Langmuir 2004, 20, 7365-7367
7365
Biological Sensing Using Transmission Surface Plasmon Resonance Spectroscopy Michal Lahav, Alexander Vaskevich, and Israel Rubinstein* Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel Received May 3, 2004. In Final Form: July 7, 2004 Ultrathin gold island films evaporated on transparent substrates offer promising transducers for chemical and biological sensing in the transmission surface plasmon resonance (T-SPR) mode. In the present work, the applicability of T-SPR-based systems to biosensing is demonstrated, using a well-established biological model system. Au island films were evaporated on polystyrene slides and modified with a biotinylated monolayer via a multistep surface reaction, the latter assisted by the good adhesion of metal islands to polystyrene. The biotin-derivatized Au island film was then used as a biological recognition surface for selective sensing of avidin binding, distinguishing between specific and nonspecific binding to the substrate. Transduction of the binding event into an optical signal was achieved by T-SPR spectroscopy, using plasmon intensity measurements, rather than wavelength change, for maximal sensitivity and convenience. T-SPR spectroscopy of Au island films is shown to be an effective tool for monitoring the binding of biological molecules to receptor layers on the Au surface and a promising approach to label-free optical biosensing.
Surface plasmon resonance spectroscopy (SPR) in the Kretschmann (reflection) configuration using continuous Au films, combined in some cases with immobilized nanoparticles, has been to date the major approach to biosensing based on resonant extinction of light by gold surface plasmons (SPs).1 Transmission surface plasmon resonance (T-SPR) spectroscopy was introduced by us as a means of monitoring changes in the SP absorbance of discontinuous Au films in the transmission configuration.2 The method involves preparation of ultrathin (e10 nm nominal thickness), semi-transparent Au island films by evaporation onto inert transparent substrates (e.g., mica, quartz, glass, polystyrene). Such Au films show a localized SP extinction peak in the visible to near-infrared range, which depends on the island morphology and is sensitive to the dielectric properties of the contacting medium.2,3 Changes in the Au SP extinction induced by binding of various analytes to the Au island surface or to a molecular layer on the surface were monitored using transmission UV-vis spectroscopy.2 Van Duyne and co-workers developed a similar approach based on Ag island films prepared by evaporation through a mask of polystyrene nanobeads.4 Recently transmission spectroscopy with immobilized Au * To whom correspondence should be addressed. Fax: +972 8 9344137. Tel.: +972 8 9342678. E-mail: israel.rubinstein@ weizmann.ac.il. (1) (a) Homola, J.; Yee, S. S.; Gauglitz, G. Sens. Actuators, B 1999, 54, 3. (b) He, L.; Musick, M. D.; Nicewarner, S. R.; Salinas, F. G.; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 9071. (2) (a) Kalyuzhny, G.; Vaskevich, A.; Ashkenasy, G.; Shanzer, A.; Rubinstein, I. J. Phys. Chem. B 2000, 104, 8238. (b) Kalyuzhny, G.; Schneeweiss, M. A.; Shanzer, A.; Vaskevich, A.; Rubinstein, I. J. Am. Chem. Soc. 2001, 123, 3177. (c) Kalyuzhny, G.; Schneeweiss, M. A.; Shanzer, A.; Vaskevich, A.; Rubinstein, I. Chem.sEur. J. 2002, 8, 3850. (d) Doron-Mor, I.; Barkay, Z.; Filip-Granit, N.; Vaskevich, A.; Rubinstein, I. Chem. Mater., in press. (e) Doron-Mor, I.; Cohen, H.; Barkay, Z.; Shanzer, A.; Vaskevich, A.; Rubinstein, I. J. Am. Chem. Soc., submitted for publication. (3) (a) Kreibig, U.; Vollmer, M. Optical Properties of Metal Clusters; Springer: Berlin, 1995. (b) Norman, S.; Andeson, T.; Granqvist, C. G.; Hunderi, O. Phys. Rev. B 1978, 18, 674. (c) Hunderi, O. Surf. Sci. 1980, 96, 1. (d) Gluodenis, M.; Manley, C.; Foss, C. A. Anal. Chem. 1999, 71, 4554. (e) Orfanides, P.; Buckner, T. F.; Buncick, M. C.; Meriaudeau, F.; Ferrell, T. L. Am. J. Phys. 2000, 68, 936. (4) Malinsky, M. D.; Kelly, K. L.; Schatz, G. C.; Van Duyne, R. P. J. Am. Chem. Soc. 2001, 123, 1471.
nanoparticles and evaporated Ag films was used to follow biotin-avidin interaction5 and DNA hybridization to complementary DNA functionalized with gold nanoparticles.6 In the present work, application of T-SPR spectroscopy of evaporated Au island films to biosensing is explored using biotin-avidin interactions, separating specific and nonspecific binding. The substrates were 2.5-nm Au island films evaporated on transparent polystyrene slides. The choice of polystyrene was motivated by the fact that Au island films on transparent oxide substrates (mica, glass, quartz) exhibit poor stability in water and other solvents,7 while the same films evaporated on polystyrene show excellent adhesion in various solvents, enabling multistep preparation of sensing interfaces. The Au island films were modified with a self-assembled monolayer of cystamine and then reacted with biotin-NHS (N-hydroxy succinimide). The resulting biotinylated surfaces were incubated in a BSA (bovine serum albumin) blocking solution to preclude nonspecific binding and then exposed to a solution of avidin, Figure 1(i). Figure 2A shows T-SPR spectra of a biotin-BSA modified Au island surface before and after exposure to an avidin solution, as in Figure 1(i). The biotin-avidin interaction, promoting specific binding of avidin to the biotinylated surface, leads to an increase in the intensity of the Au SP band at λmax ) 645 nm by about 0.005 au. The use of BSA as a blocking agent prior to avidin binding is essential for preventing nonspecific adsorption of avidin, because proteins such as avidin and BSA (negatively charged at the working pH, ∼7.0) adsorb nonspecifically to unreacted (positively charged) cystamine molecules or to bare Au pinholes on the surface.8 Figure 2B shows T-SPR spectra of a biotin-modified Au island surface before and after avidin treatment, without BSA blocking, as in Figure 1(ii). In the absence of the blocking step, a larger increase (ca. 0.013 au) of the Au SP absorbance is observed, indicating the contribution of (5) (a) Nath N.; Chilkoti, A. Anal. Chem. 2002, 74, 504. (b) Haes, A. J.; Van Duyne, R. P. J. Am. Chem. Soc. 2002, 124, 10596. (6) Hutter, E.; Pileni, M. P. J. Phys. Chem. B 2003, 107, 6497. (7) (a) Ishikawa, H.; Kimura, K. Nanostruct. Mater. 1997, 9, 555. (b) Mosier-Boss, P. A.; Lieberman, S. H. Appl. Spectrosc. 1999, 53, 862. (8) Huang, T. T.; Sturgis, J.; Gomez, R.; Geng, T.; Bashir, R.; Bhunia, A. K.; Ladisch, M. R. Biotechnol. Bioeng. 2003, 81, 618.
10.1021/la0489054 CCC: $27.50 © 2004 American Chemical Society Published on Web 08/03/2004
7366
Langmuir, Vol. 20, No. 18, 2004
Letters
Figure 1. Schematic presentation of the different steps in the preparation of the various interfaces.
Figure 2. Transmission UV-vis spectra (taken ex situ) of 2.5-nm Au island films evaporated on polystyrene, before (dashed lines) and after (solid lines) exposure to avidin solution: (A) Biotinylated surface, interacted with BSA prior to avidin exposure [Figure 1, route (i)]. (B) Same as part A, without the BSA blocking step [Figure 1, route (ii)]. (C) Cystamine-terminated surface, interacted with BSA prior to avidin exposure [Figure 1, route (iii)].
nonspecific avidin adsorption. Hence, BSA blocking leads to a decrease in the sensitivity to avidin binding, but the specificity of the system is significantly improved. Treatment of a cystamine-modified, nonbiotinylated Au island surface with avidin solution without BSA blocking also leads to an increase of the Au SP band intensity (data not shown), indicating nonspecific adsorption of avidin to the positively charged amine groups of cystamine as well as at bare Au pinholes. The role of BSA blocking is emphasized by testing a similar cystamine-modified, nonbiotinylated Au surface, treated with BSA prior to avidin treatment, Figure 1(iii). As seen in Figure 2C, the T-SPR spectra of the BSA-blocked, cystamine-modified Au island film before and after treatment in avidin solution are practically identical. This latter result shows the effective blocking capability of BSA toward the nonspecific adsorption of avidin, thus, supporting the notion that the absorbance change in Figure 2A is attributed to specific avidin binding. In conclusion, T-SPR spectroscopy of Au island films evaporated on an adhesive, transparent substrate (polystyrene) was shown to be a promising tool for label-free sensing of biological interactions, demonstrated here by specific binding of avidin to a biotinylated surface. Pretreatment with BSA introduced efficient blocking of nonspecific adsorption of avidin, thus, providing a specific sensing interface. The same approach can be applied to sensing schemes in other biological systems, such as antibody-antigen, receptor-hormone and DNA/DNA interactions, and other systems. We emphasize plasmon intensity change measurements,2a rather than the commonly used wavelength shift, as the method of choice for achieving maximal sensitivity. Protein binding to the transducer surface (Figure 2A,B) causes a minute change in the position of the SP band maximum, while the intensity change is easily detectable. Considering the linearity between the surface coverage of the analyte and change in the SP band intensity, shown by us previously,2a-c intensity measurements are promising for quantification of the amount of surface-bound target biomolecules. The simplicity of the experimental setup (a regular UV-vis spectrophotometer) is a major advantage of T-SPR spectroscopy compared to the commonly used attenuated total reflection-SPR method. Quantitative comparison of the two techniques requires additional study.
Letters
Langmuir, Vol. 20, No. 18, 2004 7367
Experimental Section Materials. Cystamine-hydrochloride‚2H2O (Aldrich), biotinNHS (Sigma), BSA (Sigma), avidin (from egg white, Sigma), ethanol (Merck, AR), and dimethylsulfoxide (DMSO; Merck, AR) were used without further purification. Water was triply distilled. The gas used was house nitrogen (99%). Au Island Film Preparation. Ultrathin (2.5 nm nominal thickness) Au island films were prepared by mounting cleaned (sonicated in ethanol) polystyrene slides (10 × 20 mm2, cut from Petri dishes) in a cryo-HV evaporator (Key High Vacuum) equipped with a Maxtek TM-100 thickness monitor. Homogeneous deposition was achieved by moderate rotation of the substrate plate. Gold (99.99%) was evaporated from a tungsten boat at (2-3) × 10-6 Torr at a deposition rate of 0.01 nm/s. Preparation of the Various Interfaces. A cystamine monolayer was self-assembled on the Au island films by immersing the slides in a solution of 10 mM cystamine in water for 2 h. Biotinylated surfaces were obtained by reaction with
biotin-NHS (200 µg/mL in DMSO) for 1.5 h. BSA blocking was carried out by reacting with an aqueous BSA solution (400 µg/ mL) for 5 h. Exposure to avidin was carried out by immersion in 50 µg/mL avidin in Tris buffer, 0.01 M, for 1.5 h. UV-Vis Spectroscopy. Measurements were carried out with a Cary-50 spectrophotometer in air, using a specially designed cell. The scan speed and averaging time were, respectively, 300 nm/min and 0.1 s/point. A baseline correction procedure was executed prior to each measurement session.
Acknowledgment. We wish to thank the Israel Ministry of Science (Tashtiot Infrastructure Project) and the Edward D. and Anna Mitchell Research Fund, Weizmann Institute, for financial support. A.V. is partially supported by a KAMEA Fellowship, the Israel Ministry of Immigrant Absorption. LA0489054
