12824
J. Phys. Chem. B 2008, 112, 12824–12826
Streptavidin Reduces Oxygen Quenching of Biotinylated Ruthenium(II) and Palladium(II) Complexes T. Soller,† M. Ringler,† M. Wunderlich,† T. A. Klar,*,† J. Feldmann,† H.-P. Josel,‡ J. Koci,‡ Y. Markert,‡ A. Nichtl,‡ and K. Ku¨rzinger‡ Photonics and Optoelectronics Group, Department of Physics and CeNS, Ludwig-Maximilians-UniVersita¨t Mu¨nchen, Amalienstrasse 54, 80799 Munich, Germany, and Roche Diagnostics GmbH, Nonnenwald 2, 82371 Penzberg, Germany ReceiVed: May 19, 2008; ReVised Manuscript ReceiVed: July 16, 2008
Transition metal complexes such as biotinylated ruthenium(II) tris(bipyridyl) and palladium(II) porphyrin show an increase in luminescence intensity and lifetime upon binding to streptavidin in aqueous solution. Here we show that this increase of luminescence lifetime and intensity are caused by the shielding of the transition metal complexes from dissolved oxygen through streptavidin rather than by hydrophobicity effects as recently claimed. The very strong binding affinity of biotin toward avidins and in particular to streptavidin has found a vast spectrum of applications in biophysics, biochemistry, immunology and clinical diagnostics.1,2 Besides the strength of the interaction, the streptavidin-biotin system is particularly useful because the four binding sites of streptavidin allow for the cross-linking of different biotinylated compounds. However, the most straightforward approach to a fluorescence-based streptavidin-biotin assay, i.e. the use of a fluorophore-biotin conjugate bound to streptavidin, suffers from a severe drawback: Many biotinylated organic dyes lose their fluorescence upon binding to streptavidin,3 because of the formation of nonfluorescent ground-state complexes with the amino acid tryptophan inside each biotin binding pocket of streptavidin.4,5 Differently, transition metal complexes are bulky, sterically demanding molecules. Therefore, steric hindrance prevents the formation of ground-state complexes. Further, long-lived metal complexes are particularly suitable for biosensing applications, as they allow one to cut off autofluorescence of biological samples by time-gated luminescence detection and they allow for time-resolved measurements using instrumentation with lower demand on time resolution compared to fluorescent labels.6,7 In contrast to fluorophores, recently developed conjugates of transition metal complexes and biotin show increased luminescence intensity and lifetimes upon binding to avidin.8-11 This effect has so far been solely attributed to the hydrophobicity of the avidin binding pocket, since the quantum yields and lifetimes of the investigated complexes increase when they are transferred from more polar to nonpolar environments.12 Due to these observations, it has been concluded that streptavidin increases the hydrophobicity in its immediate environment and therefore the luminescence of biotinylated transition metal complexes is increased upon binding to streptavidin.8,12 In contrast, we reveal in this contribution, that the increase of luminescence intensity and lifetime of two transition metal * Corresponding author. Present address: Institute of Physics and Institute of Micro- and Nanotechnologies, Technical University of Ilmenau, 98684 Ilmenau, Germany. † Photonics and Optoelectronics Group, Department of Physics and CeNS, Ludwig-Maximilians-Universita¨t Mu¨nchen. ‡ Roche Diagnostics GmbH.
SCHEME 1: Structure of Ru(bpy)3Bi and Pd-Por-Bi
complexes upon binding to streptavidin is caused by a reduced oxygen quenching of the triplet states due to shielding through streptavidin. The transition metal complexes are a biotinylated ruthenium(II) tris(bipyridyl) complex (Ru(bpy)3Bi) and a biotinylated palladium(II) porphyrin (Pd-Por-Bi). Both biotinylated complexes are sketched in Scheme 1. Ru(bpy)3Bi has been synthesized by reaction of [4-(N-succinimidyloxycarbonylpropyl)-4′-methyl-2,2′-bipyridine]bis(2,2′-bipyridine)ruthenium(II)13,14 with biotinyl-3,6-dioxaoctanediamine according to a standard NHS-ester/amine reaction, as described e.g. in ref 15. Pd-Por-Bi has been synthesized according to the following procedure. After the synthesis of the porphyrin derivate (4-[10,15,20-tris(4-sulfophenyl)-21H,23H-porphin-5yl]-benzoic acid)sas described in ref 16sthe palladium complex of the porphyrin derivative is synthesized by refluxing a solution of 10 mg of the complex with 11 mg PdCl2 in 3 mL of DMF for 8 h. After filtration, the complex is isolated by evaporation. The reaction of the product with biotinyl-3,6-dioxaoctanediamine is performed by using a standard conjugation procedure with HBTU, triethylamine in DMF, room temperature, over-
10.1021/jp8044065 CCC: $40.75 2008 American Chemical Society Published on Web 09/11/2008
Streptavidin Reduces Oxygen Quenching
J. Phys. Chem. B, Vol. 112, No. 40, 2008 12825
Figure 1. (a, b) Comparison of the absorption (OD) (a) and photoluminescence (PL) (b) spectra of two equally concentrated solutions of Ru(bpy)3Bi (black) and nonbiotinylated Ru(bpy)3 (cyan) in aerated solution. c) Time-resolved luminescence transients of Ru(bpy)3Bi (black) and Ru(bpy)3 (cyan) in aerated solution. Green line: PL transient of Ru(bpy)3Bi under deaerated conditions.
night, N2, and purification on silica gel with dichloromethane and methanol as eluents. Biotinylation does not significantly change the photophysical properties of Ru(bpy)3Bi, as can be seen from Figure 1. Ru(bpy)3Bi (black lines) shows the same absorption and emission intensities as nonfunctionalized Ru(bpy)3 (cyan lines), apart from a minor 7 nm red shift of the absorption and the emission spectra (Figure 1a,b). Time-resolved measurements of Ru(bpy)3Bi and Ru(bpy)3 in air-saturated aqueous solution reveal identical monoexponential decay profiles with a lifetime τ ) 380 ns (Figure 1c, black and cyan lines, respectively). The quantum yield η of Ru(bpy)3Bi in aerated aqueous solution amounts to 0.027. Under deaerated conditions, which were achieved by bubbling the solution with Argon for 15 min., the luminescence lifetime of Ru(bpy)3Bi increases to 592 ns (Figure 1c, green line), which is in good agreement with the corresponding published luminescence lifetime of Ru(bpy)3.17 Thus, the photophysical properties of Ru(bpy)3 are preserved upon functionalization with biotin. Similarly, the spectroscopic data of the biotinylated Pd-Por-Bi used in this work correspond to published data for unbiotinylated Pd-Por.18 We note here that Pd-Por-Bi is more sensitive to oxygen quenching than Ru(bpy)3Bi. In a degassed aqueous solution, Pd-Por-Bi possesses a monoexponential lifetime of 450 µs and a quantum yield of 0.028. In O2-saturated solution, the lifetime shortens to 3 µs and the quantum yield decreases correspondingly.19 Parts a and b of Figure 2 show the luminescence spectra before binding to streptavidin (black curves) and after binding to streptavidin (red curves). Both phosphorescent transition metal complexes Pd-Por-Bi and Ru(bpy)3Bi show enhanced luminescence when interacting with streptavidin in air-saturated aqueous solution. The luminescence of Pd-Por-Bi and Ru(bpy)3Bi increase by a factor 5.7 and 1.1, respectively, upon binding to streptavidin. We note in passing that in the case of Pd-Por-Bi only the long-wavelength triplett-emission bands are affected, while the emission from the short wavelength singlet emission bands remain unaffected upon streptavidin binding. Our observations are in line with previous reports on luminescence enhancement of Ru(bpy)3 upon streptavidin binding.8-11 Specifically, an increase by a factor of 1.4 was found in case of a 4 atom linker between Ru(bpy)3 and the biotin moiety and an increase of 1.2 was found in case of an 8 atom spacer.10 This increase has been attributed to a hydrophobicity effect of the avidin binding pocket.8 In contrast to this explanation, we show in the following that the reduction of oxygen quenching is the true nature of luminescence enhancement when complexes Ru(bpy)3Bi and Pd-Por-Bi are bound to streptavidin in aerated aqueous solution. To show this, we have performed time-resolved luminescence spectroscopy.
Figure 2. Photoluminescence (PL) spectra (a, b) and transients (c-f) in absence (black curves) and presence (red curves) of streptavidin (SA). Left hand side: Results for Pd-Por-Bi: photoluminescencence (a) and transients in aerated solution (c). (e) transients in deaerated solution (e). Right hand side: Results for Ru(bpy)3Bi: photoluminescencence (b) and transients in aerated solution (d). (f) transients in deaerated solution. In aerated solutions, the luminescence lifetimes are increased upon SA binding (c, d). No such effect is observed in deareated solutions (e, f).
Figure 2c shows time-resolved luminescence transients of Pd-Por-Bi in an aerated solution in the absence (black line) and presence (red line) of streptavidin. We observe an elongation of the luminescence lifetime from 3 to 15 µs in the presence of streptavidin. This can be explained by a weakening of the nonradiative decay channels upon streptavidin binding. Most important, a control experiment under deaerated conditions shows no increase in the phosphorescence intensity and lifetime after the addition of streptavidin (Figure 2e). Similar results are obtained in case of Ru(bpy)3Bi, where in an aearated solution an increase of the luminescence lifetime from 380 to 408 ns is found when streptavidin is added (Figure 2d). Again, the fluorescence intensity and lifetime remain unchanged upon addition of streptavidin in deaerated solution (Figure 2f). These results clearly show that the luminescence enhancement emanates from the shielding of Ru(bpy)3Bi and Pd-Por-Bi from dissolved oxygen through the bulky streptavidin molecule. Streptavidin renders the complex partly inaccessible to dissolved O2. Oxygen quenching of Ru(bpy)3Bi and Pd-Por-Bi is reduced, resulting in the observed lifetime elongation and luminescence enhancement. As noted above, a luminescence increase of metal complexes upon binding to avidin was recently attributed to the hydrophobicity of the avidin binding pocket.12 The examined transition metal complex - biotin conjugates revealed an increase in phosphorescence intensity and lifetime when transferred from
12826 J. Phys. Chem. B, Vol. 112, No. 40, 2008 more polar to nonpolar solvents with lower electron acceptor number.20 However, our control studies of streptavidin binding in deaerated solution (Figure 2d,f) clearly show that a change of local hydrophobicity due to streptavidin binding cannot explain the luminescence enhancement upon streptavidin binding. If that was true, then this effect should also be observable in deaerated solutions, a fact that we do not observe (Figure 2d,f). Therefore, we deduce that the luminescence enhancement is not caused by a diminished charge transfer rate from the excited-state to solvent molecules due to increased hydrophobicity upon streptavidin binding.20 Instead, it is caused by a reduction of oxygen quenching due to a shielding from oxygen by streptavidin. Our findings are also supported by other studies, where a decrease in the oxygen quenching of metal complexes upon direct attachment to albumin or immunglobulin,21 to BSA or amino-dextran22 or by the use of dendritic bpy ligands23 has been reported. In conclusion, we have shown that the phosphorescence intensities and lifetimes of both Ru(bpy)3Bi and Pd-Por-Bi increase when the complexes bind to streptavidin in O2-saturated solution, but no such increase is found in deaerated solution. Therefore, the enhancements are attributed to the shielding of the phosphorescent complexes from dissolved oxygen through streptavidin. Our findings are relevant for the design and optimization of assays for biotinylated biomolecules based on luminescent transition metal complexes.24 In particular, such long-lived luminescence sensor molecules have an intrinsic advantage in biosensing because they allow to cut off autofluorescence of biological samples by time-gated luminescence detection.6,7 As our results give a detailed explanation of luminescence enhancement and lifetime elongation of transition metal complexes in the presence of streptavidin, improved biosensors may become feasible, which for example rely on a lifetime change upon streptavidin binding. Acknowledgment. We thank A. Helfrich and W. Stadler for excellent technical assistance. Financial support by the Bayerische Forschungsstiftung and by the German Excellence Initiative via the “Nanosystems Initiative Munich (NIM)” is gratefully acknowledged.
Soller et al. References and Notes (1) Hermanson, G. T. Bioconjugate techniques; Academic Press: San Diego, CA, 2006. (2) Wilchek, M.; Bayer, E. A. Methods Enzymol.; Academic Press: San Diego, CA, 1990; Vol. 184. (3) Gruber, H. J.; Marek, M.; Schindler, H.; Kaiser, K. Bioconjugate Chem. 1997, 8, 552–559. (4) Marme, N.; Knemeyer, J. P.; Sauer, M.; Wolfrum, J. Bioconjugate Chem. 2003, 14, 1133–1139. (5) Freitag, S.; LeTrong, I.; Klumb, L.; Stayton, P. S.; Stenkamp, R. E. Protein Sci. 1997, 6, 1157–1166. (6) Beverloo, H. B.; van Schadewijk, A.; van Gelderen-Boele, S.; Tanke, H. J. Cytometry 1990, 11, 784–792. (7) de Haas, R. R.; van Gijlswijk, R. P. M.; van der Tol, E. B.; Veuskens, J.; van Gijssel, H. E.; Tijdens, R. B.; Bonnet, J.; Verwoerd, N. P.; Tanke, H. J. J. Histochem. Cytochem. 1999, 47, 183–196. (8) Lo, K. K. W.; Hui, W. K.; Ng, D. C. M. J. Am. Chem. Soc. 2002, 124, 9344–9345. (9) Lo, K. K. W.; Chan, J. S. W.; Lui, L. H.; Chung, C. K. Organometallics 2004, 23, 3108–3116. (10) Lo, K. K. W.; Lee, T. K. M. Inorg. Chem. 2004, 43, 5275–5282. (11) Slim, M.; Sleiman, H. F. Bioconjugate Chem. 2004, 15, 949–953. (12) Lo, K. K. W.; Hui, W. K.; Chung, C. K.; Tsang, K. H. K.; Lee, T. K. M.; Li, C. K.; Lau, J. S. Y.; Ng, D. C. M. Coord. Chem. ReV. 2006, 250, 1724–1736. (13) Massey, R. J.; Powell, M. J.; Mied, P. A.; Feng, P.; Della Ciana, L. Electrochemiluminescent Assays; 1987;Patent WO 87/06706. (14) Staffilani, M.; Ho¨ss, E.; Giesen, U.; Schneider, E.; Hartl, F.; Josel, H. P.; De Cola, L. Inorg. Chem. 2003, 42, 7789–7798. (15) Sekine, M.; Okada, K.; Seio, K.; Obata, T.; Sasaki, T.; Kakeya, H.; Osada, H. Bioorg. Med. Chem. 2004, 12, 6343–6349. (16) Schmidt, D.; Steffen, H., Labelled molecules for fluorescence immunoassays and processes and intermediates for their preparation; 1984; Patent EP 127797 B1. (17) Roundhill, D. M. Photochemistry and Photophysics of Metal Complexes; Plenum Press: New York, 1994. (18) Kalyanasundaram, K.; Neumann-Spallart, M. J. Phys. Chem. 1982, 86, 5163–5169. (19) Gewehr, P. M.; Delpy, D. T. Med. Biol. Eng. Comput. 1993, 31, 11–21. (20) Nakamaru, K. Bull. Chem. Soc. Jpn. 1982, 55, 1639–1640. (21) Terpetschnig, E.; Dattelbaum, J. D.; Szmacinski, H.; Lakowicz, J. R. Anal. Biochem. 1997, 251, 241–245. (22) O’Donovan, C.; Hynes, J.; Yashunski, D.; Papkovsky, D. B. J. Mater. Chem. 2005, 15, 2946–2951. (23) Issberner, J.; Vögtle, F.; DeCola, L.; Balzani, V. Chem.sEur. J. 1997, 3, 706–712. (24) Soller, T.; Ringler, M.; Wunderlich, M.; Klar, T. A.; Feldmann, J.; Josel, H. P.; Markert, Y.; Nichtl, A.; Ku¨rzinger, K. Nano Lett. 2007, 7, 1941–1946.
JP8044065
