Anal. Chem. 1998, 70, 5111-5113
Correspondence
Protein Adsorption on Nanocrystalline TiO2 Films: An Immobilization Strategy for Bioanalytical Devices Emmanuel Topoglidis, Anthony E. G. Cass, Gianfranco Gilardi, Sheila Sadeghi, Nicholas Beaumont, and James R. Durrant*
Department of Biochemistry, Imperial College of Science, Technology and Medicine, South Kensington, London SW7 2AY, U.K.
We have investigated the use of optically transparent, nanoporous TiO2 films as substrates for protein immobilization. Immobilization on such films may be readily achieved from aqueous solutions at 4 °C. The nanoporous structure of the film greatly enhances the active surface area available for protein binding (by a factor of 150 for a 4-µm-thick film). We demonstrate that the redox state of immobilized cytochrome c may be modulated by the application of an electrical bias potential to the TiO2 film and that the fluorescence yield of immobilized fluorophore-labeled maltose-binding protein may be used to monitor specifically maltose concentration. We conclude that nanoporous TiO2 films may be useful both for basic studies of protein/electrode interactions and for the development of array-based bioanalytical devices employing both optical and electrochemical signal transduction methodologies. There is currently considerable interest in the use of sol-gel methods for the encapsulation of biological macromolecules, aimed primarily at the development of optical bioanalytical devices,1-4 but also at providing a controlled environment for studies of biomolecular function.5-7 These studies have focused upon the encapsulation of a range of proteins in silica glasses, where the porosity of such glasses allows the diffusion of small analyte molecules to the immobilized protein. The high biomolecule loading per unit area and the optical transparency of the glass makes this approach particularly suitable for optical signal * Corresponding author: (e-mail)
[email protected]; (fax) +44 171 2250960. (1) Dave, B. C.; Dunn, B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994, 66, 1120 A-1127 A. (2) Ellerby, L. M.; Nishida, C. R.; Nishida, F.; Yamanaka, S. A.; Dunn, B.; Valentine, J. S.; Zink, J. I. Science 1992, 255, 1113. (3) Dunn, B.; Miller, J. M.; Dave, B. C.; Valentine, J. S.; Zinc, J. I. Acta Mater. 1998, 46, 737-741. (4) Lev, O.; Tsionsky, M.; Rabinovich, L.; Glezer, V.; Sampath, S.; Pankratov, I.; Gun, J. Anal. Chem. 1995, 67, 22A-30A. (5) Ji, Q.; Lloyd, C. R.; Ellis, W. R.; Eyring, E. M. J. Am. Chem. Soc. 1998, 120, 221-222. (6) Shen, C.; Kostic, N. M. J. Am. Chem. Soc. 1997, 119, 1304-1312. (7) Miller, J. M.; Dunn, B.; Valentine, J. S.; Zinc, J. I. J. Non-Cryst. Solids 1996, 202, 279-289. 10.1021/ac980764l CCC: $15.00 Published on Web 10/22/1998
© 1998 American Chemical Society
transduction methodologies. The encapsulation of biological molecules in sol-gel materials requires stringent control over gelation processes because the biomolecules tend to denature in environments with high alcohol concentration and/or extreme pH values as well as elevated temperatures.8 This has motivated the development of modified versions of the gelation processes, which employ milder environmental conditions to minimize denaturation of the encapsulated biomolecules.3 In this paper we consider an alternative substrate for protein immobilization: preformed nanoporous titanium dioxide films. TiO2 is a wide band-gap (∼3 eV) and therefore optically transparent, semiconductor. It is environmentally benign, with for example TiO2 particles being widely used as additives in toothpaste and white paint. Nanoporous TiO2 films can be readily prepared by low-cost screen-printing technologies.9 Such films typically comprise 10-20-nm crystalline particles of TiO2 which are densely packed to form a porous structure with a high active surface area (∼300 times greater than a flat surface for a 4-µm-thick film). In addition to their optical transparency and high surface area, these films exhibit excellent stability and good electrical conductivity (at potentials above the conduction band edge). Such films are currently being widely studied for applications ranging from photoelectrochemical solar cells to lithium batteries.9,10 The high surface area, conductivity, and optical transparency suggest they may also provide a suitable surface for bioanalytical applications. However, we are not aware of any previous reports of the use of such films for protein immobilization. Previous studies have demonstrated that dyes may be readily adsorbed from solution to the surface of nanoporous TiO2 films.9 Strong adsorption may, for example, be achieved by the addition of carboxylate groups to the dye, which are thought to chelate to Ti4+ centers on the oxide surface. In this study, we consider the analogous adsorption of two proteins to these films: horse heart cytochrome c (Fe Cyt-c) and maltose-binding protein (MBP) site (8) Lapanje, S. Physicochemical Aspects of Protein Denaturation; Wiley: New York, 1978; p 331. (9) O’Regan, B.; Gra¨tzel, M. Nature 1991, 353, 737-3740. (10) Hagfeldt, A.; Vlachopoulos, N.; Gra¨tzel, M. J. Electrochem. Soc. 1994, 141, L82.
Analytical Chemistry, Vol. 70, No. 23, December 1, 1998 5111
Figure 1. Absorption spectrum of a 4-µm-thick nanoporous TiO2 film before and after the immobilization of Fe Cyt c. Spectra are shown for both oxidized Fe(III) and electrochemically reduced Fe(II) immobilized Cyt c/TiO2 films.
specifically labeled with the fluorophore IANBD (S337C-NBD).11 This labeled MBP is of particular interest for the development of reagentless fluorescence biosensors; the fluorescence yield of the attached fluorophore exhibiting a strong increase following binding of maltose or β-cyclodextrin.11-13 Nanoporous TiO2 films were prepared from an aqueous suspension of 13-nm TiO2 colloids purchased from Solaronix S. A. After the addition of Carbowax, this suspension was spread onto glass slides, dried in air, and then heated to 450 °C for 25 min, resulting in 4-µm-thick films, as described previously.9 Protein immobilization was achieved by the immersion of 1-cm2 TiO2 films in aqueous protein solutions at 4 °C for 1-5 days. The immobilization solutions comprised 0.035 mg mL-1 Fe Cyt-c in 10 mM NaH2PO4 buffer solution (pH 6.5) and 0.025 mg mL-1 labeled MBP in 10 mM KH2PO4 buffer solution (pH 5.0). Prior to all spectroscopic measurements, films were removed from the immobilization solution and rinsed in buffer solution to remove all nonimmobilized protein. Storage of the resulting films in protein-free buffer solutions for up to 1 week resulted in no detectable desorption of the protein from the film. Immobilization of both Fe Cyt-c and MBP on the TiO2 films reached a limiting amount after incubation of the film in the protein solutions for 5 days at 4 °C (∼50% of the final adsorption was achieved within 1.5 days). Figure 1 shows the absorption spectra of an Fe Cyt-c/TiO2 film following 5 days of incubation. Also shown is the absorption spectrum of the TiO2 film prior to protein immobilization, showing the characteristic absorption increase below 400 nm due to the onset of TiO2 band gap excitation. The spectrum of Fe(II) Cyt-c/TiO2 film shows the characteristic heme absorption bands at 416, 521, and 550 nm, in good agreement with the solution spectrum of this protein14 and indicating that immobilization of the cytochrome in the film does not cause denaturation (denaturation of the Fe Cyt-c causes a blue (11) Gilardi, G.; Zhou, L. Q.; Hibbert, L.; Cass, A. E. G. Anal. Chem. 1994, 66, 3840-3847. (12) Gilardi, G.; Mei, G.; Rosato, N.; Argo, A. F.; Cass, A. E. G. Protein Eng. 1997, 10, 479-486. (13) Marvin, J. S.; Corcoran, E. E.; Hattangadi, N. A.; Zhang, J. V.; Gere, S. A.; Hellinga, H. W. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4366-4371. (14) Wood, P. M. Biochim. Biophys. Acta 1984, 768, 293-317.
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shift of the Soret peak to 408 nm15). The immobilized cytochrome moreover retained its redox activity: it could be readily cycled between its Fe(II) and Fe(III) states by, respectively, the addition of sodium diothionite and bubbling the buffer solution with oxygen. It could moreover be readily reduced electrochemically by the incorporation of the Cyt c/TiO2 film as the working electrode of a photoelectrochemical cell. The application of a negative potential (e-0.3 V versus SCE) to the electrode resulted in the reversible reduction of Fe(III) cytochrome to Fe(II), as illustrated in Figure 1. Employing an extinction coefficient of 129 100 M-1 cm-1 at 416 nm for Fe(II) Cyt-c, we estimate from Figure 1 a protein loading of 3.5 nmol in the 4-µm-thick, 1-cm2 film. Assuming a cross-sectional area for Cyt-c of 7 nm2, this indicates a protein loading 150 times greater than that achieved for monolayer coverage of a flat electrode surface, which we attribute to protein immobilization in the pores of nanocrystalline TiO2 film. Consistent with this conclusion, we find that protein adsorption after 5 days is proportional to film thickness, with the use of a thicker, 8-µm film resulting in an ∼2-fold increase in the amount of protein adsorbed. Comparison with dye-binding studies suggests the Cyt c achieves a 60 ( 10% coverage of the internal surface area of the film available for dye binding. The somewhat lower binding achieved with the protein is most probably due to the larger dimensions of Cyt-c (diameter ∼3 nm), which will prevent access to smaller pores of the film. Analogous studies of the binding of MBP, achieved by monitoring the IANBD label absorption at 500 nm, indicated a 20 ( 4% coverage, consistent with the larger dimensions, (diameter ∼7 nm) of this protein. The functional integrity of the immobilized fluorophore-labeled MBP protein was assessed by determination of its fluorescence yield as a function of substrate concentration. Previous solution studies have demonstrated that the fluorescence yield of the labeled MBP increases as a function of maltose or β-cyclodextrin concentration.11,12 This fluorescence increase is specific to these sugars, the addition of, for example, sucrose to labeled MBP in solution resulting in no detectable change in fluorescence yield. Figure 2 shows the corresponding data obtained for labeled MBP immobilized upon the TiO2 film. A large increase in fluorescence intensity of MBP/TiO2 was observed following the addition of maltose (41%) and β-cyclodextrin (35%), while the intensity was essentially independent of the addition of sucrose. The fluorescence increase was fully reversible; rinsing in buffer solution resulted in complete maltose unbinding with only minor MBP desorption (
