Biological Sensing and Interface Design in Gold Island Film Based

Anal. Chem. 2008, 80, 7487–7498

Biological Sensing and Interface Design in Gold Island Film Based Localized Plasmon Transducers Tatyana A. Bendikov,† Aharon Rabinkov,‡ Tanya Karakouz,† Alexander Vaskevich,*,† and Israel Rubinstein*,† Departments of Materials and Interfaces and Biological Services, Weizmann Institute of Science, Rehovot 76100, Israel Discontinuous, island-type gold films (typically e10 nm nominal thickness) prepared by evaporation of the metal on transparent substrates show a localized surface plasmon resonance (LSPR) extinction in the visible-to-NIR range and can be used as optical transducers for monitoring local refractive index change. Such transducers, operated in the transmission configuration, provide an effective scheme for label-free biological sensing using basic spectrophotometric equipment. Optimization of the sensitivity of LPSR transducers requires consideration of the distance between the metal island surface and the bound analyte, strongly affecting the optical response due to the fast decay of the evanescent field of localized plasmons. In the present work Au island based LSPR transducers were used to monitor antibody-antigen interactions, demonstrating the effect of the biorecognition interface thickness. Evaporated Au island films derivatized with IgG or hCG antigens were used as biological recognition elements for selective sensing of antibody binding, distinguishing between specific and nonspecific interactions. The LSPR results are supported by XPS and ellipsometry data as well as by AFM and HRSEM imaging, the latter providing actual visualization of the two protein binding steps. Increase of the recognition interface thickness leads to a concomitant decrease in the extinction and wavelength sensitivity, generally conforming to a model of an exponentially decaying surface plasmon (SP) evanescent field. Optical methods based on interaction of electromagnetic radiation with electrons at the interface between a metal and a dielectric medium, resulting in resonance excitation of surface plasmon (SP) polaritons, have become popular in studies and applications relating to label-free biosensing. In the case of the widely used surface plasmon resonance (SPR) method,1-3 SPs generated in the total-internal-reflection mode propagate along the * To whom correspondence should be addressed. Phone: 972-8-9342574 (A.V.); 972-8-9342678 (I.R.). Fax: 972-8-9344137. E-mail: alexander.vaskevich@ weizmann.ac.il (A.V); [email protected] (I.R.). † Department of Materials and Interfaces. ‡ Department of Biological Services. (1) Brockman, J. M.; Nelson, B. P.; Corn, R. M. Annu. Rev. Phys. Chem. 2000, 51, 41–63. (2) Homola, J. Chem. Rev. 2008, 108, 462–493. (3) Boozer, C.; Kim, G.; Cong, S. X.; Guan, H. W.; Londergan, T. Curr. Opin. Biotechnol. 2006, 17, 400–405. 10.1021/ac8013466 CCC: $40.75  2008 American Chemical Society Published on Web 08/29/2008

surface of a planar metal-dielectric interface. In the case of localized surface plasmon resonance (LSPR),4,5 the charge-density oscillations (SPs) are confined to metal nanostructures or sharp roughness on the nanometer scale. In the latter case the SPs can be excited and monitored in the transmission mode. Both methods are based on refractive index (RI) variation in the vicinity of the interface upon analyte binding, resulting in change in the resonance conditions. While propagating SPR is well established and commercial instruments for biosensing applications based on SPR are available, the LSPR approach is considerably less developed. The best sensitivity values reported for LSPR biosensors5 are comparable to those achieved in SPR biosensing.2 The last statement calls for some consideration. It is known that the RI sensitivity of Au film based SPR systems6,7 is 50-100 times larger than the RI sensitivity of LSPR systems comprising various nanostructured Au films.5 This, however, is largely compensated for by the distance sensitivity, i.e., the decay length of the SP evanescent field, in the two methods. Assuming an exponential decay of the SP evanescent field and a constant RI sensitivity m, Campbell and co-workers8 derived the expression for the SPR (or LSPR) response to the formation of a layer of a thickness d on the metal surface: R ) m(ηa - ηs)[1 - exp(-2d ⁄ Ld)]

(1)

where R is the response (as SP intensity change or wavelength shift), ηa and ηs are, respectively, the refractive indexes of the adsorbate layer and the bulk medium, and Ld is the decay length of the evanescent field, characteristic of the specific transducer. From eq 1 it is clear that the response decreases rapidly as the decay length Ld becomes larger. Results on different LSPR systems with a variable overlayer thickness can be found in the literature, including nanoparticles in solution,9 nanoparticles immobilized on transparent substrates,10-13 and metal island films.14-17 While the experimental data (4) Hutter, E.; Fendler, J. H. Adv. Mater. 2004, 16, 1685–1706. (5) Vaskevich, A.; Rubinstein, I. In Handbook of Biosensors and Biochips; Marks, R., Cullen, D., Lowe, C., Weetall, H. H., Karube, I. , Eds.; Wiley: Chichester, U.K., 2007; Vol. 1. (6) Zhang, L. M.; Uttamchandani, D. Electron. Lett. 1988, 24, 1469–1470. (7) Urbanczyk, A.; VanHook, W. A. J. Chem. Thermodyn. 1996, 28, 987–1008. (8) Jung, L. S.; Campbell, C. T.; Chinowsky, T. M.; Mar, M. N.; Yee, S. S. Langmuir 1998, 14, 5636–5648. (9) Liz-Marzán, L. M.; Giersig, M.; Mulvaney, P. Langmuir 1996, 12, 4329– 4335. (10) Okamoto, T.; Yamaguchi, I.; Kobayashi, T. Opt. Lett. 2000, 25, 372–374.

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are generally consistent with eq 1, direct fitting and estimation of Ld may be problematic, as the thickness of the overlayer in many cases was not measured directly. Typical values of Ld in LSPR systems vary between ∼10-30 nm, while the (wavelength dependent) values of Ld in SPR are typically an order-of-magnitude (or more) larger. Therefore, although (as noted above) the RI sensitivity of SPR systems is much larger than that of LSPR systems, the decay length acts as a compensating factor in favor of LSPR transducers. Moreover, the very high sensitivity of SPR to refractive index changes in the solution bulk can be a disadvantage, as it may result in false signals arising from the effect of temperature inhomogeneity and fluctuations on the refractive index. An important consequence of the large difference of the decay lengths in SPR and LSPR concerns the structure of the biological recognition interface. In the case where a biological analyte of a thickness da binds to an SPR or LSPR transducer covered with a recognition interface of a thickness di, eq 1 will take the form (assuming that both layers have the same refractive indexes ηa)18 R ) m(ηa - ηs)exp(-2di ⁄ Ld)[1 - exp(-2da ⁄ Ld)]

(2)

Equation 2 indicates that, from the sensitivity point-of-view, changes in the biological interface thickness di will make little difference in SPR measurements, while they are expected to critically affect the sensitivity of LSPR systems. Hence, while traditionally little or no attention has been paid to the dimensions of SPR recognition interfaces, the latter should be a major consideration in LSPR biosensing. Systems based on LSPR spectroscopy mostly comprise gold or silver nanostructures (nanoparticles,10-13 nanoislands,14-17 and nanoholes19). A biorecognition interface consisting of specific receptors is constructed on the metal nanostructures, and binding of an analyte is detected as a change in the SP band intensity or shift of the wavelength of maximum absorbance.4,5,19-28 The rapidly increasing interest in recent years in biological recognition systems based on localized plasmon transducers is largely attributed to the relatively simple and inexpensive technology compared to that of propagating SPR. However, a prevalent drawback of LSPR systems based on nanoparticle or nanoisland films is instability of the metal nanostructure morphology and optical properties upon immersion in solvents and drying. In certain cases the problem can be overcome by preconditioning the transducer in the respective solvent, while in other cases the metal nanostructures are stabilized upon binding of the biological receptor layer. Both approaches, however, do not provide a general solution. (11) Eck, D.; Helm, C. A.; Wagner, N. J.; Vaynberg, K. A. Langmuir 2001, 17, 957–960. (12) Eck, D.; Helm, C. A.; Wagner, N. J.; Vaynberg, A. Langmuir 2007, 23, 9522–9522. (13) Nath, N.; Chilkoti, A. Anal. Chem. 2004, 76, 5370–5378. (14) Doron-Mor, I.; Barkay, Z.; Filip-Granit, N.; Vaskevich, A.; Rubinstein, I. Chem. Mater. 2004, 16, 3476–3483. (15) Doron-Mor, I.; Cohen, H.; Barkay, Z.; Shanzer, A.; Vaskevich, A.; Rubinstein, I. Chem.sEur. J. 2005, 11, 5555–5562. (16) Haes, A. J.; Zou, S. L.; Schatz, G. C.; Van Duyne, R. P. J. Phys. Chem. 2004, 108, 109–116. (17) Whitney, A. V.; Elam, J. W.; Zou, S. L.; Zinovev, A. V.; Stair, P. C.; Schatz, G. C.; Van Duyne, R. P. J. Phys. Chem. 2005, 109, 20522–20528. (18) Haemers, S.; Koper, G. J. M.; van der Leeden, M. C.; Frens, G. Langmuir 2002, 18, 2069–2074.

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We have concentrated on the development of LSPR systems based on Au island films prepared by evaporation of a subpercolation amount of Au on transparent substrates, such as mica, polystyrene, and amino- or mercaptosilane pretreated glass slides.14,15,29-32 Biosensing was demonstrated with a biotin-avidin model system;27 in this case the island film optical response was stabilized by solvent preconditioning. Recently we have introduced a general approach to stabilizing the Au island film based LSPR systems, by coating the islands with an ∼2 nm sol-gel derived silica layer.33 While the method is highly effective in terms of structure and optical signal stabilization, the silica layer adds to the recognition interface thickness and hence lowers the system sensitivity, as discussed above. In the present study we use Au nanoisland film transducers (5 nm nominal thickness, evaporated on silanized glass and annealed) to demonstrate label-free immuno-recognition, namely, specific antibody-antigen interactions under conditions of full analyte coverage. Under these experimental conditions, an increase in the incubation time or analyte concentration does not affect the surface coverage by the analyte. The step-by-step preparation of the protein recognition interface (antigen terminated) and detection of specific antibody binding were followed by monitoring changes in the LSPR band. The optical data are supported by ellipsometry and X-ray photoelectron spectroscopy (XPS) measurements. Protein binding is imaged by high-resolution scanning electron microscopy (HRSEM) and atomic force microscopy (AFM), providing direct visualization of the different steps of protein binding to the Au surfaces. To demonstrate the effect of the recognition interface thickness on the sensitivity, three cases are presented in order of increasing thickness: (i) antigen binding directly to the Au islands; (ii) antigen binding to a carboxylate-functionalized mercaptosilane monolayer on the Au islands;33 and (iii) antigen binding to a carboxylate-functionalized silica coating on the Au islands.33 The results are discussed in terms of the model presented in eqs 1 and 2. (19) Dahlin, A. B.; Tegenfeldt, J. O.; Höök, F. Anal. Chem. 2006, 78, 4416– 4423. (20) Willets, K. A.; Van Duyne, R. P. Annu. Rev. Phys. Chem. 2007, 58, 267– 297. (21) Genet, C.; Ebbesen, T. W. Nature 2007, 445, 39–46. (22) Endo, T.; Kerman, K.; Nagatani, N.; Takamura, Y.; Tamiya, E. Anal. Chem. 2005, 77, 6976–6984. (23) Endo, T.; Kerman, K.; Nagatani, N.; Hiepa, H. M.; Kim, D.-K.; Yonezawa, Y.; Nakano, K.; Tamiya, E. Anal. Chem. 2006, 78, 6465–6475. (24) Larsson, E. M.; Alegret, J.; Kaell, M.; Sutherland, D. S. Nano Lett. 2007, 7, 1256–1263. (25) Gish, D. A.; Nsiah, F.; McDermott, M. T.; Brett, M. J. Anal. Chem. 2007, 79, 4228–4232. (26) Kim, S.; Jung, J.-M.; Choi, D.-G.; Jung, H.-T.; Yang, S.-M. Langmuir 2006, 22, 7109–7112. (27) Lahav, M.; Vaskevich, A.; Rubinstein, I. Langmuir 2004, 20, 7365–7367. (28) Stewart, M. E.; Anderton, C. R.; Thompson, L. B.; Maria, J.; Gray, S. K.; Rogers, J. A.; Nuzzo, R. G. Chem. Rev. 2008, 108, 494–521. (29) Kalyuzhny, G.; Vaskevich, A.; Matlis, S.; Rubinstein, I. Rev. Anal. Chem. 1999, 18, 237–242. (30) Kalyuzhny, G.; Vaskevich, A.; Ashkenasy, G.; Shanzer, A.; Rubinstein, I. J. Phys. Chem. B 2000, 104, 8238–8244. (31) Kalyuzhny, G.; Schneeweiss, M. A.; Shanzer, A.; Vaskevich, A.; Rubinstein, I. J. Am. Chem. Soc. 2001, 123, 3177–3178. (32) Kalyuzhny, G.; Vaskevich, A.; Schneeweiss, M. A.; Rubinstein, I. Chem.sEur. J. 2002, 8, 3849–3857. (33) Ruach-Nir, I.; Bendikov, T. A.; Doron-Mor, I.; Barkay, Z.; Vaskevich, A.; Rubinstein, I. J. Am. Chem. Soc. 2007, 129, 84–92.

Figure 1. Schematic representation of the stepwise procedures for preparation of functional interfaces on Au island films, followed by antigen binding and antibody recognition. The three routes leading to the different recognition interfaces are numbered.

EXPERIMENTAL SECTION Gold Film Preparation. Glass substrates for Au evaporation were cut from glass microscope cover slides (cover glass no. 3, Menzel-Glaser) into 22 mm × 9 mm slides and cleaned using the following procedure: the slides were immersed in fresh “piranha” solution (1:2 H2O2/H2SO4) for 1 h, followed by treatment in hot (70 °C) solution of 1:1:5 H2O2/NH4OH/H2O for 1 h and extensive rinsing in methanol. (Caution: pirahna solution reacts violently with organic materials and should be handled with extreme care.) After cleaning, the glass slides were modified with 3-aminopropyl trimethoxysilane (APTS, Aldrich) by overnight immersion in a 10 vol % APTS solution in methanol. The silanized glass slides were sonicated three times (5 min each) in methanol, washed with ethanol, and dried under a stream of nitrogen. Au evaporation was carried out 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. Au (99.99%, Holland-Moran, Israel) was evaporated from a tungsten boat at 2-4 µTorr. Discontinuous, 5-nm-thick and continuous, 20-nm-thick Au films were deposited on the APTStreated glass slides at the rates of 0.01 and 0.1 nm s-1, respectively.33,34 Postdeposition annealing of Au-covered slides was carried out in air at 200 °C for 20 h using a Ney Vulcan 3-550 furnace. The heating rate was 5 °C min-1, and the annealed slides were left to cool in air to room-temperature. The annealing temperature was chosen to maintain good adhesion of the Au to the silane-treated substrates. Continuous, 20 nm Au films, annealed, provide smooth (111)-textured surfaces suitable for common characterization techniques (ellipsomertry, XPS, AFM, etc.).34 (34) Wanunu, M.; Vaskevich, A.; Rubinstein, I. J. Am. Chem. Soc. 2004, 126, 5569–5576.


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Figure 2. HRSEM images of discontinuous (5 nm) and continuous (20 nm) Au films on glass, annealed, before and after protein binding. (a) A 5 nm Au island film, annealed. (b) Au island film as in part a, coated with an ∼2 nm silica layer. (c) Au island film as in part a, after direct antigen (rabbit IgG protein) immobilization; the arrow points to a single protein. (d) Au island film as in part c, after antibody (anti-rabbit IgG protein) binding. (e) A 20 nm Au film, annealed. (f) Au film as in part e, after antigen (rabbit IgG protein) and antibody (anti-rabbit IgG protein) binding. Sample preparation for imaging is detailed in the Experimental Section. All samples (including bare Au) were coated with a 2-3 nm Cr layer.

Functionalization of the Interface. Prior to any modification, annealed Au-coated glass slides (continuous Au films only) were exposed for 10 min to UV-ozone treatment (UVOCS model T10×10/OES/E), washed in ethanol for 20 min, and dried under a stream of nitrogen.35 Mercaptosilane-Modified Au Films. The slides were immersed for 1 h in a freshly prepared solution of 2 mM 3-mercaptopropyl trimethoxysilane (MPTS, Aldrich) in ethanol, washed for 20 min in ethanol, and dried under a stream of nitrogen to obtain an MPTS monolayer on the Au. Silica Modified Au Films. The slides were primed with MPTS as described above. Approximately 2 nm silica layers were then deposited by generally following the method of Liz-Marza´n et al.9 Sodium silicate solution (∼1.5 wt % SiO2, pH ∼12) was prepared (35) Ron, H.; Matlis, S.; Rubinstein, I. Langmuir 1998, 14, 1116–1121.

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by dilution of 2 mL of the original sodium silicate solution (Aldrich, 27 wt % SiO2) with triply distilled water to a final volume of 50 mL. The strongly acidic cation exchanger Amberlite IR-120 (Merck) was used for adjustment of the solution pH to 8.5-9. Silica deposition was carried out by dipping the slides for 2 h in the 1.5% sodium silicate solution at 90 °C.33 After removal from the sodium silicate solution, the silica-coated Au substrates were washed with water and dried under a stream of nitrogen. Carboxylate-Derivatized Surfaces. MPTS and silica coated Au substrates were extensively washed in methanol and modified with APTS by overnight immersion in a 10 vol % APTS solution in methanol. The slides were then sonicated three times (5 min each) in methanol, washed with ethanol, and dried under a stream of nitrogen. To obtain carboxylate functionalities, APTS-derivatized Au slides were washed 20 min in tetrahydrofuran (THF) and then

immersed for 4-5 h in deaerated, freshly prepared basic solution of succinic anhydride (the reaction solution consisted of 0.1 g of iosuccinic anhydride (Acros) and 400 µL of N,N-diisopropylethylamine (Aldrich) in 10 mL of dry THF). The slides were washed for 20 min in THF, dried under a stream of nitrogen, and conditioned overnight in phosphate buffer saline (PBS) solution prepared by 10-fold dilution of commercial reagent GIBCO D-PBS (10X)-CaCl2, -MgCl2 (Invitrogen) with triply distilled water. Unless otherwise specified, a similar PBS solution was used as a solvent throughout the antigen and antibody immobilization procedures. Immobilization of the Receptor (Antigen). On Bare Au Films (Route 1). Stock solutions of 1 mg mL-1 immunoglobulin G (IgG) proteins from rabbit and murine (mouse) serum (Sigma) were diluted with 0.3 M acetate buffer, pH ) 4.6, to a final concentration of 100 µg mL-1. For human chorionic gonadotropin (hCG) protein (Fitzgerald), a stock solution of 2 mg mL-1 was diluted with PBS to a final concentration of 500 µg mL-1. A volume of 30 µL of these solutions (rabbit IgG, mouse IgG, or hCG) were spread directly on the surface of Au-coated slides (working area, ∼1 cm2) and left for 20 min (rabbit and mouse IgG proteins) or for 1 h (hCG protein) in air at room temperature (22-23 °C). The slides were then washed 20 min in PBS solution and dried under a stream of nitrogen. To prevent nonspecific binding during subsequent reaction with the antibody, antigen-derivatized Au films were treated with bovine serum albumin (BSA) solution (Pentex). A volume of 30 µL of 100 µg mL-1 BSA solution was spread on the Au surface as described above and left for 1 h in air at room temperature, followed by 20 min of washing in PBS solution and drying under a stream of nitrogen. On Mercaptosilane and Silica Modified Au Films (Routes 2 and 3). The antigen was immobilized by reaction of carboxylic groups of the derivatized interface with amine groups of the protein to form amide bonds. The reaction was performed using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC) (Danyel Biotech) and N-hydroxysuccinimide (NHS) (Danyel Biotech). A 30 µL mixture of EDC and NHS of final concentrations of 37.5 and 5.75 mg mL-1, respectively, was spread on ∼1 cm2 of the carboxylatefunctionalized (mercaptosilane or silica modified) Au slide and left for 1 h in air at room temperature. The slides were then washed 20 min in PBS and dried under a stream of nitrogen. The antigen (rabbit or mouse IgG protein) was immobilized on the surface by interacting the antigen solution with the modified Au surface following the same procedure as described above for bare Au films. To block unreacted carboxylic groups, the antigenderivatized slides were treated with a 1 M solution of ethanolamine hydrochloride, pH ) 8.5 (Danyel Biotech), by spreading 60 µL of the solution on the slide surface as previously described, leaving in air for 20 min at room temperature, followed by washing 20 min in PBS solution, and drying under a N2 stream. The slides were also treated with BSA as described above to prevent nonspecific antibody binding. Binding of the Antibody. Antibody binding was carried out similarly with the three kinds of interfaces. For reaction with interfaces derivatized with rabbit IgG or mouse IgG antigens, a stock solution of 1 mg mL-1 anti-rabbit IgG antibody produced in goat (Sigma) and a stock solutions of 2 mg mL-1 anti-mouse IgG antibody produced in goat (Sigma) were diluted with PBS to a final concentration of ∼1 × 10-6 M. For reaction with interfaces

Figure 3. Sequential transmission UV-vis spectra taken after construction of each layer in a typical experiment performed according to route 3 (Figure 1). The first spectrum corresponds to a 5 nm Au island film, annealed. For details see the Experimental Section.

derivatized with hCG hormone, a 5 mg mL-1 stock solution of anti-hCG antibody (human chorionic gonadotropin β (hCGb) antibody produced in mouse, Fitzgerald) and a stock solution of 1 mg mL-1 anti-rabbit IgG antibody were diluted with PBS to a final concentration of 100 µg mL-1. An amount of 30 µL of the antibody solutions (anti-rabbit IgG, anti-mouse IgG, and anti-hCG) were spread on the surface of the antigen-derivatized Au surfaces (working area, ∼1 cm2) and left for 30 min (for anti-rabbit and anti-mouse IgG proteins) or 1.5 h (for anti-hCG or anti-rabbit IgG proteins) in air at room temperature. The slides were then washed for 20 min in PBS solution and dried under a stream of nitrogen. UV-Visible Spectroscopy. Extinction spectra at normal incidence were measured in air with a Varian Cary 50 UV-vis spectrophotometer. Spectra were recorded in the range 350-1000 nm at a scan speed of 600 nm min-1 using air as the baseline. Samples to be examined were dried from solvent under a stream of nitrogen and then placed in a special holder enabling transmission measurements of the same spot on the slide during all experimental stages. In the case of samples that were washed in PBS, before drying the slides were rinsed with triply distilled water. Rinsing with water before the measurement is essential; without rinsing a salt layer (from the buffer) crystallizes on the surface and interferes with the measurement. The stability of bound protein molecules toward rinsing of the sample was checked in a series of control experiments. After measurement of the LSPR response in situ (i.e., in the protein-containing solution) the slide was washed, dried, and immersed in a proteinfree solution. Transmission UV-vis spectra taken in situ before and after rinsing were indistinguishable, attesting to the stability of the protein binding. X-Ray Photoelectron Spectroscopy (XPS). XPS measurements were carried out using continuous, 20-nm-thick Au substrates to ensure good electrical conductivity. The Au slides were modified with protein layers as described above. XPS data were obtained with a Kratos Axis HS XPS system, using a monochromatized Al (KR) X-ray source (hυ ) 1486.6 eV). Ellipsometry. Ellipsometric measurements were carried out on continuous, 20-nm-thick Au substrates using a Rudolf Auto ELIV null ellipsometer, at an angle of incidence φ ) 70° and a Analytical Chemistry, Vol. 80, No. 19, October 1, 2008

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Figure 4. AFM images of continuous, 20 nm Au films, annealed, before and after protein binding. (a) Bare Au film. (b) Au film as in part a, after antigen (rabbit IgG protein) immobilization. (c) Au film as in part b, after antibody (anti-rabbit IgG protein) binding. (d) A 800 × 800 nm2 window prepared as described in the Experimental Section in a sample as in part c; the line corresponds to a cross-sectional profilometry (top) used for determination of the protein layer thickness.

wavelength λ ) 632.8 nm. The same four points were measured on each sample before and after film formation. High-Resolution Scanning Electron Microscopy (HRSEM). Prior to imaging, the samples were treated with 3 wt % paraformadehyde (PFA) (Merck) + 2 vol % glutaraldehyde (GA) (EMS) in cocadylate buffer for 1 h at room temperature. Cocadylate buffer, pH ) 7.4, consists of 0.1 M sodium cocadylate (Merck) + 5 mM CaCl2 (Merck) + 1 wt % sucrose (J. T. Baker). The samples were then washed 3 × 5 min with cocadylate buffer. The samples were further treated with 1 vol % solution of OsO4 (EMS) in cocadilate buffer for 1 h at room temperature and washed with cocadilate buffer 2 × 5 min followed by washing with H2O 2 × 5 min. The samples were incubated in the dark with filtered aqueous solution of 1 wt % tannic acid (Merck) for 5 min, washed with H2O 2 × 5 min, and then incubated 0.5 h in the dark with filtered aqueous solution of 1 wt % uranyl acetate (EMS) followed by washing with H2O 2 × 5 min. Dehydration was performed using mixtures of ethanol and H2O with increasing ethanol content, by washing 2 × 5 min in 25%, 50%, 70%, and 96% ethanol, followed by 2 × 10 min in 100% absolute anhydrous ethanol (Gadot). The samples were then dried using a critical point dryer (CPD30, BALTEC). Imaging was carried out using a high-resolution SEM (Ultra 7492


55, Zeiss). Prior to imaging, the samples were coated with a 2-3 nm Cr layer, either by e-beam evaporation (ODEM Scientific Applications) or by sputtering (EMITECH K575X). Atomic Force Microscopy (AFM). AFM images were recorded in air using a PicoSPM instrument (Molecular Imaging) operated in the acoustic ac (ACC) mode. The cantilevers used were NSC 12 series of ultrasharp silicon (MikroMasch, Estonia), with a resonant frequency of 100-200 kHz and an average tip radius of e10 nm. Abrasion of 800 × 800 nm2 windows was achieved by multiple scanning in the contact mode using NSC 36 ultrasharp silicon cantilevers (MikroMasch, Estonia) with a resonant frequency of 60-100 kHz, as previously described.36 RESULTS AND DISCUSSION Immunoglobulin G (IgG) proteins and their functions have been well studied.37,38 Dominating other immunoglobulins in the (36) Wanunu, M.; Vaskevich, A.; Cohen, S. R.; Cohen, H.; Arad-Yellin, R.; Shanzer, A.; Rubinstein, I. J. Am. Chem. Soc. 2005, 127, 17877–17887. (37) Janeway, C. A. Immunobiology: The Immune System in Health and Disease, 6th ed.; Garland Science: New York, 2005. (38) Abbas, A. K.; Lichtman, A. H. Cellular and Molecular Immunology, 5th ed.; Elsevier Saunders: Philadelphia, PA, 2005.

blood and lymph fluids, IgG antibodies play a major role in antigen recognition, providing the majority of antibody-based immunity against hostile bacteria and viruses. In this study rabbit and mouse IgG proteins (antigens) and anti-rabbit and anti-mouse IgG proteins (antibodies) were used to demonstrate specific protein-protein recognition. The Au island transducers comprised 5 nm (nominal thickness) Au island films evaporated on silanized glass and annealed 20 h at 200 °C, subsequently derivatized with a biological recognition interface for IgG antibody binding. Schematic representation of the stepwise procedure for functionalization of Au island films is shown in Figure 1 and described in detail in the Experimental Section. To demonstrate the effect of the recognition interface structure and dimensions, three types of interfaces were studied, presented as routes 1, 2, and 3 in Figure 1. In route 1, the specific receptor (antigen) was immobilized directly on the bare Au island surface.39 In route 2 the Au islands were coated with a monolayer of 3-mercaptopropyl trimethoxysilane (MPTS),33 while in route 3 the MPTS was further modified with an ∼2 nm silica layer.33 The silica coating on the Au islands is visualized in the HRSEM image shown in Figure 2b. In routes 2 and 3, the silane or silica surface, respectively, was chemically modified in a stepwise manner to expose carboxylate functionalities, capable of covalently linking to amino groups of the antigen by forming an amide bond.40,41 Direct adsorption of proteins on solid surfaces may result in a change in protein conformation and loss of protein activity.42,43 On the other hand, retention of protein activity on immobilized Au nanoparticles was demonstrated.44 Structural studies of individual IgG molecules show that the two Fab subunits carrying the specific binding sites are highly flexible,45-47 and it can be assumed that this flexibility is responsible for the activity preservation and specific antibody binding to antigens deposited directly on continuous or island-type Au films, as described below. An issue of major importance is the known instability of Au island films toward immersion in various solvents and drying, particularly in PBS, used extensively in protein binding. We have previously demonstrated that Au island films can be stabilized by coating with a thin (∼2 nm) sol-gel derived silica layer33 (route 3, Figure 1). In the present work it was found that all three routes lead to stable and active antigen layers, showing effective antibody recognition. Sequential UV-vis transmission spectra taken after each step in route 3 (Figure 1) starting with the Au island film and ending with antibody binding are shown in Figure 3. The sequence is divided into three parts, namely, construction of the interface, (39) Casero, E.; Vazquez, L.; Martin-Benito, J.; Morcillo, M. A.; Lorenzo, E.; Pariente, F. Langmuir 2002, 18, 5909–5920. (40) Sehgal, D.; Vijay, I. K. Anal. Biochem. 1994, 218, 87–91. (41) Patel, N.; Davies, M. C.; Hartshorne, M.; Heaton, R. J.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 1997, 13, 6485–6490. (42) Yang, M.; Chung, F. L.; Thompson, M. Anal. Chem. 1993, 65, 3713–3716. (43) Moulin, A. M.; O’Shea, S. J.; Badley, R. A.; Doyle, P.; Welland, M. E. Langmuir 1999, 15, 8776–8779. (44) Brown, K. R.; Fox, A. P.; Natan, M. J. J. Am. Chem. Soc. 1996, 118, 1154– 1157. (45) Harris, L. J.; Larson, S. B.; Hasel, K. W.; Day, J.; Greenwood, A.; McPherson, A. Nature 1992, 360, 369–372. (46) Saphire, E. O.; Parren, P. W.; Pantophlet, R.; Zwick, M. B.; Morris, G. M.; Rudd, P. M.; Dwek, R. A.; Stanfield, R. L.; Burton, D. R.; Wilson, I. A. Science 2001, 293, 1155–1159. (47) Sandin, S.; Ofverstedt, L.-G.; Wikstrom, A.-C.; Wrange, O.; Skoglund, U. Structure 2004, 12, 409–415.

Figure 5. XPS data for continuous, 20 nm Au film, annealed, coated with antigen (rabbit IgG protein) and antibody (anti-rabbit IgG protein) layers.

immobilization of a specific receptor (rabbit IgG), and proteinprotein interaction, i.e., specific antibody recognition (anti-rabbit IgG). Addition of each new layer results in an increase in the SP peak intensity and a red shift of the peak maximum, as expected from the increase in the effective refractive index.5 Exceptions are the ethanolamine hydrochloride and bovine serum albumin (BSA) layers used for blocking extra carboxylic groups and nonspecific binding sites, respectively, where no notable changes in the spectra were observed, indicating an insignificant amount of nonspecific protein binding sites. Note that in the present system the BSA treatment was not necessary for obtaining specificity, as indicated by similar experiments performed without BSA; however, to show the general applicability of the method, we followed the standard protocol which almost always includes a BSA step. Binding of the antigen results in an increase of ∼0.1 au in the SP intensity, while the recognition step shows an increase of ∼0.04 au. The considerably smaller change in plasmon intensity accompanying the antibody vs antigen binding is attributed Analytical Chemistry, Vol. 80, No. 19, October 1, 2008

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Figure 6. Specific recognition of IgG antibodies; experiments carried out according to route 3 (as in Figure 3) using 5 nm Au island films, annealed. (a-d) Absolute and difference spectra for two specific and two nonspecific antibody-antigen interactions (indicated), showing the last step of antibody binding to the antigen-derivatized surface (see Figure 3). (e-h) HRSEM images corresponding to the spectra in parts a-d, showing the final state after antibody binding.

primarily to the greater distance of the antibody from the Au surface, as well as to a somewhat lower protein density, as discussed below. Reports on SEM imaging of proteins such as IgGs on surfaces are scarce and obtained at a low resolution, showing protein spots,48 a continuous protein layer,49 or nanoparticle-labeled proteins.50 In all cases visualization of specific protein-protein binding has not been achieved. HRSEM images of Au island films before and after antigen immobilization and antibody binding (48) Lopez, G. P.; Biebuyck, H. A.; Harter, R.; Kumar, A.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10774–10781. (49) Kalele, S. A.; Ashtaputre, S. S.; Hebalkar, N. Y.; Gosavi, S. W.; Deobagkar, D. N.; Deobagkar, D. D.; Kulkarni, S. K. Chem. Phys. Lett. 2005, 404, 136– 141.

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according to route 1 (Figure 1), i.e., directly on the Au islands, are presented in Figure 2a,c,d at a single-protein resolution. Protein binding to the Au island surface (Figure 2c,d) is clearly visible, while the difference between one (Figure 2c) and two (Figure 2d) protein layers is easily recognized. Although individual antibody-antigen complexes cannot be distinguished, single protein molecules can be resolved (see arrow in Figure 2c). Hence, standard sample preparation (fixation + Cr coating, see Experimental Section) enables direct HRSEM visualization of protein recognition. (50) Wolfram, T.; Belz, F.; Schoen, T.; Spatz, J. P. Biointerphases 2007, 2, 44– 48.

Figure 2e,f and Figure 4a-c show, respectively, HRSEM and AFM images of continuous Au films before and after binding of one and two protein layers. The HRSEM image (Figure 2f) demonstrates the surface after antigen + antibody binding, viewed at a single-protein resolution, while the corresponding AFM image (Figure 4c) shows mostly aggregates, due to tip convolution. For the same reason the difference between Figure 4b (one protein layer) and Figure 4c (two protein layers) is not clear. Figure 2f indicates an essentially full protein layer under the applied experimental conditions. In order to estimate the thickness of the protein layers, samples similar to those shown in Figure 4b,c were subjected to mechanical abrasion using multiple scanning of the AFM tip operated in the contact mode, to form a 800 × 800 nm2 window in the layers,36 as shown in Figure 4d for a sample similar to that in Figure 4c. Line-scan analysis in the acoustic ac mode, carried out in air (Figure 4d), of several windows obtained in this manner resulted in average thickness values of 8.1 ± 1.2 nm for the antibody + antigen layer and 3.9 ± 0.9 nm for the antigen layer only (not shown). IgG proteins are commonly presented in the literature47,51 as disks of ∼14 nm diameter and 4 nm thickness. IgG proteins spontaneously assembled on surfaces are known to lie flat on the surface.43,51 The AFM-measured thickness values of the IgG protein layers in the present work (∼4 nm per protein layer) are consistent with a parallel orientation of the protein molecules with respect to the surface. The presence of proteins on the Au surface was verified by X-ray photoelectron spectroscopy (XPS), performed on continuous Au film substrates directly modified with rabbit + anti-rabbit IgG protein layers. High-resolution spectra of the protein elements N (1S), O (1S), and C (1S) are presented in Figure 5. The shape of the C (1S) peak (Figure 5c) showing three carbons attributed to sCdO, sCsNs, and sCsH is characteristic of protein molecules on surfaces.52 In addition to the previously discussed AFM results, the thickness of the protein layers (antibody + antigen) was estimated from the XPS data.36,53 The thickness, evaluated from attenuation of the Au peak, was 8.2 nm, in good agreement with the value derived from AFM profilometry (8.1 ± 1.2 nm). Use of the IgG antigen transducers for monitoring selective antibody binding, i.e., differentiation between specific and nonspecific interactions, was demonstrated using the two antigenantibody couples under full analyte coverage. The latter was verified by real-time analyte binding experiments showing binding saturation within several minutes. The recognition interfaces were prepared on 5 nm Au island films, annealed, according to route 3 (Figure 1). Spectroscopic and imaging results of the four cross-experiments, two specific and two nonspecific, are shown in Figure 6. In the two cases of specific binding (rabbit-anti-rabbit and mouse-anti-mouse, Figure 6a,c), the difference spectra show a substantial increase of ∼0.04 au in plasmon peak intensity, while in the two nonspecific cases (rabbit-anti-mouse and mouse-anti(51) Tronin, A.; Dubrovsky, T.; Nicolini, C. Thin Solid Films 1996, 284-285, 894–897. (52) Deligianni, D. D.; Katsala, N.; Ladas, S.; Sotiropoulou, D.; Amedee, J.; Missirlis, Y. F. Biomaterials 2001, 22, 1241–1251. (53) Yang, H. C.; Aoki, K.; Hong, H. G.; Sackett, D. D.; Arendt, M. F.; Yau, S. L.; Bell, C. M.; Mallouk, T. E. J. Am. Chem. Soc. 1993, 115, 11855– 11862.

Figure 7. LSPR recognition of human chorionic gonadotropin (hCG) antibody. Transmission UV-vis spectra for (a) specific (hCG-antihCG) and (b) nonspecific (hCG-anti-rabbit) antibody-antigen interactions are shown. hCG protein was immobilized directly on bare Au island surface.

rabbit, Figure 6b,d) the spectra before and after antibody binding are nearly indistinguishable and the difference spectra (expanded × 10) show insignificant changes in SP peak intensity, i.e., minimal nonspecific binding. The respective HRSEM images (Figure 6e-h) support the spectroscopic data. The images corresponding to specific recognition (Figure 6e,g) show Au islands heavily covered with protein molecules similar to Figure 2d, while the images corresponding to nonspecific binding (Figure 6f,h) show a thinner protein coating, similar to Figure 2c. Note that similar sensing experiments carried out in situ showed the same specificity but a smaller change in the LSPR response compared to the ex situ measurements described above, due to the difference in the refractive index of the medium. The results in the two cases are in quantitative agreement with calculations according to eqs 1 and 2. The general applicability of the present LSPR sensing scheme using evaporated Au island films was demonstrated with another antigen, human chorionic gonadotropin (hCG) protein, a hormone used as the target analyte in early detection of pregnancy. hCG is a glucoprotein with a molecular weigh of 36.7 kDa, much smaller than IgG proteins (150 kDa) and of a different structure.54 LSPR transducers were prepared by immobilization of hCG protein directly on the Au nanoisland surface, as in route 1 (Figure 1), followed by exposure to specific (anti-hCG) or nonspecific (antirabbit) antibodies. As seen in Figure 7, immobilization of hCG protein causes a significant increase of ∼0.05 au in plasmon (54) Tegoni, M.; Spinelli, S.; Verhoeyen, M.; Davis, P.; Cambillau, C. J. Mol. Biol. 1999, 289, 1375–1385.


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Figure 8. Absolute (a,b) and difference (c) LSPR extinction spectra for specific (a) and nonspecific (b) antibody-antigen interactions. Results for interfaces prepared on 5 nm Au island films, annealed, according to routes 1, 2, and 3 (Figure 1) are presented.

intensity. Binding of the specific antibody (anti-hCG, Figure 7a) leads to an increase in the maximal extinction by ∼0.03 au, while exposure to the nonspecific antibody (anti-rabbit, Figure 7b) results in almost no change (very slight decrease) in the plasmon band. As discussed in the introduction, changes in the biological interface thickness (di in eq 2) are expected to have a major effect on the sensitivity of LSPR measurements, due to the rather small characteristic decay length of the SP evanescent field (Ld in eqs 1 and 2) in LSPR systems. The effect of the recognition interface thickness in controlling the sensitivity is demonstrated here by comparing immunosensing results obtained with interfaces prepared according to routes 1-3 (Figure 1), using rabbit IgG protein as the antigen in all cases. Figure 8 presents LSPR spectra for specific (anti-rabbit) and nonspecific (anti-mouse) antibody binding to the three different recognition interfaces. In all cases the characteristic difference between specific (spectra a) and nonspecific (spectra b) binding, as shown in Figure 6, is observed. The distinction between specific and nonspecific interactions is seen most clearly in the difference spectra c in Figure 8. The maximum SP intensity difference for the antibody binding step is 7496


0.067, 0.055, and 0.039 au for routes 1, 2, and 3, respectively, in qualitative agreement with the increasing thickness of the recognition interface (di in eq 2). Additional support for the results shown in Figure 8 is provided by the ellipsometry data presented in Figure 9, obtained using continuous, 20 nm Au substrates and rabbit IgG as the antigen. Parts a-c of Figure 9 correspond, respectively, to the three schemes of interface preparation (routes 1, 2, and 3 in Figure 1). In each case two specific (treatment with anti-rabbit) and two nonspecific (treatment with anti-mouse) measurement sequences are shown. For a full coverage by a transparent film, change in the ellipsometric parameter ∆ (i.e., -δ∆) corresponds directly to thickness change. In accordance with the spectroscopic and imaging results obtained with island films (Figures 6 and 8), nonspecific antibody binding shows almost no changes in ∆ whereas specific binding results in a significant decrease in ∆ in the recognition step (Figure 9). The values of δ∆ for the antigen and antibody layers on bare Au obtained from Figure 9a are ∼-3.3° and -2.5°, respectively. These values, together with the fact that the thicknesses determined by AFM for the two protein layers on Au are similar,

Figure 9. Ellipsometric results (shown as changes in ∆) for the different steps of interface preparation and protein recognition using continuous, 20 nm Au films, annealed and rabbit IgG protein as the antigen. Parts a-c correspond to routes 1, 2, and 3 (Figure 1), respectively. The last step was exposure to specific (anti-rabbit) and nonspecific (anti-mouse) antibodies.

indicate that the second protein layer is less dense than the first. Using the AFM thicknesses of 3.9 and 4.2 nm for the antigen and antibody layers and the respective δ∆ values of -3.3° and -2.5°, one can calculate the effective refractive index ηa of the two protein layers. The values obtained are ηa ) 1.40 for the antigen layer and ηa ) 1.28 for the antibody layer. Refractive index values reported in the literature for proteins vary between 1.33 for diluted protein solutions55 and 1.6 for dry proteins.56 Taking the latter value, the refractive index of air, and the ηa values calculated for the two protein layers and using the Landau-Lifshitz/Looyenga effective medium approximation,57 a porosity of ∼32% and 51% is calculated for the antigen and antibody layers, respectively. These values correspond to a 0.72 antigen/antibody binding ratio, close to the value of 0.9 reported for the binding ratio of IgG proteins in a randomly immobilized system.58 Figures 8c (routes 1-3) show changes in the SP extinction intensity upon antibody binding. The physical model represented (55) Cole, T.; Kathman, A.; Koszelak, S.; McPherson, A. Anal. Biochem. 1995, 231, 92–98. (56) Armstrong, S. H., Jr.; Budka, M. J. E.; Morrison, K. C.; Hasson, M. J. Am. Chem. Soc. 1947, 69, 1747–1753. (57) Moretti, L.; De Stefano, L.; Rossi, A. M.; Rendina, I. Appl. Phys. Lett. 2005, 86, 061107/061101–061107/061103. (58) Kang, J. H.; Choi, H. J.; Hwang, S. Y.; Han, S. H.; Jeon, J. Y.; Lee, E. K. J. Chromatogr., A 2007, 1161, 9–14.

Figure 10. Optical response of several LSPR transducers (prepared using 5 nm Au island films, annealed) to antigen (rabbit IgG) and antibody (anti-rabbit IgG) binding on different interfaces. (a) Comparison of the SP wavelength shift and maximum extinction difference. (b) Comparison of the experimentally measured and calculated values of the maximum extinction difference. Empty and filled symbols indicate antigen and antibody binding, respectively. Red squares, green triangles, and blue circles correspond to routes 1, 2, and 3 (Figure 1), respectively.

in eqs 1 and 2 is assumed to apply to both the SP intensity change and the wavelength shift, hence both variables may respond to variations in the local refractive index. Figure 10a presents spectroscopic results (average values) for several sets of IgG recognition experiments, showing data for the antigen (rabbit) and antibody (anti-rabbit) binding steps in routes 1-3 (Figure 1). Figure 10a exhibits the experimental values of the wavelength shift associated with each step vs the corresponding maximum change in the extinction.15 The scatter in the data reflects experimental variations in the thickness of the different layers, as seen in the ellipsometric measurements in Figure 9. Figure 10a shows a generally linear relationship between the two variables; a linear correlation was previously shown for evaporated Au island films.31 Also evident in Figure 10a is the effect of the distance sensitivity and interface thickness. Although the thickness (measured by AFM) of the two protein layers is similar, the spectroscopic response to antibody binding (filled symbols) is considerably lower than the response to antigen binding (empty symbols). Moreover, in both cases the response R decreases with increasing interface thickness, i.e., route 1 > route 2 > route 3. The applicability of an exponentially decaying SP evanescent field model to our experimental system was evaluated by using Analytical Chemistry, Vol. 80, No. 19, October 1, 2008

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eqs 1 and 2 to calculate the expected LSPR response (as a maximum change in the extinction) associated with the antigen and antibody binding steps. The values used for the calculations were m ) 0.5 au/RIU and Ld ) 10 nm, typical of LSPR transducers. For simplicity, the RI of all the layers was taken as 1.4, and layer thicknesses were estimated from the AFM measurements (3.9 and 4.2 nm for the antigen and antibody layers, respectively). The calculated values shown in Figure 10b are drawn vs the same experimental values used in Figure 10a. The scatter in the data precludes the establishment of a quantitative relationship; however, qualitatively a general linear correlation is observed. The combination of m and Ld used in the model calculation to fit the experimental results is not unique, and these parameters should be determined independently. Despite these limitations, Figure 10b suggests that the model presented in eqs 1 and 2 provides a satisfactory description of the response of LSPR transducers based on random Au island films. Future improvements in our transducer technology (e.g., a flow system) will enable us to decrease the data scatter and obtain more accurate estimations of the critical parameters m and Ld as well as achieve quantitative predictions and system optimization. CONCLUSIONS The applicability of LSPR spectroscopy based on Au island film transducers to monitoring antibody-antigen interactions was demonstrated under full coverage conditions. To examine the effect of the protein recognition interface thickness on the sensitivity, three types of interfaces with increasing thickness were prepared: (i) direct adsorption of the IgG antigen on the Au (route 1); (ii) antigen binding to a carboxylate-functionalized silane monolayer on the Au (route 2); and (iii) antigen binding to a carboxylate-functionalized silica layer on the Au (route 3). Film thicknesses were determined by AFM profilometry and by ellipsometry. The antigen-derivatized Au nanoisland films were used for demonstrating LSPR sensing of antibody binding, distinguishing between specific and nonspecific interactions in cross-experiments with two IgG antigen-antibody pairs. The LSPR results, clearly showing specific protein recognition, were sup-

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ported by XPS, ellipsometry data, and direct AFM and HRSEM imaging. The latter enabled one to distinguish between one (antigen) and two (antigen + antibody) protein layers at a singleprotein resolution, providing direct evidence for specific vs nonspecific antibody binding. The general applicability of Au island transducers was further demonstrated with the hCG-anti-hCG/ anti-rabbit immunosensing system, emphasizing the potential for label-free sensing of biological molecules. The effect of the recognition interface thickness on the LSPR sensitivity was seen as a marked decrease in intensity change (from 0.067 to 0.039 au) and a corresponding decrease in the wavelength shift for antibody binding to transducers prepared according to route 1 vs route 3, respectively. Treatment of the experimental LSPR data for the protein binding steps showed qualitative agreement with a model assuming exponential decay of the SP evanescent field, using reasonable parameters for the RI sensitivity and decay length. Further quantification requires improved reproducibility of the data as well as experimental determination of the latter parameters. The thickness of the recognition interface in LSPR biosensing has a much greater effect on the detection sensitivity than in SPR sensing, due to the smaller decay length of LSPR transducers. ACKNOWLEDGMENT The authors wish to thank Dr. Hagai Cohen and Dr. Ayelet Vilan for the XPS measurements, Mrs. Orna Yeger and Mr. Alex Yoffe for assistance with sample preparation for HRSEM imaging, Dr. Irina Shin for assistance with protein handling, and Dr. Shirley Daube for helpful discussions. Support of this work by the Israel Science Foundation, Grant No. 672/07, and by EU NEST Project PROSURF, No. 028331, is gratefully acknowledged. This research is made possible in part by the historic generosity of the Harold Perlman family.

Received for review July 1, 2008. Accepted August 1, 2008. AC8013466

Biological Sensing and Interface Design in Gold Island Film Based

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