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Langmuir 1999, 15, 3920-3923
Surface Plasmon Resonance Characterization of Photoswitchable Antigen-Antibody Interactions Evgeny Kaganer,1a Roman Pogreb,1b,c Dan Davidov,1b and and Itamar Willner*,1a Institute of Chemistry and the Racah Institute of Physics, The Hebrew University of Jerusalem, Jerusalem 91904, Israel, and Research Institute, College of Judea and Samaria, Ariel 44 837, Israel Received September 20, 1998. In Final Form: March 12, 1999 Photostimulated association and dissociation of anti-dinitrophenyl antibody, anti-DNP-Ab, to and from a dinitrospiropyran photoisomerizable monolayer is probed by surface plasmon resonance (SPR). The anti-DNP-Ab binds to a dinitrospiropyran antigen monolayer, whereas the DNP-Ab dissociates from the protonated dinitromerocyanine monolayer. Reversible binding and dissociation of anti-DNP-Ab to and from the photoisomerizable monolayer was assayed by SPR spectroscopy. Analysis of the SPR data indicates that the average thickness of the antibody layer is ca. 70 Å which corresponds to a surface coverage of ca. 4.4 × 10-9 mol‚cm-2.
Photoswitchable activation of biomaterial functions is of substantial interest for the development of optobioelectronic systems.2,3 Photochemical activation of enzymes was accomplished by the covalent linkage of photoisomerizable groups to the proteins,4 reconstitution of apoflavoenzymes with a photoisomerizable FAD semisynthetic cofactor,5 the application of photoisomerizable electron mediators for the light-stimulated electrical contacting of redox enzymes and electrodes,6 and by the use of photoisomerizable monolayer-modified electrodes as photocommand surfaces that control the electron transfer between redox proteins and the conductive support.7,8 Photoinduced control of antigen-antibody interactions was accomplished by the application of photoisomerizable antigens,9 and the possibility of using the photostimulated binding interactions to develop reversible immunosensors was addressed.10 Figure 1 shows the method to control the binding interactions between an antibody and a photoisomerizable antigen associated with a surface, and the method of using the approach to assemble reusable immunosensor systems. The photoisomerizable antigen is assembled on an electronic transducer to yield an immunosensor. The antigen * Corresponding author telephone, 972-2-6585272; fax, 972-26527715; e-mail,
[email protected]. (1) (a) The Institute of Chemistry, The Hebrew University of Jerusalem. (b) Racah Institute of Physics, The Hebrew University of Jerusalem. (c) Research Institute, College of Judea and Samaria, Ariel. (2) (a) Willner, I. Acc. Chem. Res., 1997, 30, 347-356. (b) Willner, I.; Willner, B. Adv. Mater. 1995, 7, 787. (3) Willner, I.; Rubin, S. Angew. Chem., Int. Ed. Engl. 1996, 35, 367. (4) (a) Willner, I.; Rubin, S.; Riklin, A. J. Am. Chem. Soc. 1991, 113, 3321. (b) Willner, I.; Rubin, S.; Wonner, J.; Effenberger, F.; Ba¨uerle, P. J. Am. Chem. Soc. 1991, 114, 3150. (c) Willner, I.; Rubin, S.; Cohen, Y. J. Am. Chem. Soc. 1993, 115, 4937. (d) Lion-Dagan, M.; Katz, E.; Willner, I. J. Am. Chem. Soc. 1994, 116, 7913. (e) Willner, I.; Lion-Dagan, M.; Marx-Tibbon, S.; Katz, E. J. Am. Chem. Soc. 1995, 117, 6581. (5) Willner, I.; Blonder, R.; Katz, E.; Stocker, A.; Bu¨ckmann, A. F. J. Am. Chem. Soc. 1996, 118, 5310. (6) Lion-Dagan, M.; Marx-Tibbon, S.; Katz, E.; Willner, I. Angew. Chem., Int. Ed. Engl. 1995, 34, 1604. (7) Lion-Dagan, M.; Katz, E.; Willner, I. J. Chem. Soc., Chem. Commun. 1994, 2741. (8) Willner, I.; Doron, A.; Katz, E.; Levi, S.; Frank, A. J. Langmuir 1996, 12, 946. (9) Willner, I.; Blonder, R.; Dagan, A. J. Am. Chem. Soc. 1994, 116, 3121. (10) (a) Willner, I.; Blonder, R.; Dagan, A. J. Am. Chem. Soc. 1994, 116, 9365. (b) Blonder, R.; Levi, S.; Tao, G.; Ben-Dov, I.; Willner, I. J. Am. Chem. Soc. 1997, 119, 10467.
Figure 1. Cyclic photoswitchable binding and dissociation of an antibody to and from a photoisomerizable antigen monolayer modified surface, respectively.
in state “A” exhibits affinity for the antibody, and interaction with the antibody results in the antigenantibody complex on the transducer. The formation of the affinity complex is transduced as an electronic or photonic signal. After completion of the first sensing cycle, the sensing interface is photoisomerized to state “B”. The latter isomer lacks antigen features, or affinity, for the antibody. This enables the washing-off of the antibody. By a secondary irradiation process, the original active sensing layer of the antigen form “A” is regenerated, establishing a general route for the reuse of the immunosensor. A dinitrospiropyran monolayer assembled onto a Au electrode revealed photoisomerizable properties and reversible sensing of the anti-dinitrophenyl antibody DNP-Ab.10 The cyclic sensing of the DNP-Ab by the photoisomerizable antigen monolayer was electronically transduced using electrochemical means, e.g., amperometric10 or impedometric11 signals, or piezoelectric quartz crystal microbalance (QCM) transduction.10 Surface plasmon resonance (SPR) spectroscopy is a rapidly developing technique to characterize optical changes of the metal near metal-dielectric interfaces.12 The surface plasmon existing at a metal-dielectric interface is resonantly excited by p-polarized light when the surface wave vectors of incident radiation and the surface plasmon are matched. This results in a minimum in the reflected light intensity. Thus, any perturbation in (11) Patolsky, F.; Filanovsky, B.; Katz, E.; Willner, I. J. Phys. Chem. B 1998, 102, 10359. (12) Frutos, A. G.; Corn, R. M. Anal. Chem. 1998, 70, 449A.
10.1021/la981147v CCC: $18.00 © 1999 American Chemical Society Published on Web 05/07/1999
Photoswitchable Antigen-Antibody Interactions
the dielectric properties of the metal-solution interface, i.e., as a result of the adsorption or binding of chemicals on the metal support, is expected to alter the SPR conditions. Indeed, SPR has been extensively used to characterize the formation of monolayers13 or thin films14 on metal supports. The binding of proteins and other biomaterials on metal surfaces was examined by SPR.15 Specifically, SPR was used to characterize antigenantibody affinity complexes on the metal support16,17 and SPR affinity immunosensors were assembled.18 Here we wish to report on the characterization of photostimulated binding and dissociation of the DNP-Ab to and from a photoisomerizable antigen monolayer using SPR spectroscopy. Experimental Section Mercaptobutyl dinitrospiropyran, (1a), was prepared as reported in the literature.8 Anti-dinitrophenyl antibody, antiDNP-Ab, (Sigma) was used without further purification. Gold was evaporated onto a glass substrate El-OP, SF10, (refractive index n ) 1.72 at λ ) 0.63 µm) to form a thin film (ca. 500 Å). Film thickness was determined by specular X-ray reflectivity or microgravimetric quartz crystal microbalance analysis. The SPR experiments were performed on a gold-coated glass substrate mounted onto a flow cell (Teflon) equipped with a quartz window for in situ irradiation of the gold substrate. Onto the gold/glass substrate a prism (n ) 1.72) was mounted and an immersion liquid was used to adjust the refractive indexes. The surface plasmon resonance was recorded by attenuated total reflection using Kretschmann geometry. The cell was mounted on a two-stage goniometer table that enables the independent rotation of the sample or the detector with a resolution of ca. 1 × 10-3 degree. The rotation of the sample and data accumulation was computer controlled. A He-Ne laser (λ ) 0.63 µm) was used as a light source. The laser light was passed through a polarizer prior to interaction with the cell, and SPR curves were recorded by following the reflected light intensity at different incident angles. The experimental curves were analyzed by the transfer matrix method and fitted using the layer thickness and dielectric permittivity as variables. The gold layer was modified with mercaptobutyl dinitrospiropyran, (1a), by the introduction of a 2 × 10-3 M solution of (1a) in CH2Cl2, and interaction for 1 h, until no change in the SPR could be detected. Interaction of the (1a) antigen monolayer with anti-DNP-Ab was performed by initial rinsing of the cell with phosphate buffer, pH ) 7.4, followed by the interaction of the surface with a phosphate buffer solution, pH ) 7.4, that included anti-DNP-Ab 24 × 10-6 g‚mL-1. Photoisomerization of the monolayer and photoswitchable binding and dissociation of the DNP-Ab to and from the monolayer was performed by the in situ irradiation of the antigen layer (13) (a) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11, 4383. (b) Peterlinz, K. A.; Georgiadis, R. Langmuir 1996, 12, 4731. (c) Lang, H.; Duschl, C.; Gra¨tzel, M.; Vogel, H. Thin Solid Films 1992, 210/211, 818. (14) (a) Pogreb, R.; Cohen, G.; Tarabia, M.; Davidov, D.; Levine, M.; Sandomirsky, V. J. Appl. Phys. 1995, 78, 3323. (b) Cohen, G.; Pogreb, R.; Tarabia, M.; Davidov, D.; Keller, P. Ferroelectrics 1996, 181, 227. (c) Hockel, W.; Knoll, W. Thin Solid Films 1990, 187, 349. (d) Barnes, W. L.; Sambles, J. R. Surf. Sci. 1987, 183, 189. (15) (a) Spinke, J.; Liley, M.; Guder, H.-J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821. (b) Spinke, J.; Yang, J.; Wolf, H.; Liley, M.; Ringsdorf, H.; Knoll, W. Biophys. J. 1992, 63, 1667. (c) Jordan, C. E.; Corn, R. M. Anal. Chem. 1992, 69, 1449. (d) Jordan, C. E.; Frey, B. L.; Kornguth, S.; Corn, R. M. Langmuir 1994, 10, 3642. (e) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993, 9, 1361. (16) Lukosz, W. Biosens. Bioelectron 1991, 6, 215. (17) (a) Kooyman, R. P. H.; Kolkman, H.; Van Gent, J.; Greve, J. Anal. Chim. Acta 1988, 213, 35. (b) Malmqvist, M. Curr. Opin. Immunol. 1993, 5, 282. (c) Flanagan, M. T.; Pautell, R. H. Electron. Lett. 1984, 20, 968. (18) Lo¨fa´s, S.; Malmqvist, M.; Ro¨nnberg, I.; Stenberg, E.; Lieberg, B.; Lundstro¨m Sens. Actuators, B 1991, 5, 79. (19) Cohen, G.; Pogreb, R.; Tarabia, M.; Davidov, D.; Keller, P. Proceedings of the 5th International Conference on Ferroelectric Liquid Crystals, Ferrolectrics 1996, 181, 227.
Langmuir, Vol. 15, No. 11, 1999 3921 through the quartz window. The dinitromerocyanine monolayer was generated by irradiation at the surface with a Hg pencil lamp source (Oriel 6042 long-wave filter), 360 nm < λ < 380 nm. The dinitrospiropyran monolayer was generated by the irradiation of the surface with a 150 W Xe-arc lamp (Oriel), λ > 495 nm. Photoswitchable dissociation of the DNP-Ab from the interface was performed by the initial photoisomerization of the monolayer that included the dinitrospiropyran/DNP-Ab complex to the protonated dinitromerocyanine state, followed by rinsing of the cell with water for 40 min.
Results and Discussion A photoisomerizable dinitrospiropyran monolayer was assembled on a Au thin film (ca. 500 Å) evaporated onto a glass support by the interaction of the support with (1a) solubilized in dichloromethane, Figure 2. This monolayer had been previously characterized,10b and a surface coverage corresponding to 2 × 10-9 mol‚cm-2 was determined. It was also demonstrated that the monolayer exhibits reversible photoisomerizable properties. UV irradiation of the dinitrospiropyran monolayer, SP state, 360 nm < λ < 380 nm generates the protonated merocyanine, MRH+ state, whereas visible light illumination of the latter monolayer restores the SP monolayer state. Figure 3 shows the SPR curve of the dinitrospiropyran monolayer-modified surface (curve b) as compared to the bare, unmodified Au surface (curve a). The angle for minimum reflectivity is shifted by ∆θ ) 0.12°. While the points shown in Figure 3 (curves a and b) correspond to the experimental data, the solid lines appearing in the curves corresponds to the Fresnel numerical fits.19 From these fits we estimate that the index of refraction of the monolayer is n ) 1.53 and its thickness is 11.0 Å. The index of refraction is consistent with the value of other organic adsorbates on Au surfaces.20 Figure 4 shows the minimized energy structure of the mercaptobutyl dinitrospiropyran calculated by the PC model molecular mechanics program. The longitudinal dimension of the molecule is ca. 11 Å. Although the assembly of the dinitrospiropyran does not necessarily include the perpendicular orientation of the molecular longitudinal axis in respect to the surface, our results are consistent with the formation of a monolayer assembly on the Au support. It should be noted that we were unable to detect reflectivity differences of the SP- and MRH+-monolayer states. Thus, the SPR is not sufficiently sensitive to probe the photoisomerization process. Previous studies10 have indicated that the photoisomerizable dinitrospiropyran monolayer exhibits photoswitchable affinity interactions for the anti-DNP-Ab, Figure 2. It has been demonstrated that DNP-Ab associates to the dinitrospiropyran monolayer assembled on Au supports that acts as an antigen layer. Formation of the antigen-DNP-Ab complex on the Au support was probed electrochemically, using cyclic voltammetry or impedance spectroscopy, as well as by the application of microgravimetric quartz crystal microbalance analyses.10,11 Using QCM measurements, the saturated surface coverage of the dinitrospiropyran antigen monolayer with the DNPAb was estimated to be 3.8 × 10-12 mol‚cm-2. Photoisomerization of the monolayer to the protonated dinitromerocyanine followed by rinsing-off the monolayer interface resulted in the dissociation of DNP-Ab from the monolayer interface. By the secondary irradiation of the protonated dinitromerocyanine layer, λ > 495 nm, the sensing antigen layer was regenerated and the cyclic, (20) Knoll, W. Makromol. Chem. 1991, 192, 2827. (21) Lawrence, C. R.; Geddes, N. J.; Furlong, D. N.; Sambles, J. R. Biosens. Bioelectron. 1996, 11, 389.
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Figure 2. Assembly of a dinitrospiropyran antigen monolayer on a Au support and cyclic photostimulated binding and dissociation of the anti-DNP-Ab to and from the dinitrospiropyran/protonated dinitromerocyanine monolayer interface.
Figure 3. SPR curves of (a) the bare Au film; (b) the Au film after deposition of the (1a)-dinitrospiropyran thiolated monolayer. Solid lines linking experimental points correspond to the theoretical fits.
reversible analysis of the DNP-Ab was feasible, Figure 2. Figure 5 shows the SPR curves of the dinitrospiropyran monolayer modified surface before (curve a) and after interaction with the DNP-Ab. A substantial shift in the minimum reflectivity angle, ∆θ ) 0.54°, is observed, indicating that DNP-Ab binds to the metal surface. Control experiments using BSA as a foreign protein induce a minute change in the reflectivity angle. Similarly, the interaction of the dinitrospiropyran monolayer with foreign antibodies, e.g., anti-fluorescein antibody or goat anti-Fc mouse antibody, does not yield any shift in the minimum reflectivity angle, implying that these latter antibodies do not bind to the antigen layer or the Au surface. These results indicate that the association of DNPAb to the dinitrospiropyran layer originates from specific antigen-antibody affinity interactions. The solid lines in the SPR curves correspond to the calculated Fresnel numerical fits. The derived thickness of the DNP-Ab layer is 70 Å, and the refractive index of the layer corresponds
Figure 4. Minimized energy structure of mercaptobutyl dinitrospiropyran (1a).
to 1.41. The refractive index of the anti-DNP-Ab layer is in excellent agreement with the values observed for other antibody systems.21 The dimensions of the antibody layer are very similar to the thickness determined by SPR for other antibody layers (e.g., for IgG-Ab, 66 Å; anti-IgG, 88 Å). Thus, the surface is probably saturated in a dense configuration by the antibody. As the surface coverage of (22) (a) Boudillon, C.; Demaille, C.; Gueris, J.; Moiroux, J.; Saveant, J. M. J. Am. Chem. Soc. 1993, 115, 12265. (b) Neibel, M. K.; Bright, H. J. J. Biol. Chem. 1971, 246, 2734.
Photoswitchable Antigen-Antibody Interactions
Figure 5. SPR curves of: (a) the dinitrospiropyran monolayer assembled on the Au-glass support; (b) the dinitrospiropyran monolayer after interaction with the anti-DNP-Ab, 24 × 10-6 g‚mL-1. Data recorded in a CH2Cl2 solution.
a dense protein layer is ca. 60% of a closely packed dense monolayer,22 we estimate the surface coverage of the DNPAb on the antigen monolayer to be 4.4 × 10-9 mol‚cm-2. This value is consistent with the surface coverage of the DNP-Ab detected by QCM analyses. Photoirradiation of the dinitrospiropyran anti-DNPAb affinity complex monolayer, 360 nm < λ < 380 nm, followed by rinsing the SPR cell restores the initial SPR curve, characteristic of the dinitrospiropyran or protonated dinitromerocyanine monolayer interface. Thus, photoisomerization of the dinitrospiropyran DNP-Ab complex monolayer results in the protonated dinitromerocyanine layer that lacks antigen properties for the antibody. This enables washing-off of the antibody as reflected by the SPR curve. The DNP antibody can be dissociated from the functionalized interface only upon irradiation of the monolayer with UV light, 360 nm < λ < 380 nm, and formation of the protonated dinitromerocyanine monolayer state. Physical rinsing of the dinitrospiropyran/DNP-Ab complex associated with the Au support does not effect the dissociation of the antibody. Addition of the DNP-Ab to the dinitromerocyanine monolayer state does not alter the SPR curve, indicating that no affinity complex between this photoisomer state and the antibody exists. Further isomerization of the monolayer to the dinitrospiropyran state and subsequent addition of the DNP-Ab regenerated the shift of ca. ∆θ ) 0.5° in the minimum reflectivity angle, implying the association of the antibody to the antigen monolayer. By cyclic photoisomerization of the monolayer between the SP and MRH+ states in the presence of the DNP-Ab, the minimum reflectivity angle can be reversibly switched between a shifted angle, ∆θ ) 0.45°, and a nonshifted angle. That is, SPR provides an effective spectroscopic method to probe the binding of DNP-Ab to the dinitrospiropyran antigen monolayer, the dissociation of the antibody from the monolayer, upon photoisomerization of the monolayer to the protonated dinitromerocyanine state, and the regeneration of the sensing interface, Figure 6. The time-dependent changes in the layer thickness upon interaction of the dinitrospiropyran layer with the antiDNP-Ab were followed by SPR, Figure 7. This corresponds to the kinetics of association of the antibody to the antigen monolayer. The time-dependent changes in the layer thickness follow a pseudo first-order kinetics, k ) 10-3
Langmuir, Vol. 15, No. 11, 1999 3923
Figure 6. Cyclic photoswitchable SPR transduction of the interaction of anti-DNP-Ab with the photoisomerizable antigen monolayer-functionalized Au-glass substrate: (a) Monolayer in dinitrospiropyran state. (b) Monolayer in dinitrospiropyran state after treatment with anti-DNP-Ab. (c) Monolayer after illumination to form the protonated dinitromerocyanine state and rinsing-off of the DNP-Ab.
Figure 7. Dynamics of the anti-DNP-Ab association of the dinitrospiropyran interface. Data are elucidated by the estimation of the layer thickness from the respective SPR curves at time intervals of interaction with the antigen monolayer.
s-1. The time-constant for the binding of the DNP-Ab to the photoisomerizable antigen layer is very similar to that elucidated by microgravimetric quartz crystal microbalance experiments.10 Conclusions In conclusion, we have demonstrated that SPR provides an effective means to probe the photoswitchable association and dissociation of the anti-DNP antibody to and from the photoisomerizable dinitrospiropyran monolayer, respectively. The method complements other physical transduction means of photostimulated antigen-antibody interactions such as amperometric, impedance, and quartz crystal microbalance probing. The SPR technique reveals certain advantages as it is possible to follow the thickness of the antibody layer accompanying its binding to the monolayer, as well as the kinetics of binding to the monolayer interface. Acknowledgment. This research is supported by The Israel Science Foundation. LA981147V
