LETTER pubs.acs.org/Langmuir
Protein-Based SERS Technology Monitoring the Chemical Reactivity on an α-Synuclein-Mediated Two-Dimensional Array of Gold Nanoparticles Daekyun Lee,† Young-Jun Choe,† Minwoo Lee,‡ Dae H. Jeong,‡ and Seung R. Paik*,† †
School of Chemical and Biological Engineering, College of Engineering, and ‡Department of Chemistry Education, Seoul National University, Seoul 151-744, Republic of Korea
bS Supporting Information ABSTRACT: The enhancement of weak Raman signals has been challenged to obtain high-quality signals of surface-enhanced Raman scattering (SERS). By employing the Parkinson’s disease-related protein of α-synuclein, we introduce SERS-active gold nanoparticles (AuNPs) individually isolated with an ultrathin α-synuclein shell and their 2-D array into a tightly packed monolayer on a glass support, which permits a quantitative SERS measurement of phthalocyanine tetrasulfonate (PcTS), a chemical ligand of the pathological protein. Subsequently, the PcTS-bound SERS substrate was also shown to be capable of discriminating two biologically important metal ions of iron and copper by detecting copper ion to the sub-ppm level in a highly selective manner via the in situ chemical reaction of metal chelation to PcTS. The strategy of using the protein-based 2-D AuNP SERS platform, therefore, could be further developed into a custom-made protein-based biosensor system for the detection of not only specific chemical/biological ligands of the immobilized coat proteins but also their biochemical reactivities.
’ INTRODUCTION SERS provides a very sensitive, nondestructive target-monitoring platform technology that reveals structural information about analytes.1 3 Raman scattering signals of analytes have been amplified via direct adsorption to roughened metal surfaces or the surfaces of colloidal metal nanoparticles.4 A 2-D array of surfactant-functionalized AuNPs and silver nanoparticle embedded layer-by-layer (LBL) films have been developed to increase the SERS signals by creating a substrate geometry called “hot spots” where Raman signals are enhanced via plasmonic coupling between the noble metal nanoparticles.5,6 For more practical molecular sensing techniques, tip-enhanced Raman spectroscopy (TERS) that utilizes a gold metal tip to function as the Raman signal amplifier has been devised to detect substances with high spatial resolution and to avoid the heterogeneity of hot spots on the SERS substrate.7,8 Shell-isolated, nanoparticle-enhanced Raman spectroscopy (SHINERS) has been also introduced; it employs a monolayer of AuNPs individually coated with ultrathin silica or an alumina shell.9 By replacing the Au tip in TERS with a number of shell-isolated nanoparticles, the SERS signals were significantly amplified. Despite the merits these methods have offered, both approaches are inadequate for targeting biomolecules or chemicals present in multicomponent mixtures of biological fluids because of the nonspecific adsorption of other macromolecules to the surfaces.3 Moreover, the selective adsorption of analytes onto the SERS-active surface cannot be guaranteed because none of the r 2011 American Chemical Society
SERS substrates described above have appropriate sites to encourage the specific analyte interaction. A few efforts have been made to obtain specificity by modifying SERS-active metal substrates with target-specific agents, which results in high SERS cross-sections and alterations in the vibrational spectra upon analyte binding. Dithiocarbamate and 4-mercaptobenzoic acid were used as capping agents on the gold surface to detect inorganic metal ions such as Zn2+ and Hg2+/Pb2+, respectively.10,11 For biosensor development, SERS-active metal substrates were coated with antibodies or aptamers. Cocaine metabolites were successfully detected by the immobilization of their specific antibodies on silver-coated carbon nanotubes, and the geometrical change in the adenosine-specific DNA aptamer was demonstrated to amplify the SERS effect of AuNP coated with a Raman reporter molecule of 4-aminobenzenethiol.12,13 On extension of applying the SERS technology to analyze biologically important molecules and their structural information, we have prepared AuNPs directly coated with an ultrathin layer of α-synuclein and aligned them into a 2-D array to obtain an amplified high-quality SERS signals toward its chemical ligand of phthalocyanine tetrasulfonate (PcTS). The SERS substrate in turn was specifically utilized to detect metal ions with a high Received: August 11, 2011 Revised: September 15, 2011 Published: September 26, 2011 12782
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Scheme 1. Schematic Representation of the Fabrication Procedure of an α-Synuclein-Mediated 2-D Array of AuNPs and Its Use as a SERS Substrate for the Detection of PcTS and Metal Chelation
sensitivity. α-Synuclein is a well-known amyloidogenic protein forming β-sheet-enriched protein nanofibrils via a specific molecular self-assembly process, which plays a major causative role in the pathology of Parkinson’s disease.14 We have recently demonstrated that photoconductive anisotrophic AuNP chains embedded within the dielectric protein nanofibrils of α-synuclein were fabricated with α-synuclein-coated AuNPs as a building unit during their organic solvent or pH-induced assembly process.15 The hybrid conjugates of α-synuclein AuNPs have been employed here to construct a monolayered high-density 2-D assembly of AuNPs on a hydrophilic glass support. The resulting tightly packed SERS substrate must thus produce consistent high-quality SERS signals with improved sensitivity and detection reliability throughout the arrayed surface without a regional discrepancy. In addition, the protein matrix covering the nanoparticles certainly provides the following additional benefits other than the simple detection of specific ligands: (1) improved biocompatibility, (2) a matrix for functional modifications, and (3) a protective layer for both nanoparticles and analytes from the nonspecific adsorption of contaminants and chemical damage likely to be caused by the catalytic activity of the metal cores.3,16
’ EXPERIMENTAL SECTION Preparation of α-Synuclein-Coated AuNPs and Their TwoDimensional Arrays on a Glass Surface. AuNPs (∼10 nm) purchased from Sigma (cat. no. G 1527) were coated with cysteinecontaining mutants of α-synuclein (S9C, A53C, and Y136C) to prepare αsynuclein AuNPs by incubating 1 mg/mL of the mutants with 0.83A520 units/mL of colloidal AuNPs (volume ratio of 2 to 1) for 12 h at 4 °C. Following centrifugation at 16 100g for 20 min, the protein-coated AuNPs were successively washed twice with 20 mM MES (pH 6.5) to remove unbound proteins. The α-synuclein shell thickness was verified by transmission electron microscopy (TEM) (JEOL-1010 microscope, JEOL) operated at 80 kV. The 2-D arrays of α-synuclein AuNPs were fabricated by dipping a glass slide (hydroxyl-activated or alkylated) into 10 mM citrate buffer (pH 4.4) containing colloidal α-synuclein AuNPs for the indicated time periods. The slides were washed thoroughly with distilled water. Surface morphologies of the array surface were analyzed with FE-SEM (SUPRA 55VP FE-SEM system, Carl Zeiss) operated at 2.0 kV after platinum coating of the slides for 150 s using a sputter coater (SCD 005, BAL-TEC). Hydroxylation of a
Figure 1. SERS activity of α-synuclein(A53C) AuNPs on the PcTSlayered glass surface. The PcTS-layered surface was made by loading 0.5 mM PcTS on the glass surface to which wild-type α-synuclein was previously adsorbed in advance in 10 mM citrate (pH 4.4). Following thorough wash-off of the unbound PcTS, Raman spectra were recorded for the surfaces without (spectrum 1) and treated with either A53C AuNPs (spectrum 2) or double-coated AuNPs with A53C and wild-type α-synuclein (spectrum 3). Transmission electron microscope (TEM) images of the α-synuclein AuNPs. glass surface was carried out in pirahna solution, and further alkylation was accomplished by submerging the slide into 0.5% (v/v) 1H,1H, 2H,2H-perfluoredecylmethyldichlorosilane (Alfa Aesar) in iso-octane for 2 h at room temperature, followed by successive sonications in hexane and acetone.
SERS Detection of PcTS and Subsequent Binding of Transition-Metal Ions. The indicated concentrations of PcTS (cat. no. 194909, MP Biomedicals) were incubated with the substrate of 2-D arrays of α-synuclein AuNPs for 1 h at 37 °C in 20 mM MES (pH 6.5), and then the substrate was washed with the buffer and vacuum dried thoroughly before the SERS measurement. For experiments to detect transition-metal ions, the metal ion dissolved in 20 mM MES (pH 6.5) to different concentrations was further incubated with the substrate of PcTS-bound 2-D arrays of α-synuclein AuNPs. The SERS spectra were recorded on a LabRam 300 micro-Raman system (JY-Horiba) equipped with a CCD detector (Andor DU-401) and a 647 nm Kr ion laser (Innova 300C). The laser power on the sample was maintained at around 2 mW, and the acquisition time was 1 s in all cases. 12783
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Figure 2. Fabrication of 2-D array of α-synuclein AuNPs on a glass surface. (a) FE-SEM images of the 2-D arrays of AuNPs coated with three mutant α-synucleins (S9C, A53C, and Y136C) and a cross-section of the Y136C AuNP 2-D array. The inset in the Y136C AuNP array is a magnified image. The scale bars represent 500 nm. (b) Extinction spectrum of a 2-D array of Y136C AuNPs. The spectrum of colloidal Y136C AuNPs is also provided. (c) Extinction spectra of Y136C AuNP 2-D arrays at different pH values. The color development of the AuNP-array glass surfaces at the pH indicated is revealed in the upper panel. (d) FE-SEM images of the Y136C AuNP 2-D arrays prepared on alkylated and hydroxyl-activated glass surfaces. The scale bars represent 200 nm. All of the arrays were prepared by simply dipping the glass for 20 min into 10 mM citrate (pH 4.4) containing colloidal αsynuclein AuNPs at room temperature.
’ RESULTS AND DISCUSSION For the chemical-reactivity-based SERS monitoring system involving the PcTS and metal chelation, the α-synuclein-coated AuNPs have been aligned into a 2-D array (Scheme 1). After a monodisperse, tightly packed 2-D array of AuNPs is prepared, specific PcTS binding to the out-layered α-synuclein is evaluated with SERS signals that could be further altered with subsequent metal chelation because it would cause a structural change in the bound PcTS. The protein-shell metal nanoparticles were prepared by incubating AuNPs (∼10 nm) with three separate cysteinecontaining mutants of α-synuclein to replace serine (S9C), alanine (A53C), and tyrosine (Y136C). The covalent bonding of Au and sulfur allows the nanoparticles to have an ultrathin protein shell with an average thickness of 2.97 ( 0.59 nm. The target-specific SERS activity of the α-synuclein AuNP conjugates was evaluated with PcTS that was previously demonstrated to be a specific chemical ligand of α-synuclein.17 The light-absorbing and electron-donating properties of PcTS in addition to its metal binding and self-organizing characteristics designate it a multipotential compound to be applied in various areas requiring its photocatalytic, photoelectric, and photodynamic activities.18 In solution,
however, the Raman signals of PcTS were hardly amplified with the conjugated particles (Supporting Information Figure S1). The enhancement of the Raman signal became evident only when PcTS was layered on the glass surface, and it was challenged with the A53C AuNP conjugates, which gave rise to the doublet signals at around 700 cm 1 (Figure 1); the signals at 685 and 725 cm 1 correspond to isoindole deformation and isoindole breathing, respectively (Supporting Information Table S1). The enrichment of AuNPs toward PcTS on the surface ought to be responsible for the SERS activity. Intriguingly, however, the enhanced Raman signals disappeared with the AuNPs coated with a double layer of A53C and wild-type α-synuclein, which increased the shell thickness to 4.2 nm.15 The results clearly indicate that the shell thickness of ∼3 nm is critical to the SERS activity, which agrees well with the previous observation showing that the 2 to 3 nm shell thickness was crucial to the SERS activity of silica/ alumina-shell AuNPs.9 The shell thickness, therefore, should be carefully controlled when a custom-made protein-based ligand detection and reactivity monitoring system is planned to be developed by assessing the SERS activity. In addition, the results also suggest that the protein-coated noble metal nanoparticles 12784
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Figure 3. SERS detection of α-synuclein-interactive PcTS on the Y136C AuNP 2-D arrays. (a) SERS spectra of PcTS bound to the Y136C AuNP arrays. PcTS (0.5 mM) was incubated with the 2-Darrayed slide for 1 h at 37 °C in 20 mM MES (pH 6.5), and then the slide was washed and vacuum dried completely before SERS monitoring. As controls, a bare glass suface and a wild-type α-synuclein-coated surface were also monitored. (b) Two-dimensional SERS mapping of PcTS on the Y136C AuNP 2-D array. Two-dimensional SERS mapping was carried out for the area of 20 μm 20 μm, and SERS spectra were collected point by point with a step of 1 μm. One major Raman band at 685 cm 1 was plotted. The slides used in a and b were prepared by dipping overnight. (c) Effect of the AuNP-to-AuNP interaction on the SERS intensity. The Raman intensities of PcTS per one AuNP on the Y136C AuNP 2-D arrays prepared with different particle densities were estimated by dividing the total Raman intensity at 685 cm 1 by the actual number of AuNPs over an area of 1 μm2. The Raman intensity was recorded over three separate areas. The scale bars represent 200 nm.
could be employed as selective probes to detect specific chemical ligands of the corresponding proteins found on various surfaces.
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For the purpose of preparing the SERS-active substrate with AuNPs, their 2-D array formation was performed with the three α-synuclein AuNP conjugates by dipping the glass slides separately into 10 mM citrate at pH 4.4. The tightly packed, uniformly dispersed 2-D array of AuNPs was achieved only with Y136C AuNP conjugates whereas S9C AuNPs and A53C AuNPs yielded either a scattered or a locally clustered distribution of nanoparticles as examined with field-emission scanning electron microscopy (FE-SEM) (Figure 2a). The cross-sectional image of the 2-D array clearly confirmed the monolayer state of Y136C AuNPs as the embossed layer was revealed on top of the glass support with a thickness of 22 27 nm comprising AuNP, αsynuclein, and the platinum coat (∼10 nm) for FE-SEM analysis. Even with the high density enrichment of AuNPs on the surface, the surface plasmon resonance (SPR) spectrum was barely altered from that of colloidal Y136 AuNPs, only with a slight red shift of the peak maximum by 7 nm from 525 to 532 nm, unlike the AuNP agglomerates that generally broaden the SPR spectrum (Figure 2b). It is the unique self-assembly property of α-synuclein that is responsible for the tight packing and monodispersion of AuNPs in 2-D. In other words, the specific αsynuclein α-synuclein interactions enable AuNPs to be regularly spaced and uniformly distributed, which would be crucial to obtaining reliable SERS signals in homogeneity throughout the surface without regional bias. Because the 2-D array formation was mediated by α-synuclein and its self-interactive property, the pH influence on array formation was evaluated. In fact, our previous study indicated that pH played a pivotal role in aligning the α-synuclein-coated AuNPs into 1-D anisotropic pea-pod-type multichains by altering the chemical nature of α-synulcein on the AuNP surface to a more associative state.15 The extinction spectra of Y136C AuNP-coated slides obtained at different pH values for the same quantity of conjugates showed that the most effective adsorption occurred at pH 4.4, which is slightly lower than the isoelectric point of α-synuclein, pI 4.7 (Figure 2c). The high-density adsorption of AuNPs at pH 4.4 was also visualized as a winered surface on a piece of glass after 20 min of adsorption. At all other pH values, however, the AuNP enrichment was alleviated as estimated with the SPR spectra and the direct visualization of the glass surfaces. This pH sensitivity of AuNP enrichment also illustrates the involvement of protein in the process of AuNP 2-D alignment. In addition, the monodisperse 2-D array formation of the Y136C AuNPs was demonstrated to be dependent upon the chemical nature of the glass surface. The hydroxyl-activated hydrophilic surface was superior for the AuNP alignment to the alkylated hydrophobic surface (Figure 2d). The adsorbed AuNPs were very stable and thus hardly detached from the slide under harsh conditions employing various organic solvents and high salt concentrations (Supporting Information Figure S2). The SERS activity of the α-synuclein-mediated AuNP 2-D array was monitored with PcTS. The glass slides pretreated with α-synuclein in the presence or absence of AuNPs were immersed in PcTS at 0.5 mM for 1 h at 37 °C and then extensively washed and vacuum dried to remove the unbound compound completely. Strong SERS signals of PcTS were produced with the array of AuNPs coated with Y136C, but no signals from the protein-coated surface without AuNPs were produced (Figure 3a). The 2-D SERS mapping suggested that homogeneous SERS signals were acquired over the entire mapping area of 400 μm2 of the substrate; a mesh plot against one major SERS band at 684 cm 1 displayed the homogeneous intensity profile 12785
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Figure 4. SERS-based selective detection of copper ions on the PcTS-bound Y136C AuNP 2-D arrays. (a) PcTS-bound 2-D array prepared by incubating a slide of the Y136C AuNP 2-D array with 1 mM PcTS. Fe3+ and Cu2+ (500 μM) were incubated with the PcTS-bound slide for 1 h at 37 °C in 20 mM MES (pH 6.5). After complete washing and vacuum drying, Raman spectra were recorded. (b) Detection limit of Cu2+ on PcTS-bound Y136C AuNP 2-D arrays. Various concentrations of Cu2+ were incubated with the slide. The inset consists of enlarged spectra ranging from 800 to 1000 cm 1.
with a standard deviation of 8.39% (Figure 3b). This signal uniformity is the important parameter in building SERS substrates on a solid support, which is essential to securing the quality of the SERS signals. Another SERS mapping was performed for the A53C AuNP-arrayed slide as a control, which showed considerable variations in the signal intensities (Supporting Information Figure S3). These results, therefore, emphasize the notion of using the Y136C AuNP 2-D array for target-specific SERS applications. Moreover, the tight packing of the Y136 AuNP conjugates allowing optimal particle particle interactions was demonstrated to be another important contributing factor to the Raman signal amplification. The Raman intensity per nanoparticle (RINP) was estimated with the SERS substrates coated with Y136C AuNP at different particle densities obtained by differentiating the dipping periods of glass slides with respect to the colloidal solution of Y136C AuNP (Figure 3c and Supporting Information Figure S4). The RINP increased by more than 3-fold as the AuNP density increased with the dipping period from 20 min to 5 h, indicating that the interactions of AuNPs surrounded by covalently bound α-synuclein in 3 nm thickness certainly contributed to the production of SERS signals in addition to the simple increase in the number of particles. This enhancement, therefore, could also be attributable to the plasmon coupling phenomenon between the protein-shelled AuNPs. In fact, in our previous study, we noticed the coupling between AuNPs aligned within dielectric protein nanofibrils made of α-synuclein as evidenced by the photoelectric conductivity of the AuNP-embedded protein fibrils upon exposure to visible light. The chemical reactivity of bound PcTS on the surface of the Y136C AuNP 2-D array was evaluated with metal chelation by monitoring the altered SERS signals because specific metal binding to the tetrapyrrole compound would change the chemical structure of PcTS to a characteristic state.19 Among the transition-metal ions, Fe3+and Cu2+ were chosen for chemical reactivity-based metal detection because of their biological
importance. The addition of iron ion did not alter the SERS spectrum of PcTS, but intriguingly, the copper ion showed two extra vibrational bands at 748 and 954 cm 1 corresponding to isoindole breathing and benzene breathing, respectively (Figure 4a and Supporting Information Table S1). These two bands were observed in the PcTS copper derivative (PcTSCu2+), and the SERS spectrum of PcTS-Fe3+ also appeared to differ from that of PcTS (Supporting Information Figure S5). These results indicated the effective intercalation of the copper ion into the protein-bound PcTS. The detection limit of the copper ion was determined by measuring the vibrational band at 748 cm 1 with the monodisperse Y136C AuNP array as it was challenged with serially diluted copper solutions (Figure 4b). It turns out that the copper ion was detected to 1 μM (0.17 ppm). More importantly, the PcTS-mediated metal ion sensing system was able to discriminate iron and copper at the 0.17 ppm level because these two metal ions are the most redox-active ions actively involved in producing reactive oxygen species via Fenton-type reactions in the presence of hydrogen peroxide, thus often causing biologically devastating conditions. The results, therefore, demonstrate that the PcTS-bound α-synucleinmediated AuNP array is a promising SERS platform for detecting biologically important metal ions down to the sub-ppm level with high selectivity. In addition, this study represents an example of chemical-reactivity-based SERS biosensor development, which could permit an expansion of powerful SERS technology to biological systems.
’ CONCLUSIONS For the development of a protein-based SERS biosensor, the successful fabrication of the tightly packed monodisperse AuNP 2-D array is pivotal, where α-synuclein has played an essential role as it has been involved in the highly specific molecular selfassembly phenomenon, especially during the amyloid fibrillation process. Although the exact molecular mechanism for the protein 12786
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Langmuir assembly is still elusive, specific protein protein interactions should exist between the C-terminally fixed α-synucleins (Y136C) on the surfaces of AuNPs because we have witnessed regularly spaced AuNPs in the 2-D array. Consequently, the uniformly distributed and closely spaced nanoparticles result in increased sensitivity via the SERS activity, signal uniformity throughout the surface, and thus improved detection reliability. Moreover, in addition to the advantage of the α-synuclein coat in preventing the direct contact of analytes with the SERS-active metal cores, the protein could provide a biologically compatible matrix that can be further modified via the immobilization of various components including chemicals and biological macromolecules. The resulting functionalized α-synuclein-mediated 2-D array of AuNPs, therefore, could diversify the SERS biosensor to detect various target molecules. In this respect, the localization of the multipotential compound of PcTS on the αsynuclein coat of the AuNP 2-D array can be appreciated because the modified AuNP array could be employed in areas involving the photoelectroconductive, photocatalytic, and photodynamic effects of PcTS, although this investigation has focused on its metal chelation activity as an example of exhibiting chemical reactivity on the protein surface. The chemical reactivity to be assessed with the SERS signals may include any molecular interactions leading to structural changes in the Raman-active targets and actual chemical reactions resulting in the generation and degradation of Raman-active reaction products/substrates. In addition, the custom-made SERS biosensors could be generalized with various AuNP coat proteins capable of forming tightly packed 2-D array of AuNPs, where specific chemicals and biological molecules could be recognized by the individual coat proteins. The protein-based SERS sensor system, therefore, could further evolve into the areas of high-throughput screening of drug candidates or chemicals specific to disease-related or biologically important proteins.
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’ ASSOCIATED CONTENT
bS
Supporting Information. SERS activity of colloidal αsynuclein AuNPs upon PcTS binding, solvent stability of the Y136C AuNP 2-D array, 2-D SERS mapping of PcTS on the A53C AuNP-arrayed substrate, time-dependent surface coverage of Y136C AuNPs, SERS spectra of metal derivatives of PcTS-Fe2+ and PcTS-Cu2+, and the results of Raman assignments. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Tel: 82-2-880-7402. Fax: 82-2-888-1604. E-mail:
[email protected].
’ ACKNOWLEDGMENT This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2010-0009809). ’ REFERENCES (1) Hossain, M. K.; Kitahama, Y.; Huang, G. G.; Han, X.; Ozaki, Y. Anal. Bioanal. Chem. 2009, 394, 1747–1760. (2) Lal, S.; Link, S.; Halas, N. J. Nat. Photonics 2007, 1, 641–648. 12787
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