Anal. Chem. 2004, 76, 6915-6920
Quantatitive Assessment of Enzyme Immobilization Capacity in Porous Silicon Lisa A. DeLouise and Benjamin L. Miller*
Department of Dermatology and the Center for Future Health, University of Rochester Medical Center, Rochester, New York 14642
Immobilized enzyme systems are important in a broad range of applications, from biological sensing to the industrial-scale biocatalytic synthesis of chiral products. We demonstrate the ability to systematically vary and quantitatively assess the immobilization capacity of porous silicon thin films for the enzyme glutathione-S-transferase in a manner predicted by a simple geometric model of the porous silicon matrix. We find that the immobilization capacity quantatitively correlates with systematic changes in the device thickness. These results are significant since, despite the wide range over which porous silicon morphology and surface area can be varied, few attempts have been made to systematically characterize surface binding capacity. Our findings suggest that porous silicon can be an ideal matrix, where immobilization of a predictable quantity of biological material is desired. Immobilized enzymes are widely used in fields ranging from biosensing to chemical synthesis to manufacturing. Progress in the development of new immobilized enzyme systems requires advances in our understanding of the materials available for enzyme immobilization and the various ways in which the immobilization process can affect performance of the enzyme.1 The discovery and testing of predictive models for the behavior of these systems is a particularly important goal. Porous silicon is produced by the electrochemical dissolution of a single-crystal silicon wafer in a HF-containing electrolyte.2,3 It is a versatile material in which the 3D microstructure (pore diameter and porosity) can be varied over a wide range by changing the silicon wafer resistivity, electrolyte composition, and applied current density.4,5 The surface reactivity and biodegradable properties of porous silicon are also easily modified using a wide range of established surface derivatization chemistry.6,7 Because * To whom correspondence should be addressed. Benjamin_miller@ futurehealth.rochester.edu. (1) (a) Cao, R.; Gu, Z.; Patterson, G. D.; Armitage, B. A. J. Am. Chem. Soc. 2004, 126, 726-727. (b) Kumar, C. V.; Chaudhari, A. J. Am. Chem. Soc. 2000, 122, 830-837. (c) Lei, C.; Shin, Y.; Liu, J.; Ackerman, E. J. J. Am. Chem. Soc. 2002, 124, 11242-11243. (d) Luckarift, H. R.; Spain, J. C.; Naik, R. R.; Stone, M. O. Nat. Biotechnol. 2004, 22, 211-213. (2) Vinegoni, C.; Cazzanelli, M.; Pavesi, L. In Silicon Based Materials and Devices; Nalwa, H. S., Ed.; Academic Press: New York, 2001; Vol. 2, pp 124-188. (3) Chan, S.; Tsybeskov, L.; Fauchet, P. M. Mater. Res. Soc. Symp. Proc. 1999, 536, 117-122. (4) Zhang, X. G. J. Electrochem. Soc. 2004, 151, c69-c80. (5) Lehmann, V.; Stengl, R.; Luigart, A. Mater. Sci. Eng. B 2000, 69-70, 1122. (6) Buriak, J. M.; Allen, M. J. J. Am. Chem. Soc. 1998, 120, 1339-1340. 10.1021/ac0488208 CCC: $27.50 Published on Web 11/02/2004
© 2004 American Chemical Society
of these versatile attributes, porous silicon has emerged as a promising material for various biomedical device applications. The high surface area (>200 m2/cm3) characteristic of mesoporous silicon (pore diameter 2-50 nm) makes this a particularly advantageous substrate for biosensor8 or enzyme reactor9 applications where a high concentration of immobilized bioreagent can be leveraged to enhance device sensitivity and activity. The utility of porous silicon in a biodevice application hinges, however, on a requirement that the microstructure be of a sufficient pore diameter to accommodate the facile diffusion and binding of biomolecular reagents through the matrix. Dilute solutions of ethanolic KOH have been used to systematically enhance the pore diameter of mesoporous devices.10,11 Pore size dependence on the infiltration of whole antibodies and large proteins has been reported.11-13 However, few attempts have been made to quantify the relationship between PSi film morphology and the immobilization capacity for a biomacromolecule. In this paper, we present a spectrophotometric enzyme conjugation assay utilizing glutathione-S-transferase (GST) to quantify the amount of enzyme bound in a porous silicon matrix. We find that the immobilization capacity quantatitively correlates with systematic changes in the device thickness. These results are consistent with the immobilization capacity predicted by a simple geometric model of surface area. These data suggest that the immobilized GST enzyme assay can be used to quantify changes in relative surface area between different preparations of meso- and macroporous silicon, thus providing a useful guide for further work in this area particularly for optimizing PSi morphology for biosensing applications. EXPERIMENTAL SECTION General Information. Unless otherwise stated, all materials were obtained from Sigma-Aldrich and used without additional (7) Stewart, M. P.; Robins, E. G.; Geders, T. W.; Allen, M. J.; Cheul Choi, H.; Buriak, J. M. Phys. Status Solidi A 2000, 182, 109-115. (8) (a) Sohn, H.; Letant, S.; Sailor, M. J.; Trogler, W. C. J. Am. Chem. Soc. 2000, 122, 5399-5340. (b) Chan, S.; Horner, S. R.; Miller, B. L.; Fauchet, P. M. J. Am. Chem. Soc. 2001, 123, 11797-11798. (9) Karlsson, L. M.; Tengvall, P.; Lundstro ¨m, I.; Arwin, H. J. Colloid Interface Sci. 2003, 266, 40-47. (10) Hamm, D.; Sakka, T.; Ogata, Y. H. Phys. Status Solidi A 2003, 197, 175179. (11) Tinsley-Bown, A. M.; Canham, L. T.; Hollings, M.; Anderson, M. H.; Reeves, C. L.; Cox, T. I.; Nicklin, S.; Squirrell, D. J.; Perkins, E.; Hutchinson, A.; Sailor, M. J.; Wun, A. Phys. Status Solidi A 2000, 182, 547-553. (12) Arwin, H.; Gavutis, M.; Gustafsson, J.; Schultzberg, M.; Zangooie, S.; Tengvall, P. Physica Status Solidi A 2000, 182, 515-520. (13) Karlsson, L. M.; Tengvall, P.; Lundstro ¨m, I.; Arwin, H. Phys. Status Solidi A 2003, 197, 326-330.
Analytical Chemistry, Vol. 76, No. 23, December 1, 2004 6915
purification. Stock solutions of 2 mg/mL glutathione-S-transferase (equine liver; G6511) and 40 mM reduced glutathione (GSH; G4251) were prepared in 100 mM phosphate buffer containing 1 mM EDTA. A 200 mM stock solution of 1-chloro-2,4-dinitrobenzene (CDNB; C6396) was prepared in 95% ethanol. All reagents were mixed fresh each day and stored on ice between use. Porous Silicon Thin-Flim Preparation. Mesoporous silicon thin films with a chip diameter of 1.3 cm were prepared from highly doped (boron) p+ 〈100〉 silicon wafers obtained from the PCA Corp. with a resistivity of 0.01 Ω‚cm. Films were electrochemically etched at room temperature in a standard Teflon etch cell2,14 using an electrolyte solution containing ethanol (70%) and 48% hydrofluoric acid (30%) yielding a total HF concentration of 14.4%. During etching, the electrolyte was gently mixed using a manual pipet pump. After etching, the porous silicon sample was removed from the etch cell, rinsed with ethanol and then water, and dried under a stream of N2 gas. Samples of various thickness were prepared for this study using a constant current density of 50 mA/cm2 and varying the etch time from 1 to 300 s. Gravimetric measurements made on samples etched for 300 s indicated a porosity value of 84.8% and an etch depth of 8.9 µm, yielding an etch rate of 29.5 nm/s. This value is in good agreement with the etch rate determined by us from SEM micrographs (29.0 nm/s). This preparation yields a pore diameter ranging between 10 and 20 nm. Pore channels grow anisotropically along the 〈100〉 direction. KOH Postetch Modification. To remove the nanostructured features that fill the pore channels in p+ mesoporous films, we used a KOH postetch process previously described.15,16 In that work, we established that a quantitative relation exists between the amount of material removed with exposure time, and we showed that KOH treatment was essential to enable GST pore infiltration. Briefly, the procedure involves treating PSi samples with a dilute 1.5 mM KOH solution15,16 prepared from aqueous 7.7 mM KOH diluted 1:5 with 95% ethanol. Samples were immersed in this solution for 2 min. This exposure is sufficient to remove the nanostructures that give rise to photoluminescence, but only a slight increase (
