PPAni andAg/PPAni/trypsin samples. Fig. 3(a) is the XPS survey spectra for PPAniand Ag/PPAni/trypsin samples. Both the samples have peaks for C at285 eV, N at 399.8 eV and O at 531.0 eV where as Ag/PPAni/trypsinhas Ag peak at 367.5 eV, which is absent for PPAni film. Quantitative

Fig. 2. FESEM images showing the surfacemorphologyof (a) PPAni, (b) Ag/PPAni, (c) PPAni/trypsin and (d) Ag/PPAni/trypsin samples. Inset represents the images at highermagnificationsand correspondingly the EDX results are presented, (e) TEMmicrograph of Ag/PPAni/trypsin sample with SAED patterns.

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variation in atomic percentage and atomic ratio is presented in Table S2(supplementary data). The values of atomic ratio C/N, C/O and O/N forAg/PPAni/trypsin sample are found to bemore over PPAni. This is possi-ble due to the efficient interaction betweenAg/PPAnimatrix and trypsinduring immobilization process.

Fig. S1 (a)–(c) (supplementary data) indicates the deconvolution ofC1s, N1s and O1s peaks for PPAni film. The Gaussian and Lorentz mixedline shape after the treatment of background by the Shirley function isused to determine accurate peak positions. The details of the peakfittingare presented in the Table S3 (a)–(c) (supplementary data). C1s spec-trum is deconvoluted into six peaks that are assigned to C\C, C_C,C\H/C\N, C_N, C\O/C\O\C and COOH/COOR bonds, having bind-ing energies at 285.1 eV, 284.8 eV, 287.8 eV, 285.6 eV, 286.3 eV and289.3 eV respectively. It is evident from the C1s deconvolution thatthe C_N content is more than C\N indicating the partial degradationof benzenoid ring and producing of volatile functional groups CO, CO2,N2, etc. The C\O peak is associated with the partial surface oxidationof PPAni film [13]. N1s spectrum is deconvoluted into three peaksN\C, N_C/N_H and N\C_O/N\H at 400.5 eV, 398.0 eV and399.2 eV. Peak for O1s is deconvoluted into four peaks such asN\C_O, C_O\O, O\C\O and O_C having binding energy at531.6 eV, 532.6 eV, 533.5 eV and 533.8 eV respectively. The peakswith a binding energy of 532.9 eV for O1s and 289.3 eV for C1s spec-trums can be generally assigned as the absorbed oxygenated speciessuch as free water and carbon hydrated contaminations from atmo-sphere [12]. Fig. 3(b)–(d) is the representative deconvoluted spectraof C1s, O1s and Ag-3d of trypsin immobilized Ag/PPAni film. As revealedfrom Fig. 3(d) single Gaussian–Lorentzian peak function is not appliedto fit each Ag-3d level, therefore Ag-3d level can be deconvoluted intotwo splitting peaks present at binging energy of 367.5 eV and367.9 eV. AgxO splits into AgO and Ag2O oxide state respectively as pre-sented in Table S3 (d) (supplementary data). From the XPS Ag-3d spec-trum (Fig. 3(d)) the ratio of Ag+ to Ag2+ is calculated to be 1.17:1,which suggests that in the nano-composite film, the oxidation state ofAg+ is dominant. Similarly the oxygen anion to Ag cation ratio forAgxO is 1:1.84, which is slightly higher than what would be expectedfor a pure Ag2O face-centered cubic structure i.e. 1:2. Finally it can be

suggested that the AgxO tends to form some defective structure at thetime of plasma sputtering and composite deposition.

C1s spectra have been fitted by six peaks corresponding to differentchemical states for carbon corresponding to C\C (285.1 eV), C_C(284.4 eV), C\H/C\N (287.7 eV), C_N (285.6 eV), C\O/C\O\C(286.3 eV) and COOH/COOR (289.3 eV) units. PPAni film with benze-noid unit gets degraded, since C_N content ismore than C\N function-al unit. The effect of immobilization on the composite film is confirmedfrom the increased percentage of COOH/COOR (289.3 eV) unit whichwas initially negligible or absent for PPAni film. The increased percent-age of this polar group along with all other forms of carbon implies thatfree radicals generated on plasma polymer films support high affinity topeptide bonds and provide good entrapment to enzyme. Similarly N1speak for Ag/PPAni/trypsin is fitted with three peaks corresponding toN\C, N_C/N_H and N\C_O/N\H at 400.5 eV, 398.0 eV and399.2 eV. Lastly, O1s deconvoluted spectrum provides some of the im-portantfindings such as the presence of Ag in oxide form in the compos-ite film and attachment of polar components as evident from theTable S3 (b) (supplementary data). O1s spectrum is fitted to six differ-ent peaks of N\C_O, C_O\O, O\C\O, O_C, Ag2O and AgO havingbinding energies at 531.6 eV, 532.6 eV, 533.5 eV, 533.8 eV, 528.4 eVand 529.4 eV respectively. Appearance of the new peaks of Ag2O andAgO is contributed by single-bonded O interacted with Ag. The electrontransfer from the silver to the oxygen led to the formation of new peaksas revealed from O1s deconvolution. The increase in the percentage ofO\C\O and O_C may be explained by the bonding of oxygen in theimmobilization process. A large number of free radicals created duringplasma polymerization remain trapped in the film during its growth.When thisfilmmatrix is exposed to the enzyme, due to highmass trans-fer most of the peptide bonds are entrapped in these free radicals andsupport the immobilization process.

3.5. Hydrolyzing capacity

The amount of trypsin immobilized on PPAni and Ag/PPAni matrixsupport is measured by Lowry's method and presented in Table 1 [19].As compared to PPAni matrix, Ag/PPAni has more trypsin immobilized

Fig. 3. (a) XPS survey spectra of PPAni and Ag/PPAni/trypsin samples, and deconvoluted spectra of (b) C1s, (c) O1s and (d) Ag-3d of Ag/PPAni/trypsin sample.

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due to the large surface area provided by AgNPs for immobilization pro-cess [6]. The profile of hydrolysis product of immobilized trypsin withBSA protein is determined by SDS-PAGE analysis. It is observed fromFig. 4 that the enzymatic capacity of immobilized trypsin on Ag/PPAnimatrix to hydrolyze BSA is found to be higher than that of the nativetrypsin. The hydrolytic capacity of free trypsin and trypsin immobilizedonto PPAnimatrix is 41.0± 0.3% and 39.0± 0.5% respectively. Remark-ably, the immobilized trypsin on Ag/PPAni matrix shows 58.0 ± 0.2% ofhydrolytic capacity for same incubation conditions as presented inTable 1. Native trypsin has comparability low hydrolytic capacity com-pared to Ag/PPAni/trypsin, may be due to the incomplete proteolyticprocess which provides the steric hindrance in insufficient incubationtime (50 min). Decrease in hydrolysis capacity of PPAni/trypsin as com-pared to native trypsin is due to the blockage of some of the active sitesof trypsin in the PPAni polymermatrix. The exceptionally high digestionefficiency of Ag/PPAni/trypsin is related to the high amount of proteindigestion without steric hindrance at the active site of the immobilizedenzyme. Increase in digestion efficiency of Ag/PPAni/trypsin is possiblydue to the incorporation of AgNPs in PPAnimatrixwhichprovides largersurface area for higher enzyme-to-protein substrate ratio and subse-quently accelerates the digestion rate.

Table 1Quantitativemeasurement of trypsin units immobilized onto PPAni andAg/PPAni supportmatrices evaluated by Lowry's method and the percentage of hydrolysing capacity to BSAprotein.

Sample Trypsin immobilized(units)

Hydrolyzing capacity(%)

Native trypsin – 41 ± 0.3PPAni – 4 ± 0.2Ag/PPAni – 6 ± 0.1PPAni/trypsin 284 ± 0.57 39 ± 0.5Ag/PPAni/trypsin 372 ± 0.67 58 ± 0.2

4. Conclusion

Ag/PPAni nanocomposite matrix prepared by the combined processof plasma polymerization and sputtering is found to be better supportivematerial for enzyme immobilization. Trypsin is covalently attached toPPAni and Ag/PPAni matrices without any surface activator or binder,which is confirmed from FTIR and XPS analyses. Immobilized trypsin onAg/PPAni matrix shows greater hydrolytic capacity than the native en-zyme. Plasma prepared nanocomposite offers promising possibilities asideal enzyme supportive matrix for protein digestion for application inproteomics and also for development of efficient enzymatic biosensors.

Fig. 4. SDS-PAGE of peptides produced by native and immobilized trypsin lane 1: Marker(P7703S); lane 2: BSA; lane 3: Protein extracts digested by free trypsin; lane 4: protein ex-tracts digested by immobilized trypsin onto PPAni matrix; lane 5: protein extractsdigested by immobilized trypsin onto Ag/PPAni matrix; lane 6: protein extracts digestedby PPAni only; and lane 7: protein extracts digested Ag/PPAni matrix only.

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Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.msec.2014.07.025.

Acknowledgements

Thiswork is supported by Institute of Advanced Study in Science andTechnology (IASST), Department of Science and Technology, Govern-ment of India. Dolly Gogoi thanks CSIR-India for providing the researchfellowship under the award no. 9/835(0011)/2013-EMR-I. Authors ac-knowledge SAIF-NEHU-Shillong andNCL-Pune for providing the samplecharacterization facilities.

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