Anal. Chem. 2005, 77, 1950-1956
Enzyme Immobilization in Porous Silicon: Quantitative Analysis of the Kinetic Parameters for Glutathione-S-transferases 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
Porous silicon matrixes are attractive materials for the construction of biosensors and may also have utility for the production of immobilized enzyme bioreactors. In an effort to gain a quantitative understanding of the effects of immobilization on enzyme activity, we compared the activity of glutathione-S-transferase immobilized in electrochemically etched porous silicon films (∼6.5 µm thick) with the enzyme in solution. Kinetic measurements were made by varying the glutathione concentration while maintaining a fixed saturating concentration of 1-chloro2,4-dinitrobenzene. The reaction kinetics follow steadystate equilibrium behavior. The specific activity of the free enzyme in solution is ∼4× higher than the immobilized enzyme, for which we measured an apparent K′mGSH value of 1.0 ( 0.3. The maximum velocity, V′max, is linearly proportional to immobilized enzyme concentration, but the magnitude is ∼20 times lower than that in solution. Results suggest ∼25% of the enzyme is bound with the catalytic site in an inactive conformation or in a hindered orientation. Finally, the effects of hydration and exposure to denaturants on the immobilized enzyme activity are presented. Immobilization of proteins on solid supports constitutes a research area of considerable importance in emerging technologies employing biocatalytic and biorecognition events. Such technologies include biosensors for pathogen detection,1,2 microarrays for proteomic analysis,3-7 and enzyme bioreactors8,9 used, * To whom correspondence should be addressed. E-mail: Benjamin_Miller@ futurehealth.rochester.edu. (1) 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, 547-53. (2) Chan, S.; Horner, S. R.; Fauchet, P. M.; Miller, B. L. J. Am. Chem. Soc. 2001, 123, 11797-11798. (3) Turner, A. M. P.; Dowell, N.; Turner, S. W. P.; Kam, L.; Isaacson, M.; Turner, J. N.; Craighead, H. G.; Shain W. J. Biomed. Mater. Res. 2000, 51, 430441. (4) Rowe, C. A.; Tender, L. M.; Feldstein, M. J.; Golden, J. P.; Scruggs, S. B.; MacCraith, B. D.; Cras, J. J.; Ligler, F. S. Anal. Chem. 1999, 71, 38463852. (5) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763. (6) MacBeath, G. Nat. Biotechnol. 2001, 19, 828-829. (7) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R. Biebuyck, H. Langmuir 1998, 14, 2225. (8) Burton, S. G. Pure Appl. Chem., 2001, 73, 77-83. (9) Davison, B. H.; Barton, J. W.; Petersen, G. Biotechnol. Prog. 1997, 13, 3, 512-518.
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for example, in alcohol fermentation10 and penicillin production.11 The ability to quantitatively immobilize functionally stable proteins is of paramount importance in achieving high sensitivity and sustained operating efficiency in these applications. Insight into the effects of immobilization on enzyme activity has largely been gleaned from studies of enzymes encapsulated within the 3D matrixes of polymers,12-14 inorganic sol gels,15-18 and hydrogels.19-21 The microstructure of polymeric materials is typically amorphous; however, a high enzyme load can be accommodated, which can help alleviate process inefficiency due to deformation of the catalytic site. Enzymes have also been successfully immobilized on solid surfaces including derivatized glass22-24 and silicon wafers.24-26 Silicon is an attractive support because it offers the potential for integration into arrayed devices fabricated using standard IC and MEMs process technology.27 The sensitivity of planar devices may, however, be limited by the maximum concentration of functional enzyme that can be im(10) Held, M.; Schmid, A.; van Beilen, J. B.; Witholt, B. Pure Appl. Chem.. 2000, 72, 1337-1343. (11) Bruggink, A.; Roos, E. R.; de Vroom, E. Org. Proc. Res. Dev. 1998, 2, 128133. (12) Trevan, M. D. Immobilized Enzymes: An Introduction and Applications in Biotechnology; John Wiley & Sons: Chichester, U.K., 1980; Section 4 (ISBN 0-471-27826-2). (13) Berlin, P.; Klemm, D.; Jung, A.; Liebegott, H.; Rieseler, R.; Tiller, J. Cellulose 2003, 10, 343-367. (14) Butterfield, D. A.; Bhattacharyya, D.; Daunert, S.; Bachas L. G. J. Membr. Sci, 2001, 181, 29-37. (15) Park, C. B.; Clark, D. S. Biotechnol. Bioeng. 2002, 78, 229-235. (16) Kim, Y. D.; Park, C. B.; Clark, D. S. Biotechnol. Bioeng. 2001, 73, 331337. (17) Luckarift, H. R.; Spain, J. C.; Naik, R. R.; Stone, M. O. Nat. Biotechnol. 2004, 22, 211-213. (18) Lei, C.; Shin, Y.; Liu, J.; Ackerman, E. J. J. Am. Chem. Soc. 2002, 124, 11242-11243. (19) Demers, N.; Agostinelli E.; Averill-Bates, D. A.; Fortier, G. Appl. Biochem. 2001, 33, 201-207. (20) Peppas, N. A.; Huang, Y.; Torres-Lugo, M.; Ward, J. H.; Zhang, J. Annu. Rev. Biomed. Eng., 2000, 2, 9-29. (21) Sakiyama-Elbert, S. E.; Hubbell, J. A. Annu. Rev. Mater. Res., 2001, 31, 183-201. (22) Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloshchuk, S.; Chupeeva, V.; Mirzabekov, A. Anal. Biochem. 2000, 278, 123-131. (23) Lee, M. Y.; Srinivasan, A.; Ku, B.; Dordick, J. S.; Biotechnol. Bioeng. 2003, 83, 20-28. (24) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Langmuir 1998, 14, 2225-2229. (25) Laurell, T.; Rosengren, L. Sens. Actuators, B 1994, 19, 614-617. (26) Laurell, T.; Rosengren L.; Drott, J. Biosens. Bioelectron. 1995, 10, 289299. (27) Beebe, D. J.; Moore, J. S.; Yu, Q.; Liu, R. H.; Kraft, M. L.; Jo, B. H.; Devadoss, C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13488-13493. 10.1021/ac0486185 CCC: $30.25
© 2005 American Chemical Society Published on Web 03/02/2005
mobilized within the designated 2D surface area of the device in addition to the deformation of the catalytic site. A promising method to enhance the sensitivity of silicon-based supports is the electrochemical fabrication of porous silicon.28-30 Porous silicon is an advantageous material because of its characteristically high surface area and the ability to fabricate a wide range of pore morphologies, including diameters from 10 nm to >1 µm, and channel structures (highly anisotropic to dendritic) by varying etch conditions.31,32 Recently, we demonstrated that the enzyme immobilization capacity of p+ mesoporous silicon films (pore diameter ∼10-20 nm) is quantifiable and readily controlled with film depth.33 Because of these attributes, derivatized porous silicon is being increasingly considered in biomedical applications including optical biosensing,2,34-36 drug delivery,35,37,38 and tissue engineering.39-41 However, little quantitative data exist as yet on the activity of enzymes immobilized in electrochemically produced porous silicon thin films. We therefore set out to characterize the activity of a prototypical immobilized enzyme, glutathione-S-transferase (GST),42,43 in electrochemically etched porous silicon thin films, and to quantitatively contrast the kinetics of the immobilized enzyme with the free enzyme in solution. GSTs comprise an important family of dimeric cytosolic enzymes where the catalytic substrate-binding site is formed at the interface between the two monomeric units.43 Because of this unique structural aspect of GST (i.e., enzymatic activity depends on quaternary structure, as well as on orientation and maintenance of tertiary structure), it comprises an ideal system to probe the effects of immobilization on activity.42-45 Successful immobilization of GST in a chip-based format opens up a range of possible applications. For example, a portable microfluidic device containing a detoxifying enzyme chip could be exploited in the continuous monitoring of environmental water for trace levels of toxins. More importantly, with regard to longer (28) Drott, J.; Lindstro ¨m, K.; Rosengren, L.; Laurell,T. J. Micromech. Microeng. 1997, 7, 14-23. (29) Drott, J.; Rosengren, L.; Lindstro ¨m, K.; Laurell, T. Thin Solid Films 1998, 330, 161-166. (30) Le´tant, S. E.; Hart, B. R.; Kane, S. R.; Hadi, M. Z.; Shields, S. J.; Reynolds, J. G. Adv. Mater. 2004, 16 (8), 689-693. (31) Zhang, X. G. J. Electrochem. Soc. 2004, 151 c69-c80. (32) Lehmann, V.; Stengl, R.; Luigart, A. Mater. Sci. Eng. 2000, B69, 11-22. (33) DeLouise, L. A.; Miller, B. L. Anal. Chem. 2004, 76, 6915-6920. (34) Chan, S.; Fauchet, P. M.; Li, Y.; Rothberg, L. J. Proc. SPIE 2000, 39123923. (35) Li, Y. Y.; Cunin, F.; Link, J. R.; Gao, T.; Betts, R. E.; Reiver, S. H.; Chin, V.; Bhatia, S. N.; Sailor, M. J. Science 2003, 299, 2045-2047. (36) Cunin, F.; Schmedake, T. A.; Link, J. R.; Li, Y. Y.; Koh, J.; Bhatia, S. N.; Sailor, M. J. Nat. Mater. 2002, 1, 39-41. (37) Rosengren, A.;. Wallman, L.; Bengtsson, M.; Laurell, T.;. Danielsen, N.; Bjursten, L. M. Phys. Status Solidi A 2000, 182, 527-531. (38) Xin, L.; John, St. J.; Coffer, J. L.; Chen, Y.; Pinizzotto, R. F.; Newey, J.; Reeves, C.; Canham, L. T Biomed. Microdevices 2000, 2, 265-272. (39) Tirrell, M.; Kokkoli, E.; Biesalski, M. Surf. Sci. 2002, 500, 61-83. (40) Kleps, I.; Mihaela, M.; Simion, M.; Neghina; T.; Petrescu, S.; Moldovan, N.; Paduraru, C.; Raducanu, A. Rev. Adv. Mater. Sci. 2003, 5, 440-449. (41) Canham, L. T.; Reeves, C. L.; Newey, J. P.; Houlton, M. R.; Cox, T. I.; Buriak, J. M.; Stewart, M. P. Adv. Mater. 1999, 11, 1505-1507. (42) Ortiz-Salmero´n, E.; Yassin, Z.; Clemente-Jimenez, M. J.; Javier, F.; Las HerasVazquez, L.; Rodriguez-Vico, F.; Baro´n, C.; Garcı´a-Fuentes, L. Eur. J. Biochem., 2001, 268, 4307-4314. (43) Allardyce C. S.; Mcdonagh, P. D.; Lu-Yun, L.; Wolf, C. R.; Roberts G. C. K. Biochem. J. 1999, 343, 525-531. (44) Vargo, M. A.; Nguyen, L.; Colman, R. F. Biochemistry 2004, 43 (12), 33273335. (45) Hornby, J. A.; Luo, J. K.; Stevens, J. M.; Wallace, L. A.; Kaplan, W.; Armstrong, R. N.; Dirr, H. W. Biochemistry 2000, 39, 12336-12344.
term goals of exploiting porous silicon for biosensing and proteomics applications, the enzymatic activity of GST functions as a secondary analytical check on the effect of changes in device microstructure on protein infiltration, immobilization capacity, and retention of activity. This is particularly advantageous in developing PSi optical biosensors, which are typically characterized by a change in the optical response following immobilization of a probe molecule and then in the presence of a target analyte. However, the optical signal generated provides only an incomplete picture of the device, since some of the immobilized protein may be inactive (i.e., producing an optical response, but not able to bind an analyte or participate in an enzymatic reaction). Previously, we successfully immobilized functional GST in the 3D matrix of porous silicon using conventional attachment chemistries.33 Sophisticated site-directed immobilization strategies are not required,14 although it is conceivable that such strategies might produce a device with higher specific enzyme activity. To interpret the immobilized enzyme results generated in this study, we first characterized the free enzyme in solution. The apparent kinetic parameters K′mGSH, kcat, V′max, and activity for the free enzyme are reported in GST Solution Kinetics Results. In Characterization of Immobilized Enzyme Stability, we demonstrate that covalently bonded GST is functional and immobilization is quantitative. The impact of hydration level and exposure to denaturants on enzyme activity are briefly explored. In Immobilized GST Kinetics: K′m and V′max, the immobilized kinetic parameters are reported and contrasted to the free enzyme values. Results indicate a decrease in the specific activity and in the steady-state kinetic parameters for the immobilized enzyme relative to the enzyme in solution. EXPERIMENTAL SECTION Porous Silicon Thin-Film Preparation. The methods employed to produce the mesoporous silicon films (pore diameters ∼10-20 nm) used in this study have been described in detail elsewhere.2,34,46 In brief, porous silicon films were fabricated from p+ 〈100〉 silicon wafers (0.01 Ω‚cm) using an electrochemical etch process. Etching was conducted at room temperature using electrolyte containing ethanol (70%) and 48% hydrofluoric acid (30%). After the silicon chip was removed from the Teflon etch cell, the sample was rinsed in ethanol and then water and dried under a stream of nitrogen. The films were thermally oxidized in dry O2 at 900 °C using a three-zone Lindberg tube furnace to enhance the stability of porous silicon in buffer solutions containing salt and to create hydrophilic pore channels. Samples were slowly shuttled into the center zone where they were annealed for 3 min. The entire oxidization cycle took ∼15-16 min to complete. Unlike our previous work, which focused on single-layer mesoporous silicon devices,33 the films prepared for this study comprised a λ/2 microcavity microstructure. Although the multilayer microcavity structure is not needed for a simple bioreactor application, it is essential for the optical sensing applications (genomics, proteomics, point-of-care diagnostics, etc.) that are our primary long-term focus. Here the microcavity device is sensitive (46) DeLouise, L. A.; Miller, B. L. Mater. Res. Soc. Symp. Proc. 2004, 782, A5.3.1A5.3.1.
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to changes in refractive index, which provides the foundation for label-free detection of essentially any desired biomolecule. The microstructure of the λ/2 microcavity used in the study consisted of 28 layers of alternating 71 and 84% porosity. The estimated film thickness of the multilayer device was ∼6.5 µm. Given the high porosity of the multilayers and the fact that the pore diameter (∼10-20 nm) is primarily a function of wafer doping and etchant composition, the enzyme immobilization capacity of single layer and multilayer devices is mainly determined by film thickness.33 Assuming an average interpore distance of 30 nm, the surface area, predicted from a simple geometric model of porosity,33 is more than an order of magnitude larger than that needed to accommodate the amount of GST immobilized in this study. General Information. Unless otherwise stated, all materials were obtained from Sigma-Aldrich and used without further purification. Stock solutions of GST (EC 2.5.1.18, equine liver; G6511) and reduced glutathione (GSH; G4251) were prepared in 100 mM phosphate buffer containing 1 mM EDTA (PBE, pH 6.5). 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. Surface Derivatization and Enzyme Immobilization. Porous silicon films were equivalently derivatized for enzyme immobilization using standard aminosilane and glutaraldehyde coupling chemistry described in detail elsewhere.47 Immobilization of GST (∼50 000 MW) was realized by applying to 50 µL of a GST solution. After 1.5 h, 25 µL of the residual supernatant was recovered from the chip and assayed for enzyme activity using the 100-µL solution-phase procedure described in detail below. The amount of GST present in the 25-µL residual supernatant could range from negligible (all bound to the silicon) to half of the initial amount applied (no binding). In all cases, no product was formed using the residual supernatant, suggesting that all of the GST was immobilized in the porous silicon matrix. After removing the enzyme supernatant, the porous silicon samples were washed with 2-3 mL of PBE buffer and soaked in ∼200 µL of buffer for a minimum of 30 min prior to conducting kinetic studies. GST Enzyme Activity Assay. To probe GST activity, a standard assay involving the conjugation of GSH to CDNB was used. CDNB is a substrate of choice, since most GST isozymes display high activity toward it. The conjugation product was monitored spectrophotometrically at 340 nm. Stock solutions of GSH and GST were prepared in PBE at a pH of 6.5 where studies indicate the activity of the free enzyme in solution is at a maximum.48-50 The final concentration of ethanol in the reaction solution remained 250 µg/mL), the dilution effect slowed the enzyme reaction sufficiently to enable an accurate determination of absorbance. A standard deviation error of ∼5% was typically observed when absorbance values were acquired using method 2. Error was evaluated by conducting three to four independent measurements (typically 1-min reactions) employing fresh stock solutions. Although the mechanics of obtaining the product yield-time course plots by method 2 were more cumbersome than the continuous monitoring of the product yield by method 1, results from both methods compared favorably for equivalent reaction conditions. Immobilized Enzyme Activity Procedure. Immobilized enzyme reactions were conducted by applying 75 µL of the substrate stock solution directly onto the derivitized porous silicon thin films. This volume was completely confined to the porous silicon area. Excess buffer was removed from the porous silicon film by shaking the sample immediately before applying the substrate stock solution. Approximately 25 µL of buffer was retained within the porous matrix, yielding a final reaction volume of ∼100 µL (analogous to solution method 2). Plots of absorbance versus time were acquired by a procedure similar to that described above. The immobilized conjugation reaction was allowed to proceed for a specific time, after which 40 µL of reaction solution supernatant was recovered from the porous silicon sample. This was diluted with buffer to a final volume of 600 µL and the absorbance at 340 nm recorded. Residual solution was washed from the porous silicon with copious amounts of buffer, and the chip was allowed to soak in buffer for g10 min before running another immobilized enzyme reaction. Absorbance values from chip reactions are reported with a standard deviation error ranging between 8 and 13%.
Figure 1. GST conjugation product-time course plots using method 1 as a function of [GSH] for two different enzyme reaction concentrations: (a) 4 and (b) 333 µg/mL.
rapid equilibrium random ordered bi-bi mechanism.53,55,56 Both substrates must bind the enzyme, but the product formation rate is independent of the substrate binding order.56 Kinetic parameters (Km and Vmax) for the sequential bi-bi mechanism are amenable to experimental determination using a double-reciprocal plotting methodology similar to Lineweaver-Burk.56 Typically, however, initial velocity measurements are made as a function of one substrate43,51-55 under fixed and saturating conditions of the other. In this way, the kinetic equations simplify considerably, enabling the determination of apparent steady-state parameters. In this study, we determined the apparent kinetic parameters for GSH under saturating conditions of CDNB using the relationship defined in eq 1, where v is the reaction velocity, K′m is an apparent Figure 2. Specific activity plotted as a function of GST concentration for various GSH concentrations and a fixed [CDNB] of 1.0 mM.
Enzyme Kinetic Parameter Analysis. Initial reaction velocities were determined from the product yield time course plots in the presence of a saturating 1-3 mM CDNB concentration (∼310K′mCDNB ) by varying the concentration of GSH in the range of 0.05-5.0 mM (0.2-20K′mGSH).43,51-55 Since 1 mM CDNB is saturating, the use of concentrations greater than this will not affect the experimental results. Details of the kinetic equations describing the enzyme mechanism are presented below. The apparent kinetic parameters were determined using a steady-state LineweaverBurk approach and nonlinear regression analysis. GST SOLUTION KINETICS RESULTS Each GST subunit is composed of two structurally distinct domains. The highly conserved N-terminal domain, a four-stranded β-sheet and two R-helices, forms the glutathione-binding site (G-site). The C-terminal domain is almost entirely helical. It forms the binding site for the hydrophobic substrate (H-site) and shows greater variability among family members. Hence, GSTs are multisubstrate enzymes exhibiting kinetics consistent with the (51) Tang, S. S.; Chang, G. G. J. Biochem. 1996, 119, 1182-1188. (52) Tang, S. S.; Chang, G. G. Biochem. J. 1996, 315, 599-606. (53) Lo Bello, M.; Oakley, A. J.; Battistoni, A.; Mazzetti, A. P.; Nuccetelli, M.; Mazzarese, G.; Rossjohn, J.; Parker, M. W.; Ricci, G. Biochemistry 1997, 36, 6207-6217. (54) Liu, L. F.; Liaw, Y. C.; Tam, M. F. Biochem. J. 1997, 327, 593-600. (55) Labrou N. E.; Mello, L. V.; Clonis, Y. D. Biochem J. 2001, 358, 101-110.
v ) V′max[GSH]/(K′m + [GSH])
(1)
Michaelis-Menton constant for GSH, and V′max (µmol/min) is the apparent maximum velocity obtainable under saturating conditions of both [GSH] and [CDNB]. V′max is proportional to the total enzyme concentration, [Et], as given in eq 2, where kcat is a first-
V′max ) kcat [Et]
(2)
order rate constant having units of reciprocal time (s-1). This rate constant encompasses the chemical transformation events leading to product formation from the ternary enzyme complex.56 When the [GSH] equals K′m, eq 1 reduces to
v ) V′max /2
(3)
and the reaction velocity is half the apparent maximum value. Solution Activity of GST. Enzyme specific activity (µmol/ min‚mg) is determined from the slope of the absorbance versus time plot. Typical product time course plots as a function of GSH concentration are shown in Figure 1 for two different enzyme concentrations. Figure 1a, [GST] ) 4 µg/mL, shows product (56) Copeland, R. A. Enzymes: a practical introduction to structure, mechanism, and data analysis, 2nd ed.;Wiley-VCH: New York, 2000 (ISBN 1-56081-9030).
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Figure 3. (a) Reaction velocity versus GSH concentration for different GST enzyme concentrations. The total reaction volume was 600 µL, and the CDNB concentration was fixed at 1.0 mM. (b) A Lineweaver-Burk plot as a function of GSH concentration derived from the data in (a). Legend indicated applies to both (a) and (b). Table 1. Kinetic Parameters for Free GST in Solution (600 µL Total Volume) as a Function of Enzyme Concentration kcat/K′m V′max GST ∼[GST] K′mGSH (µM) (nmol/min) kcat(s-1) (M-1 s-1) × 10-4 (µg/mL) (nM) 16.7 8.3 6.7 4.0 0.7 0.3 av SD
334.0 166.0 134.0 80.0 13.4 6.6
296.6 239.4 250.5 239.2 239.9 242.0 251.3 22.6
97.6 56.1 49.4 34.0 5.3 2.6
8.1 9.4 10.2 11.8 10.9 10.9 10.2 1.3
2.74 3.92 4.09 4.93 4.56 4.51 4.12 0.77 Figure 4. Maximum velocity versus the reaction GST concentration.
formation increases linearly over several minutes, whereas in Figure 1b, [GST] ) 333 µg/mL, the product formation rate becomes nonlinear after ∼20 s. By ∼75 s, product formation ceases as the reaction becomes substrate limited. Specific activity was determined for various enzyme (0.2-666 µg/mL) and GSH (0.05-5 mM) concentrations with a fixed saturating concentration of CDNB (1.0 mM). These conditions were chosen so that the enzyme concentration is 3 orders of magnitude lower than the substrate concentrations. The results from these measurements (Figure 2) show that specific activity depends on both the concentration of GSH and of GST. Specific activity increases with [GSH] and decreases with [GST]. Potential reasons for the dependence on [GST] may include a dependence on solution viscosity57 or the effect of enzyme aggregation.58 For GSH concentrations between 0.05 and 5.0 mM, enzyme activity is highest when the GST concentration is
