Plasmon-Tuned Silver Colloids for SERRS Analysis of Methemoglobin

Sep 9, 2012 - (10, 11) Label-free direct and Raman-dye-labeled indirect methods are the two major SERS-based detection protocols for ..... metHb by SE...
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Plasmon-Tuned Silver Colloids for SERRS Analysis of Methemoglobin with Preserved Nativity Govindasamy Kalaivani,† Arumugam Sivanesan,‡ Ayyadurai Kannan,† N. S. Venkata Narayanan,§ Agnieszka Kaminska,‡ and Ranganathan Sevvel*,† †

Department of Chemistry, Vivekananda College, Tiruvedagam West, Madurai-625 214, Tamilnadu, India Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland § Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom ‡

S Supporting Information *

ABSTRACT: Optically tuned silver nanoparticles (AgNP's) functionalized with ω-mercaptoalkanoic acids are synthesized and used as a signal amplifier for the surface-enhanced resonance Raman scattering (SERRS) study of heme cofactor in methemoglobin (metHb). Even though both mercaptopropionic acid (MPA)- and mercaptononanoic acid (MNA)-functionalized AgNP's exemplify vastly enhanced SERRS signal of metHb, MNA-AgNP's amplify the SERRS signal amid preservation of the nativity of the heme pocket, unlike MPA-AgNP's. The electrostatic interaction between MNA-AgNP's and metHb leads to instant signal enhancement with a Raman enhancement factor (EFSERS) of 4.2 × 103. Additionally, a Langmuir adsorption isotherm has been employed for the adsorption of metHb on the MNA-AgNP surface, which provides the real surface coverage and equilibrium constant (K) of metHb as 139 nM and 3.6 × 108 M−1, respectively. The lowest detection limit of 10 nM for metHb has been demonstrated using MNA-AgNP's besides retaining the nativity of the heme pocket. spectrum of metalloprotein.10,11 Label-free direct and Ramandye-labeled indirect methods are the two major SERS-based detection protocols for proteins.10−12,21−23 The label-free strategy detects the proteins of interest by directly adsorbing them on a SERS-active substrate and analyzes the vibrational pattern acquired from the protein itself. The Raman-dyelabeled method detects protein indirectly by SERS of Raman dyes that are linked to probes. Noble metal surfaces, in particular, roughened nanostructure silver substrates, have been used extensively as SERS-active substrates. However, the above substrate is associated with a few shortcomings such as poor reproducibility, dissimilar orientation of proteins due to an inhomogeneous surface, and importantly, the solid static surfaces are not appropriate for in vivo studies. To overcome these drawbacks, the direct detection of proteins in aqueous solution is more desired because it has high reproducibility and the possibility for in vivo measurement. Additionally, the complexity of the protein structure makes the direct label-free SERS detection of biomolecules even more challenging to carry out; however, it can gives rise to measurements with better reliability, reproducibility, and control. Until now, few reports have been published on the SERSbased direct detection of proteins in aqueous solution using silver nanoparticles (AgNP's) as a SERS amplifier.23−26

1. INTRODUCTION Surface-enhanced Raman scattering (SERS) is a well-known phenomenon associated with the electric field enhancement of incident light upon interaction with the surface plasmon resonance (SPR) of noble metal nanostructures. The scattering of molecules in close proximity to this resonant electric field is enhanced by several orders of magnitude, which has been exploited by SERS to obtain structural information about the molecules.1−5 The selectivity and sensitivity of this method can be improved further by combining the surface enhancement with the molecular resonance Raman (RR) effect (i.e., the excitation line in resonance with both the SPR and electronic transition of the close molecules), and this approach is recognized as surface-enhanced resonance Raman scattering (SERRS).6−9 The growing importance of biomedical research exploits SERRS for the structural study, diagnosis, and detection of various biomolecules and microorganisms such as proteins, peptides, DNA and RNA, tissues, living cells, antigens, bacteria, and viruses.10−21 Proteins are the fundamental building blocks of living species whose identification and detection have been very crucial in various fields ranging from medicine to proteomics. SERRS is a nondestructive technique that provides rich structural and molecular information about proteins. SERRS selectively enhances the signal of the protein cofactor and largely gets rid of the protein backbone signal. Thus, information such as the ligation number, spin, and oxidation state of the metal ion can be acquired from the SERRS © 2012 American Chemical Society

Received: August 2, 2012 Revised: September 8, 2012 Published: September 9, 2012 14357

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413 nm excitation line of a Kr+ laser using a Renishaw Raman microscope in back-scattering mode. 2.3. Nanoparticle Synthesis and metHb Immobilization. The synthesis of MPA- or MNA-functionalized AgNP's was carried out as a three-step procedure.27 In the first step, citrate-capped AgNP seeds were prepared as follows: 1 mL of 1% AgNO3 and 2 mL of 38.8 mM sodium citrate were added to 100 mL of ice-cold Millipore water with magnetic stirring, followed by the dropwise addition of 2 mL of freshly prepared 0.005% NaBH4 under vigorous stirring. The resulting darkyellow colloidal solution was stirred for an additional 1 h and stored at 4 °C. The second step is the seeding process, and here 50 mL of the above AgNP seed solution was diluted with 50 mL of Millipore water, followed by the addition of 0.5 mL of 1 mM AA under constant stirring. After 2 min, 0.5 mL of 1 mM AgNO3 was slowly added with vigorous stirring. The stirring was continued for 1 h under ice-cold conditions. The final step is the ligand-exchange reaction where 4 mL of 20 mM MPA or MNA in 1 M KOH was added with continuous stirring. The solution was stirred overnight and then centrifuged. The deposited MPA- or MNA-capped AgNP's were washed with KOH and water several times until the colloidal solution was free of excess thiocarboxylic acid molecules and KOH. After purification, the particles were stored at 4 °C prior to the SERRS measurement. MetHb was immobilized on MPA- or MNA-capped AgNP's by mixing the required concentration of metHb aqueous solution with 12 nM AgNP's in a rotating cuvette.

Nevertheless, they have some drawbacks: The nanoparticles produced by the reported methods23−26 are very polydisperse, and weak ligands such as citrate/cetyltrimethylammonium bromide on the surface of AgNP's may not be protective enough to prevent the protein from losing its nativity. In addition, the AgNP's were aggregated using inorganic salts to induce SERS activity, which again makes one to think seriously about the reproducibility and biocompatibility.25 Moreover, there has been a report26 in the literature of Raman-dye-tagged AgNP's for the indirect detection of proteins. Recently, the authors of this article published work on a SERRS study of cytochrome c using functionalized silver colloids, which incidentally is a label-free method for the detection of cytochrome c.27−29 On the basis of this knowledge, we have synthesized reasonably more monodisperse AgNP's with the precise tuning of their SPR to match the excitation laser line and the molecular electronic transition of the ferric heme protein, methemoglobin (metHb). metHb is an alternative form of oxygen-carrying protein hemoglobin (Hb), where the heme iron center is in the +3 oxidation state as opposed to the +2 oxidation state in normal hemoglobin. Hb in a normal human being contains about 1 to 2% methemoglobin. Any excess amount of metHb is converted back to Hb using an enzyme called methemoglobin reductase. A deficiency in the production of methemoglobin reductase can lead to various complications, including death. Therefore, it is very important to determine the concentration of metHb in a human body, and it is absolutely essential to develop a suitable label-free method of detection for metHb. In the conventional surfaceenhanced Raman scattering method of detection of metHb,30 even though a large enhancement of the protein signal is achieved, the process of protein detection itself results in the denaturation of metHb because of the proximity of protein to the SERS-active substrate, which will ultimately deprive us from gaining valuable inside knowledge of the protein nativity. Therefore, SERRS spectroscopy with a suitably developed substrate with the optimum distance between the protein and the SERS-active substrate could be a potential label-free method for the detection of methemoglobin with the preserved nativity. In this article, we have used metHb as a model protein to test the SERRS enhancement and biocompatibility of newly synthesized silver colloids by seed-mediated methodology as a Raman-active substrate. To the best of our knowledge, so far no work has been published concerning the SERRS study of metHb using silver colloids with the preserved nativity of the heme cofactor. MetHb shows a strong electron absorption Soret band at around 410 nm. In addition, metHb has an overall net positive charge on its surface. To match these criteria, we have synthesized two different alkyl lengths of carboxylic acid-functionalized AgNP's with precisely tuned SPR and have carried out a SERRS study of metHb.

3. RESULTS AND DISCUSSION 3.1. Characterization of Silver Nanoparticle. The preparation of plasmon-tuned MPA- or MNA-functionalized AgNP's involves the synthesis of smaller (10 nm) citratecapped silver seeds, followed by the stepwise addition of the growth solution, AgNO3, and ascorbic acid (AA).31 Here, AA works as a mild reducing agent and initiates particle enlargement by reducing the silver ions to their atomic state. The surface concentration of citrate ions is crucial to the enlargement of particle size. Additionally, secondary nucleation is prevented in the presence of silver seeds.32 AgNP's exhibits a strong surface plasmon absorption band that prominently depends on the size and shape of the particle.33 For example, approximately 10 nm AgNP's have a plasmonic band at around 390 nm. In general, an increase in the particle size or aspect ratio (the ratio between longer and shorter axes of the particle) or a change in the capping agent will lead to a shift in the plasmonic band. Figure 1 shows the UV−visible absorption spectra of differently sized AgNP's with different capping agents. The borohydride-reduced citrate-capped AgNP seeds depict a sharp absorption maximum at 390 nm (curve a). The enlarged AgNP's shows a red-shifted plasmonic band at around 404 nm (curve b). This suggests that the particle size or aspect ratio is increased upon seeding (vide infra). Furthermore, this red shift is not due to particle aggregation because aggregation would show a pronounced band at around 600 nm34 and hence the particles are well dispersed. A closer examination of these two spectra reveals that, apart from a red shift of 14 nm, the seeded AgNP's show a marginally broadened plasmonic band, in contrast to seed AgNP's. This broadening is due to the introduction of polydispersity, which generally happens during seeding. The seeding of citrate-capped AgNP's is followed by the synthesis of carboxylic acid-functionalized AgNP's by a ligand-exchange reaction with MNA. This leads to a further red shift of 8−10 nm (curve c) that is due to the increase in the external dielectric constant of AgNP's by the ligand-exchange reaction.35 Moreover, the ligand-exchanged MNA-capped AgNP's shows only a red shift without any appearance of a new band at longer

2. MATERIAL AND METHODS 2.1. Chemicals. AgNO3, trisodium citrate, NaBH4, ascorbic acid (AA), 3-mercaptopropionic acid (MPA), 9-mercaptononanoic acid (MNA), and methemoglobin (metHb) were obtained from SigmaAldrich and used without further purification. All solutions were prepared with Millipore water. 2.2. Instrumentation. UV−visible spectra were recorded with a Jasco V-530 spectrophotometer. High-resolution transmission electron microscopy (HR-TEM) images of AgNP's were obtained from a Jeol JEM 3010 operating at 200 kV. The samples were prepared by dropping 2 μL of colloidal solutions onto a carbon-coated copper grid. RR and SERRS spectra were measured in a rotating cuvette with the 14358

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alkyl chain backbone there would be a red shift of about 3 nm in the SPR band.35 Altogether, changes in the dielectric constant introduced by ligand exchange as well as the presence of a long alkyl chain in the MNA capping ligand induce the red shift, and hence the surface plasmon of 18.5 ± 3.5 nm MNAAgNP's was observed at around 413 nm. 3.2. RR Spectrum of metHb. Prior to SERRS measurement, the RR spectrum of 50 μM metHb was measured in aqueous solution (Figure 3a). The vibrational bands of the

Figure 1. UV−vis absorption spectra of (a) sodium borohydridereduced citrate-capped, (b) seeded citrate-capped, and (c) seeded MNA-AgNP's.

wavelength around 600−700 nm. This in turn confirms that particles are not aggregated after the ligand-exchange reaction. To support the UV−visible results and to confirm the particle morphology, a TEM measurement was performed for the AgNP's. Figure 2 depicts the TEM image of borohydrideFigure 3. Comparison of normalized (a) native RR and non-native time-dependent (1, 10, and 25 min in b−d, respectively) SERRS spectra of metHb bound to MPA-AgNP's.

heme cofactor has a unique fingerprint region in the frequency range of 1300−1600 cm−1 corresponding to the oxidation, spin, and coordination states of the iron atom, therefore referred to as the “marker band” region for the heme protein.37−40 The details of all vibrational modes in the fingerprint region of the heme cofactor are presented in Table 1. The oxidation state Table 1. Raman “Marker Band” Band Region of Heme Protein

Figure 2. TEM images of (a) sodium borohydride-reduced citratecapped seed AgNP's and (b) MNA-AgNP's.

Raman shift (cm−1)

vibrational modes 3+

oxidation state (ν4) - Fe oxidation state (ν4) - Fe2+ 6cHS (ν3) 5cHS (ν3) 6cLS (ν3) 6cHS (ν2) 6cLS (ν2)

reduced citrate-capped AgNP's (i.e., (a) AgNP seeds and (b) MNA-capped AgNP's). The sizes of the AgNP seeds are almost spherical with an aspect ratio of 1.08 ± 0.007. The average diameter of the particle calculated from the longer axis of the particles is 12 ± 0.7 nm. However, the average longer-axis particle diameter and aspect ratio of MNA-capped AgNP's were found to be 18.5 ± 3.5 and 1.6 ± 4, respectively (Figure S1). Furthermore, the TEM images confirm that the particles are well isolated in accordance with UV−visible results. On the basis of the Mie theory calculation, 10 and 20 nm citratecapped AgNP's are expected to show the surface plasmon resonance (SPR) band at around 390 and 400 nm, respectively.36 Additionally, in the present case, changes in the external dielectric constant caused by the ligand exchange of citrate with thiocarboxylic acid leads to an additional red shift of the SPR band of AgNP's.35 Even though there is no systematic study available on the optical response of thiocarboxylic acidfunctionalized AgNP's with respect to the chain length, studies on chain-length-varied, alkanethiol-functionalized silver nanoparticles reveal that for every addition of a carbon atom to the

1368−1377 1344−1364 1470−1480 1490−1500 1500−1511 ∼1565 ∼1585

vibrational band, ν4 mode, falls in the range of 1368−1377 cm−1 for ferric hemes and in the range of 1344−1364 cm−1 for ferrous hemes. The ν3 and ν2 vibrational modes appearing in the range of 1470−1585 cm−1 are sensitive to the spin state and the coordination state of the iron atom. The RR spectrum of metHb shows a strong ν4 vibrational mode at 1374 cm−1 corresponding to the ferric heme of metHb. The appearance of ν3 and ν2 bands at 1479 and 1565 cm−1 is associated with the six-coordinate high-spin (6cHS) heme. In addition, metHb also displays characteristic vibrational bands at 1506 (ν3) and 1582 cm−1 (ν2), corresponding to the six-coordinate low-spin (6cLS) heme. Because the energy level between the low and high spin 14359

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states is sufficiently small, there is a probability for the electrons to populate the two energy states accordingly at room temperature; therefore, Raman bands for both spins are observed.41,42 The vibrational band at 1425 cm−1 corresponds to the vinyl substituent in the porphyrin ring.30 3.3. Non-Native and Native SERRS Spectrum of metHb. The SERRS enhancement along with the protein nativity was examined with carboxylic acid-functionalized AgNP's with varied chain lengths (i.e., MPA- and MNA-capped AgNP's). Figure 3b shows the SERRS spectrum of metHb recorded immediately after the addition of MPA-AgNP's to an aqueous solution of 1 μM metHb. Compared to the RR spectrum of metHb, the SERRS spectrum with MPA-AgNP's shows distinguished changes leading to a dramatic conformational change in the heme pocket. The ν3 bands at 1479 and 1506 cm−1 corresponding to 6cHS and 6cLS started merging and appear as a new band at 1492 cm−1 (Figure 3b). Additionally, the ν2 bands at 1565 and 1582 cm−1 jointly appears as a broad band at 1571 cm−1. These spectral features obviously indicate that the SERRS spectrum with MPA-AgNP's is from a five-coordinate high-spin heme.30,40 Thus, in contradiction to the six-coordinate mixed-spin state in the RR spectrum, the SERRS spectrum of metHb displays a fivecoordinate high-spin (5cHS) state in combination with MPAAgNP's. An earlier study authenticated that the direct contact of Hb with the silver surface leads to the expulsion of the dioxygen ligand from Hb, apparently as a superoxide leaving the 5cHS ferric heme.30 Therefore, in accordance with the literature, it is clear that metHb is in direct contact with a bare silver surface, leading to a five-coordinate high-spin species indicating that MPA is not protective enough to prevent metHb from reaching the underlying silver surface. To confirm this, a SERRS study of metHb was carried out with seeded citrate-capped AgNP's that also showed a band at 1492 cm−1 corresponding to 5cHS (data not shown). Furthermore, the SERRS spectrum was also recorded for a solution of metHb and MPA-AgNP's with respect to increasing contact time (Figure 3c,d). As the contact time between the protein and MPA-AgNP increases, the intensity of the band at 1492 cm−1 correlating to the 5cHS state also increases, demonstrating that more and more metHb’s were in direct contact with the silver surface.30 The study of a self-assembled monolayer (SAM) of molecules with thiol headgroups on noble metal surfaces such as gold and silver revealed that the molecules were anchored more vertically and the compactness of the monolayer was also increased by the van der Waals interaction between the nearby carbon chains.43,44 Therefore, to increase the compactness of carboxylic acid-capped AgNP's we increased the carbon chain length (i.e., 9-mercaptononanoic acid (MNA) was used in place of MPA). Figure 4b shows the SERRS spectrum of 1 μM metHb in the presence of MNAAgNP's. The intensity of the SERRS spectrum was monitored with respect to the ν3 band. A drastic signal enhancement was achieved immediately after the addition of MNA-AgNPS to the protein solution, and the enhancement was saturated after 5 min, which is presented in Figure 4b. The SERRS spectrum thus obtained is identical to that of the RR spectrum of 50 μM metHb (Figure 4a) except for the intensity difference. All of the band positions and band widths of SERRS spectra were in perfect agreement with the RR spectra of metHb. This clearly demonstrates that MNA on the surface of AgNP prevents metHb from reaching the bare Ag surface. Thus, as expected, MNA forms a more compact and protective layer on the AgNP

Figure 4. Comparison of (a) the native RR spectrum of metHb (50 μM) with (b) the SERRS spectrum of metHb (1 μM) bound to MNAAgNP's.

surface. However, the preservation of nativity with MNAAgNP's has decreased the intensity of the metHb signal in contrast to that of MPA-AgNP's (Figure S2). This observation is explained by the increased protein distance from the AgNP surface due to the longer carbon chain in MNA. 3.4. Raman Enhancement Factor. The surface concentration of metHb on MNA-AgNP particles was determined by inspecting the SERRS intensity of the ν4 band in relation to the increasing concentration of metHb with respect to a fixed concentration of MNA-AgNP's (Figure S3). Here, the concentration of MNA-AgNP's in a rotating cuvette is 12 nM, and the metHb concentration was increased slowly from 10 to 180 nM with the simultaneous recording of the SERRS spectra. It is worth pointing out at this juncture that a SERRS detection of as low as 10 nM metHb is possible while preserving its native form using MNA-AgNP's as a signal amplifier. The relative surface concentration of metHb is given by θ = Γ/ΓS, where Γ and ΓS, respectively, refer to the surface coverage of metHb at a particular concentration and the saturation surface coverage. Because the surface coverage is directly related to the SERRS intensity, θ was calculated from θ = ISERRS/Imax SERRS. The above-calculated θ was plotted against the initial solution concentration of metHb (Figure 5). The plot increases linearly at the low concentration of metHb that can be well fitted by the Langmuir adsorption isotherm Kc θ= (1) 1 + Kc where K is the equilibrium constant and c corresponds to the metHb concentration in solution after adsorption equilibrium is established (i.e., c = co − θΓS*). Here, ΓS* correlates to the real surface coverage of metHb on the nanoparticle surface. The fit of eq 1 to the data in Figure 5 provides a real surface coverage of 139 nM and K = 3.6 × 108 M−1 (Figure S4). On the basis of these data, the Raman enhancement factor (EFSERS) is determined according to eq 2 I c EFSERS = SERRS RR IRR cSERRSk (2) 14360

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compact on the AgNP surface and are strong enough to prevent metHb from reaching the bare silver surface.

4. CONCLUSIONS In this work, we have demonstrated the precise tuning of the surface plasmon of citrate-capped silver nanoparticle via seeding methodology, followed by carboxylic acid functionalization through the ligand-exchange reaction. Both MPA- and MNAfunctionalized AgNP's showed vast SERRS amplification for metHb. However, MPA-AgNP's lead to the structural deformation of the heme pocket whereas MNA-AgNP's preserved the nativity of the heme pocket. The Raman enhancement factor of metHb using MNA-AgNP's as an amplifier was estimated to be 4.2 × 103. Furthermore, the UV− visible spectra for the mixture of MNA-AgNP's and metHb with respect to increasing contact time and spinning speed of the cuvette remain identical, confirming that MNA-AgNP's were very stable even after interacting with the protein. Therefore, this model system could be used as a direct label -free detection system in aqueous solution for identifying and analyzing metHb by SERRS without compromising the native heme structure.

Figure 5. Relative surface coverage (θ) of metHb on MNA-AgNP's as a function of solution metHb concentration.

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where CRR is the concentration of metHb used in the RR experiment and CSERRS is the surface concentration of metHb in the SERRS experiments. k is a shielding factor that is assumed to be 0.25 as discussed previously.45 Here, the ν4 band is used as a reference band to determine the intensity of RR and SERRS spectra. Thus, EFSERS was determined to be 4.2 × 103. 3.5. Protein−Nanoparticle Interaction and Stability. In SERRS studies, a strong chemical interaction between the analyte and surface is very important to a better enhancement. In this respect, most of the earlier SERRS studies were carried out by incubating the protein along with AgNP solution for certain period of time to enhance the signal intensity.27,46,47 Nonetheless, the structure of protein after incubation and also the nature of the interaction between the protein and enhancing surface are to be addressed. Thus, the solution containing metHb and MNA-AgNP's was examined with respect to increases in time by both SERRS and UV−visible spectroscopy to understand the nature of the protein structure and MNA-AgNP's, respectively. Figure S5 shows the SERRS spectra of the metHb/MNA-AgNP solution with respect to increasing time. A closer examination of the spectra in Figure S5 reveals that almost 85% of the enhancement was achieved immediately after adding AgNP's to protein (spectrum a). The intensity increased up to 5 min, and after that it was almost saturated (b−f). This observation implies that there is a strong interaction, most likely electrostatic, between metHb and MNA-AgNP's.27 Additionally, except for the intensity, the spectral skeleton and band positions remain identical in all of the spectra. This clearly confirms that the protein structure is never altered at the MNA-AgNP surface and remains native even after 1 h. The nativity of the protein was further examined by increasing the spinning speed of the cuvette, which also resulted in identical spectra as shown in Figure 4b. The stability of the MNA-AgNP's after interaction with metHb was monitored by UV−visible spectroscopy (Figure S6). The addition of metHb to MNA-AgNP solution leads to an immediate red shift of 9−10 nm, indicating an electrostatic interaction between the protein and nanoparticle.27 After this small red shift, the plasmonic band was stable with neither a decrease in intensity nor the appearance of new shoulder or band at higher wavelengths between 600 and 700 nm. These observations clearly confirm that the MNA SAMs are more

ASSOCIATED CONTENT

S Supporting Information *

Histograms of citrate- and MNA-capped AgNP's. SERRS spectrum of metHb with MPA-AgNP's. SERRS spectrum of metHb with increasing concentration and time using MNAAgNP's. Langmuir adsorption isotherm plot and UV−vis absorption spectra of MNA-AgNP's before and after adding metHb. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +91 9865708536. Fax: +91 4543 258358. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS G.K., A.K. (Kannan), and R.S. sincerely thank the management and the principal of Vivekananda College for their support and encouragement. A.S. acknowledge European Union 7.FP under grant REGPOT-CT-2011-285949-NOBLESSE for support. A.K. (Kamińska) thanks the foundation for Polish Science POMOST programme cofinanced by the European Union within the European Regional Development Fund.

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dx.doi.org/10.1021/la303136v | Langmuir 2012, 28, 14357−14363