NANO LETTERS
Enzyme−Polymer−Single Walled Carbon Nanotube Composites as Biocatalytic Films
2003 Vol. 3, No. 6 829-832
Kaushal Rege,† Nachiket R. Raravikar,‡ Dae-Yun Kim,† Linda S. Schadler,‡ Pulickel M. Ajayan,‡ and Jonathan S. Dordick*,† Departments of Chemical Engineering and Materials Science and Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180 Received March 5, 2003; Revised Manuscript Received April 7, 2003
ABSTRACT Enzyme-containing polymer−single walled carbon nanotube (SWNT) composites have been explored as unique biocatalytic materials. The biocatalytic composites were prepared by suspending SWNT and r-chymotrypsin (CT) directly into a poly(methyl methacrylate) solution in toluene. The activity of the resulting CT−polymer−nanotube films was observed to be higher than both polymer−CT and polymer−graphite−CT films. The effect of SWNT loading on biocatalytic activity was also studied, and the optimal SWNT loading was used to compare the biocatalytic performance of different polymer−SWNT systems. In all polymers tested, SWNT-containing composites show higher enzyme activity than the non-SWNT preparations. This was most likely due to a reduction in the leaching of enzyme from the film in the former, perhaps caused by the adsorption of enzyme molecules onto highly structured and high energy SWNTs within the polymer matrix. Investigation of different polymer composites indicates that SWNTs may be used to improve the performance of a wide range of biocatalytic composites for applications ranging from antifouling surface coatings to sensor elements.
The incorporation of enzymes into polymeric composites has led to the development of biocatalytic paints, coatings, and films with potential use as selective biocatalysts for biotransformations,1,2 antifouling surfaces,3-5 and biosensors.6-9 Although biocatalytic films have been generated from a wide range of polymeric materials,10-13 unless significant crosslinking of the enzyme or polymer is performed, enzyme leaching is often substantial and restricts the lifetime of these materials.14-18 Thus, there is a need to develop reactive systems that are stable and active in a polymeric environment. Single-walled carbon nanotubes (SWNTs) are slender molecules having a diameter of ca. 1.3-2.0 nm and a length of the order of a few tens of microns, thus having very high surface area-to-volume ratios.19 We reasoned, therefore, that the incorporation of SWNTs to a polymer matrix might offer a high surface area for interaction with suspended enzyme particles or the polymer chains that comprise the film. This would enable a higher retention of the enzyme in the polymeric film as compared to films without SWNT. This paper describes the first report of the generation of biocatalytic polymer-SWNT composites. We prepared three types of enzyme-containing polymeric films: polymer-CT, polymer-graphite-CT, and polymer* Corresponding author. E-mail:
[email protected]. Phone: (518) 2762899. † Department of Chemical Engineering. ‡ Department of Materials Science and Engineering. 10.1021/nl034131k CCC: $25.00 Published on Web 04/22/2003
© 2003 American Chemical Society
SWNT-CT. The general method of film preparation20 is depicted in Figure 1 for the polymer-SWNT-CT film. The first preparation consisted of 1.0 mg CT suspended in 1.0 g poly(methyl methacrylate), (Mw ) 75 000). Enzyme activity in the film was calculated by measuring the total activity of the preparation and subtracting the activity of the supernatant.21 The latter was presumed to contain active enzyme that leaches out of the biocatalytic film. Table 1 provides a summary of the activity of the leachate and the calculated activity of the film after 1.0 and 48 h. As expected, essentially all of the measured biocatalytic activity was due to enzyme that leached out of the pMMA film (note the lack of activity calculated to be due to the enzyme in the film). This result is consistent with those of our previous investigations of CT incorporated into pMMA materials22 and indicates that the likely phase separation of enzyme from the pMMA results in a preparation where enzyme is easily removed by placing the film in an aqueous solution. A second preparation was generated that was similar to the first one except that 1.0 mg of graphite was added. As in the case without the graphite, there was substantial enzyme leaching, and the film again showed essentially no biocatalytic activity (Table 1). Finally, a third composite was prepared and contained 1.0 mg CT and 1.0 mg SWNT in 1.0 g pMMA. The SEM micrograph of this composite (Figure 1) shows a highly mixed matrix containing both SWNT (hair-like projections) and pMMA (as the continuum).
Figure 2. Comparison of biocatalytic film activities of PMMAR-chymotrypsin-SWNT composites. (light gray bar) 1 mg SWNT; (medium gray bar), 2 mg SWNT; (dark gray bar), 5 mg SWNT, each with 1 mg CT and 1.0 g pMMA.
Figure 1. Preparation of enzyme-SWNT-polymer composite films along with an SEM image of a typical composite. Table 1. Comparison of Wash (leaching) and Actual Film Activities of PMMA-R-chymotrypsin (PMMA), PMMA-R-chymotrypsin-graphite (PMMA + Graphite), and PMMA-R-chymotrypsin-SWNT (PMMA + SWNT) Composites (see text and refs 20 and 21 for details)a activity of film (µmol/cm2/s)
activity of leachate (µmol/cm2/s)
time pMMA + pMMA + pMMA + pMMA + (h) pMMA graphite SWNT pMMA graphite SWNT 1 48
0.026 0.110
0 0.101
0.012 0.015
* *
0.008 *
0.043 0.068
a Values marked with an asterisk indicate activity that was nearly indistinguishable from the leachate values, and therefore, the activity of the film was small compared to the activity of the enzyme that is removed from the polymer film.
Interestingly, this composite showed higher biocatalytic activity in the film than did the other two preparations (Table 1). Furthermore, the SWNT-containing biocatalytic composite showed relatively little enzyme leaching after 48 h; hence, this preparation gave both active and stable (to leaching) biocatalytic films. The difference between the graphite and SWNT preparations was particularly significant and indicates that the enhanced enzyme activity in biocatalytic polymer composite films (and reduced enzyme leaching) was not strictly dependent on the chemistry of the carbon material. Rather, the high radius of curvature and chemical activity of the nanotube appear to play a major role in 830
endowing the biocatalytic composite film with activity and stability. Having established that SWNTs improve the activity and stability of enzyme-containing polymer films, it was of interest to examine whether the concentration of SWNT influenced the catalytic properties of the films. To that end, we varied the SWNT concentration in the pMMA films from 1 to 5 mg/g pMMA. CT was maintained at 1 mg/g pMMA. The film activity was measured as a function of incubation time (Figure 2). Minimal leaching of CT was observed with any of the three preparations, even after 48 h (data not shown). Differences were evident, however, in the biocatalytic activity of the films. After 1 h, both the 2 and 5 mg SWNT/g pMMA preparations showed higher activity than the 1 mg SWNT/g pMMA preparation, and this was also the case after 48 h. The activities of the two higher SWNT loaded preparations increased substantially in the 48 h incubations. The time-dependence of enzyme activity is intriguing. One possible mechanism for this phenomenon is the slow dissolution of enzyme from the solid enzyme particles embedded in the pMMA matrix as a result of the partitioning of water into the polymer continuum. Hence, an increasing amount of soluble enzyme molecules would be generated over time and these enzyme molecules would have three fates. In the absence of SWNT or graphite, the polymer matrix is sufficiently porous to allow water to pass through the polymer film and allow soluble enzyme to easily escape into the bulk aqueous solution, thereby resulting in leaching. However, in the presence of the SWNT or graphite, soluble enzyme molecules can either bind to the carbon of the additives (and hence remain within the film), or they may be retained in the film because of a restructuring of the polymer molecules in the vicinity of the additive and resulting in changes in polymer mobility. To distinguish between these two fates, we used thermal analysis to examine the pMMA-CT composites with and Nano Lett., Vol. 3, No. 6, 2003
Figure 3. Influence of polymer type on biocatalytic film activity. See text for experimental details.
without either graphite or SWNT. Films were prepared with enzyme as described above,20 and the Tg of the films were measured after 48 h in aqueous buffer solution.23 The Tg values of pMMA and pMMA-SWNT composites were similar: 75.3 and 71.8 °C, respectively. The Tg of the pMMA-graphite composite, however, was 57.4 °C, indicating that graphite acts as a significant plasticizer of the pMMA. In the case of SWNT-containing composites, such a plasticizing effect is not observed. The plasticizing effect may change the rate of water transport through the polymer, but because the pMMA-SWNT did not experience a change in Tg, it seems more likely that the SWNT are trapping the enzyme that is being leached out of the composite. This is likely due to the high surface energy of the SWNT compared to sheet graphite, which would result in greater protein adsorption to the surface of the additive. Different Polymer-SWNT Systems. To test whether this behavior can be translated to other polymer systems, we examined the dependence of film activity and leaching with several different polymers, including polystyrene (PS) and poly(lactic acid) (PLA) [PS, bimodal MW distribution of 4000 and 200 000 and PLA, MW of 75 000-120 000]. PSCT-SWNT and PLA-CT-SWNT films were prepared identically to pMMA-based materials, and 2.0 mg SWNT and 1.0 mg CT per g polymer was used. The results are shown in Figure 3 (pMMA added for comparison). As expected, significant leaching occurred in the absence of SWNT, thereby resulting in low film activities. Only in the SWNT-containing preparations for the two more hydrophobic polymers was observable film activity evident (e.g., pMMA and PS). Furthermore, a time-dependent increase of enzyme activity of the film activity was observed, as was seen with pMMA (the drop for PLA may not be statistically significant). Finally, the film activity was strongly dependent on the nature of the polymer used. Enzyme activity in PLA was not significantly affected by the SWNT. This could be because of the high rate of water transport through PLA, which washed away enzyme molecules even when they are Nano Lett., Vol. 3, No. 6, 2003
attracted to the SWNT. The lower enzymatic activity in PS compared to the PMMA could be the result of very low water solubility in PS, thereby preventing dissolution of the enzyme into the bulk material. In conclusion, we have discovered that the incorporation of SWNTs into enzyme-polymer composites result in active and leaching-stable biocatalytic polymer films. This is likely due to the chemical interaction of hydrophobic carbon with polymers and/or the high surface area of the SWNT. This reduces enzyme leaching from the film, yet provides effective pore sizes large enough to allow the enzymic substrate to diffuse to the biocatalyst, thereby allowing the film to be biocatalytically active. Increasing the amount of SWNTs to the film increases film activity, and this is likely due to an increased exposed hydrophobic and structured surface area of the SWNTs incorporated into the polymer film. We are currently examining the precise role of SWNTs in stabilizing enzyme-polymer composite films, including the specific adsorption of protein molecules onto SWNT surfaces. We are also examining applications of these composites as active and stable surface materials, ranging from antifouling paints and coatings to biosensors. Acknowledgment. This work was supported through the Nanoscale Science and Engineering Initiative of the National Science Foundation (NSF) under NSF Award Number DMR0117792. The authors thank Karunya Ramasamy and Steven M. Cramer for helpful discussions. References (1) Appendini, P.; Hotchkiss, J. H. Packag. Technol. Sci. 1997, 10, 271. (2) Wang, P.; Sergeeva, M. V.; Lim, L.; Dordick, J. S. Nature Biotechnol. 1997, 15, 789. (3) Novick, S. J.; Dordick, J. S. Chem. Mater. (Communication), 1998, 10, 955. (4) Kim, J.; Delio, R.; Dordick, J. S. Biotechnol. Prog. 2002, 18, 551. (5) Kim, Y.-D.; Dordick, J. S.; Clark, D. S. Biotechnol. Bioeng. 2001, 72, 475. (6) Abel, P U.; von Woedtke, T.; Schulz, B.; Bergann, T.; Schwock, A. J. Molec. Cataly. B: Enzymatic 1999, 7, 93. (7) Jin, W.; Brennan, J. D. Analytica Chimica Acta 2002, 461, 1. (8) Kurzawa, C.; Hengstenberg, A.; Schuhmann, W. Analytical Chemistry 2002, 74, 355. (9) Niculescu, M.; Erichsen, T.; Sukharev, V.; Kerenyi, Z.; Cso¨regi, E.; Schuhmannb, W. Analytica Chimica Acta 2002, 463, 39. (10) Liu, H.; Li, H.; Ying, T.; Sun, K.; Qin, Y.; Qi, D. Anal. Chim. Acta 1998, 358, 137. (11) Gill, I.; Pastor, E.; Ballesteros, A. J. Am. Chem. Soc. 1999, 121, 9487. (12) Jia, H.; Zhu, G.; Vugrinovich, B.; Kataphinan, W.; Reneker, D. H.; Wang, P.; Biotechnol. Prog. 2002, 18, 1027. (13) Betigeri, S. S.; Neau, S. H. Biomaterials 2002, 23, 3627. (14) Elc¸ in, Y. M. Biomaterials 1995, 16, 1157. (15) Uhlich, T.; Ulbricht, M.; Tomaschewski, G. Enzyme Microb. Technol. 1996, 19, 124. (16) Vazquez-Duhalt, R.; Tinoco, R.; D’Antonio, P.; Topoleski, L. D. T.; Payne, G. F.; Bioconjugate Chem. 2001, 12, 301. (17) Kim, J.; Kosto T. J.; Manimala, J C.; Nauman, E. B.; Dordick, J. S. AIChE J. 2001, 47, 240. (18) Marconi, W.; Faiola, F.; Piozzi, A. J. Molec. Catal. B 2001, 15, 93. (19) Ajayan, P. M. Chem. ReV. 1999, 99, 1787. (20) One gram of polymer was dissolved in 3.0 g (∼3.5 mL) toluene by ultrasonicating for 5 min and allowed to cool to room temperature. A predetermined weight of SWNT (Carbon Nanotechnologies, Inc., Houston, TX) was added to 1.0 g toluene and the dispersion was sonicated until a uniform suspension of the SWNT was obtained and allowed to cool to room temperature. One milligram of R-chymotrypsin (Sigma Chemical Co., St. Louis, MO) was added, and the 831
resulting suspension was sonicated for 1 min to ensure a uniform dispersion of both the enzyme and the SWNT. After cooling to room temperature, the polymer-toluene solution and the enzyme-SWNTtoluene suspensions were mixed together and drop cast into a 5-cm Petri dish to form a composite film. Similar approaches were used for the graphite (avg. particle size e 100 µm from Fluka Chemie AG, Switzerland) and additive-free enzyme-pMMA films or for other polymer films. (21) CT was chosen as the model enzyme for all films studied and N-succinyl Ala-Ala-Pro-Phe-p-nitroanilide was used as a chromogenic substrate. The tetrapeptide substrate is cleaved by CT to yield the p-nitroaniline chromophore, which was detected spectrophotometrically at 410 nm (Perkin-Elmer Lambda 6.0 UV-vis spectrophotometer (Norwalk, CT)). To study the leaching of CT from the films, repeated washing was performed with 4 mL of 20 mM Tris buffer, pH 7.4. Each wash carried out for 12 min in an orbital shaker at 135 rpm and 25 °C. After successive washes, the supernatants were collected and filtered, and 25 µL of the tetrapeptide substrate solution was added at a final concentration of 60 µM. The films were washed until no further increase in leachate enzyme activity was obtained or
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a stable release of enzyme activity was measured. To measure enzyme activity specifically in the film, the leached enzyme solution was replaced with a fresh substrate solution in aqueous buffer and the observed enzyme activity determined. The “final film activity” or “film activity” was calculated by subtracting the final wash activity from the observed film activity. (22) Novick, S. J.; Dordick, J. S. Biomaterials 2002, 23, 441. (23) Tg was determined by differential scanning calorimetry (DSC, differential thermal analyzer from Mettler-Toledo). The samples were heated and cooled over a range from 25 to 180 °C in two cycles under N2. An identical heating and cooling rate of 10 °C/min was used. The Tg was determined during the second heating cycle. The films were prepared as described above20 and then washed five times with DI water by pouring water onto the films and shaken at 100 rpm for 10 min. This was followed by cutting a center section from the film for DSC analysis. The remaining part of each film was placed in water for 48 h and then subjected to an identical sequence of washing and DSC analysis as described above.
NL034131K
Nano Lett., Vol. 3, No. 6, 2003
