Effect of coupled graphene oxide on the sensitivity of surface plasmon resonance detection Yeonsoo Ryu,1,† Seyoung Moon,2,† Youngjin Oh,1 Yonghwi Kim,1 Taewoong Lee,1 Dong Ha Kim,3 and Donghyun Kim1,* 1
School of Electrical and Electronic Engineering Yonsei University, Seoul 120-749, South Korea
2
Korea Institute of Science & Technology Evaluation and Planning, Seoul 137-717, South Korea 3
Department of Chemistry and Nano Science, College of Natural Sciences, Ewha Womans University, Seoul 120-750, South Korea *Corresponding author: [email protected]
Received 19 November 2013; revised 26 January 2014; accepted 29 January 2014; posted 30 January 2014 (Doc. ID 201623); published 27 February 2014
We investigated graphene-oxide-(GO-) coupled surface plasmon resonance (SPR) detection sensitivity for sandwiched antigen-antibody interaction between human and antihuman immunoglobulin G molecules. GO was prepared in a Langmuir–Blodgett solution on gold and dielectric surfaces. Theoretical and experimental data suggest that an increased dielectric spacer thickness reduces resonance shifts for GO-coupled SPR detection as dielectric properties of GO appear to prevail. In general, a metal-enhanced structure was shown to provide a larger resonance shift by plasmonic field enhancement. The far-field properties were described in terms of near-field overlap. The peak resonance shift that was obtained with GO-coupled SPR detection was enhanced to 113% of the resonance shift obtained by conventional thin-film-based SPR detection and may further be improved by GO stacking. © 2014 Optical Society of America OCIS codes: (240.6680) Surface plasmons; (280.1415) Biological sensing and sensors; (310.0310) Thin films. http://dx.doi.org/10.1364/AO.53.001419
1. Introduction
Surface plasmon (SP) refers to a longitudinal wave of electron concentration in which electron density oscillates in the direction of propagation at the dielectric–metal interface with p-polarized light incidence. Resonance arising from the creation of SP is sensitive to surface states and thus has been used as a label-free biosensing technique. Despite advantages of surface plasmon resonance (SPR) detection, it suffers from weaknesses, most notably, relatively poor binding capacity using traditional thin-filmbased plasmon sensing on the order of 1 pg∕mm2 [1]. For this reason, numerous approaches have been attempted to improve detection sensitivity. For 1559-128X/14/071419-08$15.00/0 © 2014 Optical Society of America
example, mediation of molecular interactions with metal nanoparticles (NPs) for amplification of optical signatures [2–4], combination of complementary detection techniques [5,6], localization of evanescent waves [7–9], detection of phase changes in an interaction [10–12], and colocalization of target interactions and surface fields [13–16]. Despite these efforts, sensitivity remains as a major issue in SPR detection. Recently, there have been growing interests in graphene which is known to be metallic despite its molecular composition even for an atomic monolayer and highly transparent with transmittance exceeding 97% [17]. Graphene oxide (GO) is often used as an intermediary in the creation of a graphene monolayer because functional groups attached to graphene sheets can modify electrical and optical properties of graphene or allow facilitated specific 1 March 2014 / Vol. 53, No. 7 / APPLIED OPTICS
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bindings on the surface through rich π-π interactions [18]. Graphene and GO have drawn tremendous attention because of their unique electronic and optical properties for improving the detection characteristics of SPR biosensors. For example, sensitive SPR detection was performed by modifying a GO surface with gold nanorods or NPs [19,20]. Also, protein adsorption and desorption were measured in realtime on a graphene surface by SPR detection [21]. Many studies of the graphene effects on SPR detection sensitivity were attempted from numerical aspects [22–25]. In this work, we investigate the effect of GO on the sensitivity of SPR detection by modulating the coupling of plasmonic fields to GO. We have restricted our attention to conventional thin-film-based detection without adopting any nanostructures. Therefore, a dielectric spacer that separates GO from underlying SP was adjusted as a way to modulate the degree of plasmonic coupling to GO, as shown in the optical schematic of Fig. 1(a). GO-coupled SPR detection was compared with metal-enhanced SPR structure in which GO film is replaced with gold and plasmonic fields created in the top and bottom metal layer can be coupled. The metal-enhanced structure is similar to waveguide-coupled SPR that uses metallic thin films for anisotropic molecular characterization [26,27], bimetallic detection [28,29], or electro-optic modulation [30–32]. In this study, an intermediate dielectric layer was not used
as a waveguide, i.e., a waveguide mode is not excited because total internal reflection condition is maintained. Even if a waveguide mode would exist, GO is weakly lossy and light power coupled to the waveguide mode should be negligible. The intermediate dielectric layer was employed to investigate the effect of GO or metal on the SPR detection by varying the coupled field strength in the GO-coupled and metal-enhanced SPR structure. On the other hand, coupling of plasmonic fields weakens with distance from SP, which may reduce resonance shift as a result of an interaction that takes place on the dielectric layer. The drastic differences between the dielectric and metallic coupling with underlying SP may help understand the nature of GO in the SPR detection. The characteristics of GO-coupled SPR detection were evaluated by measuring the sandwiched immunoassay of human immunoglobulin G (h-IgG) and antihuman IgG (a-h-IgG). Sandwich-type assays of IgG were used for colloidal enhancement of SPR signatures [33] and in various optical biosensors to reduce the effect of nonspecific adsorption [34,35]. 2. Materials and Methods A. Numerical Simulation
The schematic model of sandwiched antibody interactions of a-h-IgG, h-IgG, and a-h-IgG on GO presented in Fig. 1(a) assumed the GO layer to be
Fig. 1. (a) Optical schematic of GO-coupled SPR detection. The underlying gold film is 50 nm thick. The thickness of a SiO2 dielectric spacer is varied. For metal-enhanced SPR structure, the GO layer is replaced with gold. The interaction between a-h-IgG and h-IgG is modeled as a change of thickness. The thickness after the completion of sandwiched assay is 25 nm. (b) SEM image of GO flakes deposited by the Langmuir–Blodgett assembly method. Scale bar: 1 μm. The SEM image was taken on a silicon wafer to avoid the dielectric charging effect. A picture of a sample is shown in the inset. (c) AFM image and height profiles of GO flakes deposited on a glass substrate. The profiles along the dashed lines show that the thickness is approximately between 5 and 10 nm, indicating the deposition of multiple GO flakes. 1420
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formed on SiO2 ∕Au films and an SF10 glass substrate. For simplicity, an adhesion chrome layer was ignored. Abeles transfer matrix method was used for numerical computation to calculate axial field distribution. Material optical constants for gold and glass were taken from [36]. The refractive index of GO was taken to be 1.7 i0.17 [37]. It has been reported that the refractive index of h-IgG and ah-IgG falls in the range of 1.41–1.49 [38,39]. In this work, both refractive indices were assumed to be 1.41. The refractive indices of h-IgG and a-h-IgG being identical allows the complex biomolecular chemistry that takes place in the sandwiched assay to be simplified as an optical model of Fig. 1(a), because the resonance shifts measured as a result of an immune-reaction between h-IgG and a-h-IgG can be described as a change in the layer thickness [40]. h-IgG and a-h-IgG layers were assumed to be 7 and 9 nm, respectively, in thickness in line with [41]. Considering that both h-IgG and a-h-IgG are essentially identical immunoglobulin molecules with the same shape, molecular weight and dimension, except for the paratope sequence in the Fab region of the antibody, this assumption is more about slightly denser a-h-IgG, than h-IgG. Thus, optical path length is longer associated with a-h-IgG than with h-IgG. The effective refractive index of the buffer ambience was 1.33 in the model. All thin film layers were modeled as optically homogeneous and isotropic. The light source was p-polarized at a wavelength of λ 632.8 nm. B.
Optical Setup
SPR detection of sandwiched immunoassays employed two concentric rotation stages to implement θ∕2θ measurement. Light from a He–Ne laser (36 mW, λ 632.8 nm, Melles Griot, Carlsbad, California, USA) was first p-polarized through a polarizer to illuminate on a sample index-matched to an SF10 prism substrate. The photocurrent was measured by a p-i-n photodiode (818-UV, Newport, Irvine, California, USA) and fed to a low-noise lock-in amplifier (SR830DSP, Stanford Research Technology Inc., Sunnyvale, California, USA). The lock-in amplifier was synchronized with a chopper for intensity modulation. The measurement procedure was fully computer-controlled. C.
Sample Fabrication
Sample fabrication started with thermal evaporation of chrome (thickness: 2 nm) and gold (thickness: 50 nm) on an SF10 prism. A SiO2 layer was deposited by sputtering. The thickness of the SiO2 layer works as the separation between the layers of gold and GO and was thus varied as a way to change the degree of coupling in the range of 0–100 nm. For each set of geometrical parameters, 2–4 samples were fabricated and measured. A GO layer was prepared from solutions (Graphene Labs Inc., Calverton, New York, USA) and deposited onto the SiO2 layer using the Langmuir–Blodgett assembly method [42,43]. A
scanning electron microscope (SEM) image of the deposited GO is shown in Fig. 1(b). In the inset is a picture of a fabricated sample. The layer thickness of deposited GO flakes was measured by atomic force microscopy (AFM) to be on the order of a few nanometers, as shown in Fig. 1(c), indicating that the measured GO consisted of multiple flakes in a thickness between 5 and 10 nm. D.
Preparation of Sandwiched Immunoassays
For experimental confirmation, we have measured resonance shifts in response to sandwiched assays of immune-interactions. In the sandwiched assay, adsorption of a-h-IgG was followed by binding of h-IgG, and a-h-IgG in sequence. The procedure to produce sandwiched antibody-antigen assays is shown in Fig. 2, where h-IgG and a-h-IgG were used as antigen and antibody. h-IgG purified from serum and a-h-IgG (Fc specific) antiserum were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Au films used in the control experiment were modified with alkanethiols (MPA, 10 mM ethanolic). Carboxylate-modified (MPA-coated or GO) surfaces were first prepared so as to attach antibodies covalently via traditional carbodiimide. MPA was not coated on GO, which has its own carboxyl group on the surface. Protein immobilization was aided by active ester at the surface by adding 100 μL of a 100 mM, 1-ethyl-3-(3·(dimethylamino)propyl)carbodiimide hydrochloride (EDC, pH 5.5) solution with the carboxylated surface for 15 min. Injection of 50 μL sulfo-N, hydroxysuccinimide (S-NHS: 40 mM, pH 7) onto the surface was then followed for stabilization. The cell was rinsed with 1 mL of PBS (85 mM phosphate, pH 7.4). Protein immobilization was then performed by injecting 1 mL solution of a-h-IgG (120 μg∕mL) at 0.1 mL∕ min. Prior to immunochemical reaction, the surface was treated with 1 mL solution of bovine serum albumin (BSA, 10 mg∕mL) to block the unreacted chemical moieties, eliminating noise from nonspecific bindings. Immunochemical reaction was performed by injecting 1 mL h-IgG (1 mg∕mL) at 0.1 mL∕ min. Another 1 mL of a-h-IgG solution was then injected to form the sandwich configuration. All SPR curves were acquired in PBS before and after each immobilization and immunochemistry process after thorough rinsing with 1 mL PBS at 0.5 mL∕ min. Each measurement was taken three times for statistical analysis. 3. Results and Discussion
Figure 3 shows the theoretically calculated resonance shift that is produced by the GO-coupled SPR structure, as the thickness of a SiO2 spacer (tSiO2 ) is varied. The relative resonance shift (RRS), relative to the shift without GO at an identical SiO2 layer thickness is presented in Fig. 3, i.e., RRS ΔθGO ∕ΔθNo GO , as a black line. In other words, RRS > 1 represents resonance enhancement of optical signatures. The shifts concern the completion of sandwiched interactions: what was calculated as 1 March 2014 / Vol. 53, No. 7 / APPLIED OPTICS
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Fig. 2. Procedure of the sandwiched immunoassay. (a) The sample surface is initially carboxylate-modified. For preparation on GO, carboxyl modification was omitted. (b) Protein immobilization to the surface is followed by antibody binding of a-h-IgG. (c) The surface is treated with BSA to reduce nonspecific adsorption by reaction blocking. This is followed by (d) antigen binding of h-IgG and (e) subsequent antibody binding of a-h-IgG.
a result of intermediate steps produced almost identical trends in general, although shifts were not as large. In Fig. 3, a thicker SiO2 dielectric spacer decreases resonance shifts for GO because plasmonic fields are more weakly overlapped and the optical signature is reduced. The effect of field-matter overlap on the optical signatures in SPR detection was discussed in more detail elsewhere [44–46]. The resonance shift is the largest without a dielectric spacer (tSiO2 0)
Fig. 3. RRS calculated for GO-coupled and metal-enhanced SPR detection of sandwiched immunoassay as the dielectric spacer (SiO2 ) thickness is varied (GO-coupled detection, black line; metal-enhanced detection, red line). The arrows represent the increase of GO and gold layer thickness. 1422
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for all GO thicknesses. When dielectric separation was less than 40 nm (tSiO2 < 40 nm), a larger shift was obtained with thicker GO: at GO 10 nm, RRS 1.24, i.e., resonance shift is enhanced by 24%, compared to not using a GO layer. The RRS was also found to increase almost linearly with the thickness of GO in this case. For example, thicker GO increases RRS most efficiently in the absence of a SiO2 layer (tSiO2 0 nm) with a linear coefficient of 2.4%/nm (correlation coefficient R 0.9994). This indicates that GO-coupled SPR detection enhances resonance shift by 2.4% per each nanometer of GO over conventional SPR detection without GO. Although not presented explicitly in Fig. 3, an extremely thick GO layer, approximately above 10 nm, induces significant damping and thus leads to broad SPR curves that are inappropriate for specific detection. When the dielectric separation was approximately 40 nm, RRS was not affected by the GO layer thickness as a result of nonlinear dependence of optical signatures of GO on the spacer thickness tSiO2. It is also interesting to consider the effect of operating wavelengths. Although we focus on λ 632.8 nm, it was found that the resonance shift decreases as the wavelength increases and, at all wavelengths, the shift increases with the GO thickness almost proportionately [24]. For reference, we have compared the GO-coupled resonance shift with the shift produced by metalenhanced structure where gold is used in place of GO at an identical thickness of a SiO2 dielectric spacer. The RRS in this case is defined as ΔθAu ∕ΔθNo Au , shown as the red line in Fig. 3, and is
much larger than RRS obtained with GO, due presumably to the plasmonic fields amplified between the two gold films. If we compare RRS at an identical spacer thickness tSiO2, RRS of the metal-enhanced structure may not be larger than that of the GOcoupled structure for all the dielectric thicknesses and is larger only for tSiO2 > 20 nm. Interestingly, for metal-enhanced SPR detection, a larger resonance shift was obtained as the separation increased: resonance enhancement was 68% at tSiO2 100 nm. RRS was also almost linear with gold thickness: RRS increased with gold with a linear coefficient of 6.9%/ nm (R 0.9992) at tSiO2 100 nm. For better understanding, near-field profiles of tangential field amplitude jEx j are presented in Fig. 4 and generally support the far-field results shown in Fig. 3. Clearly, thicker GO tends to reduce the nearfield intensity, which is a direct consequence of nonzero attenuation in the refractive index of GO at the wavelength of interest. On the other hand, while the near-field intensity also decreases for the metal-enhanced SPR structure, it shows field enhancement associated with the coupling of SPs formed in the metal–dielectric interfaces. In fact, the field profiles presented in Figs. 4(e) and 4(f) are similar to those observed with the excitation of long-range SP. Larger RSS for the metal-enhanced structure than that of GO-coupled SPR detection appears to be related to a more drastic increase of fields in the coupling layer, i.e., δEx ∕δzgold > δEx ∕δzGO, the effect of which is more pronounced when the coupling layer is thick. The ratio between the slopes jδEx ∕δzgoldj∕jδEx ∕δzGOj in the coupling layer is approximately equal to 17 and 2 at tSiO2 40 and
80 nm, respectively, indicating stronger fields enhanced by gold and thus larger resonance shifts. The theoretical results suggest that GO-coupled SPR detection provides enhancement, although it may not be as large as that of metal-enhanced SPR. The result largely reflects the nature of GO that is much more dielectric than graphene, which in turn provides functional groups to host specific interactions on the surface or to be combined with metal NPs for amplification of optical signatures [47]. In this sense, it is obvious that the enhancement of GO decreases with the distance from plasmonic fields, i.e., the distance of GO from the fields, if increased, decreases the optical signatures produced by GO. Experimental GO-coupled SPR detection of sandwiched immunoassay was performed with samples of a dielectric spacer with thicknesses tSiO2 0, 10, 20, and 30 nm. Control samples without GO were also measured for tSiO2 0. Figures 5(a) and 5(b) present SPR characteristics measured at tSiO2 10 and 20 nm, where resonance occurs at a larger angle of incidence as the immunoassay reaction proceeds. RRS at tSiO2 0 was measured to be RRS 0.69∕0.61 1.13, in good agreement with theoretical results. Also, the measured resonance shift with GO-coupled detection decreased with dielectric separation, as Fig. 5(c) shows. RRS normalized by RRS (tSiO2 0 nm) presented in Fig. 5(c) indicates that RRS decreases with tSiO2 . In general, the decrease is more significant for the GO-coupled structure than metal-enhanced coupling and takes place more quickly if GO thickness increases. The metal-enhanced structure shows an opposite trend,
Fig. 4. Tangential near-field amplitude profiles of jEx j calculated for GO-coupled SPR detection: tSiO2 = (a) 0, (b) 40, and (c) 80 nm. The field amplitude was normalized by that of an incident light field. Also, for metal-enhanced SPR detection of sandwiched immunoassays: tSiO2 = (d) 0, (e) 40, and (f) 80 nm. Thickness of GO or gold: 0–10 nm. SI stands for sandwiched interaction. 1 March 2014 / Vol. 53, No. 7 / APPLIED OPTICS
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Fig. 5. GO-coupled SPR characteristics measured as the interactions progress in the sandwiched assay: tSiO2 = (a) 10 nm and (b) 20 nm. Each step made a positive resonance shift as shown in the insets. (1) a-h-IgG and (2) a-h-IgG represent the initial adsorption of IgG and the final binding of a-h-IgG to h-IgG, respectively. (c) Relative resonance shift normalized by that of a spacerfree (tSiO2 0 nm) structure. The decrease of RRS was less severe for metal-enhanced SPR detection (red) than for GO-coupled SPR (black). For the metal-enhanced structure, a thicker metal layer increased RRS. In contrast, thicker GO reduced RRS, i.e., RRS is more localized with thicker GO. Experimental data fitted in a quadratic polynomial (blue, R 0.95118) show the decreasing trend of RRS, which is in fair agreement with theoretical results. The error bar represents standard deviation.
i.e., the decrease of RRS is less significant for thicker gold in the coupling layer. In other words, for GOcoupled SPR detection, evanescent fields become more localized near the SiO2 ∕gold interface with thicker GO. For the metal-enhanced structure, this is the opposite because of the plasmonic field enhancement in the interface between the coupling and interaction layer. Experimental results generally confirm the trend that RRS decreases with SiO2 spacer thickness for both GO-coupled and metal-enhanced structures. The experimentally observed decrease of normalized RRS is clearer than theoretical results at an identical GO thickness, possibly because the extinction of GO flakes is higher than the theoretical parameter and simultaneously the deposited GO may have been much thicker by flake overlaps than a monolayer GO, as shown in Fig. 1(c). It was also found that GO-coupled SPR detection has larger standard deviation than what is typically observed in conventional SPR measurements, which may reflect relatively nonuniform deposition of GO flakes using the Langmuir– 1424
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Blodgett method. The deviation decreases at a thicker spacer because the effect of the variation in the individual flake thickness and also the flake overlap is less pronounced. While the enhancement in the resonance shift that is directly associated with GO-coupled SPR detection is modest at 13%, i.e., 113% of the resonance shift obtained by conventional thin-film-based SPR detection, the findings here suggest that the enhancement may be amplified proportionately by increasing GO thickness. In addition, use of GO in SPR detection may be combined with other approaches, e.g., amplification by metallic nanostructures, for more efficient resonance enhancement. The modest improvement of sensitivity by GO-coupled SPR detection is related to the loss of metallic nature for GO, which instead has functional groups for sensor applications. In this regard, metallic properties of graphene should be more directly capitalized, possibly by optical reduction of GO through heating for enhanced resonance characteristics. GO-coupled SPR can be made adaptable for many biosensor applications, based on chemically modified graphene products derived from functionalized GO, for example, by way of organic covalent functionalization to increase dispersion in solvents [48]. 4. Concluding Remarks
We have investigated detection characteristics of a GO-coupled SPR structure for sandwiched immunoassay. GO was prepared on gold and SiO2 using the Langmuir–Blodgett assembly method. It was found both theoretically and experimentally that a dielectric spacer would reduce resonance shift if the spacer is thick, as the near-field overlap with GO decreases. Also, thick GO tends to increase sensitivity in a linear fashion. GO-coupled SPR detection enhances resonant shift by at least 13% experimentally compared to conventional SPR detection, i.e., 113% of the shift that would be obtained by thin-film-based SPR detection, as the dielectric nature of GO prevails and makes it less effective than resonant coupling based on a metal-enhanced SPR structure. The sensitivity can be increased by GO stacking and other possibilities, such as reduction of GO, were discussed. The authors acknowledge Jae Eun Jang at DGIST for SiO2 sputtering. This work was supported by the National Research Foundation (NRF) grants funded by the Korean Government (2011-0017500 and NRF2012R1A4A1029061). †These authors contributed equally to this work. References 1. C. T. Campbell and G. Kim, “SPR microscopy and its applications to high-throughput analyses of biomolecular binding events and their kinetics,” Biomaterials 28, 2380–2392 (2007). 2. L. He, M. D. Musick, S. R. Nicewarner, F. G. Salinas, S. J. Benkovic, M. J. Natan, and C. D. Keating, “Colloidal Au-enhanced surface plasmon resonance for ultrasensitive detection of DNA hybridization,” J. Am. Chem. Soc. 122, 9071–9077 (2000).
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46. W. Lee and D. Kim, “Field-matter integral overlap to estimate the sensitivity of surface plasmon resonance biosensors,” J. Opt. Soc. Am. A 29, 1367–1376 (2012). 47. Y. Wan, Y. Wang, J. Wu, and D. Zhang, “Graphene oxide sheet-mediated silver enhancement for application to electrochemical biosensors,” Anal. Chem. 83, 648–653 (2011). 48. V. Georgakilas, M. Otyepka, A. B. Bourlinos, V. Chandra, N. Kim, K. C. Kemp, P. Hobza, R. Zboril, and K. S. Kim, “Functionalization of graphene: covalent and non-covalent approaches, derivatives and applications,” Chem. Rev. 112, 6156–6214 (2012).
School of Electrical and Electronic Engineering Yonsei University, Seoul 120-749, South Korea
2
Korea Institute of Science & Technology Evaluation and Planning, Seoul 137-717, South Korea 3
Department of Chemistry and Nano Science, College of Natural Sciences, Ewha Womans University, Seoul 120-750, South Korea *Corresponding author: [email protected]
Received 19 November 2013; revised 26 January 2014; accepted 29 January 2014; posted 30 January 2014 (Doc. ID 201623); published 27 February 2014
We investigated graphene-oxide-(GO-) coupled surface plasmon resonance (SPR) detection sensitivity for sandwiched antigen-antibody interaction between human and antihuman immunoglobulin G molecules. GO was prepared in a Langmuir–Blodgett solution on gold and dielectric surfaces. Theoretical and experimental data suggest that an increased dielectric spacer thickness reduces resonance shifts for GO-coupled SPR detection as dielectric properties of GO appear to prevail. In general, a metal-enhanced structure was shown to provide a larger resonance shift by plasmonic field enhancement. The far-field properties were described in terms of near-field overlap. The peak resonance shift that was obtained with GO-coupled SPR detection was enhanced to 113% of the resonance shift obtained by conventional thin-film-based SPR detection and may further be improved by GO stacking. © 2014 Optical Society of America OCIS codes: (240.6680) Surface plasmons; (280.1415) Biological sensing and sensors; (310.0310) Thin films. http://dx.doi.org/10.1364/AO.53.001419
1. Introduction
Surface plasmon (SP) refers to a longitudinal wave of electron concentration in which electron density oscillates in the direction of propagation at the dielectric–metal interface with p-polarized light incidence. Resonance arising from the creation of SP is sensitive to surface states and thus has been used as a label-free biosensing technique. Despite advantages of surface plasmon resonance (SPR) detection, it suffers from weaknesses, most notably, relatively poor binding capacity using traditional thin-filmbased plasmon sensing on the order of 1 pg∕mm2 [1]. For this reason, numerous approaches have been attempted to improve detection sensitivity. For 1559-128X/14/071419-08$15.00/0 © 2014 Optical Society of America
example, mediation of molecular interactions with metal nanoparticles (NPs) for amplification of optical signatures [2–4], combination of complementary detection techniques [5,6], localization of evanescent waves [7–9], detection of phase changes in an interaction [10–12], and colocalization of target interactions and surface fields [13–16]. Despite these efforts, sensitivity remains as a major issue in SPR detection. Recently, there have been growing interests in graphene which is known to be metallic despite its molecular composition even for an atomic monolayer and highly transparent with transmittance exceeding 97% [17]. Graphene oxide (GO) is often used as an intermediary in the creation of a graphene monolayer because functional groups attached to graphene sheets can modify electrical and optical properties of graphene or allow facilitated specific 1 March 2014 / Vol. 53, No. 7 / APPLIED OPTICS
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bindings on the surface through rich π-π interactions [18]. Graphene and GO have drawn tremendous attention because of their unique electronic and optical properties for improving the detection characteristics of SPR biosensors. For example, sensitive SPR detection was performed by modifying a GO surface with gold nanorods or NPs [19,20]. Also, protein adsorption and desorption were measured in realtime on a graphene surface by SPR detection [21]. Many studies of the graphene effects on SPR detection sensitivity were attempted from numerical aspects [22–25]. In this work, we investigate the effect of GO on the sensitivity of SPR detection by modulating the coupling of plasmonic fields to GO. We have restricted our attention to conventional thin-film-based detection without adopting any nanostructures. Therefore, a dielectric spacer that separates GO from underlying SP was adjusted as a way to modulate the degree of plasmonic coupling to GO, as shown in the optical schematic of Fig. 1(a). GO-coupled SPR detection was compared with metal-enhanced SPR structure in which GO film is replaced with gold and plasmonic fields created in the top and bottom metal layer can be coupled. The metal-enhanced structure is similar to waveguide-coupled SPR that uses metallic thin films for anisotropic molecular characterization [26,27], bimetallic detection [28,29], or electro-optic modulation [30–32]. In this study, an intermediate dielectric layer was not used
as a waveguide, i.e., a waveguide mode is not excited because total internal reflection condition is maintained. Even if a waveguide mode would exist, GO is weakly lossy and light power coupled to the waveguide mode should be negligible. The intermediate dielectric layer was employed to investigate the effect of GO or metal on the SPR detection by varying the coupled field strength in the GO-coupled and metal-enhanced SPR structure. On the other hand, coupling of plasmonic fields weakens with distance from SP, which may reduce resonance shift as a result of an interaction that takes place on the dielectric layer. The drastic differences between the dielectric and metallic coupling with underlying SP may help understand the nature of GO in the SPR detection. The characteristics of GO-coupled SPR detection were evaluated by measuring the sandwiched immunoassay of human immunoglobulin G (h-IgG) and antihuman IgG (a-h-IgG). Sandwich-type assays of IgG were used for colloidal enhancement of SPR signatures [33] and in various optical biosensors to reduce the effect of nonspecific adsorption [34,35]. 2. Materials and Methods A. Numerical Simulation
The schematic model of sandwiched antibody interactions of a-h-IgG, h-IgG, and a-h-IgG on GO presented in Fig. 1(a) assumed the GO layer to be
Fig. 1. (a) Optical schematic of GO-coupled SPR detection. The underlying gold film is 50 nm thick. The thickness of a SiO2 dielectric spacer is varied. For metal-enhanced SPR structure, the GO layer is replaced with gold. The interaction between a-h-IgG and h-IgG is modeled as a change of thickness. The thickness after the completion of sandwiched assay is 25 nm. (b) SEM image of GO flakes deposited by the Langmuir–Blodgett assembly method. Scale bar: 1 μm. The SEM image was taken on a silicon wafer to avoid the dielectric charging effect. A picture of a sample is shown in the inset. (c) AFM image and height profiles of GO flakes deposited on a glass substrate. The profiles along the dashed lines show that the thickness is approximately between 5 and 10 nm, indicating the deposition of multiple GO flakes. 1420
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formed on SiO2 ∕Au films and an SF10 glass substrate. For simplicity, an adhesion chrome layer was ignored. Abeles transfer matrix method was used for numerical computation to calculate axial field distribution. Material optical constants for gold and glass were taken from [36]. The refractive index of GO was taken to be 1.7 i0.17 [37]. It has been reported that the refractive index of h-IgG and ah-IgG falls in the range of 1.41–1.49 [38,39]. In this work, both refractive indices were assumed to be 1.41. The refractive indices of h-IgG and a-h-IgG being identical allows the complex biomolecular chemistry that takes place in the sandwiched assay to be simplified as an optical model of Fig. 1(a), because the resonance shifts measured as a result of an immune-reaction between h-IgG and a-h-IgG can be described as a change in the layer thickness [40]. h-IgG and a-h-IgG layers were assumed to be 7 and 9 nm, respectively, in thickness in line with [41]. Considering that both h-IgG and a-h-IgG are essentially identical immunoglobulin molecules with the same shape, molecular weight and dimension, except for the paratope sequence in the Fab region of the antibody, this assumption is more about slightly denser a-h-IgG, than h-IgG. Thus, optical path length is longer associated with a-h-IgG than with h-IgG. The effective refractive index of the buffer ambience was 1.33 in the model. All thin film layers were modeled as optically homogeneous and isotropic. The light source was p-polarized at a wavelength of λ 632.8 nm. B.
Optical Setup
SPR detection of sandwiched immunoassays employed two concentric rotation stages to implement θ∕2θ measurement. Light from a He–Ne laser (36 mW, λ 632.8 nm, Melles Griot, Carlsbad, California, USA) was first p-polarized through a polarizer to illuminate on a sample index-matched to an SF10 prism substrate. The photocurrent was measured by a p-i-n photodiode (818-UV, Newport, Irvine, California, USA) and fed to a low-noise lock-in amplifier (SR830DSP, Stanford Research Technology Inc., Sunnyvale, California, USA). The lock-in amplifier was synchronized with a chopper for intensity modulation. The measurement procedure was fully computer-controlled. C.
Sample Fabrication
Sample fabrication started with thermal evaporation of chrome (thickness: 2 nm) and gold (thickness: 50 nm) on an SF10 prism. A SiO2 layer was deposited by sputtering. The thickness of the SiO2 layer works as the separation between the layers of gold and GO and was thus varied as a way to change the degree of coupling in the range of 0–100 nm. For each set of geometrical parameters, 2–4 samples were fabricated and measured. A GO layer was prepared from solutions (Graphene Labs Inc., Calverton, New York, USA) and deposited onto the SiO2 layer using the Langmuir–Blodgett assembly method [42,43]. A
scanning electron microscope (SEM) image of the deposited GO is shown in Fig. 1(b). In the inset is a picture of a fabricated sample. The layer thickness of deposited GO flakes was measured by atomic force microscopy (AFM) to be on the order of a few nanometers, as shown in Fig. 1(c), indicating that the measured GO consisted of multiple flakes in a thickness between 5 and 10 nm. D.
Preparation of Sandwiched Immunoassays
For experimental confirmation, we have measured resonance shifts in response to sandwiched assays of immune-interactions. In the sandwiched assay, adsorption of a-h-IgG was followed by binding of h-IgG, and a-h-IgG in sequence. The procedure to produce sandwiched antibody-antigen assays is shown in Fig. 2, where h-IgG and a-h-IgG were used as antigen and antibody. h-IgG purified from serum and a-h-IgG (Fc specific) antiserum were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Au films used in the control experiment were modified with alkanethiols (MPA, 10 mM ethanolic). Carboxylate-modified (MPA-coated or GO) surfaces were first prepared so as to attach antibodies covalently via traditional carbodiimide. MPA was not coated on GO, which has its own carboxyl group on the surface. Protein immobilization was aided by active ester at the surface by adding 100 μL of a 100 mM, 1-ethyl-3-(3·(dimethylamino)propyl)carbodiimide hydrochloride (EDC, pH 5.5) solution with the carboxylated surface for 15 min. Injection of 50 μL sulfo-N, hydroxysuccinimide (S-NHS: 40 mM, pH 7) onto the surface was then followed for stabilization. The cell was rinsed with 1 mL of PBS (85 mM phosphate, pH 7.4). Protein immobilization was then performed by injecting 1 mL solution of a-h-IgG (120 μg∕mL) at 0.1 mL∕ min. Prior to immunochemical reaction, the surface was treated with 1 mL solution of bovine serum albumin (BSA, 10 mg∕mL) to block the unreacted chemical moieties, eliminating noise from nonspecific bindings. Immunochemical reaction was performed by injecting 1 mL h-IgG (1 mg∕mL) at 0.1 mL∕ min. Another 1 mL of a-h-IgG solution was then injected to form the sandwich configuration. All SPR curves were acquired in PBS before and after each immobilization and immunochemistry process after thorough rinsing with 1 mL PBS at 0.5 mL∕ min. Each measurement was taken three times for statistical analysis. 3. Results and Discussion
Figure 3 shows the theoretically calculated resonance shift that is produced by the GO-coupled SPR structure, as the thickness of a SiO2 spacer (tSiO2 ) is varied. The relative resonance shift (RRS), relative to the shift without GO at an identical SiO2 layer thickness is presented in Fig. 3, i.e., RRS ΔθGO ∕ΔθNo GO , as a black line. In other words, RRS > 1 represents resonance enhancement of optical signatures. The shifts concern the completion of sandwiched interactions: what was calculated as 1 March 2014 / Vol. 53, No. 7 / APPLIED OPTICS
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Fig. 2. Procedure of the sandwiched immunoassay. (a) The sample surface is initially carboxylate-modified. For preparation on GO, carboxyl modification was omitted. (b) Protein immobilization to the surface is followed by antibody binding of a-h-IgG. (c) The surface is treated with BSA to reduce nonspecific adsorption by reaction blocking. This is followed by (d) antigen binding of h-IgG and (e) subsequent antibody binding of a-h-IgG.
a result of intermediate steps produced almost identical trends in general, although shifts were not as large. In Fig. 3, a thicker SiO2 dielectric spacer decreases resonance shifts for GO because plasmonic fields are more weakly overlapped and the optical signature is reduced. The effect of field-matter overlap on the optical signatures in SPR detection was discussed in more detail elsewhere [44–46]. The resonance shift is the largest without a dielectric spacer (tSiO2 0)
Fig. 3. RRS calculated for GO-coupled and metal-enhanced SPR detection of sandwiched immunoassay as the dielectric spacer (SiO2 ) thickness is varied (GO-coupled detection, black line; metal-enhanced detection, red line). The arrows represent the increase of GO and gold layer thickness. 1422
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for all GO thicknesses. When dielectric separation was less than 40 nm (tSiO2 < 40 nm), a larger shift was obtained with thicker GO: at GO 10 nm, RRS 1.24, i.e., resonance shift is enhanced by 24%, compared to not using a GO layer. The RRS was also found to increase almost linearly with the thickness of GO in this case. For example, thicker GO increases RRS most efficiently in the absence of a SiO2 layer (tSiO2 0 nm) with a linear coefficient of 2.4%/nm (correlation coefficient R 0.9994). This indicates that GO-coupled SPR detection enhances resonance shift by 2.4% per each nanometer of GO over conventional SPR detection without GO. Although not presented explicitly in Fig. 3, an extremely thick GO layer, approximately above 10 nm, induces significant damping and thus leads to broad SPR curves that are inappropriate for specific detection. When the dielectric separation was approximately 40 nm, RRS was not affected by the GO layer thickness as a result of nonlinear dependence of optical signatures of GO on the spacer thickness tSiO2. It is also interesting to consider the effect of operating wavelengths. Although we focus on λ 632.8 nm, it was found that the resonance shift decreases as the wavelength increases and, at all wavelengths, the shift increases with the GO thickness almost proportionately [24]. For reference, we have compared the GO-coupled resonance shift with the shift produced by metalenhanced structure where gold is used in place of GO at an identical thickness of a SiO2 dielectric spacer. The RRS in this case is defined as ΔθAu ∕ΔθNo Au , shown as the red line in Fig. 3, and is
much larger than RRS obtained with GO, due presumably to the plasmonic fields amplified between the two gold films. If we compare RRS at an identical spacer thickness tSiO2, RRS of the metal-enhanced structure may not be larger than that of the GOcoupled structure for all the dielectric thicknesses and is larger only for tSiO2 > 20 nm. Interestingly, for metal-enhanced SPR detection, a larger resonance shift was obtained as the separation increased: resonance enhancement was 68% at tSiO2 100 nm. RRS was also almost linear with gold thickness: RRS increased with gold with a linear coefficient of 6.9%/ nm (R 0.9992) at tSiO2 100 nm. For better understanding, near-field profiles of tangential field amplitude jEx j are presented in Fig. 4 and generally support the far-field results shown in Fig. 3. Clearly, thicker GO tends to reduce the nearfield intensity, which is a direct consequence of nonzero attenuation in the refractive index of GO at the wavelength of interest. On the other hand, while the near-field intensity also decreases for the metal-enhanced SPR structure, it shows field enhancement associated with the coupling of SPs formed in the metal–dielectric interfaces. In fact, the field profiles presented in Figs. 4(e) and 4(f) are similar to those observed with the excitation of long-range SP. Larger RSS for the metal-enhanced structure than that of GO-coupled SPR detection appears to be related to a more drastic increase of fields in the coupling layer, i.e., δEx ∕δzgold > δEx ∕δzGO, the effect of which is more pronounced when the coupling layer is thick. The ratio between the slopes jδEx ∕δzgoldj∕jδEx ∕δzGOj in the coupling layer is approximately equal to 17 and 2 at tSiO2 40 and
80 nm, respectively, indicating stronger fields enhanced by gold and thus larger resonance shifts. The theoretical results suggest that GO-coupled SPR detection provides enhancement, although it may not be as large as that of metal-enhanced SPR. The result largely reflects the nature of GO that is much more dielectric than graphene, which in turn provides functional groups to host specific interactions on the surface or to be combined with metal NPs for amplification of optical signatures [47]. In this sense, it is obvious that the enhancement of GO decreases with the distance from plasmonic fields, i.e., the distance of GO from the fields, if increased, decreases the optical signatures produced by GO. Experimental GO-coupled SPR detection of sandwiched immunoassay was performed with samples of a dielectric spacer with thicknesses tSiO2 0, 10, 20, and 30 nm. Control samples without GO were also measured for tSiO2 0. Figures 5(a) and 5(b) present SPR characteristics measured at tSiO2 10 and 20 nm, where resonance occurs at a larger angle of incidence as the immunoassay reaction proceeds. RRS at tSiO2 0 was measured to be RRS 0.69∕0.61 1.13, in good agreement with theoretical results. Also, the measured resonance shift with GO-coupled detection decreased with dielectric separation, as Fig. 5(c) shows. RRS normalized by RRS (tSiO2 0 nm) presented in Fig. 5(c) indicates that RRS decreases with tSiO2 . In general, the decrease is more significant for the GO-coupled structure than metal-enhanced coupling and takes place more quickly if GO thickness increases. The metal-enhanced structure shows an opposite trend,
Fig. 4. Tangential near-field amplitude profiles of jEx j calculated for GO-coupled SPR detection: tSiO2 = (a) 0, (b) 40, and (c) 80 nm. The field amplitude was normalized by that of an incident light field. Also, for metal-enhanced SPR detection of sandwiched immunoassays: tSiO2 = (d) 0, (e) 40, and (f) 80 nm. Thickness of GO or gold: 0–10 nm. SI stands for sandwiched interaction. 1 March 2014 / Vol. 53, No. 7 / APPLIED OPTICS
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Fig. 5. GO-coupled SPR characteristics measured as the interactions progress in the sandwiched assay: tSiO2 = (a) 10 nm and (b) 20 nm. Each step made a positive resonance shift as shown in the insets. (1) a-h-IgG and (2) a-h-IgG represent the initial adsorption of IgG and the final binding of a-h-IgG to h-IgG, respectively. (c) Relative resonance shift normalized by that of a spacerfree (tSiO2 0 nm) structure. The decrease of RRS was less severe for metal-enhanced SPR detection (red) than for GO-coupled SPR (black). For the metal-enhanced structure, a thicker metal layer increased RRS. In contrast, thicker GO reduced RRS, i.e., RRS is more localized with thicker GO. Experimental data fitted in a quadratic polynomial (blue, R 0.95118) show the decreasing trend of RRS, which is in fair agreement with theoretical results. The error bar represents standard deviation.
i.e., the decrease of RRS is less significant for thicker gold in the coupling layer. In other words, for GOcoupled SPR detection, evanescent fields become more localized near the SiO2 ∕gold interface with thicker GO. For the metal-enhanced structure, this is the opposite because of the plasmonic field enhancement in the interface between the coupling and interaction layer. Experimental results generally confirm the trend that RRS decreases with SiO2 spacer thickness for both GO-coupled and metal-enhanced structures. The experimentally observed decrease of normalized RRS is clearer than theoretical results at an identical GO thickness, possibly because the extinction of GO flakes is higher than the theoretical parameter and simultaneously the deposited GO may have been much thicker by flake overlaps than a monolayer GO, as shown in Fig. 1(c). It was also found that GO-coupled SPR detection has larger standard deviation than what is typically observed in conventional SPR measurements, which may reflect relatively nonuniform deposition of GO flakes using the Langmuir– 1424
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Blodgett method. The deviation decreases at a thicker spacer because the effect of the variation in the individual flake thickness and also the flake overlap is less pronounced. While the enhancement in the resonance shift that is directly associated with GO-coupled SPR detection is modest at 13%, i.e., 113% of the resonance shift obtained by conventional thin-film-based SPR detection, the findings here suggest that the enhancement may be amplified proportionately by increasing GO thickness. In addition, use of GO in SPR detection may be combined with other approaches, e.g., amplification by metallic nanostructures, for more efficient resonance enhancement. The modest improvement of sensitivity by GO-coupled SPR detection is related to the loss of metallic nature for GO, which instead has functional groups for sensor applications. In this regard, metallic properties of graphene should be more directly capitalized, possibly by optical reduction of GO through heating for enhanced resonance characteristics. GO-coupled SPR can be made adaptable for many biosensor applications, based on chemically modified graphene products derived from functionalized GO, for example, by way of organic covalent functionalization to increase dispersion in solvents [48]. 4. Concluding Remarks
We have investigated detection characteristics of a GO-coupled SPR structure for sandwiched immunoassay. GO was prepared on gold and SiO2 using the Langmuir–Blodgett assembly method. It was found both theoretically and experimentally that a dielectric spacer would reduce resonance shift if the spacer is thick, as the near-field overlap with GO decreases. Also, thick GO tends to increase sensitivity in a linear fashion. GO-coupled SPR detection enhances resonant shift by at least 13% experimentally compared to conventional SPR detection, i.e., 113% of the shift that would be obtained by thin-film-based SPR detection, as the dielectric nature of GO prevails and makes it less effective than resonant coupling based on a metal-enhanced SPR structure. The sensitivity can be increased by GO stacking and other possibilities, such as reduction of GO, were discussed. The authors acknowledge Jae Eun Jang at DGIST for SiO2 sputtering. This work was supported by the National Research Foundation (NRF) grants funded by the Korean Government (2011-0017500 and NRF2012R1A4A1029061). †These authors contributed equally to this work. References 1. C. T. Campbell and G. Kim, “SPR microscopy and its applications to high-throughput analyses of biomolecular binding events and their kinetics,” Biomaterials 28, 2380–2392 (2007). 2. L. He, M. D. Musick, S. R. Nicewarner, F. G. Salinas, S. J. Benkovic, M. J. Natan, and C. D. Keating, “Colloidal Au-enhanced surface plasmon resonance for ultrasensitive detection of DNA hybridization,” J. Am. Chem. Soc. 122, 9071–9077 (2000).
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