J. MATER. CHEM., 1991, 1(3), 447-450 Matrix Surface Modification by Plasma Polymerization for Enzyme Im mo bi Iization Mehmet Mutlu,a*b Selma Mutlu,"Bb Mark F. RosenberglaPc John Kane,*' Malcolm N. Jonesc and Pankaj Vadgama" " University of Manchester, Medical School, Dept. of Clinical Biochemistry, Hope Hospital, Manchester M6 8HD, UK Hacettepe University, Chemical Engineering Dept., Beytepe Campus, 06532 Ankara, Turkey University of Manchester, Medical School, Dept. of Biochemistry and Molecular Biology, Manchester M13 9PT, UK Polycarbonate (PC) membranes have been treated with dimethylamine (DMA), mpentylamine (PA) or mheptylamine (HA) in a glow-discharge apparatus. Amino group concentrations on plasma-polymerized PC membranes were then assayed by binding radiolabelled [l-'4C]acetic anhydride to the membrane, followed by scintillation counting.For the different plasma-polymerized membranes different degrees of radiolabelled (1251) glucose oxidase or rennet binding were observed and this was found to be directly related to the surface amino-group concentrations for the appropriate membrane. Keywords: Surface modification; Plasma polymerization; Enzyme immobilization; Radiolabelled enzyme; [ 7-'4C]acetic anhydride Enzymes are biocatalytically active entities upon which the metabolism of all living organisms is based. With the technical methods in common use it is generally impossible to separate efficiently the dissolved or finally suspended native biocata- lysts from the products of bioconversion. Therefore, the loss of these valuable materials results in high product cost.The problem can be resolved by the use of immobilized biocatalysts, which, in addition, allow the process to be carried out continuously. A biocatalyst is termed 'immobilized' if its mobility has been restricted by chemical or physical means. This artificial limitation of mobility may be achieved by widely differing methods, such as binding the biocatalysts to one another or to carrier substances, by entrapment in a network of a polymer matrix or by membrane confinement. A variety of techniques have been developed for the immobi- lization of enzymes. 1-4 These methods find practical appli- cation in the fields of medicine, food, agriculture, chemistry and other disciplines.Covalent binding is one of the most popular immobilization techniques, as it is expected to pro- duce a close association between one biocatalyst and another, or between a biocatalyst and a carrier. Covalent binding is usually employed for coupling enzymes but not whole cells,5 the latter being too sensitive to the harsh reagent conditions demanded. A range of functional groups can be used for immobilization including, amino groups, as well as carboxy, mercapto, hydroxy, imidazolyl and phenolic groups. Some of these groups, e.g. SH and amino groups can react directly with appropriate groups of the carrier. Others, e.g. OH groups, have to be activated before they can react with a carrier group. The active site of an enzyme is often located deep within the molecule, and a solid phase prepared by coupling small ligands directly to a matrix can exhibit a low capacity due to steric hindrance between the matrix and the substance binding to the ligand.In these circumstances a 'spacer arm' can be interposed between matrix and ligand to facilitate effective binding. The aim of this study was to modify the matrix surfaces of PC membranes by exposure to plasma glow discharge of amino-group-containing entities (DMA, PA, HA) and to immobilize enzymes (glucose oxidase and rennet) using glutar- aldehyde as a coupling agent. Experimental Surface Modification of PC Membranes by Plasma Polymerization High-density PC membranes (HDPCM) with nominal pore sizes ranging from 0.01 to 0.05 pm (rated by manufacturer, 8 x 10' pores cm-2) were supplied by Poretics (Livermore, CA).DMA, PA and HA were obtained from Sigma (Poole, Dorset). The plasma polymerization was carried out in the system shown schematically in Fig. 1. The apparatus consisted of a glass-tube reactor with two copper electrodes mounted on the outside and a radiofrequency generator (RFG).The reactor was evacuated to 10-3-10-4 mbar. A radiofrequency of 13.6 MHz was applied, its power ranging from 0 to 100 W. Power losses were kept to a minimum by a matching network. The membranes were exposed to plasma media for 5 min at 10 W with DMA or PA or HA monomer fed at a flow rate of 80 cm3 min-'. Preparation of Radiolabelled Enzymes Glucose oxidase (E.C.1.1.3.4. from Aspergillus niger: 292 IU mg-' protein) and rennet [extracted from calf stomach, containing 80% NaCl] were obtained from Sigma (Poole, Dorset). The enzymes were labelled by 1251to a specific activity of 7 mCi mg-' by the Chloramine-T method described by Hunter and Greenwood.6 The radiolabelled needle mopnrerelectrode valve /--S I meter plasma reactor \ -=-vacuum electrode pump Fig. 1 Schematic representation of plasma-polymerization apparatus enzyme was purified by Sephadex-G25 column chromotogra- phy. A series of different concentrations for both enzymes (0.002, 0.02, 0.2, 0.6 and 2.0 mg cm-3) was prepared and 12'1- glucose oxidase and 1251-rennet were added to these solutions as a tracer just before commencement of the experiments.Determination of the Surface Amino-group Concentrations For each amine-modified PC membrane and a control mem- brane of unmodified PC, three sections of equal weight and surface area were placed in glass containers (7 cm3) taking care to ensure the membranes were as dry as possible. An equal volume of dry dimethyl sulphoxide (3 cm3) was added to each membrane. For each membrane type a range of [l-14C]acetic anhydride (Radiochemical Centre, Amersham, Bucks., UK; specific activity 30 mCi mmol -I) activities were added: 2.5, 5, 10 and 15 pCi corresponding to 0.008, 0.016, 0.032 and 0.048 mg of acetic anhydride, respectively. The membranes were agitated continuously for 3 h and washed exhaustively by rinsing with water overnight to remove any non-specifically adsorbed acetic anhydride.The extent of acetylation on each membrane, which should in turn reflect the availability of amino groups, was assessed by counting the 14C attached to the washed membrane. This was under- taken by adding 4 cm3 of scintillation fluid to each membrane followed by counting on a scintillation counter. To allow for [1-14C]acetic anhydride non-specifically adsorbed to the membranes, the counts bound to the control membrane were subtracted from those bound to the activated membranes prior to the calculation of the amino-group concentrations. The calculation involved relating the number of disinte-grations per minute (DPM) to the specific activity of the [1-'4C]acetic anhydride from which the number of moles of bound acetic anhydride and therefore the number of moles of amino group could be established.The calculation took into account that only one side of the membrane was coated with the amine and that only one of the terminal carbons of the [1-'4C]acetic anhydride was radiolabelled. Immobilization of Enzymes The mechanism of the immobilization of the enzymes on the plasma-treated surfaces is shown schematically in Fig. 2. The plasma-treated PC membrane surfaces (16 cm2 surface area) were activated by incubation with 10cm3 of 12.5% (v/v) glutaraldehyde solution for 24 h at 37 "C. In order to prevent stagnant film formation on the membrane surfaces, activation tubes were continuously mixed (Luckham, Rotatest Shaker, Model R 100, UK).The surface-activated membranes were then washed with distilled water to remove any unbound glutaraldehyde present on the surfaces. A series of enzyme solutions were prepared with different enzyme contents (0.02, 0.2, 2, 6 and 20 mg) in 10cm3 of buffer solution, and after the addition of the radiolabelled tracers ('251-glucose oxidase or 1251-rennet) the membranes were placed into the tubes and left to interact for 24 h at 37 "C. Again, in order to prevent stagnant film formation on the membrane surfaces, activation tubes were continuously mixed during the coupling process. At the end of the incubation period the membranes were removed and washed with a sequence of buffers, 0.1 mol dm-3 acetate buffer (pH 4), 0.1 mol dmP3 borate buffer (pH 8), 0.1 mol dm-3 acetate buffer (pH 4) and 0.5 mol dm-3 NaCl, to remove non-specifically bound protein.Quantification of Immobilized Enzyme on the Surface The assessment of the immobilized enzyme on the plasma- treated surfaces was carried out by counting the radioactivity J. MATER. CHEM., 1991, VOL. 1 DMA or -[-PAplain membrane or HA plasma polymerization a membrane glutaralde hyde surface activation nu membrane enzyme enzyme attachment nU membrane enzyme immobilized enzyme Fig. 2 Mechanism of enzyme immobilization on plasma-treated surfaces of each membrane on a y-scintillation counter (LKB, Wallac, Finland). In addition the activity of each enzyme solution was counted and the amount of the enzyme bound on eachcm2 of the membrane was thereby calculable.Results Determination of Surface Amino-group Concentration for Plasma-polymerization-treatedPC Membranes The binding of [1-14C]acetic anhydride of increasing concen- trations to various plasma-polymerization(P1zP)-treatedmem-branes is shown in Fig. 3. It should be stressed that at saturation the surface density of amino groups in PlzP(HA) and PlzP(AA) surfaces are ca. 3 and 4 times higher than the background levels, respectively. The results also indicate that 10 pCi of [l-14C]acetic anhy- dride was sufficient to saturate the reactive sites and therefore this activity was used to evaluate the amino-group concen- trations for subsequent membranes.The number of moles of acetic anhydride bound to the membrane was calculated and thus the number of moles of amino group present on each membrane was established after correction for non-specific binding to plain PC membranes. The results are shown in Table 1. It is clearly shown in the table that, the amino-group density is highest for the PlzP(DMA)-treated surface and lowest for the PlzP(HA) surfaces. 449J. MATER. CHEM., 1991, VOL. 1 I . . . I...I..._ 4 8 12 16 activity of [1-"'CJacetic anhydride/pCi Fig. 3 The effect of initial activity of [l-14C]acetic anhydride on disintegration rate for plasma-polymerization-treatedsurfaces. Type of surface: 0,control; 0,PlzP(DMA); 0,PlzP(PA); A, PlzP(HA) Table 1 The concentration of amine groups bound to various plasma- polymerized PC membranes plasma-polymerized PC membrane NHJmmol cm-2 HA 1.6 x lo-' PA 3.0 x lo-' DMA 16.0 x lo-* Immobilization of Enzymes The amount of two different enzymes (glucose oxidase and rennet) immobilized on the plasma-polymerization-treated surfaces are shown in Fig.4(a) and (b), respectively. For all types of surface, it can clearly be seen that the amount of bound enzyme increases as the initial enzyme content is increased without evidence of saturation. Fig. 4(a) shows that the plasma-polymerized DMA, PlzP(DMA), treated surface has the largest capacity to bind glucose oxidase. This capacity is ca. 18 times greater than for the plain mem- brane at the highest level of initial enzyme content (20 000 pg).N I E 0)a \8 E *-OI1.50 Q) R0 3-0 W 000L 3 250 initial glucose oxidase contentlpg 5.0 N I 4.5 ;(b) E 4.0 .-B , , , , . , , , ,a::-, .I o.oo 5000 10000 15000 20000 25000 However, this capacity is only ca. 3.2 times greater for rennet 7000, 1 [Fig. 4(b)]. In addition, rennet has a higher tendency to bind non-specifically to the plain PC surface than has glucose oxidase [Fig. 4(a)and (b)]. The correlation between the surface amine concentration of the various membranes at saturation with the amount of bound glucose oxidase at the surface (20 000 pg initial enzyme content) is shown in Fig. 5. In addition, Fig. 5 shows the percentage of available amino groups occupied by glucose oxidase.It can be seen that the amount of glucose oxidase bound increases with amino concentration. However, the glucose oxidase bound to the membrane, expressed as a percentage of the total available amino-group binding sites, decreases with increasing amine concentration. Discussion Plasma-plymerization-treated PC Membranes PC membranes are commonly used in biosensor technology as selective barriers to contaminating species. For this reason, these membranes were selected for surface modification by plasma polymerization and subsequent enzyme immobil-ization. In order to investigate the effect of the chain length, three different types of amino-group-containing monomers, DMA, PA and HA, were selected. In the coupling processes of enzymes or different types of protein, the length of the spacer arm is critical.If it is too short, the arm may be ineffective and the ligand may fail to bind substances in the sample. If it is too long, non-specific effects become pronounced and reduce the ~electivity.~ Plasma polymerization was selected as the method of surface modification for enzyme immobilization. The advan- tage of plasma polymerization is that a simple derivation step with high surface enzyme loading can be achieved without the use of complicated chemistry or the use of destructive reagents, which might compromise the structural integrity of the membrane. A satisfactory application of plasma polymeriz- ation for surface modification has been elaborated by one of the authors'.' and These studies conducted with blood-plasma proteins showed that plasma polymerization can be used to modify the surface of polyurethane bio- materials.Determination of Surface Amino-group Concentration of Plasma-plymerization-treatedPC Membranes Many organic species can form surface polymers under 'glow discharge' conditions, so-called 'plasma polymerization'. The application of this technique for coating metal surfaces was N-'E lo-' (400 0D surface amino-group density/mmol cm-* initial rennet content/pg Fig. 5 The relationship between surface amino-group density and (-) the amount of bound glucose oxidase per unit area of mem- Fig. 4 The effect of the type of animated surfaces by plasma polymer- brane and (---) percentage occupancy of amino groups by glucose ization on (a) glucose oxidase immobilization, (b) rennet immobiliz- oxidase (GOD) (at 20 000 pg for 24 h reaction).0,HA; .,AA; A, ation. Symbols as for Fig. 3 DMA initiated about two decades ag0.’~9l~ However, little is known about the mechanism of plasma polymerization, and this is still under intense investigation. Commonly accepted mechan- isms involve the formation of radicals which initiate polymer- ization and subsequent attachment to the surface and/or attachement of the radicals directly to the surfaces and formation of highly cross-linked polymer^.'^ The studies shown here (Fig. 3 and Table I), involving analysis of free amino groups on the plasma-treated surfaces suggest that plasma polymerization may involve C-H bonds, rather than N-H bonds, in the formation of radicals as suggested by the presence of free amino groups on the membrane surfaces.The higher level of amine immobilization found for DMA compared to PA and HA suggests that the secondary amine reacts more readily with the PC surface, possibly because of increased charge-transfer effects which would be favoured by the inductive effect of the methyl groups. It is also possible that the primary amines might react more via the N-H bonds and so render the residues unreactive to acetic anhydride. The concentration of primary amino groups on an appro- priate plasma-polymerized membrane was calculated to assess whether there was any relationship between this value and the degree of glucose oxidase binding.Initially the reagent 2,4,6-trinitrobenzenesulphonicacid (TNBS) was employed, since this compound has been found to react specifically and under mild conditions with amino groups to give trinitro- phenyl derivatives.” Unfortunately, however, these derivatives are formed on the membrane surface making assessment of the amino-group concentrations difficult by absorbance measurements. For this reason an alternative technique was investigated involving acetylation of the amino groups on the membrane with radiolabelled [l-14C]acetic anhydride fol- lowed by heterogeneous scintillation counting of the respective membranes. Adams et ~1.’~have acetylated lysozyme to enable the behaviour of this protein at an air/water interface to be evaluated.A similar procedure was employed for the acety- lation of the membranes, although in this case separation of the non-bound from the bound acetic anhydride was made easier since the unbound material could simply be washed Off. Immobilization of Enzymes The choice of the enzyme proteins used was dictated by availability and low cost. To study the efficacy of the washing step in removing non-specifically bound enzyme, plain mem- branes were counted before and after the washing cycle. For glucose oxidase 85% of the bound radioactivity (i.e. enzyme) could be removed. However, for rennet this was only 20%. Since, only 0.24 and 0.19% of the glucose oxidase enzyme was attached to the PlzP(DMA)-treated PC membrane at an initial enzyme content of 6000 and 20 000 pg, respectively, [Fig.4(a)] and the surface did not saturate, it follows that the reaction of glucose oxidase with the membrane is very slow and possibly does not reach equilibrium during the 24 h reaction time. The amount of enzyme binding increases with, but is not proportional to, the surface amino-group density (Fig. 5). This may be due to steric hindrance inhibiting maximal binding of glucose oxidase to higher membrane densities of amino J. MATER. CHEM., 1991, VOL. 1 groups. However, the percentage of available amino groups occupied by glucose oxidase is greater for the surfaces treated with larger spacer arms.This may be due to the fact that these amino groups are separated from the surface by the spacer arm and therefore less susceptible to steric hindrance. Conclusion Fig. 4(a) and (b)indicate kinetically slow bulk-concentration- driven reactions. The surface reaction is not zero order with respect to the bulk reactants, despite the extended incubation periods employed. However, binding of the proteins does reflect the concentrations of the original surface amino groups and confirms that increased amounts of enzyme can be bound to membranes by increasing the surface amino-group density. The reduced binding efficiency associated with the decreased length of the spacer arm (Fig. 5) could have been due to steric limitation of glucose oxidase molecules on the membrane surface.It may also be possible that surfaces that contain longer spacer arms, such as PlzP(HA)-treated surfaces, have a proportionally higher tendency to bind very large enzyme molecules because of the increased availability of amino groups for coupling. Rennet attachment, both specific and non-specific, is greater than that of glucose oxidase. Both effects may have been due to the greater availability of binding sites for the smaller, and therefore sterically less hindered rennet molecule. It is unlikely that the membrane pores had a significant quantitative effect on this differential binding. M. M. gratefully acknowledges financial support from the International Atomic Energy Agency (IAEA) (Grant No: C6/TUR/88 13).References 1 W. Hartmeier, Immobilized Biocatalysts, Springer-Verlag, Berlin, Heidelberg, 1988. 2 D. R. Zaborsky, Immobilized Enzymes, CRC Press, Cleveland, 1973. 3 T. M. S. Chang, Biomedical Applications of Immobilized Enzymes and Proteins, Plenum Press, New York, 1976, vol. I and 11. 4 B. Mattison, Immobilized Cells and Organelles, CRC Press, Florida, 1983. 5 D. Thomas and J. P. Kervenez, Analysis of Control oflmmobilized Enzyme Systems, North-Holland, Amsterdam, 1976. 6 W. M. Hunter and F. C. Greenwood, Nature (London), 1962, 194, 495. 7 P. O’Carra, S. Barry and T. Griffin, Biochem. SOC. 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