Protein Integrated, Functionally Active Silver Nanoplanar Structures for

Jan 17, 2013 - (a) Digital photographs of different stages of HNT growth (b) the UV–vis ... The signature vibrational modes of the reduced porphyrin...
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Protein Integrated, Functionally Active Silver Nanoplanar Structures for Enhanced SPR Shibsekhar Roy,† Chandra K. Dixit,‡ Ramprasad Gandhiraman,§ Una Prendergast,† Stephen Daniels,§,∥ Richard O’Kennedy,‡,§ and Colette McDonagh*,†,§ †

National Biophotonics and Imaging Platform, School of Physical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland National Biophotonics and Imaging Platform, School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland § Biomedical Diagnostics Institute, Dublin City University, Glasnevin, Dublin 9, Ireland ∥ School of Electronic Engineering, Dublin City University, Dublin 9, Ireland ‡

S Supporting Information *

ABSTRACT: In an alternative approach to achieving nanoparticle-based enhanced surface plasmon resonance (SPR), we present a simple protein seeding-based method for the synthesis of a protein integrated, surface active silver nanoplanar structure, where the protein is cocrystallized with silver. The growth kinetics of this integrated structure has been probed by X-ray diffraction and Raman spectroscopy, which show a spherical to prismatic transition within a three minute growth span. The SPR activity is directly proportional to the tip sharpness of the nanostructure due to the localized surface plasmon resonance (LSPR) phenomenon. This biometallization process depends on the size, shape, and chemical nature of the interacting protein as demonstrated by comparing the results for different model proteins. Moreover, the protein remains functionally intact when embedded in the nanostructured SPR surface. Taking horseradish peroxidase (HRP) as a model protein, we have demonstrated a very high SPR binding profile against anti-HRP compared to direct binding of HRP to the gold surface.

1. INTRODUCTION

In this article, we present the synthesis and potential application of silver planar structures, embedded with specific proteins to develop a functional nanosurface for SPR and other assay platforms. This is the first report to our knowledge where a bioactive, nanostructured surface is produced, which bypasses the complex steps of surface biofunctional linking chemistry for assays. The key features of this work are first that the functionality of the embedded protein is kept intact such that the planar structure remains functionally active and second, the surface enhances the SPR detectability of the protein. We have chosen horseradish peroxidise (HRP) as the model protein as the peroxidase activity is a convenient measure of its functionality. To assess the effect of the shape of the protein on various surface properties, we have also used the globular protein bovine serum albumin (BSA) and the fibrous protein fibrinogen. The synthesis of the protein-conjugated nanostructure was performed in two steps, the first step being the seed preparation and the second step the generation of planar structures from the seeds. The seed was synthesized by slightly modifying the method of Aherne et al.13

Noble metal nanostructures are used in many applications due to their unique optical and surface properties, which are tunable by altering their size, shape, and aspect ratio.1−3 During the past decade, the emergence of noble metal planar structures has led to the development of new sensor platforms for plasmonic enhancement of luminescence, Raman enhancement, and surface plasmon resonance (SPR).4−6 The progression from small spherical structures to larger and more complex geometrical nanoforms gives rise to new plasmon properties. For a small spherical nanoparticle, a sharp surface plasmon peak is observed, which represents the dipole mode of the multipolar expansion. As the radius increases, a red shift takes place and higher order terms become more significant.7 These higher order multipoles have been reported for noble metal spherical nanostructures by Halas et al and Krenn et al.8,9 This multipolar spectral property, is also associated with particles of very high aspect ratio, for example metal planar structures due to their variable height and edge length.7,10 The work by the Schatz and Mirkin group described the effect of aspect ratio on multipolar excitation in great detail for both gold and silver planar structures assigning various in-plane and out of plane excitation wavelengths.11 These optical properties and their corresponding surface roughness have allowed the design of various sensing platforms with very high detection efficiency; for example Kwon et al. has shown enhanced SPR for thrombin linked with gold nanoforms of various shapes.12 © 2013 American Chemical Society

2. EXPERIMENTAL SECTION 2.1. Synthesis of Silver Nanoplanar Structures. All of the reagents described in this section have been purchased from Received: October 16, 2012 Revised: December 7, 2012 Published: January 17, 2013 3078

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(3:1 of sulfuric acid and hydrogen peroxide, respectively) treatment. Afterward, chips were extensively washed with deionized water to remove any traces of piranha as it could interfere with the plasmon resonance by changing the resultant refractive index. A BIAcore 3000 instrument was employed for analyzing the plasmonic properties of AgNT. Prior to use the instrument was desorbed and primed as per manufacturer’s recommendations. The piranha-treated chip was then docked into the chip holder of the BIAcore 3000. Analysis of Plasmon Behavior of AgNT Samples (HNT1, 2, and 3). Each AgNT sample (50μl) was flushed into the microfluidic channels such that one channel is dedicated to one sample only. Prior to injection, a stable baseline was obtained for each NT sample sensorgram. The SPR response for each sample was registered in arbitrary response units (RU). The SPR response units are related to the shift of the plasmon band in such a way that for each 1000 RU, the plasmon angle shifts by 0.1 degree. Therefore, the higher the plasmonic shift, the greater will be the change in RU namely the SPR response of the AgNT sample. Analysis of Plasmon Behavior of AgNT-HRP Conjugates in an Immunoassay Format. To analyze the SPR behavior of HNTs, the anti-HRP antibody was immobilized in the four flow cells. The antibodies were chemisorbed on the piranha activated gold chip. The first two cells were used to analyze HNTs, whereas the other two were used as controls. The conjugates were assayed at five concentrations between 0.002 to 20 μg/mL at 1/10 dilution, whereas the controls (AgNTHRP conjugates and free HRP with no metal particle) were assayed at five concentrations between 1 to 50 μg/mL.

Sigma Aldrich and used without further modification unless mentioned otherwise. The synthesis is a two step process as described below: A. Metal Modified Ag Seed Synthesis. Into the mixture of aqueous trisodium citrate (5 mL, 2.5 mM), 500 μL HRP solution (in 10 mM pH 7.4 phosphate buffer) was added followed by aqueous poly(sodium styrenesulphonate) of 1000 kDa (250 μL of 500 μg/mL) and 300 μL 10 mM freshly prepared aqueous NaBH4. After that, 5 mL of 0.5 mM aqueous AgNO3 (was added to the solution at a rate of 2 mL/minute with continuous stirring). The protein modified Ag seeds were used for the next step as prepared. For various proteins, identical conditions were maintained as described above. B. Growth of Nanoplanar Structure. A reaction mixture was prepared with 5 mL distilled water, 75 mL of 10 mM aqueous ascorbic acid (75 mL, 10 mM), and various quantities of seed solution starting from 100 μL to 1 mL. To this mixture, 3 mL of 0.5 mM aqueous AgNO3 solution was added at a rate of 1 mL/minute with continuous stirring. So, the reaction took 3 min to complete. The three resultant planar structures obtained from three different seed concentrations (100 μL, 500 μL, and 1 mL) are referred to as HNT1, HNT2, and HNT3, respectively. 2.2. Characterization. 2.2.1. Spectroscopy. For the UV− vis spectroscopy to determine the plasmonic bands of the nanoplanar structures, a Cary 50 scan UV−vis spectrophotometer (Varian) was used. The Raman spectra were collected on a Horiba Jobin-Yvon Labram HR instrument with a 532 nm laser using a 40× objective, a 600 lines/mm grating and a 300 μm hole. An exposure time of 2 s was used with 5 accumulations, for each spectrum. 2.2.2. Microscopy. Regarding the TEM measurement, a Hitachi 7000 transmission electron microscope was operated at 100 kV. Image capture was performed digitally by a Megaview 2 CCD camera. Specimens were prepared by putting a drop of the aqueous solutions of the nanoforms on to a Formvar carbon-coated copper grid (Agar Scientific). 2.2.3. BCA Assay. HRP-conjugated HNT were subjected to bicinchoninic acid (BCA) in order to quantify amount of HRP that is conjugated to them. Simultaneously, a standard curve was generated with eight concentrations of free HRP (at onehundredth dilution). The samples and standards (50 μL each) were mixed with 950 μL of BCA reagent mix according to the guidelines of manufacturer. This solution was later incubated at 37 °C for 30 min until the blue color changes to deep purple. Optical density of each sample was measured using a Tecan spectrophotometer (Tecan GmbH,Austria). 2.2.4. Peroxidase Assay. Activity of the HNT-conjugated HRP was also assessed by subjecting the samples to a peroxidase enzymatic assay with peroxide along with tetraethylbenzene (TMB) chromogen. Simultaneously, a standard HRP activity curve was also generated with free HRP (at one-tenth stock dilution). Similarly, each HNT solution was prepared at the same HRP dilutions as for standards in order to obtain a fair activity comparison. Each of the samples and standards were incubated with HRP substrate and chromogen mixture for 3 min at room temp in a microtiter plate. Later the plate was read with a Tecan plate reader (GmbH, Austria) at a measurement wavelength of 450 nm with a standard wavelength of 650 nm. 2.2.5. SPR Assay. Surface interaction analysis customized gold chips (BIAcore SIA kit) were activated by acid piranha

3. RESULT AND DISCUSSION 3.1. Basic Characterization. The absorption spectra of the structures are shown in Figure 1, which indicates the expected blue shift of the plasmon peak with increasing seed concentration. According to the calculation by Schatz et al., the major peak is the in plane dipole resonance peak (HNT1 = 625 nm, HNT2 = 560 nm, HNT3 = 500 nm).14 The blue shift of the dipole peak with the increase of seed concentration from HNT1 to HNT3 is very clear in the image. This was also

Figure 1. UV−vis spectroscopy results of control silver nanotriangle (NT) and HRP-doped nanotriangles. The typical out of plane octupole resonance peak is highlighted in the image. 3079

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reported by Aherne et al.13 It also signifies the fact that the more the peak is blue-shifted from 770 nm, the more likely the structure deviates from the perfect triangular nanoplate.11 Hence, the sharp tip nanoprism to circular nanoplate transition from HNT1 to HNT3 is again proved by the corresponding blueshift of the dipole peak. The peak at ca. 430 nm represents the out of plane dipole resonance peak, whereas the peak at ca. 350 nm represents the out of plane quadrupole resonance peak. It should also be noted that from HNT1 to HNT3 the relative intensity of the 430 nm peak has increased, which is indicative of the increased fraction of spherical population. The presence of the protein on the surface was determined quantitatively and its functionality was confirmed. Prior to the experiments, the nanostructures were washed well with dilute phosphate buffered solution (1 mM, pH 7.4) to prevent interference from the free protein. The amount of surface bound protein was determined by the standard BCA assay (Figure S1 of the Supporting Information). The values obtained from the test were 0.1, 0.25, and 20 μg/mL for HNT1, HNT2, and HNT3, respectively. Additionally, the structural integrity of the protein was found to be intact, as shown by the antibody-based ELISA performed with anti-BSA antibody (Figure S2 of the Supporting Information). Finally, the functionality of the protein was tested with respect to its peroxidise activity, which is a function of both the availability of the heme group to the reaction plane as well as the structural integrity of the protein. The peroxidise assay (Figure S3 of the Supporting Information) shows very high peroxidise activity for all three structures. HNT1 seed-based conjugates were found to have the most preserved protein functionality, which was approximately 89% while for HNT2 and HNT3 the functionality was 79 and 73% respectively, relative to the control, where the control was the substrate reaction free enzyme (details of the assay can be obtained in the Supporting Information). 3.2. Structural Polymorphism of Modified Seeds. There was an interesting effect of HRP on the seed morphology. Unmodified seeds have been found to be heterogeneous/irregular in shape ranging from spherical to polygonal (Figure S4 of the Supporting Information), whereas HRP modified seeds have been found to be much more regularly spherical. Possibly, protein functions as an additional stabilizer for the nanoform. To explore the detailed role of the protein in the synthesis, we have used X-ray diffraction (XRD) and Raman Spectroscopy. In Figure 2, we present the XRD of

the seed doped with the three different proteins and compare with the undoped silver seed. For Ag nanoprisms and triangles, the most dominant growth plane is reported as {111} because of the higher organic material binding affinity of less stable peaks like {100}, {110}, and so forth.13 This is confirmed for our protein conjugated structures as all show a dominant {111} plane and weak {200} and {311} planes as seen in Figure 2. However, there are some differences which are proteindependent. The XRD profile shows that both the undoped (protein free) and protein-doped Ag seeds show a mixture of crystal growth with various planes of symmetry based on different oxidation states of Ag. The undoped Ag seed shows three strong peaks of monoclinic AgO {111}, fcc Ag {111}, and cubic Ag2O {311} with two relatively weaker peaks of monoclinic Ag3O4 {211} and fcc Ag {111}. When comparing different proteins, the monoclinic AgO {111}, fcc Ag {111}, and fcc Ag {311} planes are found to be common in all the seeds with the emergence of a new common fcc {200} plane, which is probably the initiating crystal plane for protein interaction. For globular proteins BSA and HRP the monoclinic {211} and cubic {311} planes are absent but are present for fibrinogen. Additionally, the BSA structure shows monoclinic {002} and {122} planes. These observations are indicative of the dependence of the nanostructure evolution on the size, shape and chemical nature of the protein (The detailed sources of JCPDS files are tabulated in Table S2 in the Supporting Information). 3.3. Growth Kinetics of the Nanoplanar Structures. The nanostructure forms during a 3 min chemical reaction. To probe the kinetics, the reaction was stopped after every minute (0, 1, 2, and 3 min) and then centrifuged and resuspended in Milli-Q water for further processing. The spectral description of HNT growth is described in Figure 3. The digital images of various stages of HNT growth is presented in part a of Figure 3. The UV−vis spectroscopy data, shown in part b of Figure 3 describes the systematic red shift of the dipole plasmon resonance peak with time with concomitant decrease of the relative absorbance of the out of plane dipole resonance peak (ca. 430 nm) indicative of spherical to planar transition of the silver nanoform. The TEM images for different stages of growth for the HNT1 particle are presented in part a of Figure 4. It can be seen that the spherical Ag seeds are gradually transformed into sharp triangular and prismatic planar structures within the 3 min. The corresponding XRD profile, shown in part b of Figure 4, shows the disappearance of the monoclinic {111} plane and the increase of the fcc {111} plane over time. However, the fcc {200} plane remains unchanged. The detailed TEM image analysis has shown ca. 90% conversion from spherical to planar geometry (triangle and hexagonal prism) representative of a very efficient transition process (Figure S5 of the Supporting Information). 3.4. Effect of Protein Concentration on Nanoplanar Crystal Growth. The HNT particles with the three different seed concentrations are compared in Figure 5 to determine the role of protein/silver stoichiometry on the particle properties. Part a of Figure 5 shows schematically the protein seed concentration-dependent formation of the particles. It is suggested that the integrated protein plays a dual role in determining both the morphology and bioactivity of the nanostructures. It is proposed that chemical reactions between the amino acids integrated near the edge of the structure will etch the fcc {111} surface component thus decreasing the

Figure 2. XRD profile of Ag nanoseeds doped with the three different proteins compared to undoped Ag seeds. 3080

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Figure 3. (a) Digital photographs of different stages of HNT growth (b) the UV−vis spectroscopy result shows the systematic evolution and red shift of the dipole plasmon peak.

Figure 4. (a) TEM images showing the growth kinetics of HNT1 particles from HRP seeds. (b) XRD profile of the growth kinetics.

sharpness of the tip stoichiometrically. This decrease in tip sharpness with increasing protein seed concentration is clear from the TEM images in part b of Figure 5. Interestingly, this result also confirms the earlier spectroscopic prediction of increasing fraction of spherical population with increasing seed population, made on the basis of increased relative intensity of the out of plane dipole resonance peak (430 nm). The Raman properties of the structures are shown in part c of Figure 5. The signature vibrational modes of the reduced porphyrin (i.e., the heme moiety) group are present15 With increasing protein concentration, multiple layers of protein are attached to the nanosurface (the 2D boxes in part a of Figure 5) causing a large increase in the Raman intensity as shown in the insert of part c of Figure 5 but significantly compromising the resolution of the fine structure due to the etching effect discussed above. However, strong modes like ferrous (1600 cm−1), Amide III (1220 cm−1), Tyr (1180 cm−1), Phe (1000 cm−1), carboxyl wagging (640 cm−1) are still present in HNT3. A detailed account can be found in Table S1 of the Supporting Information. The Raman data is further supported by the sharp decrease of the fcc Ag {111} XRD peak with increasing seed concentration (Figure S6 of the Supporting Information). This was also observed for BSA but to a lesser extent, which is probably due to the relative redox properties of the amino acids (of the conjugating protein) integrated to the Ag surface, which is differentially etched by the different chemical environment.

3.5. Effect of Protein Conformation on Nanoplanar Assembly. However, the role of individual amino acids or amino-acid chains cannot be ascertained as the three-dimensional conformation of the protein plays an important role in modifying the Ag seed and forming the nanostructure. The role of the protein structure in determining particle morphology is further explored by observing the formation of the fibrinogen integrated structures under identical conditions described for the other proteins (HRP, BSA). Figure 6 shows TEM images of fibrinogen conjugated Ag seeds and nanoplanes. As observed in part a of Figure 6, the fibrinogen conjugated seeds are very different compared to that of HRP and BSA (Figure S7 of the Supporting Information) regarding their self-assembly property. The fibrinogen modified seeds have been identified mainly in doublets and triplets having a common connection point of around 50 nm. This length is indicative of the longitudinal distance of a fibrinogen molecule that is 47 nm, which suggests the connecting role of the protein molecule to assemble Ag seeds. During the second stage of nanoplane synthesis, when further reactions of seeds with Ag+ occur, a micrometer scale assembly of planar structures is formed indicating the selfassembly property of Ag conjugated fibrinogen, which is observed in part b of Figure 6. When the Ag seeds were prepared in the presence of a very high concentration of BSA (10 mg/mL), the AgNO3 addition resulted in the formation of large macro structures in the shape 3081

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Figure 5. (a) Schematic of the suggested formation of the three HNT particles from the three seed concentrations. The 2D view shows the effect of higher protein concentration that leads to the decrease of tip sharpness as well as loss of resolution of the Raman peaks. (b) TEM images of HNT1, HNT2, and HNT3. (c) Raman peaks for HNT1, HNT2, and HNT3 and their relative intensities shown in the insert.

of a star and scissors (Figure S7 of the Supporting Information) arising from larger globular seeds. In addition, different stages of the formation of these giant structures have also been identified by investigating the TEM image in detail. However, these structures were absent for HRP and fibrinogen at similarly elevated protein concentrations. 3.6. SPR-Based Assay Platform. The importance of this noble metal integration to protein molecules was further evaluated on a surface plasmon resonance (SPR) biosensing platform. The binding result obtained from the direct binding assay, which involves the binding of the HNT particles to antiHRP conjugated to the gold surface, is described in Figure 7, where the SPR binding affinity is represented as the mean RU (less than 5% standard deviation) for all the HNTs as a function of their concentration. This clearly shows a very high affinity for HNT1 particles and decreased response for the other two structures. This result directly correlates with the sharpness of the nanoplane tips. With gradual etching of the tips, the localized surface plasmon resonance (LSPR) decreases significantly resulting in the decrease of the RU value.16 Hence, protein concentration plays a key role to determine the optimized nanoplanar structure with maximum SPR activity having a smaller dynamic activity range. The importance of direct contact between protein and nanosurface has further been emphasized by comparing these results with the control AgNT (Figure S7 of the Supporting Information) that is physically conjugated with HRP. In Figure 7, it clearly shows that HRP-AgNT shows much less SPR response in the whole protein concentration range. So, nanoplane integration of protein significantly enhances the SPR property compared to the conventional surface attachment mode. Hence, at this

Figure 6. (a) Proposed model of fibrinogen binding with silver seeds (dimer or trimer), (b) TEM image of micrometer scale assembly of fibrinogen seeded nanoplanes (nanodisks). 3082

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ACKNOWLEDGMENTS This work was supported through the National Biophotonics and Imaging Platform, Ireland, and funded by the Irish Government’s Programme for Research in Third Level Institutions, Cycle 4, National Development Plan 2007-2013.

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(1) Lee, K. S.; El-Sayed, M. A. J. Phys. Chem. B 2006, 110, 19220− 19225. (2) Zhai, Y.; Han, L .; Wang, P.; Li, G.; Ren, W.; Liu, L.; Wang, E.; Dong, S. ACS Nano 2011, 5, 8562−8570. (3) Mahmoud, M. A.; El-Sayed, M. A. J. Am. Chem. Soc. 2010, 132, 12704−12710. (4) Dutta Choudhury, S.; Badugu, R.; Ray, K.; Lakowicz, J. R. J. Phys. Chem. C 2012, 116, 5042−5048. (5) Li, M.; Cushing, S. K.; Zhang, J.; Lankford, J.; Aguilar, Z. P.; Ma, D.; Wu, N. Nanotechnology 2012, 23, 115501. (6) Zheng, J.; Ding, Y .; Tian, B.; Wang, Z. L.; Zhuang, X. J. Am. Chem. Soc. 2008, 130, 10472−10473. (7) Shuford, K. L.; Ratner, M. A.; Schatz, G. C. J. Chem. Phys. 2005, 123, 114713. (8) Le, F.; Brandl, D. W.; Urzhumov, Y. A.; Wang, H.; Kundu, J.; Halas, N. J.; Aizpurua, J.; Nordlander, P. ACS Nano 2008, 2, 707−718. (9) Félidj, N.; Grand, J.; Laurent, G.; Aubard, J.; Lévi, G.; Hohenau, A.; Galler, N.; Aussenegg, F. R.; Krenn, J. R. J. Chem. Phys. 2008, 128, 094702. (10) Millstone, J. E.; Park, S.; Shuford, K. L.; Qin, L.; Schatz, G. C.; Mirkin, C. A. J. Am. Chem. Soc. 2005, 127, 5312. (11) Jin, R.; Cao, Y,W.; Mirkin, C. A.; Kelly, K. L.; Schatz, G. C.; Zheng, J. G. Science 2001, 294, 1901−1903. (12) Kwon, M. J.; Lee, J.; Wark, A. W.; Lee, H. J. Anal. Chem. 2012, 84 (3), 1702−1707. (13) Aherne, D.; Ledwith, D. M.; Gara, M.; Kelly, J. M. Adv. Funct. Mater. 2008, 18, 2005−2016. (14) Schatz, G. C.; Van Duyne, R. P. In Handbook of Vibrational Spectroscopy; Chalmers, J. M., Griffiths, P. R., Eds.; Wiley: New York, 2002. (15) Palaniappan, V.; Terner, J. J. Biol. Chem. 1989, 16046−16053. (16) Sherry, L. J.; Jin, R.; Mirkin, C. A.; Schatz, G. C.; Van Duyne, R. P. Nano Lett. 2006, 6, 2060−2065.

Figure 7. SPR-based sensing by HNT1, HNT2, HNT3 and free HRP against anti-HRP.

protein concentration the enzymatic activity and antibody binding affinity of HRP remains intact making the SPR-based sensing platform a multifunctional one. Also shown in Figure 7 is a second control result using free HRP with no metal particle, which further re-enforces the enhancement obtained using the HNT particles.

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CONCLUSIONS In summary, we have demonstrated a simple method to construct protein-embedded silver nanostructures with potential application as an SPR substrate. The integration of the protein within the silver crystal plane was probed using Raman scattering and XRD. The advantage of the construct is that the structural integrity and functionalities of the whole protein are kept intact making the system a multifunctional assay platform and, more importantly, it bypasses the steps of surface functionalization and linking chemistry for biomolecular attachment. The influence of protein size, shape, and concentration is demonstrated by comparing the structural and functional aspects of the nanoplanes synthesized with three different proteins. The HRP-integrated structures were used in conjunction with a standard SPR surface and enhanced SPR response over the appropriate dynamic range was measured compared to the standard technique.

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REFERENCES

ASSOCIATED CONTENT

* Supporting Information S

Details of various protein assays and XRD detail of various protein conjugated NTs with source files, TEM images of Ag seeds, undoped AgNT, BSA conjugated NT, Raman peak assignment are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 3083

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