Letter pubs.acs.org/Langmuir

Effect of Hydration on Plasmonic Coupling of Bioconjugated Gold Nanoparticles Immobilized on a Gold Film Probed by SurfaceEnhanced Raman Spectroscopy Jeremy D. Driskell,* Carleigh G. Larrick, and Christopher Trunell Department of Chemistry, Illinois State University, Normal, Illinois 61790, United States S Supporting Information *

ABSTRACT: Gold nanoparticle (AuNP)−Au film constructs were prepared using antibody−antigen interactions or a small organic crosslinker to systematically control the gap between the AuNP and Au film. Surface-enhanced Raman spectroscopy (SERS), scanning electron micrsocopy (SEM), and atomic force microscopy (AFM) were used to characterize each construct and elucidate structure−activity relationships. Interestingly, plasmonic coupling and SERS intensity were reversibly modulated with wetting/drying cycles for the protein immobilized AuNP, and this effect was attributed to changes in protein size with hydration state. This work provides insight into fundamental limitations of AuNP-enabled SERS bioassays and will facilitate rational design of novel biospecific ligands that maximize SERS sensitivity.



INTRODUCTION Surface-enhanced Raman scattering (SERS) is a powerful analytical tool that provides molecular-specific information and high sensitivity, extending the possibilities of traditional vibrational spectroscopy. The signal enhancement results from an intensified electric field in close proximity to an appropriately designed nanostructured substrate. While enhanced electric fields are observed from isolated gold and silver nanoparticles and nanorods, it has been extensively demonstrated that the largest electric fields, and therefore SERS enhancement, are localized in the gaps between closely spaced nanoparticles1−4 or between a nanoparticle and a metal film.5−11 Several approaches to reproducibly and precisely immobilize gold nanoparticles (AuNPs) on a gold film have been instrumental in detailing the plasmonic coupling between AuNP localized surface plasmons and Au film surface plasmon polaritons that is responsible for the large electric fields. For example, polyelectrolyte layers,8 self-assembled monolayers,5 polymers,6,7 oligonucleotides,9 and oxide films10 have facilitated subnanometer control over the AuNP−Au film gap distance. These previous works consistently demonstrate a nonlinear increase in SERS enhancement with a corresponding decrease in gap distance. AuNP−Au film plasmonic coupling has been exploited by several groups, including ours, to develop SERS immunoassays.12−21 Toward this end, an antibody is immobilized on the surface of a gold film, and antigen is then captured on the surface via antibody−antigen interactions. Surface bound antigen is then labeled with a modified AuNP, e.g., extrinsic Raman label (ERL) or SERS tag. This extrinsic labeling © 2014 American Chemical Society

approach to detection is often desirable to the direct detection of intrinsic Raman signal from the antigen because Raman reporter molecules can be selected with large Raman cross sections for maximum sensitivity and unique spectral signatures to afford multiplexed detection.19 The high sensitivity in these assays is only enabled by the coupling between the ERL and the underlying Au film, as a well-dispersed suspension of nonaggregated ERLs does not provide a detectable signal.5 Importantly, the dimensions of the antibody and antigen control the AuNP−Au film gap, and any variability in gap distance is expected to have a significant impact on SERS enhancement and assay sensitivity.2,9,10 Surprisingly, the effect of antibody-modified AuNP on SERS signal has not been directly probed, considering the dimensions of an IgG molecule suggest minimal AuNP−Au film coupling based on computational and experimental evidence using precisely controlled model systems. Moreover, antibody size is dependent on its hydration state, which is anticipated to influence the AuNP−Au film gap, thereby significantly impacting the SERS enhancement. Here we use an antibody−antigen model system to quantify the diminished SERS signal resulting from the antibody modification relative to the signal of a Raman active AuNP without the antibody coating. Most significantly, we show the plasmonic coupling between bioconjugated AuNPs and an underlying Au film, and therefore SERS signal, is modulated with wetting/drying cycles. Received: February 18, 2014 Revised: May 21, 2014 Published: May 22, 2014 6309

dx.doi.org/10.1021/la500640q | Langmuir 2014, 30, 6309−6313

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Letter

EXPERIMENTAL SECTION

ERLS consisting of AuNPs modified with 4-nitrobenzenethiol (NBT) and goat antimouse IgG antibodies were prepared as detailed in the Supporting Information, following a previously reported procedure.18,21 Smooth gold film substrates (1 × 1 cm2) were first modified with goat antimouse IgG antibodies and then mouse IgG antigen to saturate the antibody binding sites. ERLs were then allowed to bind to the antibody−antigen modified Au film to form an antibody−antigen− antibody sandwich construct, which places three IgG proteins in the gap between the AuNP and Au film. Additional samples were prepared in which AuNPs were modified with a mixed monolayer of 3,3′dithiobis(sulfosuccinimidylpropionate) (DTSSP) and NBT (Supporting Information).5 These DTSSP/NBT-modified AuNPs were then allowed to bind to the surface of a 2-aminoethanethiol (AET)modified gold film. SERS spectra were acquired for each sample type, i.e., three-protein construct and AET immobilized AuNP, with an Enwave Optronics, Inc. ProRaman-L-785B Analyzer, equipped with a 785 nm diode laser as the excitation source. SERS spectra were collected from the sample surface in air and through a liquid layer. An Asylum Research MFP-3D atomic force microscope was used to image the AuNPs immobilized on a Au film via antibody−antigen−antibody interactions, i.e., threeprotein construct. The atomic force microscope (AFM) was operated in tapping mode to image the samples in air and in liquid to determine the height of the ERLs in the dry and hydrated states. An FEI-Quanta 450 scanning electron microscope (SEM) was used to image the samples in order to measure AuNP surface coverages and calculate a normalized SERS signal per AuNP for each sample.



RESULTS AND DISCUSSION Antibody−antigen interactions were exploited to place three IgG proteins in the gap between the AuNP and Au film, as is standard for SERS-based immunoassays. First, polyclonal goat antimouse IgG antibody was immobilized on a 100 nm thick gold film via cross-linking with DTSSP. The binding sites of the immobilized antibody were then saturated with antigen by exposing the substrate to a sufficiently high concentration of mouse IgG. Subsequently, the antigen captured on the Au film was labeled with ERLs consisting of 60 nm AuNPs modified with 4-nitrobenzenethiol (NBT) and goat antimouse IgG antibody. Figure 1A shows a SERS spectrum collected from the dried three-protein construct sample. The SERS spectrum is characteristic of NBT, the Raman reporter molecule. Consistent with other reports on extrinsic Raman immunoassays,19 no signal is detected for the antibody because the NBT is in more intimate contact with the nanoparticle surface than the Raman-active modes of the antibody. The intensity of the major band due to the symmetric nitro stretch centered at 1338 cm−1 was 4353 ± 687 cts/5 s for the dried three-protein construct. The same sample was then immersed in water, and SERS spectra were collected (Figure 1A). The same characteristic NBT bands were observed for the hydrated sample; however, the intensity was significantly attenuated. The band at 1338 cm−1 for the wet sample only yielded a signal of 96 ± 20 cts/5 s. To rule out the possibility that the signal drop was due to the removal of bound ERLs by disrupting the antibody− antigen interaction, the sample was dried again and analyzed with SERS. Interestingly, the original signal for the dry sample was recovered (4148 ± 346 cts/5 s; Figure 1A). The wetting/ drying cycle was repeated several times, and for each cycle the dry sample provided approximately 50-fold increase in signal (Figure 1B). To explain this phenomenon, we hypothesize that the hydration state of the proteins dictates the gap size, thereby

Figure 1. (A) SERS spectra of NBT-modified gold nanoparticles immobilized onto a gold film via antibody/antigen/antibody interactions (three-protein construct). Each spectrum represents the average measurement collected from the same substrate: in air (black), immersed in water (red), redried (green), reimmersed in water (blue). (B) SERS intensities acquired in air or immersed in water for three cycles. Each data point is the average of five spectra, and the error bars represent the standard deviation.

controlling the SERS enhancement. It has been previously established via AFM that IgG molecules are approximately 3−4 nm in size when imaged in air,22 i.e., dry state, and 7−10 nm when imaged in liquid,23 i.e., hydrated state. Thus, based on these previously reported values for IgG size as a function of hydration, the AuNP−Au film gap modulates between ∼9−12 nm and ∼21−30 nm for each dry and wet cycle, respectively. A conservative estimate suggests the gap distance increased by 9 nm from the dry to wet state, a sufficiently large difference to significantly affect plasmonic coupling and SERS intensity.1,2,8,9 Therefore, it is expected that hydration of the three-protein construct was responsible for the significant drop in SERS intensity. AFM was used to interrogate the change in size of the IgG proteins in this Ab−Ag−Ab sandwich construct. The threeprotein construct was imaged in air using tapping mode AFM. Line scans were used to measure the height of the bioconjugated particle relative to the plane defined by the underlying antigen-saturated antibody (Figure 2). This height includes that of the AuNP and two dried IgG proteins, i.e., one IgG on top and one under the AuNP, and was determined to 6310

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Letter

should increase by (20.1/10.5)5 ≈ 26. Additional independent samples were prepared and analyzed to confirm the repeatability of this result (Supporting Information). The observed data presented in Table S1 are reasonably consistent with these previous reports and corroborate our hypothesis that the change in gap distance as a function of hydration is responsible for the change in SERS intensity. A second construct was prepared as a control to confirm that spectral acquisition through a liquid layer, i.e., SERS dependency on refractive index, was not responsible for the reduced SERS signal. For this sample, DTSSP/NBT-modified AuNPs were immobilized on an AET-modified Au film. This selfassembled monolayer provides a smaller gap distance that is less susceptible to hydration effects. Thus, it is anticipated that SERS intensity should be similar when collected in air, i.e., dry state, or immersed in liquid, i.e., hydrated state. The SERS spectra for the NBT-modified AuNPs supported on an AET-modified Au film acquired in air is dominated by the Raman active modes of NBT, identical to that of the threeprotein construct (Figure 3A). The intensity of the strongest band centered at 1338 cm−1 was 2286 ± 517 cts/5 s. This sample was then immersed in water, and the SERS spectrum

Figure 2. (A) Schematic illustration of the structure and proposed effect of hydration on the antibody−antigen−antibody immobilized gold nanoparticle above a gold film (not to scale). AFM line scans measure the height of the bioconjugated nanoparticle, which includes two IgG molecules. Representative AFM images (B) and cross-section profiles (C) of antibody/antigen/antibody immobilized AuNP imaged in air (left) and in liquid (right). Each AFM image is 500 nm × 500 nm.

be 58.1 ± 1.9 nm (Figure 2). The sample was then imaged in a liquid cell, and similarly, the height of the bioconjugated AuNPs was determined (Figure 2). This measured height included that of the AuNP and two hydrated IgG molecules, and was determined to be 64.4 ± 3.3 nm (Figure 2). It is important to note that antibody orientation was not controlled during immobilization via DTSSP;24 thus, the variability in the measured heights for dehydrated and hydrated IgGs is attributed to variable orientation of the antibodies.25 These results reflect the average size of IgG molecules for a distribution of molecular orientations. From the analysis of 50 nanoparticles, it was determined that the diameter of the IgG-conjugated AuNP increased by 6.3 nm when imaged in liquid compared to air (Figure 2). Taking into account that the AuNP size is independent of hydration state, it is concluded that each IgG is 3.2 nm larger when hydrated relative to the dehydrated size, to effectively modulate the gap distance by ∼9.6 nm between the dry and wet conditions. Based on previous theoretical calculations that were experimentally supported,9 the SERS intensity should modulate as (dwet/ ddry).5 Assuming an average IgG size of 3.5 nm when dehydrated and the measured increase of 3.2 nm per IgG when rehydrated, it can be estimated that the SERS intensity

Figure 3. (A) SERS spectra of NBT-modified gold nanoparticles immobilized onto a gold film via AET. Each spectrum represents the average measurement collected from the same substrate: in air (black) and immersed in water (red). (B) SERS intensities acquired in air or immersed in water for three cycles. Each data point is the average of five spectra and the error bars represent the standard deviation. 6311

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OUTLOOK The findings herein have significant implications for optimizing SERS immunoassays with respect to molecular design, e.g., antibody versus antibody fragment versus short peptide, and spectral acquisition, e.g., wet versus dry. These data suggest that designing SERS tags with small molecular recognition elements may provide considerable improvement in assay sensitivity. Furthermore, efforts to improve assay performance by controlling antibody orientation with the use of specialized immobilization reagents, such as protein A, may be offset because of the increase in AuNP−Au film gap distance. The extended antibody size and reduced SERS signal in the hydrated state may also limit the utility of in situ measurements of binding for real-time analysis. Moreover, these findings may not be limited to SERS-based assays and will likely have significant consequences for other AuNP-enabled plasmonic assays.

was acquired through the liquid layer (Figure 3A). The same characteristic NBT bands were observed for the hydrated sample; however, unlike the three-protein construct, for this sample, the intensity of the 1338 cm−1 band increased slightly to 2390 ± 370 cts/5 s (Figure 3A). Repeated wetting/drying cycle demonstrated that the signal is ∼5% greater for the hydrated sample than the dehydrated sample (Figure 3B). This minimal, but notable increase in intensity is attributed to the difference in refractive indices of air and water. Thus, it is concluded that the refractive index of the liquid layer was not responsible for the significant drop in SERS intensity for the three-protein construct acquired through a liquid layer. Additionally, this control sample supports the hypothesis that the hydration state of the proteins dictates the gap size, thereby controlling the SERS enhancement. If the gap distance controls the SERS enhancement, the SERS signal is expected to be much greater for the AET immobilized AuNPs (∼1.3 nm gap5) than the three-protein construct (9−12 nm gap (dry) and 24−30 nm gap (wet)); however, this was not reflected in the raw SERS intensities (Figures 1A and 3A). To directly compare SERS enhancement for the three-protein and AET constructed samples, the SERS intensities were normalized with respect to AuNP surface concentration. To this end, SEM was used to measure AuNP surface concentrations and normalized SERS intensities per AuNP were calculated (Figures 4; Figure S1). As is evident, the



ASSOCIATED CONTENT

S Supporting Information *

Detailed experimental section, representative SEM images, and SERS intensities for additional independent samples. This material is available free of charge via the Internet at http:// pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the Defense Threat ReductionAgency, Basic Research Award No. HDTRA1-13-1-0028 to Illinois State University. Additional funding was provided by Illinois State University’s Department of Chemistry and College of Arts and Sciences. Partial support for the SEM was provided by NSF No. DBI-0923448. The authors would like to acknowledge Scott MacLaren at the Materials Research Laboratory Center for Microanalysis of Materials (University of Illinois at Urbana−Champaign) for assistance with AFM imaging.



Figure 4. Normalized SERS intensities with respect to nanoparticle surface density (SERS signal per nanoparticle) acquired in air or immersed in water for three cycles. Each data point is the average of five spectra and the error bars represent the standard deviation.

REFERENCES

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dx.doi.org/10.1021/la500640q | Langmuir 2014, 30, 6309−6313

Effect of hydration on plasmonic coupling of bioconjugated gold nanoparticles immobilized on a gold film probed by surface-enhanced Raman spectroscopy.

Gold nanoparticle (AuNP)-Au film constructs were prepared using antibody-antigen interactions or a small organic cross-linker to systematically contro...
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