Protein-Friendly Intercalation of Cytochrome c and Hemoglobin into

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Protein-Friendly Intercalation of Cytochrome c and Hemoglobin into Thermally Evaporated Anionic and Cationic Lipid Films: A New Approach Based on Diffusion from Solution Anand Gole, Prajakta Chaudhari, Jaspreet Kaur, and Murali Sastry* Materials Chemistry Division, National Chemical Laboratory, Pune 411 008, India Received April 17, 2001. In Final Form: June 7, 2001 Intercalation of the heme proteins, cytochrome c (Cyt c) and bovine hemoglobin in thermally evaporated fatty lipid films under protein-friendly conditions by a novel beaker-based diffusion mechanism is described. An attractive electrostatic interaction between charged groups on the protein surface and ionized lipid molecules in the film is primarily responsible for the diffusion of the proteins from the solution into the lipid matrix. To highlight the generality of the approach, Cyt c (cationic at a pH of ca. 7) and hemoglobin (anionic at pH ) 9) have been incorporated into thermally evaporated arachidic acid (anionic at pH ) 7) and octadecylamine (cationic at pH ) 9) films, respectively, by simple immersion of the films in the respective protein solutions. Quartz crystal microgravimetry measurements were used to follow the kinetics of protein diffusion into the films and was found to be strongly dependent on the protein solution pH. The electrostatic nature of coordination between the proteins and matrix lipid molecules enables reversal of the protein adsorption processsthe proteins could be “pumped out” of the composite films into solution under appropriate conditions. Fluorescence spectroscopy and Fourier transform infrared spectroscopy studies indicated little perturbation to the native protein structure, while UV-Vis spectroscopy was used to follow the redox behavior of Cyt c in arachidic acid films. Incorporation of Cyt c in the lipid matrix leads to a lamellar film structure, with a repeat distance of 58.8 Å. The advantages of this approach over other methods currently used for entrapment of proteins is briefly discussed.

Introduction The immobilization and microencapsulation of proteins and enzymes in various inert matrixes is a problem attracting considerable attention, and different techniques are continuously being developed. Such biocomposites have important applications in the food, chemical, pharmaceutical, and agricultural industries. Industrial applications require that the encapsulation process be relatively quick, inexpensive, protein-friendly, result in high loading factors, and be applicable to a large range of biomolecules. An important requirement is that the matrix for immobilizing the proteins be biocompatible and inert; i.e., it should not interfere with the native structure of the protein and thereby compromise its biological activity. Protection of the protein against microbial degradation, hydrolysis, and deamidation and accessibility of the encapsulated proteins to cofactors, substrates, and redox agents are important goals, especially in biosensing1 and biocatalytic2 applications. Furthermore, the use of immobilized biocatalysts results in additional advantages such as convenient handling, ease of separation from the product, and possibility of reuse thereby dramatically lowering the effective cost of the enzymes.3 Until now, proteins have been immobilized onto/within 2-D supports such as phospholipid/synthetic bilayers,4 silicate sol-gels,5 cross-linked crystals,6 monolayers attached to solid supports,7 Langmuir-Blodgett (LB) films,8 polymer ma* To whom correspondence should be addressed. Tel: +9120-5893044. Fax: +91-20-5893044/5893952. E-mail: sastry@ ems.ncl.res.in. (1) Avnir, D.; Braun, S. Biochemical Aspects of Sol-Gel Science and Technology; Kluwer: Hingham, MA, 1996. (2) Shabat, D.; Grynszpan, F.; Saphier, S.; Turniansky, A.; Avnir, D.; Keinan, E. Chem. Mater. 1997, 9, 2258. (3) Tischer, W.; Wedekind, F. Top. Curr. Chem. 1999, 200, 95 and references therein.

trixes,9 galleries of R-zirconium phosphates,10 hydrophobic controlled pore-glasses,11 and antibody/antigen-labeled surfaces12 as well as onto 3-D supports such as organic13 and inorganic14 colloidal particles. (4) (a) Himachi, I.; Fujita, A.; Kunitake, T. J. Am. Chem. Soc. 1994, 116, 8811. (b) Hamachi, I.; Honda, T.; Noda, S.; Kunitake, T. Chem. Lett. 1991, 1121. (c) Himachi, I.; Noda, S.; Kunitake, T. J. Am. Chem. Soc. 1990, 112, 6744. (d) Ramsden, J. J. Biosens. Bioelectron. 1998, 13, 593. (e) Hianik, T.; Snejdarkova, M.; Passechnik, V. I.; Rehak, M.; Babincova, M. Bioelectrochem. Bioenerg. 1996, 41, 221. (f) Chen, X.; Hu, N.; Zeng, Y.; Rusling, J. F.; Yang, J. Langmuir 1999, 15, 7022. (g) Nassar, A.-E. F.; Zhang, Z.; Hu, N.; Rusling, J. F.; Kumosinski, T. F. J. Phys. Chem. B 1997, 101, 2224. (h) Lu, Z.; Huang, Q.; Rusling, J. F. J. Electroanal. Chem. 1997, 423, 59. (i) Burgess, J. D.; Rhoten, M. C.; Hawkridge, F. M. Langmuir 1998, 14, 2467. (j) Salamon, Z.; Tollin, G. Biophys. J. 1996, 71, 848. (5) (a) Ellerby, L. M.; Nishida, C. R.; Nishida, F.; Yamanaka, F. A.; Dunn, B.; Valentine, J. B.; Zink, J. I. Science 1992, 255, 1113. (b) Gill, I.; Ballesteros, A. J. Am. Chem. Soc. 1998, 120, 8587. (c) Ji, Q.; Lloyd, C. R.; Ellis, W. R.; Eyring, E. M. J. Am. Chem. Soc. 1998, 120, 221. (d) Zheng, L.; Flora, K.; Brennan, J. D. Chem. Mater. 1998, 10, 3974. (e) Das, T. K.; Khan, I.; Rousseau, D. L.; Friedman, J. M. J. Am. Chem. Soc. 1998, 120, 10268. (f) Rao, M. S.; Dave, B. C. J. Am. Chem. Soc. 1998, 120, 13270. (g) Avnir, D.; Braun, S.; Lev, O.; Ottolenghi, M. Chem. Mater. 1994, 6, 1605. (h) Chen, Q.; Kenausis, G. L.; Heller, A. J. Am. Chem. Soc. 1998, 120, 4582. (i) Wang, J.; Pamidi, P.; Park, D. Anal. Chem. 1996, 68, 2705. (j) Lev, O.; Tsionsky, M.; Rabinovich, L.; Glezer, V.; Sampath, S.; Pankratov, I.; Gun, J. Anal. Chem. 1995, 67, 23A. (6) (a) Lalonde, J. J.; Govardhan, C.; Khalaf, N.; Martinez, A. G.; Visuri, K.; Margolin, A. L. J. Am. Chem. Soc. 1995, 117, 6845. (b) Zelinski, T.; Waldmann, H. Angew. Chem., Int. Ed. Engl. 1997, 36, 722. (c) Cao, L.; van Rantwijk, F.; Sheldon, R. A. Org. Lett. 2000, 2, 1361. (d) Partridge, J.; Halling, P. J.; Moore, B. D. Prog. Biotechnol. 1998, 15, 373. (e) Vilenchik, L. Z.; Griffith, J. P.; Clair, N. St.; Navia, M. A.; Margolin, A. L. J. Am. Chem. Soc. 1998, 120, 4290. (f) Browne, J. K.; Mckervey, M. A.; Pitarch, M.; Russell, J. A.; Millership, J. S. Tetrahedron Lett. 1998, 39, 1787. (7) (a) Fang, J.; Knobler, C. M. Langmuir 1996, 12, 1368. (b) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11, 4383. (c) Ferretti, S.; Paynter, S.; Russell, D. A.; Sapsford, K. E.; Richardson, D. J. TrAC, Trends Anal. Chem. 2000, 19, 530. (d) Viitala, T.; Vikholm, I.; Peltonen, J. Langmuir 2000, 16, 4953. (e) Guiomar, A. J.; Guthrie, J. T.; Evans, S. D. Langmuir 1999, 15, 1198. (f) Gooding, J. J.; Hibbert, D. B. TrAC, Trends Anal. Chem. 1999, 18, 525. (g) Yang, Z.; Yu, H. Langmuir 1999, 15, 1731.

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Scheme 1. Diagram Showing the Various Steps Involved in the Growth of the Cyt c-AA Composite Films (the Expected Microscopic Structure of the Protein-Lipid Composite Film Is Also Shown)

In this laboratory, we have developed a protocol for the electrostatically controlled entrapment of ions15 and charged inorganic nanoparticles16 in thermally evaporated ionizable lipid films by simple immersion of the films in the electrolyte/nanoparticle solution. We have recently extended this technique to charged biomacromolecules such as proteins/enzymes and have demonstrated the immobilization and enzymatic activity of the digestive (8) (a) Boussaad, A.; Dziri, L.; Arechabaleta, N. J.; Tao, N. J.; Leblanc, R. M. Langmuir 1998, 14, 6215. (b) Nicolini, C.; Erokhin, V.; Antolini, F.; Catasti, P.; Facci, P. Biochim. Biophys. Acta 1993, 1158, 273. (c) Berzina, T. S.; Piras, L.; Troitsky, V. I. Thin Solid Films 1998, 327329, 621. (d) Preininger, C.; Clausen-Schaumann, H.; Ahluwalia, A.; de Rossi, D. Talanta 2000, 52, 921. (e) Kiselyova, O. I.; Guryev, O. L.; Krivosheev, A. V.; Usanov, S. A.; Yaminsky, I. V. Langmuir 1999, 15, 1353. (f) Girard-Egrot, A. P.; Morelis, R. M.; Coulet, P. R. Langmuir 1997, 13, 6540. (g) Loescher, F.; Ruckstuhl, T.; Jaworek, T.; Wegner, G.; Seeger, S. Langmuir 1998, 14, 2786. (h) Erokhin, V.; Facci, P.; Kononenko, A.; Radicchi, G.; Nicolini, C. Thin Solid Films 1996, 284285, 805. (i) Chen, X.; Moser, C. C.; Pilloud, D. L.; Dutton, P. L. J. Phys. Chem. B 1998, 102, 6425. (j) Nicolini, C. Thin Solid Films 1996, 284285, 1. (9) (a) Yang, Z.; Mesiano, A. J.; Venkatasubramanian, S.; Gross, S. H.; Harris, J. M.; Russel, A. J. J. Am. Chem. Soc. 1995, 117, 4843. (b) Franchina, J. G.; Lackowski, W. M.; Dermody, D. L.; Crooks, R. M.; Bergbreiter, D. E.; Sirkar, K.; Russell, R. J.; Pishko, M. V. Anal. Chem. 1999, 71, 3133. (c) Umana, M.; Waller, J. Anal. Chem. 1986, 58, 2979. (d) Gill, I.; Pastor, E.; Ballesteros, A. J. Am. Chem. Soc. 1999, 120, 9487. (e) Mateo, C.; Abian, O.; Fernandez-Lafuente, R.; Guisan, J. M. Biotechnol. Bioeng. 2000, 68, 98. (f) Moustafa, A. B.; Kahil, T.; Faizalla, A. J. Appl. Polym. Sci. 2000, 76, 594. (g) Caruso, F.; Trau, D.; Moehwald, H.; Renneberg, R. Langmuir 2000, 16, 1485. (h) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3427. (i) Caruso, F.; Rodda, E.; Furlong, D. N.; Niikura, K.; Okahata, Y. Anal Chem. 1997, 69, 2043. (j) Nakayama, Y.; Matsuda, T. Langmuir 1999, 15, 5560. (k) Ma, H.; Hu, N.; Rusling, J. F. Langmuir 2000, 16, 4969. (l) Zu, X.; Lu, Z.; Zhang, Z.; Schenkman, J. B.; Rusling, J. F. Langmuir 1999, 15, 7372. (10) (a) Kumar, C. V.; Chaudhari, A. J. Am. Chem. Soc. 2000, 122, 830. (b) Kumar, C. V.; McLendon, G. L. Chem. Mater. 1997, 9, 863. (11) (a) Bosley, J. A.; Clayon, J. C. Biotechnol. Bioeng. 1994, 43, 934. (b) Bastida, A.; Sabuquillo, P.; Armisen, P.; Fernandez-Lafuente, R.; Huget, J.; Guisan, J. M. Biotechnol. Bioeng. 1998, 58, 486. (12) (a) deWildt, R. M. T.; Mundy, C. R.; Gorick, B. D.; Tomlinson, I. M. Nat. Biotechnol. 2000, 18, 989. (b) Niemeyer, C. M.; Ceyhan, B.; Blohm, D. Bioconjugate Chem. 1999, 10, 708. (c) Niemeyer, C. M.; Boldt, L.; Ceyhan, B.; Blohm, D. Anal. Biochem. 1999, 268, 54.

enzyme pepsin17a and a fungal protease17b in thermally evaporated lipid films. As part of our ongoing studies in the formation of lipid-protein biocomposites, we show herein that the metalloproteins cytochrome c (Cyt c, cationic protein at pH ) 6.8 and pI ∼ 8.8) and bovine hemoglobin (Hb, anionic protein at pH ) 9 and pI ∼ 6.8) can be intercalated via attractive electrostatic interactions into thermally evaporated arachidic acid (AA, anionic lipid) and octadecylamine (ODA, cationic lipid) films, respectively, by simple immersion of the lipid films in the appropriate protein solutions under protein-friendly conditions. On immersion of the AA film in the Cyt c solution at pH ) 6.8, attractive electrostatic interactions between the positively charged surface groups on Cyt c molecules and the negatively charged AA matrix (pKa of AA ∼ 4.5)18 drives (to a large extent) the diffusion of the protein into the lipid matrix. Similarly at pH ) 9, immersion of the ODA film (pKb of ODA ∼ 10.5) into Hb solution leads to the intercalation of the protein due to the electrostatic (13) (a) Zhu, G.; Mallery, S. R.; Schwendeman, S. P. Nat. Biotechnol. 2000, 18, 52. (b) Caruso, F.; Mohwald, H. J. Am. Chem. Soc. 1999, 121, 6039. (c) Rembaum, A.; Dreyer, W. J. Science 1980, 208, 364. (d) Schmitt, A.; Fernandez-Barbero, A.; Cabrerizo-Vilchez, M.; Hidalgo-Alvarez, R. Prog. Colloid Polym. Sci. 1997, 104, 144. (e) Elgersma, A. V.; Zsom, R. L. J.; Norde, W.; Lyklema, J. J. Colloid Interface. Sci. 1990. 138, 145. (14) (a) Keating, C. D.; Kovaleski, K. M.; Natan, M. J. J. Phys. Chem. B 1998, 102, 9404. (b) Xu, H.; Bjerneld, E. J.; Kall, M.; Borjesson, L. Phys. Rev. Lett. 1999, 83, 4357. (c) Zhao, J.; O’Daly, J. P.; Henkens, R. W.; Stonehuerner, J.; Crumbliss, A. L. Biosens. Bioelectron. 1996, 11, 493. (d) Gole, A.; Dash, C.; Ramakrishnan, V.; Sainkar, S. R.; Mandale, A. B.; Rao, M.; Sastry, M. Langmuir 2001, 17, 1674. (15) Ganguly, P.; Pal, S.; Sastry, M.; Shashikala, M. N. Langmuir 1995, 11, 1078. (16) (a) Sastry, M.; Patil, V.; Mayya, K. S. Langmuir 1997, 13, 4490. (b) Patil, V.; Sastry, M. J. Chem. Soc., Faraday Trans. 1997, 93, 4347. (c) Patil, V.; Sastry, M. Langmuir 1997, 13, 5511. (d) Sastry, M.; Patil, V.; Sainkar, S. R. J. Phys. Chem. B 1998, 102, 1404. (e) Patil, V.; Sastry, M. Langmuir 1998, 14, 2707. (f) Patil, V.; Malvankar, R. B.; Sastry, M. Langmuir 1999, 15, 8197. (g) Sastry, M. Curr. Sci. 2000, 72, 1089. (17) (a) Gole, A.; Dash, C. V.; Rao, M.; Sastry, M. J. Chem. Soc., Chem. Commun. 2000, 297. (b) Gole, A.; Dash, C. V.; Mandale, A. B.; Rao, M.; Sastry, M. Anal. Chem. 2000, 72, 4301. (18) Roser, S. J.; Lovell, M. R. J. Chem. Soc., Faraday Trans. 1995, 91, 1783.

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interaction between the oppositely charged species. Scheme 1 illustrates the procedure for Cyt c-AA composite film formation; the procedure is identical for the Hb-ODA combination with the difference that the charges on the protein and the lipid molecules (ODA) are reversed with respect to the Cyt c-AA combination (pI of Hb ∼ 6.8; pKb of ODA ∼ 10.5). The kinetics of diffusion of the proteins into (and out of) the lipid host matrix have been studied using quartz crystal microgravimetry (QCM) and the data has been analyzed in terms of a one-dimensional (1-D) diffusion model to obtain protein diffusivities under different conditions. The protein-lipid composite films have been further characterized using UV-Vis spectroscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), contact angle measurements, and X-ray diffraction studies (XRD). The fact that both positively and negatively charged proteins can be encapsulated in suitable lipid matrixes under mild conditions together with the possibility of pumping the proteins back into solution from the lipid host (accomplished by modulating the protein-lipid electrostatic interaction by solution pH variation) makes this approach potentially exciting and easily extendable to other biomacromolecules. Taking Cyt c as an example, we also show that the rate of diffusion of the protein into the lipid matrix can be controlled by variation in the thickness of the deposited lipid, through use of appropriate buffers, and by a novel three-step protocol involving preordering of the lipid film by the LB technique. This approach offers a number of advantages over techniques currently in vogue and will be discussed. Presented below are the details of the investigation. Experimental Details Chemicals. Horse heart Cyt c and Hb were obtained from Sigma Chemicals and used as received. AA (C19H39COOH), ODA (C18H37NH2), and lead chloride (PbCl2) were obtained from Aldrich Chemicals and used without further purification. All buffer salts were from standard commercial sources and of the highest quality available. Determination of Protein Isoelectric Points. The isoelectric points of Cyt c and Hb were determined by the use of a mini-scale isoelectric focusing unit built in-house19 in the pH range 3-10 (Pharmalyte). The isoelectric points of Cyt c and Hb were determined to be 8.8 and 6.8, respectively. Deposition of AA and ODA Thin Films. AA films of 1000 Å thickness each were deposited on gold-coated AT cut quartz crystals (for QCM measurements), on quartz substrates (for UVVis spectroscopy and fluorescence spectroscopy measurements), and on Si(111) substrates (for FTIR measurements) by thermal evaporation in an Edwards E308 chamber equipped with a liquid nitrogen trap (Scheme 1, step 1). Similarly, 2000 Å thick AA films were deposited on Si(111) substrates for XRD measurements. The deposition was done at a pressure of 1 × 10-7 Torr, and the film deposition rate and thickness were monitored in situ using an Edwards thickness monitor. 1000 Å thick ODA films were deposited on quartz substrates (for UV-Vis and fluorescence spectroscopy measurements) while 250 Å thick films were deposited on gold-coated AT cut quartz crystals (for QCM studies) and Si(111) substrates (for FTIR studies) as done for AA. Protein Intercalation Studies. Cyt c-AA System. Concentrated aqueous solutions of Cyt c (10-5 M) were prepared at different pH values using dilute HCl and NaOH. The protein diffusion into 1000 Å thick AA films was monitored by the immersion of the AA-covered gold-coated AT cut quartz crystals for different time intervals in the protein solution and by measuring the frequency change of the crystals ex situ after thorough washing and drying of the crystals (Scheme 1, step 2). (19) (a) Sathivel, C.; Lachke, A.; Radhakrishnan, S. J. Chromatogr., A 1995, 705, 400. (b) Gole, A.; Sathivel, C.; Lachke, A.; Sastry, M. J. Chromatogr., A 1999, 848, 485.

Gole et al. The time required for equilibration of the protein concentration in the films was estimated from the QCM studies and was used for the immersion of AA-deposited substrates such as Si(111) and quartz in the Cyt c solutions to form Cyt c-AA composite films. The films were dried in flowing nitrogen for a period of 5 min after being thoroughly rinsed in deionized water and were used for further studies. Hb-ODA System. Concentrated solutions of Hb (10-5 M) were prepared in different buffer solutions to yield varying pH values of the protein solution. The buffers used were glycineHCl (0.05 M, pH ) 3), sodium-phosphate (0.05 M, pH ) 6), and Tris-HCl (0.05 M, pH ) 9). The protein diffusion into the 250 Å thick ODA films was monitored by immersion of the ODAcovered gold-coated AT cut quartz crystals for different time intervals in the different Hb solutions and by measurement of the frequency change of the crystals ex situ after thorough washing and drying of the crystals as done for the Cyt c-AA system. QCM Frequency Counter. The frequency counter used was an Edwards FTM5 instrument operating at a frequency stability and resolution of (1 Hz. For the 6 MHz crystal used in this investigation, this translates into a mass resolution of 12 ng/ cm2. The frequency changes were converted to mass loading using the standard Sauerbrey formula.20 UV-Vis Spectroscopy Studies. Fatty acid coated quartz substrates of 1000 Å thickness each were immersed in the Cyt c solution at pH ) 6.8 (pH at which maximum protein incorporation occurs) for 50 h and dried prior to optical measurements. The redox activity of Cyt c was monitored using UV-Vis spectroscopy after immersion of the Cyt c-AA composite film in 1mM of ascorbate and ferricyanide solutions, respectively, for a period of 20 min.10b In a similar manner, 1000 Å thick ODAcoated quartz substrates were immersed in the Hb solution at pH ) 9 (pH at which maximum protein incorporation occurs) for 1 h and rinsed and dried before measurement. UV-Vis spectroscopy measurements were carried out on a Shimadzu dual beam spectrophotometer (model UV-1601 PC) operated at a resolution of 1 nm after thorough washing and drying of the films. Protein Native Structure Studies. Fluorescence studies of 1000 Å thick Cyt c-AA composite films and 1000 Å thick HbODA composite films on quartz were performed on a PerkinElmer luminescence spectrometer (model LS 50B). The tryptophan residues in the protein were excited at 295 nm, and the emission band was monitored from 300 to 400 nm. FTIR measurements of the protein-lipid composite films on Si(111) substrates were made after the immersion of 1000 Å thick AA films in the Cyt c solutions for 70 h and 250 Å thick ODA films in Hb solution for 1 h and the subsequent rinsing and drying of the films in flowing nitrogen for 5 min. A Shimadzu FTIR-8201 PC instrument operated in the diffuse reflectance mode at a resolution of 4 cm-1 was used to obtain FTIR spectra of the protein-lipid composite films. To obtain good signal-to-noise ratios, 256 scans were taken of the composite films in the range 400-4000 cm-1. Lamellar Structural Studies. XRD measurements of a 2000 Å thick Cyt c-AA composite film as well as an as-deposited AA film on Si(111) substrates were made on a Philips PW 1830 instrument operating at 40 kV voltage and a current of 30 mA with Cu KR radiation. As in previous studies, precautions were taken to clean the biocomposite film by washing and drying prior to XRD measurement. Contact Angle Measurements. Contact angle measurements of a sessile water drop (1 µL) on 100, 250, 500, and 1000 Å thick AA films and a 250 Å thick ODA film deposited on Si(111) substrates before and after intercalation of the proteins were carried out on a Rame Hart 100 goniometer. Contact angle measurements of a plain Si(111) substrate, a Si(111) substrate covered by drop-dried Cyt c/Hb films, as well as an AA/ODA film (250 Å thickness) on which a Cyt c/Hb film was deposited by drop-drying were also performed for comparison. Ellipsometry Measurements. Ellipsometry measurements of the 1000 and 250 Å thick AA films and the 250 Å thick ODA film before and after intercalation of the proteins were carried (20) Sauerbrey, G. Z. Phys. Chem. (Munich) 1959, 155, 206.

Protein-Friendly Intercalation in Lipid Films out on a Gaertner L118 null ellipsometer equipped with a HeNe laser (6328 Å, 5 mW). The ellipsometer was operated in the polarizer compensator sample analyzer (PCSA) mode and the ellipsometric angles were measured in four zones to correct for optical misalignment. The film thickness and refractive index were calculated from the ellipsometric ψ and ∆ values using an application written by M.S. using Mathcad (a commercial mathematical package available from Mathsoft Inc). Methods for Increasing the Rate of Diffusion of Cyt c into the Lipid Matrix. (a) Diffusion of Cyt c into 250 Å Thick AA Films. Three 250 Å thick AA films were deposited onto gold-coated AT cut quartz crystals by thermal evaporation as explained earlier. A Cyt c (10-5 M) solution was prepared in different buffers (glycine-HCl, 0.05 M, pH ) 3; sodiumphosphate, 0.05 M, pH ) 6.8; and Tris-HCl, 0.05 M, pH ) 9). The quartz crystals were immersed in the protein solutions for different time intervals, and the frequency changes were monitored and converted to a mass uptake as mentioned previously. (b) Diffusion of Cyt c in Preordered Lipid Films. The entrapment of Cyt c in preordered arachidic acid films was accomplished by a three-step procedure. (i) Deposition of Lead Arachidate LB Films. A 50 µL portion of arachidic acid solution (1 mg/mL) in chloroform was spread on a subphase of aqueous solution of lead chloride (10-4 M, pH ) 5.5) in a Nima 611 LB trough equipped with a Wilhelmy plate pressure sensor. Pressure-area (π-A) isotherms of the arachidic acid-lead chloride system were recorded immediately and also after 30 min of spreading of the arachidic acid monolayer. After measurement of the π-A isotherms, films of lead arachidate of thickness of 17 monolayers (375 Å thick) were formed by the LB technique21 at a surface pressure of 30 mN/m onto gold-coated AT cut quartz crystals (for QCM measurements) and Si(111) substrates (for FTIR and XRD measurements). For the LB films grown on different substrates, monolayer transfer was observed both during the upward and downward strokes of the substrate when close to a unity transfer ratio. A linear increase in mass uptake (as measured by QCM studies) was observed indicating the formation of ordered, lamellar LB films. (ii) Removal of Lead Ions from the Lead-Arachidate System. After the formation of multilayers of lead arachidate, the substrates were immersed into an aqueous solution held at pH ) 2 (pH adjusted by using HCl). QCM kinetics as a function of time of immersion of the lead arachidate multilayer film on quartz crystals showed a mass loss, indicating removal of lead ions from the LB film. (iii) Intercalation of Cyt c. After complete removal of lead ions from the lead arachidate LB film, the lipid film was further immersed into a 10-5 M Cyt c solution (0.05 M sodium phosphate buffer, pH ) 6.8) leading to the incorporation of the protein. This intercalation of the protein was followed by QCM measurements. The entire process of formation of LB films, removal of lead ions, and intercalation of the protein into this preordered lipid film was followed by QCM, FTIR, and XRD measurements. Control Experiments. A control experiment was performed wherein 250 Å thick AA and ODA-deposited quartz crystals were immersed for different time intervals in plain buffer solutions (0.05 M sodium phosphate, pH ) 6.8 and 0.05 M Tris-HCl, pH ) 9, respectively) to monitor changes in mass uptake (if any) due to possible intercalation of buffer ions.

Results and Discussion Before proceeding to the details of the intercalation of Cyt c in AA and Hb in ODA lipid films, we would like to mention that the technique that is proposed in this paper is general and should, in principle, be applicable to different ionizable biomolecules such as enzymes and (poly)nucleic acids as well. We have chosen to demonstrate its application with heme proteins such as Cyt c and Hb, the motivation arising from a purely academic point of view to realize simple model systems to better understand (21) Ulman, A. An introduction to Ultrathin Organic Films: from Langmuir-Blodgett to Self-Assembly; Academic Press: San Diego, CA, 1991.

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Figure 1. QCM mass uptake of Cyt c with time during immersion of 1000 Å thick AA films in 10-5 M Cyt c solutions at different pH values. The pH values are indicated next to the respective mass uptake curves together with the equilibrium Cyt c/AA molar ratios in parentheses. The solid lines are fits to the data using a 1-D diffusion model. The error bars to the data show roughly 10% of deviation of the data from the mean values. The inset shows the loss of Cyt c from the 1000 Å thick biocomposite film formed at pH ) 6.8 during immersion in water at pH ) 3. (See text for details.)

protein-lipid (bilayer membrane structures) interactions. Second, the presence and accessibility of Cyt c in the AA matrix to analytes in solution may readily be determined by reaction with suitable reducing/oxidizing reagents in solution. As shown below, the interaction of the proteins with an ionized lipid matrix appears to be mainly electrostatic, though it must be mentioned that other interactions also play an important role in stabilizing the protein in the hydrophilic lipid bilayer regions (Scheme 1, step 2, possible microstructure of the biocomposite). QCM Diffusion Kinetics Studies. Cyt c-AA System. As mentioned earlier, Cyt c was incorporated into thermally evaporated 1000 Å thick AA films by simple immersion of the fatty acid films in 10-5 M Cyt c solution maintained at different pH values (Scheme 1, step 2). Figure 1 shows a plot of the QCM mass uptake data of Cyt c intercalation into a 1000 Å thick AA film during immersion in Cyt c solutions at pH ) 3 (circles), pH ) 6.8 (squares), and pH ) 9 (triangles). The protein solution pH and equilibrium Cyt c/AA molar ratios are indicated next to the respective curves. In this case, it can be seen that complete incorporation of the protein takes place within 70 h of immersion of the AA film. It is observed from Figure 1 that maximum Cyt c intercalation occurs close to physiological pH with the equilibrium Cyt c loading factors being lower at pH ) 3 and pH ) 9. At solution pH ) 6.8, Cyt c would acquire a net positive charge (pI of Cyt c ) 8.8) while the AA molecules would be completely negatively charged (pKa of AA ) 5.4).18 Consequently, at this pH, maximum attractive electrostatic interaction between the protein in solution and AA molecules in the lipid matrix occurs leading to enhanced Cyt c density in the films. The fact that Cyt c incorporation in the AA matrix occurs at pH values of 9 and 3 also indicates that factors such as hydrogen bonding and hydrophobic interactions also play a role in controlling the protein diffusion process. Five separate QCM kinetics studies of the incorporation of Cyt c into 1000 Å thick AA films were carried out for different pH conditions in order to study the reproducibility of the protein diffusion process. The error bars shown in Figure 1 for the Cyt c-AA film are based on an analysis of these sets of data. It can be

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Figure 2. (A) QCM mass uptake of Cyt c with time during immersion of 250 Å thick AA films in 10-5 M Cyt c at different pH values. The pH values and equilibrium molar ratios of Cyt c/AA are indicated in parentheses next to the respective curves. The solid lines are fit to the data using 1-D diffusion model. The error bars to pH ) 6.8 show 10% of deviation of the data and for other two pH values shows 5% deviation of the data. A control experiment of mass uptake measured as a function of immersion of 250 Å thick AA film-deposited quartz crystal for different time intervals into 0.05 M sodium phosphate buffer, pH ) 6.8 (crosses). (B) QCM mass uptake kinetics of incorporation of Cyt c by a three-step protocol. The solid line is a 1-D diffusion model fit to the data, with error bars showing a 10% deviation to the data. (See text for details.)

seen that the variance in the masses during separate runs is roughly 10% and may thus be used as confidence limits in the QCM kinetics measurements of the Cyt c-AA system. The fact that electrostatic interactions dominate the protein incorporation in the lipid films implies that the adsorption process should be reversible. After the formation of the Cyt c-AA composite film at pH ) 6.8, the film was immersed in aqueous solution at pH ) 3. The inset of Figure 1 shows the QCM kinetics of leaching out of Cyt c at pH ) 3. It can be seen from the inset that the release of Cyt c is not completesca. 76% of the Cyt c molecules are retained in the matrix, and this agrees with earlier inferences that interactions other than electrostatic interactions contribute to the protein immobilization process. Increasing the Rate of Diffusion of Cyt c into the Lipid Matrix. (a) Diffusion of Protein Prepared in Buffers into 250 Å Thick Films. Figure 2A shows a plot of the QCM mass uptake data of Cyt c intercalation into a 250 Å thick AA film during immersion in 10-5 M Cyt c solutions prepared in different buffers (squares, pH ) 3; circles, pH ) 6.8; and triangles, pH ) 9). The protein solution pH and equilibrium Cyt c/AA molar ratios are indicated next to the respective curves. It can be seen that complete incorporation of the protein in this case takes place within 120 min of immersion of the AA film in protein solutions, with maximum incorporation at pH ) 6.8 and lesser incorporation at the other two pH values, as also observed in Figure 1. The solid lines are 1-D diffusion fits to the data as done in the earlier case, with the error bars of 10% deviation for the case where the pH ) 6.8 and 5% deviation for pH ) 3 and 9 (the error bars were determined from five separate QCM experiments in each case). It is important to note that by varying the thickness of the as-deposited films, the equilibrium mass loading of the protein in the film can be varied from ca. 7.6 µg for a 250 Å thick AA film (Figure 2A, pH ) 6.8) to 18 µg for a 1000 Å thick AA film (Figure 1, pH ) 6.8). The rate of diffusion into the film is also increased due to the presence of buffers in the protein solution. This might be due to a competitive

Gole et al.

diffusion process between the protein and ions from the buffer in the matrix leading to enhanced diffusion rates of the proteins. This aspect is not clear at the moment. Such a dependence of protein binding to lipids on the ionic strength of buffer solution was observed by Salamon et al.4j wherein the binding of Cyt c onto solid-supported planar phosphatidylcholine membranes under varying protein and ionic concentrations was studied. They also observed the role of secondary interactions responsible for protein immobilization, as has been observed by us. A control experiment was performed in which the 250 Å thick AA film deposited on QCM crystal was immersed for different time intervals in a buffer (0.05 M sodium phosphate, pH ) 6.8). Some amount of mass loading was observed as indicated in Figure 2A (crosses), but is negligible as compared to the overall mass uptake due to that of the protein. (b) Diffusion of the Protein into a Preordered AA Film. Figure 2B shows the QCM kinetics of diffusion of a 10-5 M Cyt c solution (0.05 M sodium phosphate buffer, pH ) 6.8) into the AA matrix preordered via a three-step process as outlined in the Experimental Section. The three steps involve the preparation of lead arachidate multilayer films via the LB technique, removal of lead ions from the interlamellar spaces via alteration of the electrostatic interaction (immersion of the lead arachidate films in pH ) 2 aqueous solution), and subsequent immersion of the films into 10-5 M Cyt c solution (0.05 M, sodium phosphate buffer, pH ) 6.8). As seen in Figure 2B, the intercalation of the protein in the AA matrix is achieved within a period of 10 min. Five such separate experiments yielded a 10% deviation in the data, as shown by the error bars. The solid line is a 1-D diffusion analysis fit to the data (to be discussed subsequently). In XRD analysis, the lead-arachidate film gave (00l) Bragg reflections with a repeat distance of 50 Å indicating lamellar ordering, which is retained on the removal of lead ions (data not shown). FTIR spectra showed the carboxylate stretch bands at 1541 and 1512 cm-1 in the lead arachidate films, and on removal of the lead ions the carbonyl stretch frequency at 1700 cm-1 is regenerated with a complete absence of the carboxylate stretch bands (data not shown). This signature was used as an indicator of complete removal of lead ions from the LB films. The intercalation of Cyt c in this film was also monitored by FTIR spectroscopy to confirm the presence of amide I and II bands (at 1548 and 1654 cm-1, data not shown). It can be clearly seen that the loading of the proteins and the variation in rate of diffusion of the protein can be achieved by judicious control of the thickness of the films, use of appropriate buffers, and by the use of preordered films that enable easy diffusion of the proteins into the lipid matrix. Hb-ODA System. Hb was incorporated into 250 Å thick thermally evaporated ODA films by simple immersion of the fatty amine films in 10-5 M Hb solution maintained at different pH values. Figure 3 shows a plot of the QCM mass uptake data of Hb into the ODA film during immersion in the Hb solutions at pH ) 3 (circles), pH ) 6 (squares), and pH ) 9 (triangles). The protein solution pH and the equilibrium Hb/ODA molar ratios are indicated next to the respective curves. It can be seen from Figure 3 that maximum Hb intercalation occurs at pH ) 9.0 with the equilibrium Hb loading factors being lower at pH ) 6 and pH ) 3. It can also be seen from Figure 3 that complete protein intercalation is achieved within a period of 30 min of immersion and is considerably better than that reported for other techniques.4,9,10 As in the case of the Cyt c-AA system, five separate QCM


Figure 3. QCM mass uptake of Hb with time during immersion of 250 Å thick ODA films in 10-5 M Hb solutions at different pH values. The pH values are indicated next to the respective curves while the equilibrium molar ratios of Hb/ODA are listed in parentheses. The error bars shown in the curves are ca. 5% deviation of the data from the mean values. The inset shows the loss of Hb from a 250 Å thick biocomposite film formed at pH ) 9 during immersion in water at pH ) 3. A control experiment of mass uptake measured as a function of immersion of 250 Å thick ODA-deposited quartz crystal for different time intervals into 0.05 M Tris-HCl, pH ) 9 buffer (crosses). (See text for details.)

kinetics studies of the incorporation of Hb into 250 Å thick ODA films (at different pH values) were made in order to study the reproducibility of the protein diffusion process. The error bars shown in Figure 3 for the Hb-ODA film are thus based on an analysis of these sets of data. It can be seen that the variance in the masses during separate runs is roughly 5 % and indicates slightly better confidence limits for this system in comparison with the Cyt c-AA system. A control experiment was also performed wherein a 250 Å thick ODA film was immersed for different time intervals in a buffer solution (0.05 M Tris-HCl, pH ) 9), and the mass uptake measured for this film is shown in Figure 3 (crosses). It is clearly seen that there is no significant mass uptake due to the buffer salts in both the cases [Cyt c (Figure 2, crosses) and Hb (Figure 3, crosses)], and so the mass uptakes measured for proteins can be safely considered to be due to that of the respective proteins. The variation in Hb uptake with protein solution pH is explained as follows. At pH ) 9, both the Hb (pI ) 6.8, anionic) and ODA molecules (pKA ) 10.5, cationic) are completely ionized leading to maximum attractive electrostatic interaction between the host and guest. At pH ) 6, the surface charge on the Hb molecules is nearly zero, and therefore, a vanishingly small attractive Coulombic interaction should exist at this pH. At pH ) 3, the ODA and the protein molecules bear the same sign of charge, i.e., both the host and guest molecules are positively charged, and therefore, a repulsive Coulombic interaction exists under these conditions. Consequently, the extent of protein uptake at these pH values is smaller than at pH ) 9 indicating that electrostatic interactions do play a crucial role in driving the diffusion of the protein molecules into the ODA host matrix. However, the fact that protein incorporation still does occur under conditions of negligible or repulsive electrostatic interactions underlines the fact that other interactions such as hydrogen bonding and hydrophobic interactions also contribute to the interactions between the protein and ODA film. It is expected that the Hb molecules are immobilized within

Langmuir, Vol. 17, No. 18, 2001 5651

the hydrophilic regions of the ODA matrix as shown in Scheme 1. It should thus be possible for analytes in solution to communicate with the protein molecules via hydrophilic water channels during immersion of the biocomposite films in different solutions. The inset of Figure 3 shows the release of Hb molecules from a 250 Å thick Hb-ODA composite film formed at pH ) 9 on immersion in water held at pH ) 3. At this pH, the Hb molecules acquire a positive charge and repel the similarly charged ODA molecules leading to their expulsion from the lipid matrix. As in the case of the leaching out of Cyt c, the release of Hb molecules from the composite film is not completesca. 28% of the Hb molecules are retained in the matrix providing additional confirmation that interactions other than electrostatic contribute to the protein immobilization process. This feature of reversible adsorption/desorption is possible in very few techniques currently in vogue. Such an approach was used for the electrostatically controlled reversible adsorption/desorption of glucose oxidase in a weak amine, polyelectrolyte hyperbranched thin film by Crooks and co-workers.9b The main difference between the approach of Crooks et al.9b and our technique (other than the use of different matrixes) is that we did not functionalize the proteins separately and we used the pH dependent charge on the native protein to drive the diffusion from solution into the lipid matrix. Functionalization of the proteins led to a reduction in the amount of active protein by ca. 35%,9b and this loss in active protein concentration may be avoided using our protocol for biocomposite formation. Dave and Rao5f have also shown the selective intake and release of proteins by organically modified silica solgels. The sol-gel was prepared by hydrolysis of bis[3(trimethoxysilyl)-propyl]ethylenediamine (enTMOS), and the porosity was adjusted so that large biomolecules could freely diffuse in and out of the bulk of the material. They observed selective release of Cyt c from an encapsulated mixture of Mb and Cyt c or Hb and Cyt c when the material was immersed in water, which they attributed to the repulsive electrostatic interaction between the Cyt c and the amino groups in the sol-gel material.5f In our method we use the same charge reversal concept to pump out the proteins, except for the fact that we have to change the water pH to vary the ionization between the charged groups on the surface of the proteins and that on the lipid matrix. The process of the leaching out of proteins from the films may be useful in applications such as protein purification. A crude mixture of proteins having different pI values can be selectively intercalated in the lipid films depending on the net charge on the protein of interest and then can be leached out at a different pH leading to highly purified protein. Thus, the reversibility of the host-guest interaction based on electrostatic interactions enhances the versatility of the approach described herein. In the comparison of the QCM kinetics results of incorporation of Cyt c with that of Hb molecules in the different lipid matrixes, it can be seen that the time involved in preparation of fully loaded biocomposites in both cases is vastly different. This is due to the different thicknesses of the lipid matrixes used for Cyt c diffusion in AA (1000 Å, Figure 1) and Hb diffusion in ODA (250 Å , Figure 3), respectively. The time involved and the rate of diffusion for the intercalation process can be altered by the use of a 250 Å thick AA matrix for diffusion of Cyt c, by the use of appropriate buffers (Figure 2A), and also via a three-step process (Figure 2B).

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Analysis of the QCM Diffusion Data. The kinetics of protein incorporation into the lipid films studied by QCM measurements (Figures 1-3) may be conveniently analyzed in terms of a 1-D diffusion model, as has been demonstrated by us for colloidal nanoparticles of silver, gold, and CdS in fatty amine films.16b,d,f The equation for 1-D diffusion is written as 2

∂ C(x,t) ∂ C(x,t) )D ∂t ∂x2 where C(x,t) is the time- and distance-dependent protein concentration in the film and D is the protein diffusivity. The boundary conditions for this equation appropriate to the problem on hand are

C(x,t) ) 0 t < 0 ) C0 t > 0 (protein concentration at interface after definite time interval of immersion) where C0 is the protein concentration at the film/protein solution interface. The second boundary condition is that the quartz crystal substrate is impervious to protein diffusion which gives ∂C(0,t)/∂x ) 0. The solution to the 1-D diffusion equation using the above two boundary conditions is given by

[ [∑ ∞

C(x,t) ) C0 1 + 4

e-D[(2n+1) π /4L ]t × 2 2

[

n)0

cos

2

]

(2n + 1)πx 2L

(-1)n+1 ×

1

]

(2n + 1)π

In QCM studies, one observes a mass uptake over the whole length of the film covering the sensing electrode. The total mass uptake recorded as a function of time, M(t), is therefore

M(t) ) m0

∫0L C(x,t)dx

where m0 is the mass of protein. With the initial values for concentration of protein and its molecular weight being known, the diffusion coefficients were calculated using a program written by M.S. in Mathcad. We would like to point out that there is considerable swelling of the films after protein incorporation and therefore, the film thickness values used in the 1-D analysis are different from the thickness values of the as-deposited films. We would like to mention here that in the ellipsometric calculations, we assumed the refractive index of all films (before and after protein uptake) to be 1.5 and calculated the thickness of the lipid films before and after protein intercalation by using the exact equation of ellipsometry.22 After incorporation of the protein, it is possible that the refractive index could change due to the presence of highly polarizable functional groups in the amino acids of the entrapped proteins. This assumption together with possible contributions from scattering by the film should be considered when analyzing the ellipsometry results. The thickness of the composite films before and after protein incorporation at different pH values as measured by ellipsometry are listed in Table 1 along with the values of the protein (22) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland: Amsterdam, 1977.

Table 1. Parameters Obtained from a 1-D Diffusion Analysis of QCM Mass Uptake Measurements during Incorporation of Cyt c and Hb in Fatty Lipid Matrixes film thickness (Å) solution lipid pH matrix

before incorpn

after incorpn

C0(molecules cm-3)

D (Å2min-1)

(A) Cyt c-AA system 1000 2000 1.3 × 1011 476 1000 2000 6.3 × 1011 413 1000 2000 1.3 × 1011 955 250 500 4.46 × 1011 1.06 × 104 250 500 6.63 × 1011 1.96 × 104 250 500 5.1 × 1011 1.09 × 104 375 750 3.91 × 1011 4.07 × 105

3 6.8 9 3 6.8 9 6.8

AA AA AA AA AA AA AAa

3 6 9

ODA ODA ODA

a

Preordered films.

(B) Hb-ODA system 250 1100 1.9 × 1010 250 1200 4.7 × 1010 250 1200 8.1 × 1011

4.4 × 105 1.4 × 105 9.3 × 104

concentration at the film-protein solution interface (C0, molecules cm-3) and the protein diffusivity (D, Å2 min-1). It is interesting to note that the film thickness after Hb incorporation into a 250 Å thick as-deposited ODA matrix is more than that in the case of Cyt c incorporation into a 250 Å thick as-deposited AA matrix. This may be due to the larger size of Hb molecules as compared to that of Cyt c. The solid lines in Figs 1-3 are due to a 1-D diffusion model fit to the QCM data. Cyt c-AA System. The parameters obtained from fits to the QCM diffusion data are listed in Table 1A for the Cyt c-AA system (data shown in Figures 1 and 2). It is seen that in the 1000 Å thick AA case, the diffusivities of the Cyt c molecules into the AA matrix are almost identical at pH ) 6.8 (pH at which maximum incorporation takes place) and at pH ) 3 (pH at which minimum mass uptake is observed). At pH ) 9, it is observed that the diffusivity is much higher than that at the other two pH values, and at this stage, we are unable to explain this result. The concentration at the film-solution interface, however, does show a physically meaningful trend with a maximum at pH ) 6.8 and a minimum at pH ) 3. The interfacial concentration of Cyt c being the highest at pH ) 6.8 may be a consequence of enhanced electrostatic interaction between the proteins and the lipid matrix as reflected in the equilibrium protein loading factors at the different pH values. Another interesting point is that the diffusivities of Cyt c are greatly enhanced by following the QCM kinetics for Cyt c into a 250 Å thick AA matrix as a function of immersion in the protein solution prepared in appropriate buffers (Table 1A). There is a trend in the diffusivities (D) and concentration at the interface (C0) of Cyt c, the maximum being at the pH at which there is maximum electrostatic attraction between the protein and the matrix (pH ) 6.8) and less at the other two pH values. Furthermore, a greatly enhanced diffusivity for Cyt c is observed for preordered film by the three-step protocol. It is also interesting to note that the preordered film (375 Å thick) gave better diffusion coefficients than the thermally evaporated less thicker (250 Å) AA film, indicating an advantage of preordering the lipid films prior to intercalation. Hb-ODA System. The parameters obtained from fits to the QCM mass uptake data for the Hb-ODA system (data shown in Figure 3) are listed in Table 1B. The interesting point to note in this case is that the diffusivity of the Hb molecules into the ODA matrix is highest under conditions where the host and guest repel one another


(pH ) 3). The diffusivities at pH ) 6 and 9 are nearly identical but less than at pH ) 3, as mentioned above. The concentration at the film-solution interface, however, does show a physically meaningful trend with a maximum at pH ) 9 and a minimum at pH ) 3. Therefore, the electrostatic interaction between the Hb and ODA molecules leads to enhanced adsorption at the interface. There is a difference in the order of magnitude between the diffusivities for Cyt c in 250 Å thick AA matrix and that for Hb in the ODA matrix. This might be due to the difference in the overall charge on the protein. However, the diffusion coefficient for Cyt c involving preordered AA films has similar values as that calculated for the HbODA system. We do not know whether the discrepancies observed in the diffusivity values in both the systems are a consequence of the very simple model being used to describe the protein incorporation process or whether they are due to the assumption of a primitive electrostatic picture for the process. We would like to emphasize that this model is highly idealized and represents the first step in the analysis of the kinetics of the protein incorporation process. Further refinement of the analysis will be done as our understanding of the different protein-lipid interactions operative in this experiment advances. It is clear that a purely electrostatic picture of the process is not correct and this conclusion supports earlier studies of polyion-ionized lipid monolayer interactions where a Poisson-Boltzmann electrostatic model was clearly shown to be inadequate in explaining the complexation observed.23 The situation on hand regarding protein-lipid interactions is expected to be far more complex. Before proceeding to the next section, we would like to point out possible errors in the interpretation of the QCM results using the simple Sauerbrey equation. Most QCM investigations assume that the film layer is rigid with no slip at the resonator-fluid boundary.24 These are valid assumptions when dealing with inorganic thin films under conditions where the Sauerbrey equation is valid. However, while dealing with lipid films that are being intercalated by biological molecules such as proteins and DNA, one would need to consider viscoelastic effects, high mass loadings, surface roughness, surface stress, interfacial slippage, and nonuniform mass distribution in any piezoelectric measurement.24 For thin films where the extent of protein loading is not high, this technique is quantitative. However, at large loadings and high film thickness, the above-mentioned factors must be borne in mind. Contact Angle Measurements. It is well-known that protein molecules spontaneously accumulate at phase boundaries,25 and therefore, adsorption of the proteins on the surface of the lipid films must be discounted. Cyt c-AA System. Contact angle measurements of a sessile water drop on 100, 250, 500, and 1000 Å thick AA films grown on Si(111) wafers (contact angle of Si ) 25°) after immersion in 10-5 M Cyt c solution at pH ) 6.8 for 75 h yielded 89°, 88°, 90°, and 90° respectively. The contact angle of an as-deposited 250 Å thick AA film was found to be 92°. Hb-ODA System. Contact angle measurements with a sessile water drop on a 250 Å thick ODA film deposited on Si(111) substrates before and after immersion in the 10-5 M Hb solution at pH ) 9 for 1 h yielded 95° and 92°, respectively. (23) Cuvillier, N.; Rondelez, F. Thin Solid Films 1998, 327-329, 19. (24) (a) Buttry, D. A.; Ward, M. D. Chem. Rev. 1992, 92, 1356. (b) Wang, J.; Frostman, L. M.; Ward, M. D. J. Phys. Chem. 1992, 96, 5224. (25) Razumovsky, L.; Damodaran, S. Langmuir 1999, 15, 1392.

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Figure 4. (A) Optical absorption spectra recorded from a 1000 Å thick Cyt c-AA composite film after immersion in 1 mM ascorbate and ferricyanide solutions for 20 min (upper and lower curves, respectively). The inset shows the as-prepared 1000 Å thick Cyt c-AA composite film after immersion in Cyt c solution at pH ) 6.8 for a period of 70 h. (B) Optical absorption spectrum recorded from a 10-5 M Hb solution in pH ) 9 buffer. The inset shows the optical absorption spectrum recorded from a 1000 Å thick Hb-ODA composite film formed at pH ) 9.

It is clear that the reduction in contact angle after protein incorporation is marginal and therefore, the protein molecules are entrapped within the lipid matrix and not on the surface. It is pertinent to mention here that the contact angles measured for the bare Si(111) surface, the protein film (both Cyt c and Hb) deposited on the Si(111) substrate by evaporation of a drop of the protein solution, and protein film (Cyt c and Hb) deposited on a 250 Å thick AA/ODA film by evaporation of a drop of the protein solution (lipid film having protein on the surface) yielded values of 19°, 15°, and 18°, respectively, further strengthening the conclusion mentioned above. The above results clearly indicate that the protein molecules are adsorbed within the lipid matrix (in the hydrophilic regions), as indicated in Scheme 1 (magnified view, expected film structure), and not on the film surface. UV-Vis Studies. Cyt c-AA System. Cyt c is a redox active heme protein and this was followed for the Cyt c-AA composite film using UV-Vis spectroscopy. The UV-Vis spectrum recorded from a 1000 Å thick AA film deposited on a quartz substrate after immersion in Cyt c solution (pH ) 6.8) for 75 h is shown in the inset of Figure 4A. The redox behavior of the protein in the lipid matrix was confirmed by reaction with ascorbate and ferricyanide solutions, and the spectra obtained under both conditions is shown in Figure 4A. The upper spectrum in Figure 4A was recorded from the 1000 Å thick Cyt c-AA composite film after reduction of the heme center, while the lower curve is the spectrum recorded after the oxidation cycle.10b While the Soret band occurs at 409 nm in both cases, important differences are observed in the Q-band absorption region (500-600 nm). In the reduced state, two fairly distinct bands are seen at 520 and 550 nm (features a and b respectively, Figure 4A), and this becomes a broad indistinguishable feature in the oxidized state. This is known to be a signature of the redox activity of the protein10b and shows that the intercalated protein is accessible to analytes in solution. The inset of Figure 4A shows the UV-Vis spectrum of the as-prepared Cyt c-AA composite film, and it is observed that the Q-band region (500-600 nm) shows two distinct peaks at about 520 and 550 nm in this case as well. This result indicates

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Figure 5. (A) FTIR spectra recorded from a 1000 Å thick asdeposited AA film (curve 1), a 1000 Å thick Cyt c-AA composite film in the reduced state (curve 2), and a 1000 Å thick Cyt c-AA composite film in the oxidized state (curve 3). The wavenumbers of the different features are mentioned in the figure (see text for assignments). (B) FTIR spectra recorded from a 250 Å thick as-deposited ODA film (curve 1) and a 250 Å thick ODA film immersed in Hb solution at pH ) 9 for 5 h (curve 2). The wavenumbers of the different features are mentioned in the figure (see text for assignments).

that the acid matrix behaves like a reducing environment, and thus, is not strictly an inert host. Hb-ODA System. The intercalation of Hb in the ODA matrix was also studied using UV-Vis spectroscopy. These measurements were carried out ex situ on a 1000 Å thick ODA film on a quartz substrate immersed in 10-5 M Hb solution at pH ) 9 for a period of 1 h. Figure 4B shows the UV-Vis spectrum of Hb solution at pH ) 9, while the inset of Figure 4B shows the spectrum measured from Hb-ODA composite film. The Soret band at 409 nm10b is clearly seen both in the solution and in the biocomposite film clearly indicating the presence of Hb in the ODA matrix. The fact that the form of the Soret band in both cases is similar also indicates that there is little distortion to the secondary structure of Hb in the encapsulated form. Protein Native Structure Studies. (A) Secondary Structure, FTIR Studies. The amide linkages between amino acid residues in polypeptides and proteins give wellknown signatures in the infrared region of the electromagnetic spectrum. The positions of the amide I and II bands in the FTIR spectra of proteins are sensitive indicators of conformational changes in the protein secondary structure10b,26 and may be used to study the protein molecules in the lipid matrix. Cyt c-AA System. Figure 5A shows the FTIR spectra recorded from a 1000 Å thick as-deposited AA film on Si(111) substrate (curve 1), a 1000 Å thick Cyt c-AA composite film in the reduced state (curve 2), and a 1000 Å thick Cyt c-AA composite film in the oxidized state (curve 3). A number of vibrational modes can be observed for the three films. The amide I band occurs at ca. 1654 cm-1 (feature b in Figure 5A) for the Cyt c-AA composite films (curves 2 and 3). This band is clearly absent in the as-deposited AA film (curve 1). The position of this band is close to that reported for the native protein in earlier reports26 and indicates that the secondary structure of the proteins in the different lipid environments is unperturbed. The amide II band, which occurs at 1548 cm-1 (feature a, Figure 5A), can also be clearly seen for the Cyt (26) Dong, A.; Huang, P.; Caughey, W. S. Biochemistry 1992, 31, 182.

Gole et al.

c-AA composite films. This band also indicates that the secondary structure of the protein is maintained in the encapsulated form.10b The band at 1699 cm-1 (feature c, Figure 5A) arises from the carbonyl stretch modes from the carboxylic acid groups in the AA matrix.27 It is difficult to track changes in the conformation of Cyt c during the redox cycle using FTIR, and this technique has primarily been used to identify changes, if any, in the protein secondary structure due to intercalation in the lipid matrix. Hb-ODA System. Figure 5B shows the FTIR spectra recorded from a 250 Å thick as-deposited ODA film (curve 1) on Si(111) substrate and a 250 Å thick ODA film after immersion in 10-5 M Hb solution at pH ) 9 for 5 h (curve 2). The amide I band, which is assigned to the carbonyl stretch in the amide linkage, occurs at ca. 1644 cm-1 (Figure 5B, feature a) for the Hb-ODA biocomposite film. Whereas a small feature at this wavenumber does occur in the as-deposited ODA film (Figure 5B, curve 1); the intensity of this band increases in curve 2 clearly showing that it originates from the Hb molecules in the composite film. The position of this band is close to that reported for native proteins in earlier papers10,26 and indicates that the secondary structure of the protein in the ODA environment is relatively unperturbed. The amide II band, which arises due to the C-N stretching modes of vibration in the amide bond, occurs at 1544 cm-1 (Figure 5B, feature c) and can also be clearly seen for the Hb-ODA composite film. The position of this band indicates that the secondary structure of the native protein is retained in the encapsulated form. The band at 1564 cm-1 (Figure 5B, feature b) arises from the ODA host and is tentatively assigned to the N-H deformation vibration from the amine groups in the ODA matrix. (B) Tertiary Structure, Fluorescence Spectroscopy. The intactness of the tertiary structure of the heme proteins in the intercalated form was studied by fluorescence spectroscopy measurements of the biocomposite films on quartz substrates. This standard method involves exciting the sample at a particular wavelength and monitoring the fluorescence emission from the tryptophan or tyrosine residues in the protein. The encapsulated heme proteins were excited at 295 nm; this wavelength corresponds to excitation of the π-π* transition in the tryptophan residues of the proteins. The wavelength of this transition is known to be a sensitive indicator of the tertiary structure of proteins.28 Cyt c-AA System. Figure 6A shows the fluorescence spectra recorded from a 10-5 M Cyt c solution at pH ) 6.8 (curve 1) and a Cyt c-AA composite (curve 2) after 50 h of immersion of a 1000 Å thick AA film on quartz in Cyt c solution at pH ) 6.8. The Cyt c solution shows an emission band centered at about 332 nm, which arises as a consequence of radiative decay of the π-π* transition from the tryptophan residues in the protein on excitation at 295 nm. The fluorescence signal from the Cyt c-AA biocomposite film occurs at the same value of 332 nm, as can be easily seen in curve 2 of Figure 6A. The spectrum from the composite film was recorded after proper baseline subtraction, using the as-deposited 1000 Å thick AA film as a blank. Since there is no detectable shift in the emission maxima, it may be concluded that the changes in the tertiary structure of Cyt c on intercalation in the AA matrix are negligible. Hb-ODA System. Figure 6B shows the fluorescence spectra recorded from 10-5 M Hb solution at pH ) 9 (curve (27) Rabolt, J. F.; Burns, F. C.; Schlotter, N. E.; Swalen, J. D. J. Chem. Phys. 1983, 78. (28) Eftink, M. R; Ghiron, C. A. Anal. Biochem. 1981, 199.


Figure 6. (A) Fluorescence spectra recorded from a 10-5 M Cyt c solution at pH ) 6.8 (curve 1) and a 1000 Å thick Cyt c-AA composite film (prepared at pH ) 6.8) on quartz (curve 2). (B) Fluorescence spectra of a 10-5M Hb solution at pH ) 9 (curve 1) and a 1000 Å thick Hb-ODA composite film (prepared at pH ) 9) on quartz (curve 2). (See text for details.)

1) and a Hb-ODA composite (curve 2) after 1 h of immersion of a 1000 Å thick ODA film on quartz in Hb solution at pH ) 9. The spectrum from the film was recorded after proper baseline correction, using an asdeposited 1000 Å thick ODA film as a blank. It can be seen that there is a red shift of ca. 5 nm of the tryptophan emission in the film when compared to that of the free protein in the solution. This shift does indicate some variation in the tertiary structure of the Hb molecules in the ODA matrix. This disturbance to the tertiary structure of Hb may be due to a stronger electrostatic interaction of Hb with the lipid matrix. That this is a possible explanation is supported by the QCM diffusion results, which show a much faster Hb diffusion into the ODA matrix in comparison with diffusion of Cyt c into the AA matrix. Fluorescence spectroscopy has also been used to study the intactness of the native structure of the heme proteins in solution at different pH values. Cyt c Solution. Figure 1A, Supporting Information, shows the fluorescence spectra of aqueous solutions of 10-5 M Cyt c at pH ) 3, 6.8, and 9 (curves 1, 2, and 3, respectively). The spectra have been shifted vertically to bring out the differences between the peak positions. The spectra clearly show maxima at 332 nm in curves 2 and 3 (pH ) 6.8 and 9 respectively). However, the spectrum at pH ) 3 (curve 1) shows a small shift of red shift of ca. 3 nm. This does indicate that there might be a small change in the tertiary structure of the protein under this pH condition. Hb Solution. Figure 1B, Supporting Information, shows the fluorescence spectra of 10-5 M Hb in different buffer solutions at pH ) 3, 6, and 9 (curves 1, 2, and 3 respectively). The spectra have been shifted vertically to bring out the differences between the peak positions. The fluorescence maxima for the solutions with pH ) 6 and 9 are observed to coincide (curves 2 and 3) while the emission from the pH ) 3 solution (curve 1) shows a small red shift of 5 nm. The fluorescence measurements for both the protein solutions at pH ) 3 indicate that there is a small perturbation to the protein tertiary structure under these conditions. Lamellar Structural Studies. XRD is a powerful tool used to determine the lamellar repetitive arrangement of electron-rich groups in protein films. The heme group in the Cyt c molecule scatters X-rays strongly and hence the

Langmuir, Vol. 17, No. 18, 2001 5655

protein-matrix composite film can be conveniently studied by XRD to determine the composite structure. Figure 2, Supporting Information, shows the XRD pattern recorded from a 2000 Å thick Cyt c-AA composite film formed at pH ) 6.8 revealing a lamellar phase with a repeat distance of 58.8 Å. In addition to the (00l) reflections arising from the Cyt c-AA lamellar phase (l being odd), the acid matrix also yielded characteristic Bragg reflections with a repeat distance of 49.02 Å. AA films with normal orientation of the hydrocarbon chains in the all-trans configuration are expected to yield a repeat distance of ca. 55 Å.29 This indicates that the as-deposited thermally evaporated AA film is organized with a ca. 27° tilt in the hydrocarbon chains with respect to the film surface normal. Assuming a dimension of ca. 35 Å for the Cyt c molecule,30 the bilayer thickness of 58.8 Å yields a large tilt of ca. 57° with respect to the film surface normal. Another possibility is interdigitation of the hydrocarbon chains in the bilayers. This is likely since the size of the protein is large and would dictate the extent of packing of the AA molecules. Such interdigitation has been observed in lamellar LB films of fatty amine films coordinated to bulky chloroplatinic acid ions.31 Each of the methods for intercalation of biomolecules has their own advantages/disadvantages. Our approach is rather similar to the work of Kunitake et al.4a-c wherein the intercalation of Cyt c and myoglobin (Mb) into multilayered films of synthetic bilayer membranes was demonstrated. They observed that the intercalation of Mb takes place between the polar inter-bilayer spaces of the membrane by attractive Coulombic interaction between local surface charges of Mb and the oppositely charged bilayer surface. They used the local charge of the protein for its diffusion into the membrane, irrespective of the overall charge of the protein.4b Electron spin resonance was used to study the orientation of heme proteins within the lipid bilayers, and it was found that the heme group is tilted at an angle of 15-20° relative to the bilayers.4a,c Since we also have used an anionic matrix for protein immobilization, the orientation of Cyt c relative to the AA bilayers is expected to be similar in this study. In our case we use the overall charge on the protein surface due to the ionization of amino acid residues as a consequence of pH of the solution, to drive the protein into the fatty lipid matrix. This gives better control over the diffusion of the protein into the lipid matrix with solution pH variation leading to facile control of the charge of the protein. On the other hand Crooks and co-workers9b use the charge on the protein by modifying its surface to drive it into a hyperbranched polymer matrix. This leads to the reduction of biocatalytic activity of the enzyme by 35%.9b Our protocol can be used to overcome this defect. Kumar et al.10 intercalated proteins into inorganic matrixes such as zirconium phosphates by electrostatic interactions. However, the time scales involved in the formation of the protein composite materials are of the order of a few days,10b in contrast to our technique where intercalation is achieved within a few minutes both for Cyt c and Hb by the use of appropriate film thickness, buffers, and preorganized films. Conclusions To conclude, it has been shown that the electrostatic interactions of a cationic protein (Cyt c) with an anionic (29) Ganguly, P.; Paranjape, D. V.; Chaudhari, S. K.; Patil, K. R. Langmuir 1992, 8, 2365. (30) Riccio, A.; Lanzi, M.; Antolini, F.; De Nitti, C.; Tavani, C.; Nicolini, C. Langmuir 1996, 12, 1545. (31) Ganguly, P.; Paranjape, D. V.; Sastry, M. J. Am. Chem. Soc. 1993, 115, 793.

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lipid matrix (AA) and that of an anionic protein (Hb) with a cationic (ODA) lipid film may be used to intercalate the proteins by a simple beaker-based immersion process. The extent of loading of the proteins in the composite films may be controlled by variation of the solution pH. The protein loading factor may also be controlled by depositing thicker lipid films with an additional degree of freedom provided by the chain length of the lipid amphiphiles. The rates of protein diffusion and the time scales involved in the biocomposite formation can be altered by the use of appropriate buffers and by preordering the lipid matrix. The electrostatic nature of the protein-lipid interaction permits facile “bleeding out” of the proteins from the composite into solution. Access of the proteins to biological analytes in solution is provided via hydrophilic channels in the films (Scheme 1) and is a consequence of lamellar ordering in the composite films. The protein molecules in the lipid films are immobilized without significant distortion to the native protein structure as evidenced by fluorescence and infrared spectroscopy studies. The elasticity of the bilayers may be primarily responsible for this and enables the matrix to adopt the contours of the protein molecule (Scheme 1). The reasonably fast time

Gole et al.

scales for the synthesis of the protein-lipid composites and the protein-friendly intercalation conditions are a major improvement over other techniques currently being investigated.4,5,7,10 The technique demonstrated above shows immense promise for the intercalation of other biomacromolecules such as DNA and extension to the generation of patterned protein-lipid composite structures and is currently being pursued in this laboratory. Acknowledgment. A.G. and J.K. thank the Council for Scientific and Industrial Research (CSIR), Government of India, for financial assistance. Partial funding of this project by the Indo-French Centre for Promotion of Advanced Scientific Research (IFCPAR) is gratefully acknowledged. Supporting Information Available: Figures corresponding to fluorescence spectra from the different protein solutions and the XRD pattern recorded from a 2000 Å thick Cyt c-AA composite film. This material is available free of charge via the Internet at http://pubs.acs.org. LA010571K

Protein-Friendly Intercalation of Cytochrome c and Hemoglobin into

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