Anal Bioanal Chem DOI 10.1007/s00216-013-7587-5

RESEARCH PAPER

Using a silver-enhanced microarray sandwich structure to improve SERS sensitivity for protein detection Xuefang Gu & Yuerong Yan & Guoqing Jiang & Jason Adkins & Jian Shi & Guomin Jiang & Shu Tian

Received: 21 September 2013 / Revised: 13 December 2013 / Accepted: 16 December 2013 # Springer-Verlag Berlin Heidelberg 2014

Abstract A simple and sensitive method, based on surfaceenhanced Raman scattering (SERS), for immunoassay and label-free protein detection is reported. A series of bowlshaped silver cavity arrays were fabricated by electrodeposition using a self-assembled polystyrene spheres template. The reflection spectra of these cavity arrays were recorded as a function of film thickness, and then correlated with SERS enhancement using sodium thiophenolate as the probe molecule. The results reveal that SERS enhancement can be maximized when the frequency of both the incident laser and the Raman scattering approach the frequency of the localized surface plasmon resonance. The optimized array was then used as the bottom layer of a silver nanoparticle–protein– bowl-shaped silver cavity array sandwich. The second layer of silver was introduced by the interactions between the proteins in the middle layer of the sandwich architecture and silver nanoparticles. Human IgG bound to the surface of this microcavity array can retain its recognition function. With the Raman reporter molecules labeled on the antibody, a detection limit down to 0.1 ng mL−1 for human IgG is easily achieved. Furthermore, the SERS spectra of label-free proteins (catalase, Electronic supplementary material The online version of this article (doi:10.1007/s00216-013-7587-5) contains supplementary material, which is available to authorized users. X. Gu : G. Jiang : J. Shi (*) : G. Jiang : S. Tian (*) School of Chemistry and Chemical Engineering, Nantong University, Nantong 226007, China e-mail: [email protected] e-mail: [email protected] Y. Yan Department of science and technology, Jiaozuo Teachers College, Jiaozuo 454000, China J. Adkins Institute of Chemical Power Sources, Soochow University, Suzhou 215006, China

cytochrome C, avidin and lysozyme) from the assembled sandwich have excellent reproducibility and high quality. The results reveal that the proposed approach has potential for use in qualitative and quantitative detection of biomolecules. Keywords SERS . Bowl-shaped silver cavity . Silver enhancement . Immunoassay . Label-free protein detection

Introduction Multiplexed measurement of proteins is important in comprehensive proteomic surveys, studies of protein networks and pathways, validation of genomic discoveries, and work on clinical biomarkers [1–3]. Mass spectroscopy (MS) and immunoassay are two general approaches used for protein detection. However, the degradation of many proteins during the process of preparation, the cost of the instrumentation, and the destructive nature of the technique are challenging disadvantages of routine use of MS detection [4, 5]. The accepted best method for single-protein measurement is immunoassay, which makes use of the diversity and specificity of an antibody to its corresponding antigen. After the formation of a single antigen–antibody or an antibody–antigen–antibody sandwich, researchers use analytical tools for the readout, including: fluorescence [6, 7], chemiluminescence, electrochemical detection [8, 9], enzymatic [10], surface plasmon resonance (SPR) [11], and surface-enhanced Raman scattering (SERS) [12–24]. Of these techniques, fluorescence spectroscopy has been one of the most widely used readout methods, primarily because of its high sensitivity. However, broad emission spectra from molecular fluorophores make multiplex immunoassay impossible, and susceptibility to photobleaching may greatly worsen the detection limits.

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Surface-enhanced Raman scattering (SERS), on the other hand, has proved to be an advantageous analytical technique in the field of biochemistry because of its nondestructive nature, ultrahigh sensitivity, and the richness of molecular information it offers. The first indicators of the rapid expansion of SERS in basic biological science were the use of SERS for nucleic acids, by Koglin et al. in 1979 [25], and the first use of surface-enhanced resonance Raman scattering (SERRS) for detecting protein with heme chromophores, by Cotton et al. [26]. Several characteristics of SERS make it a reliable readout method. First, SERS signals provide rich structural information through sharp and distinguishable vibration bands, which have contributed greatly to the technique’s popularity, especially for label-free protein detection. Second, Raman bands are narrower than fluorescence bands, and are therefore well suited for multiplexing and background subtraction. For fluorescein isothiocyanate (FITC) molecules (the labeled molecules used in this study, as discussed below), for example, the Raman band at 1618 cm−1 has a full width at half-maximum (fwhm) of 20 cm−1 or 1 nm, whereas the fluorescence emission has a fwhm of 50 nm under ambient conditions. The large widths of fluorescence bands limit the ability to distinguish such labels from each other. Third, the SERS signal from water is extremely weak, enabling the measurement of the aqueous samples under physiological conditions. Finally, the optimum excitation wavelength for SERS is mainly dependent on the SERS-active substrate, which means that one may use a single excitation source to complete a multiplex detection. The SERS effect occurs when the targeted molecules are placed in proximity to a roughened metal surface, and is caused by the electromagnetic (EM) and the chemical effect [27]. Silver and gold nanoparticles are the most frequently used SERS-active substrates in bio-application. The advantage of a silver substrate is that it provides the strongest enhancement of Raman signals, whereas the advantage of gold substrates is their excellent biocompatibility [2, 28, 29]. However, in protein detection, especially label-free detection, the SERS spectra of the same simple protein from different research groups are often significantly different. This phenomenon has two causes: one is the adsorption behavior of proteins and the distance-dependent enhancement of SERS, meaning that only amino acids bound directly to the surface of a SERS-active substrate or within a space no greater than five or six residues from the surface are enhanced; the other is the instability and the irregular aggregation of metallic nanoparticles. Therefore, it is very important to prepare homogeneous and stable SERS-active substrates for highly sensitive and reliable detection of proteins. Regular metallic gratings with micrometer or nanometerscale structures seem to be ideal substrates for SERS. The highly reproducible patterns, with a great variety of geometries and uniform-sized particles, largely overcome the

problem of poor control over particle aggregation. Moreover, such gratings can precisely confine the local EM field at the surface of the metallic structure and optimize the localized surface plasmon resonance (LSPR) to a specific wavelength, even to the region of near-infrared (NIR), which is a spectral region free from unwanted fluorescence or photoinduced sample degradation [30–32]. Bartlett et al. have developed a powerful technique for producing gold sphere segment void (SSV) arrays from PS templates through electrodeposition of metal around particles [33, 34]. These Au nanovoid arrays have been used as reproducible and tailorable substrates for several SERS applications [35–37]. More recently, through theoretical calculation, Huang et al. [38] revealed that fabrication of a particle-in-cavity (PIC) structure can result in a great increase to the SERS enhancement of the bridging molecules, caused by the coupling EM field between the bottom gold surface and the upper metallic nanoparticles. Herein, we report a highly sensitive and simple microarray method, designed for labeled immunoassay and for label-free protein detection. The proposed method is based on a sandwich structure, which was constructed by use of an immunocomplex or protein-bridged bowl-shaped silver cavity (BSSC) array and silver nanoparticles (AgNPs). A series of the BSSC arrays was fabricated by electrodeposition, using highly ordered polystyrene spheres as a template. To determine the optimum substrate for further protein detection, the plasmon resonances of such substrates were observed by reflection spectroscopy. The SERS signals of sodium thiophenolate on such substrates were collected, and correlated to the morphology of the BSSC thin films. Immunocomplexes or label-free proteins were adsorbed on the surface of these BSSC arrays, and the AgNPs were then introduced into the cavity via interaction between proteins and nanoparticles (see Scheme 1 for a schematic flow diagram). The building process of such a sandwich structure is a silver

Scheme 1 Schematic illustration of the procedures for immunoreaction and the parametrization scheme for the silver cavity

Using a silver-enhanced–microarray sandwich structure

enhancement process, which enables the entire immunocomplexes to be in the coupled EM field between the silver cavity and silver nanoparticles, thereby generating a significantly enhanced Raman signal with high reproducibility. The plasmon resonances of such substrates were observed by reflection spectroscopy. The significant enhancement, reproducibility, and application of such bowl-shaped substrates for labeled and label-free protein detection were evaluated by SERS.

pulse to a current density of 30 mA cm−2 for 100 ms, followed by a series of pulses of 5 mA cm−2 for 60 ms, separated by a rest time of 1 s (zero current). After the reaction, the assembly was rinsed and the template was removed by gently sonicating it in toluene for 5 min, to reveal Ag nanostructures on the ITO surface. The SEM images were observed on a Hitachi S4700 fieldemission scanning electron microscope. Ultraviolet–visible (UV–vis) mirror-reflection spectra were obtained on a Shimadzu UV-3600 spectrophotometer at an incident angle of 5 °.

Experimental Chemicals and materials Human immunoglobulin G (IgG), FITC-goat antihuman IgG, bovine serum albumin (BSA), cytochrome C, avidin, lysozyme, catalase, and sodium thiophenolate (TP) were obtained from Sigma and used without further purification. Silver nitrate, trisodium citrate and all other chemicals were purchased from Shanghai Chemical Reagent Company and used as received. All solutions were prepared with pure water from Millipore. The phosphate-buffered saline (PBS; 0.01 mol L−1, pH 7.2) used in this study contained 0.8 % NaCl, 0.02 % KH2PO4, 0.02 % KCl, and 0.12 % Na2HPO4·12H2O. A blocking buffer containing 1 % BSA was prepared by dissolving BSA in the PBS buffer. A washing buffer containing 0.05 % Tween-20 was prepared by adding Tween-20 to the PBS buffer. Assembly of the PS templates The monolayer of PS particles was generated by selfassembly at the air–water interface in a 5 cm diameter Petri dish, and transferred onto the gold-coated slides (more details can be found in an earlier publication [39]). The gold-coated slides used in this work were prepared by electromagnetic sputtering of a 10 nm-thick chromium layer and then a 200 nm-thick gold layer onto ITO glass slides. Bowl-shaped silver cavity array preparation Electrochemical deposition was performed in a thermostated cell at room temperature, using a conventional three-electrode configuration controlled by a CHI 660D electrochemical station. The template-coated substrate was the working electrode, and a platinum and an Hg–Hg2SO4 electrode were used as the auxiliary and reference electrodes, respectively. Silver was deposited from a cyanide-free plating bath (0.1 mol L−1 AgNO3 +0.1 mol L−1 EDTA + 0.05 mol L−1 NH4NO3 and several milliliters of concentrated ammonia were added to ensure a pH~9–10). The BSSC arrays were produced using multi-current pulse plating, with the first

Fabrication of protein-induced sandwich structures for SERS detection For the adsorption of label-free proteins, the BSSC arrays were immersed into different protein solutions (in 0.01 mol L−1 PBS buffer) for 2 h at 37 °C. After being rinsed three times with a washing buffer (0.01 mol L−1 PBS with 0.05 % tween-20), the silver substrates with adsorbed target proteins were immersed in silver colloid for 2 h at 37 °C and washed three times with ultrapure water. For the FITClabeled-protein detection, human IgG was immobilized on BSSC thin films by immersing the substrates in protein solutions of different concentrations. After being rinsed with the washing buffer, the substrates were soaked in a blocking buffer (1 % BSA in PBS solution) for 2 h at 37 °C to block the unoccupied sites of the BSSC surface, and then rinsed three times with the washing buffer. The substrates coated with human IgG were immersed in a solution of 100 μg mL−1 FITC-goat antihuman IgG for 2 h at 37 °C. Then all the substrates were rinsed three times with the washing buffer to remove any nonspecific binding antibodies. After that, the silver substrates with adsorbed target immunocomplexes were immersed in silver colloid for 2 h at 37 °C and washed three times with ultrapure water. Colloidal silver used in this work was prepared by aqueous reduction of silver nitrate with trisodium citrate, according to Lee’s protocols [40]. Different stages during the formation of the Ag–FITC labeled immunocomplex–BSSC thin film sandwich structure are illustrated in Scheme 1, and the SEM image of the silver cavity after the silver enhancement is shown as an inset.

SERS measurement SERS spectra were measured on a Jobin Yvon/HORIBA LabRam ARAMIS Raman spectrometer. The excitation sources included an air-cooled argon-ion laser (514 nm). For the detection of protein, the laser power at the sample was ~0.12 mW, and the accumulation time was 30 s. For the SERS detection of TP, the accumulation time was 10 s.

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Results and discussion Preparation and characterization of the BSSC arrays The typical SEM images of the assembled PS particles and silver cavity arrays are shown in Fig. 1. The PS particles can be spontaneously assembled into a two-dimensional monolayer with a large area at the air–water interface, and the highly ordered assembly structure of the PS particles can be preserved when they are carefully transferred onto the surface of a gold-coated ITO electrode. As can be seen, for spheres of 700 nm-diameter, the surface homogeneity is relatively good and no major defect is observed (Fig. 1a). Samples assembled from 400 to 1000 nm-diameter spheres, with high quality up to 1 cm2, were easily fabricated using this method, and PS monolayers were fabricated that contained defect-free areas as large as hundreds of μm2. During the deposition process, AgNPs fill the interstices among the PS spheres; i.e., the cavity arrays left in the silver film after removal of the PS spheres. Figure 1b shows a typical SEM image of a BSSC array of 320 nm thickness, grown using a template made of a monolayer of 700 nm-diameter PS spheres (as shown in Fig. 1a). It can be clearly seen that the resulting silver cavities match the size of the PS particles, and the array retains the two-dimensional close-packed ordering. Figures 1c–f show tilted views of the as-prepared BSSC arrays, using 980, 700, 580, and 500 nm-diameter PS spheres

Fig. 1 SEM images of (a) closely packed PS spheres of 700 nm-diameter, (b) the as-prepared BSSC arrays using template (a), and the tilted views of the as-prepared BSSC arrays using (c) 980, (d) 700, (e) 560, and (f) 500 nm-diameter PS spheres as templates

as templates, respectively. In these four situations, the charges passed to the surface of the electrodes were the same, meaning that the quantity of silver built up through electrodeposition should be the same. As can be seen, however, the thickness of the cavities differs. This is caused by the different volumes of the interstices among the PS spheres, indicating that the morphology of the silver cavities can be modulated by changing the diameter of the PS spheres. Moreover, a unique feature of this approach is that the electrochemical deposition enables fine control over the thickness of the resultant cavities through controlling the total charge passed to deposit the film (via changing the repeat time of the second pulse). The silver cavities with a gradient in thickness were fabricated on goldcoated substrates, which had been previously covered with templates made up of 410, 500, 580, 700, and 980 nmdiameter PS spheres; some of the typical SEM images are shown in Fig. S1 and S2 (see Electronic Supplementary Material). As for each set of silver cavity arrays with a specific template, the apertures of these silver cavities increased with an increase in the repeat time of the second pulse, reaching a maximum of the pore diameter of their respective PS spheres. A further increase in the number of small pulses led to a continuous increase in the thickness, until the PS template was completely covered. LSPR and SERS properties of such BSSC arrays Bartlett and co-workers have theoretically and experimentally researched the surface plasmon polariton (SPP) properties of gold cavity arrays, and revealed that the plasmonic properties of metal nanostructures are critically dependent on their shape and size [41–43]. In our previous study, we fabricated a set of BSSC thin films using a closely packed monolayer of 700 nmdiameter PS spheres as a template [44]. The reflection spectra of the films were then recorded as a function of film thickness to introduce the different SPPs of the cavity arrays; the different locations of the dips in the reflection spectra indicated the variation of the LSPR. Correlating the LSPR with the SERS intensities of p-aminothiophenol (PATP) molecules in the cavity, we revealed that the largest enhancement is obtained when the frequency of the LSPR is between the frequency range of the excitation laser and the Raman scattered light. As in this work, the templates used for silver cavity array fabrication were expanded to 410, 500, 580, 700, and 980 nmdiameter PS spheres. For clarity, the parametrization scheme for the void cavity is illustrated in Scheme 1, where R is the radius of the template PS spheres, h is the thickness of the silver cavity, and rpore is the radius of the cavity mouth. We found that these silver cavity arrays have some similarities with the 700-nm-diameter cavities reported previously. The LSPR varies with the thickness of the silver cavity (see Electronic Supplementary Material Fig. S3 and Fig. S4), and there is a specific thickness which provides the highest SERS

Using a silver-enhanced–microarray sandwich structure

enhancement. For example, when we used the 980 nmdiameter PS spheres as a template, the maximum SERS signal was obtained at 1.1R thickness; when the template used was 580 nm-diameter PS spheres, the strongest signal was obtained at 0.9R thickness. To further elucidate the dependence of LSPR on the structure of nanocavity arrays and the correlation between LSPR and SERS, we fabricated a set of semi-spherical cavities with fixed void height where h = R, and this value was found to be optimum for most of our substrates when performing SERS detection. Both reflection data and SERS spectra were obtained from the same nanocavity array; the reflection spectra were taken on clean nanocavity arrays before the SERS measurements. Figure 2 shows the SEM images, the mirror reflection spectra, and the SERS spectra of TP on five silver cavity arrays with diameters of 410, 500, 580, 700, and 980 nm. For clarity, in Fig. 2b all reflection spectra were converted to absorption spectra by the formula A=1−R (where A is absorptivity, and R is reflectivity). The absorption spectra of the nanocavity arrays are quite different, and the absorption peaks have a red-shift as the diameters of the nanocavities increase from 410 to 980 nm. The absorption spectra are well fitted by two peaks in each spectrum, as shown in Fig. 2b. The lower fitted peaks are at 499, 511, 521, 534, and 552 nm, and the higher fitted peaks are at 543, 576, 572, 571, and 580 nm, for the nanocavity arrays with the diameters of 410, 500, 580, 700, and 980 nm, respectively. Recently, Huang et al. [38] systematically researched the SPP properties of gold cavity arrays. The authors denoted the two dominant plasmonic modes as “0D” mode and “0P” mode. The “0D” mode confines light near the bottom of the void, whereas the “0P” mode

concentrates light near the center of the void. They found that these optical modes were tunable with void size, scaling almost linearly with void size at longer wavelengths. However, another mode, which they denoted as “SPP” mode, did not shift with void size. Although in this study we mainly focused on silver cavity arrays, the mechanism in the two cases could have some similarity. For silver cavity arrays, the lower fitted peak might belong to the “0D” mode, and the higher fitted peak could be the “SPP” mode. As can be seen from Fig. 3, there is an approximate linear relationship between the wavelength of the lower fitted peak and the cavity size, which agrees well with the theoretical results of Huang et al. [38]. This feature provides straightforward tunability of optical properties, which is crucial for many applications, for example SERS detection. To help us identify which peak (the higher or the lower fitted peaks) was associated with the LSPR, we further tested the SERS signal of TP on these nanocavity arrays. SERS spectra of TP are shown in Fig. 2c, and as a red curve in Fig. 4b; the predominant bands are at 419, 689, 998, 1022, 1072, and 1578 cm−1, originating from the ring-breathing mode coupled with the C–S stretching vibration. These SERS spectra for TP on silver agree well with previous literature in terms of the band positions and relative intensities [45]. TP has been chosen as a probe molecule for SERS study because it has a large scattering cross-section and forms a selfassembled monolayer on metal surfaces [46, 47]. Furthermore, the strong bands of TP molecules on silver surfaces belong to the totally symmetric a1 modes. As we know, the long-range EM enhancement and short-range chemical enhancement contribute simultaneously to the total

Fig. 2 (a) SEM images with diameters from 410 to 980 nm, as marked on upper left corner of each image. The corresponding reflection spectra and SERS spectra are listed below as (b) normalized mirror reflection spectra

of BSSC arrays, and (c) SERS spectra of TP adsorbed on five silver cavity arrays excited by a 514 nm laser

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Fig. 3 Resonant wavelengths of lower fitted peaks as a function of cavity size

enhancement. However, in the visible spectral region, only b2 modes can be enhanced via molecule-to-metal or metal-tomolecule charge transfer [48, 49]. That is, a1 modes are largely associated with the EM mechanism. The EM enhancement is strongly related to the SPR of the metal particles in the visible spectral region. Therefore, the intensity of the band at 1578 cm−1 was used as an indicator for EM enhancement on different nanostructured surfaces. We randomly collected 100 Raman spectra of TP under the 514 nm laser excitation from five different substrates (20 points from each substrate), shown in Fig. 2a; the average spectra are shown in Fig. 2c. The intensity of the band at 1578 cm−1 increases as the diameter increases from 410 to 700 nm, but decreases when the diameter of nanocavities is further increased to 980 nm. From the dependence of SERS signals on the morphology of the nanocavity arrays and the shifting trend of the lower fitted peaks, we believe that the lower fitted peaks might be associated with LSPR of nanocavity arrays. It has been well documented that large SERS signals are expected when the frequency of both the incident laser and the scattered Raman electromagnetic field approach the resonance frequency of LSPR [50, 51]. Further theoretical analysis predicts that the maximum SERS effect will occur when the LSPR wavelength, λLSPR, is equal to half the sum of the excitation (λin) and the Raman scattering (λout) wavelengths; that is, λLSPR = ½(λin + λout) [31, 52]. The incident light (in) used in our present experiments was 514 nm, and thus the Raman scattering wavelength corresponding to the band 1578 cm−1 of TP molecules was calculated to be 559 nm (out). Direct correlation can be observed between the Raman intensities shown in Fig. 4a (two-dimensional Raman maps of 50 points collected from five substrates corresponding to Fig. 2a) and the plasmonic resonances in Fig. 2b. Large signal enhancement occurs when the wavelength of the lower fitted peaks (here equivalent to the λLSPR) is close to the λin or the λout, corresponding to the desired resonance coupling between

Fig. 4 (a) Thickness-dependent spectral trajectory of TP on BSSC arrays with 514 nm excitation. The vertical axis represents different points on five different substrates, 1–10 represents silver cavity with 410 nm void diameter, and 11–20 represents 500 nm, 21–30 represents 580 nm, 31–40 represents 700 nm, and 41–50 represents 980 nm void diameter. The SERS spectrum of TP is also shown, as a red curve. (b) Variation of spectral intensity of TP on BSSC thin film excited by a 514 nm laser. The spectra were collected from five different substrates with nominally the same cavity size

incoming and outgoing radiation with the plasmons generated on BSSC surface. When the PS template used for fabricating the BSSC arrays was 700 nm, the wavelength of the lower fitted peak was 534 nm, which was almost the same as that obtained by the theoretical calculation (536 nm). As expected, the strongest SERS enhancement was obtained under these conditions. These results provide us with helpful guidelines for the design and fabrication of silver cavity arrays with specific templates, and for the choice of SERS substrates in later SERS detection. In this study, a 1618 cm−1 characteristic Raman band was chosen as the indicator for the quantitative immunoassay (as discussed below). The Raman scattering wavelength was calculated to be 561 nm, and λLSPR was then calculated to be 538 nm. On the basis of results shown in Fig. 2, a hemispheric silver cavity array with a 700 nm diameter was used as the SERS-active substrate to generate an optimized SERS signal. The SERS enhancement factors (EF) were determined by comparison to the spectrum of neat TP, as described elsewhere (normal Raman spectrum and SERS spectrum of TP are shown in Fig. S5, see Electronic Supplementary Material) [53]. The enhancement factor of the substrate with thickness R was calculated to be 1.7×107 for 1578 cm−1. The run-to-run reproducibility of the surface enhancement was assessed by

Using a silver-enhanced–microarray sandwich structure

randomly recording 50 SERS spectra of TP (Fig. 4b) for five BSSC arrays with the same nominal film thickness. The relative standard deviation of the peak intensity at 1578 cm−1 was calculated to be ca. 10.6 %. The results reveal that BSSC arrays have excellent performance in Raman enhancement, and could potentially be used to detect trace amounts of target analytes. Labeled-protein detection The proposed substrate for labeled-protein detection was based on FITC-labeling-specific recognition on the BSSC array, using antigen–antibody chemistry followed by silver enhancement and SERS detection. FITC is a commonly used fluorescent dye with an isothiocyanate (N = C = S) group, which has strong electronic transitions in the visible spectrum (maximum absorption at ca. 495 nm), so resonance Raman enhancement can be obtained using 514 nm excitation to further increase the signal intensities. In most cases, resonance Raman provides 2–3 orders of magnitude of additional enhancement, relative to surface enhancement alone [54]. Two main steps were involved in the fabrication of the biomolecules’ immobilized sandwich structures: integration of the biomolecule–BSSC array with complementary units, and deposition of AgNPs. Amino-acid residues bind to metal surfaces via hydrophobic binding, electrostatic, and covalent interactions. These strong interactions between proteins and the two layers of metal nanoparticles are necessary for constructing the sandwich structures. As shown in Scheme 1, in the inset SEM image, AgNPs are evenly distributed inside the cavity. In this case, silver enhancement is essential for improving the sensitivity of the method. The introduction of AgNPs has two advantages:

Fig. S6). The results revealed that the immobilized proteins retained their recognition function on the surface of the BSSC array and any nonspecific binding of antibodies was below the detection limit, suggesting the feasibility of using the proposed method for immunoassay. To elucidate the contributions of the AgNPs layer and the negative curvature of the cavity structure to the SERRS spectrum of the FITC-labeled immunocomplex, three SERRS spectra were collected, as depicted in Fig. 5. The SERRS intensity of the FITC-labeled complexes from the Ag–BSSC sandwich (Fig. 5a) was approximately 60 times stronger than that from BSSC thin films without silver enhancement (Fig. 5b). This result is in good agreement with the theoretical calculations by Huang [38]. Such particle-in-cavity hybrid architectures could greatly increase the SERS enhancement of the bridging molecules at the particle–cavity junctions; the additional enhancement could reach two orders of magnitude. To investigate the effect of the bowl-shaped cavity structure, control experiments were performed by comparing the spectra from an Ag–BSSC sandwich with that from a sandwich

1. a large SERS enhancement is generated across the cavity array, caused by the strong plasmon coupling between the underlying thin film and the upper AgNPs; and 2. the fluorescence emission can be efficiently quenched by the AgNPs, without interfering with Raman detection. Before the SERRS-based quantitative immunoassay, control experiments were conducted to investigate whether the immobilized proteins still had a recognition function on the surface of the BSSC array. FITC-labeled rabbit anti-human IgG and FITC-labeled rabbit anti-goat IgG were added to two separate BSSC substrates with human IgG immobilized on their surfaces. After the recognition step the substrates were rinsed thoroughly, and, after silver enhancement, the SERRS signal of the FITC was then collected. As expected, only the substrate in the FITC-labeled rabbit anti-human IgG solution could be detected, and the substrate immersed in the FITClabeled rabbit anti-goat IgG solution had no detectable SERS signal from its surface (see Electronic Supplementary Material

Fig. 5 SERS spectra of FITC-labeled immunocomplex adsorbed on BSSC thin film substrate (a) with silver enhancement and (b) without silver enhancement; and (c) SERS spectra of FITC-labeled immunocomplex adsorbed on flat Ag with silver enhancement. The concentration of human IgG is 50 ng mL−1

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structured on the surface of a flat thin silver film (on the same substrate, at the region without a PS template, as shown in Fig. 5c). Although weak SERS signals were detected on an AgNPs flat film substrate, the intensity of the 1618 cm−1 Raman band was a factor of 30 times weaker than that for the Ag–BSSC sandwich structure. Because the only difference between the two substrates is the structure of the bottom surface, we believe that it is this particular cavity structure that contributes to the order-of-magnitude enhancement . In Fig. S7 (see Electronic Supplementary Material), we measured the reflection spectrum of a flat Ag substrate. The only dip that can be seen is at ca. 410 nm, which was away from the wavelength of the incident light and the Raman scattered light, resulting in a weak Raman enhancement. The abovementioned results suggest that both the bottom Ag cavity and the upper AgNPs contribute to the SERRS signals observed in this study. Compared with flat SERS-active substrates, for example an electrodeposited silver surface without cavity structure, the sandwich structure may provide approximately three orders of magnitude of additional enhancement for the detection of the SERRS signals of the labeled FITC, thus reducing the limit of detection of the immunocomplexes. In this study, BSSC thin film and AgNPs were used to form an Ag–immunocomplex–BSSC sandwich structure for further SERS detection of labeled and label-free proteins. Quantitative FITC-labeled protein detection A set of experiments was designed to investigate the quantitative immunoassay performance and to determine the detection limits of the proposed method. Figure 6a shows the SERRS spectra of the FITC-labeled immunocomplex with different concentrations of human IgG (0.5–50 ng mL−1). As can be seen, the peak intensities for the vibrations at 1174, 1376, and 1618 cm−1 are strongly correlated with the concentration of the human IgG solution. The 1618 cm−1 characteristic Raman band, which can be attributed to xanthene ring C– C stretching vibration, was chosen as an indicator for quantitative immunoassay. The detection limit of this method was found to be as low as 0.1 ng mL−1. When the concentration was down to the detection limit (inset of Fig. 6a), the characteristic peak at 1618 cm−1 became difficult to distinguish from that of the blank spectrum. Compared with other similar projects [16, 20], we feel this limit is a satisfactory value and comparable to that of ELISA [55]. The intensity of a peak at 1618 cm−1 versus the logarithm of the concentration of human IgG was used for Gaussian curve fitting, and is plotted in Fig. 6b. Each point is an average of seven randomly selected points; each error bar is the sample-to-sample variability of the SERRS intensities. The remarkable increase in the SERRS intensities of FITC covers a large range of protein concentrations. Moreover, the observed signal has linearity between the logarithm of the human IgG concentration and the SERRS

Fig. 6 SERS spectra of (a) FITC-labeled immunocomplexes with different concentrations of human IgG, (b) SERS intensity of band at 1618 cm−1 as a function of concentration using Gaussian curve fitting. The incident laser wavelength is 514 nm

peak intensity within the range 0.5–5 ng mL−1, having a correlation coefficient of 0.9913. In comparison with fluorescence detection, the proposed SERS-based immunoassay had advantages in sensitivity and selectivity. In this study, FITC molecules served as Raman reporters. When the excitation laser was selected to match the electronic energy level of the dye molecules, additional enhancement could be achieved from a resonance effect. The relatively narrow bandwidth of the SERS signals may also provide better selectivity for the detection. The above results indicate that the proposed method could be a good candidate for a sensitive and highly selective biosensor for detection of biomolecules and for SERS-based immunoassay. Label-free protein detection In contrast with labeled protein detection, label-free detection of biomolecules provides direct spectroscopic evidence associated with the target molecule itself rather than the label. To verify the potential of the proposed method for use in label-

Using a silver-enhanced–microarray sandwich structure

free protein detection, we detected two hemoproteins (catalase and cytochrome C), and two proteins with no chromophore (lysozyme and avidin); the corresponding SERS spectra are shown in Fig. 7. A protein-bound heme gives rise to an intense visible absorption band, and thus contributes resonance Raman to the total enhancement. For each protein, the upper spectrum represents a higher concentration (50 μg mL−1 for the two hemoproteins, and 1 mg mL−1 for no-chromophore proteins) and the lower spectrum represents a tenfold-diluted concentration. As for catalase, the typical vibrations of heme, e.g. ν37Eu (1648 cm −1), ν19A2g (1593 cm −1), ν4A1g (1358 cm−1), ν14B1g (1157 cm−1), and ν42Eu (1277 cm−1) are shown in the SERS spectra [56, 57]. Similar vibrations can be found in the SERS spectra of cytochrome C. The SERS spectra of lysozyme have an amide I (1655 cm−1) band, resulting from the R-helix structure, and bands assigned to the aromatic residues of phenylalanine (1005 cm−1), tryptophan (1573, 1377, and 1342 cm−1), and tyrosine (887 cm−1)

[2]. Just like lysozyme, SERS spectra of avidin mainly have vibrations of amide bands (1621 and 1243 cm−1) and of aromatic residues including Phe (1031 and 1002 cm−1), Trp (1576 and 1377 cm−1), and Tyr (855 cm−1) [2, 20]. As a kind of biological macromolecule, the adsorption behavior of proteins on SERS-active substrates differs substantially from that of small organic molecules, because of their three-dimensional structures and large diameters. The random adsorption of proteins on SERS-active substrates leads to different orientation and partial enhancement of the target proteins, causing the run-to-run, group-to-group variation. However, from Fig. 7a and b one can observe that the SERS spectra of the target molecules had no obvious change of wavenumber at different concentrations of the sandwich substrates. We believe that in the proposed sandwich architecture, the highly ordered bottom layer of the silver cavity array has a crucial function in the enhanced spectral reproducibility. A magnified image shows that the as-prepared silver cavities mainly consist of nanoparticles in the size range of 50–80 nm; these nanoparticles further interconnect to form many aggregates. It is very likely that the target proteins are adsorbed on the surface of these aggregates or at the gap between two adjacent nanoparticles; when AgNPs are introduced into the cavities, a coupled electromagnetic field across the whole cavity array can be generated. Therefore, vibration information from whole proteins would probably be displayed in their SERS spectra with remarkable enhancement and high reproducibility. The results imply that this SERS-based detection procedure for label-free proteins combines simplicity, sensitivity, and reproducibility; it may have great potential in both qualitative and quantitative practical protein detection.

Conclusion

Fig. 7 SERS spectra of (a) cytochrome C and avidin and (b) catalase and lysozyme. The concentrations of cytochrome C and catalase are 50 μg mL−1 (upper) and 5 μg mL−1 (lower), and the concentrations of avidin and lysozyme are 1 mg mL−1 (upper) and 0.1 mg mL−1 (lower)

By using a monolayer of closely packed PS spheres as a template, followed by electrodeposition, we have developed a versatile BSSC microarray, with much enhancement and reproducibility, for SERS measurement. Combining with silver enhancement, we subsequently constructed a silver nanoparticle–protein–BSSC thin film sandwich structure. Proofof-principle experiments based on an antigen–antibody recognition system were performed to investigate the possibility of using the proposed structure for the SERS detection of proteins. We found that both the bottom BSSC thin film and the upper AgNPs contribute to the enhancement of the final SERS signals, and the detection limit for FITC-labeled protein could be as low as 0.1 ng mL−1. Furthermore, using this technique, we have also successfully detected the SERS signal of labelfree proteins. This preliminary study reveals that the proposed method is promising for the SERS detection of both labeled and label-free proteins. Also, our method might be capable of multiplexing because, in principle, the screening of several

X. Gu et al.

different protein–protein interactions could be performed simultaneously on the same structure. Acknowledgements Financial support from the Nature Science Foundation of China (Nos. 21177067,21173122, 21201105), Natural Science Foundation of Jiangsu Province (BK20131200), and Scientific and Technological Innovation Projects of Nantong City (HS2012006, BK2012012) are gratefully acknowledged. We would like to thank Dr Lei Chen for his assistance in this work.

References 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

Kingsmore SF (2006) Nat Rev Drug Discov 5:310–321 Kahraman M, Sur I, Culha M (2010) Anal Chem 82:7596–7602 Isola NR, Stokes DL, Vo-Dinh T (1998) Anal Chem 70:1352–1356 Li J, Zhang Z, Rosenzweig J, Wang YY, Chan DW (2002) Clin Chem 48:1296–1304 Washam CL, Byrum SD, Leitzel K, Ali SM, Tackett AJ, Gaddy D, Sundermann SE, Lipton A, Suva LJ (2013) Cancer Epidem Biomar 22:972–983 Royer CA (2006) Chem Rev 106:1769–1784 Cao Y-C, Hua X-F, Zhu X-X, Wang Z, Huang Z-L, Zhao Y-D, Chen H, Liu M-X (2006) J Immunol Methods 317:163–170 Barlag RE, Halsall HB, Heineman WR (2013) Anal Bioanal Chem 405:3541–3547 Lin D, Wu J, Ju H, Yan F (2013) Biosens Bioelectron 45:195–200 Wang Z, Lee J, Cossins AR, Brust M (2005) Anal Chem 77:5770– 5774 Homola J (2008) Chem Rev 108:462–493 Rohr TE, Cotton T, Fan N, Tarcha PJ (1989) Anal Biochem 182:388– 398 Cui Y, Ren B, Yao J-L, Gu R-A, Tian Z-Q (2006) J Phys Chem B 110:4002–4006 Cui Y, Ren B, Yao J-L, Gu R-A, Tian Z-Q (2007) J Raman Spectrosc 38:896–902 Manimaran M, Jana NR (2007) J Raman Spectrosc 38:1326–1331 Han XX, Cai LJ, Guo J, Wang CX, Ruan WD, Han WY, Xu WQ, Zhao B, Ozaki Y (2008) Anal Chem 80:3020–3024 Han XX, Kitahama Y, Tanaka Y, Guo J, Xu WQ, Zhao B, Ozaki Y (2008) Anal Chem 80:6567–6572 Han XX, Zhao B, Ozaki Y (2009) Anal Bioanal Chem 394:1719– 1727 Han XX, Xie Y, Zhao B, Ozaki Y (2010) Anal Chem 82:4325–4328 Chen L, Han X, Yang J, Zhou J, Song W, Zhao B, Xu W, Ozaki Y (2011) J Colloid Interface Sci 360:482–487 Chen L, Park Y, Seo H, Hong W, Jung YM, Zhao B (2011) J Raman Spectrosc 42:1963–1966 Granger JH, Granger MC, Firpo MA, Mulvihill SJ, Porter MD (2013) Analyst 138:410–416 Guarrotxena N, Liu B, Fabris L, Bazan GC (2010) Adv Mater 22: 4954–4958 Guarrotxena N, Braun G (2012) J Nanopart Res 14:1–8 Koglin E, Sequaris JM, Valenta P (1980) J Mol Struct 60:421–425

26. Cotton TM, Schultz SG, Van Duyne RP (1980) J Am Chem Soc 102: 7960–7962 27. Kneipp K, Kneipp H, Itzkan I, Dasari RR, Feld MS (1999) Chem Rev 99:2957–2976 28. Giljohann DA, Seferos DS, Daniel WL, Massich MD, Patel PC, Mirkin CA (2010) Angew Chem Int Ed 49:3280–3294 29. Saha K, Agasti SS, Kim C, Li X, Rotello VM (2012) Chem Rev 112: 2739–2779 30. Dick LA, McFarland AD, Haynes CL, Van Duyne RP (2001) J Phys Chem B 106:853–860 31. Yu Q, Guan P, Qin D, Golden G, Wallace PM (2008) Nano Lett 8: 1923–1928 32. Hong G, Li C, Limin Q (2010) Adv Funct Mater 20:3774–3783 33. Bartlett PN, Baumberg JJ, Birkin PR, Ghanem MA, Netti MC (2002) Chem Mater 14:2199–2208 34. Bartlett PN, Baumberg JJ, Coyle S, Abdelsalam ME (2004) Faraday Discuss 125:117–132 35. Abdelsalam M, Bartlett PN, Russell AE, Baumberg JJ, Calvo EJ, Tognalli NSG, Fainstein A (2008) Langmuir 24:7018–7023 36. Johnson RP, Richardson JA, Brown T, Bartlett PN (2012) J Am Chem Soc 134:14099–14107 37. Mahajan S, Richardson J, Brown T, Bartlett PN (2008) J Am Chem Soc 130:15589–15601 38. Huang FM, Wilding D, Speed JD, Russell AE, Bartlett PN, Baumberg JJ (2011) Nano Lett 11:1221–1226 39. Tian S, Zhou Q, Li C, Gu Z, Lombardi JR, Zheng J (2012) J Phys Chem C 117:556–563 40. Lee PC, Meisel D (1982) J Phys Chem 86:3391–3395 41. Baumberg JJ, Kelf TA, Sugawara Y, Cintra S, Abdelsalam ME, Bartlett PN, Russell AE (2005) Nano Lett 5:2262–2267 42. Cole RM, Baumberg JJ, Garcia de Abajo FJ, Mahajan S, Abdelsalam M, Bartlett PN (2007) Nano Lett 7:2094–2100 43. Mahajan S, Abdelsalam M, Suguwara Y, Cintra S, Russell A, Baumberg J, Bartlett P (2007) PCCP 9:104 44. Tian S, Zhou Q, Gu Z, Gu X, Zheng J (2013) Analyst 138:2604– 2612 45. Joo TH, Kim MS, Kim K (1987) J Raman Spectrosc 18:57–60 46. Han SW, Lee SJ, Kim K (2001) Langmuir 17:6981–6987 47. Szafranski CA, Tanner W, Laibinis PE, Garrell RL (1998) Langmuir 14:3570–3579 48. Osawa M, Matsuda N, Yoshii K, Uchida I (1994) J Phys Chem 98: 12702–12707 49. Lombardi JR, Birke RL (2009) Acc Chem Res 42:734–742 50. Haynes CL, Van Duyne RP (2003) J Phys Chem B 107:7426–7433 51. Abdelsalam ME, Mahajan S, Bartlett PN, Baumberg JJ, Russell AE (2007) J Am Chem Soc 129:7399–7406 52. Félidj N, Aubard J, Lévi G, Krenn JR, Salerno M, Schider G, Lamprecht B, Leitner A, Aussenegg FR (2002) Phys Rev B 65: 075419 53. Lin X-M, Cui Y, Xu Y-H, Ren B, Tian Z-Q (2009) Anal Bioanal Chem 394:1729–1745 54. Doering WE, Nie S (2003) Anal Chem 75:6171–6176 55. Rosi NL, Mirkin CA (2005) Chem Rev 105:1547–1562 56. Han XX, Huang GG, Zhao B, Ozaki Y (2009) Anal Chem 81:3329– 3333 57. Keskin S, Culha M (2012) Analyst 137:2651–2657