Prevention of Photooxidation of Deoxymyoglobin and Reduced

Apr 29, 2014 - Yasutaka Kitahama , Masatoshi Egashira , Toshiaki Suzuki , Ichiro Tanabe , Yukihiro Ozaki. The Analyst 2015 140 (17), 6147-6148 ...
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Prevention of Photooxidation of Deoxymyoglobin and Reduced Cytochrome c during Enhanced Raman Measurements: SERRS with Thiol-Protected Ag Nanoparticles and a TERS Technique Ichiro Tanabe, Masatoshi Egashira, Toshiaki Suzuki, Takeyoshi Goto, and Yukihiro Ozaki* Department of Chemistry, School of Science and Technology, Kwansei Gakuin University, Gakuen 2-1, Sanda, Hyogo, Japan ABSTRACT: Resonance Raman, surface-enhanced resonance Raman scattering (SERRS), and tip-enhanced Raman scattering (TERS) spectra were acquired for deoxymyoglobin (deoxy-Mb) and reduced cytochrome c (reduced Cyt c). Two kinds of Ag nanoparticles (citrate-reduced Ag and thiol-protected) were used for the SERRS measurements. The SERRS spectra could be obtained even when the concentration and laser power were respectively less than a thousandth part and hundredth part of those of the corresponding resonance Raman spectral measurements, demonstrative of the remarkably high sensitivity of SERRS spectroscopy. However, the oxidation of deoxy-Mb (Fe2+) and reduced Cyt c to met-Mb (Fe3+) and oxidized Cyt c, respectively, during the acquisition of the SERRS spectra using citrate-reduced Ag nanoparticles limited the analysis of the proteins in their initial oxidation state. Oxidation was induced by the photocatalytic activity of the citratereduced Ag nanoparticles upon irradiation with incident laser light. Oxidation was effectively prevented by using thiol-protected Ag nanoparticles for the SERRS measurement or by employing TERS measurements, where the molecules and Ag nanoparticles did not make direct contact, thus preventing the oxidation reactions. The function of heme proteins is strongly related to their oxidation states; thus, it is crucial to obtain Raman spectra in the native oxidation states while circumventing the possibility of photooxidation. From this perspective, SERRS employing thiol-protected Ag nanoparticles and TERS are essential techniques for Raman measurements. In particular, TERS is a developing technique, and this study demonstrates its suitability for acquiring enhanced Raman spectra of heme proteins with nanoscale spatial resolution while preventing photooxidation.

1. INTRODUCTION Surface-enhanced resonance Raman scattering (SERRS) spectroscopy is sensitive enough for the acquisition of a Raman spectrum from even a single molecule adsorbed on noble metal nanoparticles because of its enormous enhancement factor of 1011−1014 for a Raman cross section.1−3 SERRS has been applied in a wide range of fields.1−11 In particular, SERRS measurements of biological molecules with metal nanoparticles have been employed for the past three decades.9−11 The SERRS enhancement factor depends on various elements such as the metal particle size,12 shape,13 and interparticle distance.14 During SERRS measurements, a sample consisting of metal nanoparticles and target molecules is irradiated with laser light. When the laser light wavelength corresponds to a localized surface plasmon resonance (LSPR) wavelength of the metal nanoparticles, the SERRS intensity is markedly increased.15 Ag nanoparticles are also known to exert catalytic effects. Under irradiation with LSPR wavelength light, hot electrons and holes are respectively generated above and below the Fermi level of the metal nanoparticles.16 The photoinduced charge separation promotes chemical reactions such as oxidation of citrate molecules17 and dissociation of H2 molecules.18 Therefore, plasmonic metal nanoparticles exhibit both SERRS and catalytic capacity upon laser irradiation. © 2014 American Chemical Society

Tip-enhanced Raman scattering (TERS) is one of the most attractive, novel analytical techniques, particularly from the perspective of its nanoscale spatial resolution power.8,19−23 TERS employs a metal-nanoparticle-coated tip and is based on enhancement principles similar to SERRS. TERS has been used in a wide range of research fields for the analysis of biological molecules,24 polymer blends,25 graphene,26 carbon nanotubes (CNT),27 CNT, and polystyrene nanocomposites.28 Deckert’s group utilized this technique to evaluate the distribution of oxidation states of Cyt c molecules in mitochondria.29 Wood and co-workers analyzed Hb nanocrystals with TERS and revealed that nanoscale oxidation occurred at the crystal edge.30 They also found that TERS enabled selective enhancement of heme, protein, and amino acid bands, which could not be achieved with resonance Raman and SERRS.30 This extremely high spatial resolution (down to ∼10 nm) is one of the advantages of TERS. In addition, TERS measurements do not require the adsorption of target molecules on the metal surface, which is a prominent virtue of TERS compared with SERRS. Received: March 25, 2014 Revised: April 26, 2014 Published: April 29, 2014 10329

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Absorption spectra and resonance Raman spectra of a met-Mb aqueous solution (0.1 mM) were acquired before and after addition of sodium borohydride (0.5 mg, as a reducing agent). Resonance Raman spectra of a reduced Cyt c aqueous solution (0.3 mM) were also obtained. These resonance Raman spectra were collected using a reflection-mode Raman microscope (Photon design, Nanostar NFRSM800) employing an objective lens (Photon Design, SLWD Plan (APO) 9×) and a 514.5 nm argon ion laser (Spectra-Physics, Stabilite 2017-06S). The laser intensity at the sample position was about 50 mW. Absorption spectra of met-Mb, deoxy-Mb, and Cyt c solutions were acquired via a UV−vis spectrometer (Shimadzu, UV-3101PC). 2.2. SERRS Measurements with Citrate-Reduced Ag Nanoparticles. Citrate-reduced Ag nanoparticles were prepared by the Lee−Meisel method.37 Briefly, silver nitrate (30 mg) was dissolved in water (150 mL) and brought to boil, and sodium citrate (30 mg) was added to the boiling solution. The solution was allowed to boil for about 1 h to get citrate-reduced Ag nanoparticle colloids. Subsequently, the citrate-reduced Ag colloids (1 mL), aqueous sodium hydrate (100 mM, 0.5 mL), and the reduced Mb solution (0.1 mM, 1 μL) or the Cyt c solution (0.3 mM, 1 μL) were mixed, and SERRS spectra were acquired. The instruments for the SERRS measurements were the same as those used for the resonance Raman measurements, but the laser intensity was about 0.5 mW. 2.3. SERRS Measurements with Thiol-Protected Ag Nanoparticles. Two types of carboxyl-terminated alkanethiols (6-mercaptohexanoic acid, COOH−(CH2)5−SH, and 3mercaptopropionic acid, COOH−(CH2)2−SH) were purchased from Sigma Chemical Co. and used as purchased. The citrate-reduced Ag nanoparticle colloids were cast on aminosilane-coated glass substrates (100−200 μL cm−2) and left for about 1 h. The substrates were rinsed with ultrapure water, and the Ag nanoparticles were electrostatically adsorbed on the substrates. The ethanolic thiol solutions (20 mM) were then cast on the Ag-adsorbed substrates (100−200 μL cm−2) and left for 1 h. The substrates were rinsed with ethanol, and selfassembled monolayers (SAM) were formed on the Ag surface according to previous reports.38,39 The reduced Mb solution (0.1 mM) or the Cyt c solution (100 nM) was then cast on the substrates (∼10 μL cm−2), and SERRS spectra were acquired. The instruments for the SERRS measurements are the same as those used for the resonance Raman measurements. 2.4. TERS Measurements of Myoglobin and Cytochrome c. For the TERS measurements, a deoxy-Mb solution (0.1 mM) or a reduced Cyt c solution (0.3 mM) was cast on a glass substrate (∼10 μL cm−2), and TERS spectra were acquired. The setup for the TERS analysis consisted of a reflection mode Raman microscope and an atomic force microscope (Photon Design, Nanostar NFRSM800) with a 514.5 nm laser (the same excitation wavelength as the SERRS measurement). An objective lens (Photon Design, SLWD Plan (APO) 90×) was used for the TERS measurements. A TERS needle coated with silver (tip radius: ∼100 nm, UNISOKU Co. Ltd.) was attached to a quartz tuning fork of a shear-force-based AFM at an angle of 45°. Raman spectra of the samples were obtained under tip-retracted and tip-approaching conditions at the same point. The TERS spectrum was calculated by subtracting the spectrum observed under the tip-retracted conditions from that collected under the tip-approaching conditions; the details are described in our previous reports.28,40

Both SERRS and TERS are promising analytical techniques and have been utilized in a variety of ways. For example, it is well-known that a Raman mode enhanced by SERRS depends on the orientation of the molecule on the metal surface and the excitation laser wavelength.4−6 It has been found that when Ag nanoparticles covered with alkanethiol self-assembled monolayers are used for SERRS measurements,31−33 the SERRS intensity varies according to the carbon chain length of the alkanethiol.32,33 Analysis of the oxidation states of heme proteins is highly important to understand their function. For instance, in the case of Cyt c, the oxidation−reduction reaction of a heme iron ion (Fe2+ or Fe3+) plays a key role in the expression of the Cyt c function;34 thus, determination of its oxidation state is essential. Many other metal proteins, nonheme proteins, and copper proteins35 have a central metal ion, and it is desirable to detect their natural oxidation states with high sensitivity. However, there is a possibility that the laser light in conjunction with the nanoparticles used in the analytical techniques may induce oxidation reactions of the target molecules. Consequently, it must be ensured that the oxidation states of the molecules do not change during SERRS measurements. Although there are a few papers referring to the oxidation of Cyt c during SERRS measurements,36 the case of Mb has not been reported. In addition, TERS is a currently developing technique, and possible oxidation during TERS measurement remains completely undiscussed. In the present study, the resonance Raman, SERRS, and TERS spectra of deoxy-Mb and reduced Cyt c are acquired and compared. We used two kinds of Ag nanoparticles for SERRS measurements: one was citrate-reduced Ag nanoparticles, and the other was thiol-protected Ag nanoparticles. Citrate-reduced Ag nanoparticles are mostly used for SERRS measurements, and we also used them at first. However, it was revealed that target molecules were oxidized during the measurements with the citrate-reduced Ag nanoparticles. Therefore, thiol-protected Ag nanoparticles were subsequently employed in this study so as to elucidate the mechanism of the photooxidation reactions occurring during the SERRS measurements. In a TERS measurement, the tip and sample are, in general, separated by several nanometers. SERRS spectroscopy allows the acquisition of a Raman spectrum with highly sensitive detection (i.e., with a low concentration of analyte and low laser power) compared with resonance Raman spectroscopy. However, the oxidation of the sample on the metal is possible during SERRS measurements employing citrate-reduced Ag nanoparticles. On the other hand, the oxidation of the molecules may be suppressed by using thiol-protected Ag nanoparticles or TERS. In order to obtain Raman spectra of the molecules in their natural oxidation states, oxidation must be prevented during spectral acquisition. Therefore, for some investigations of redox molecules such as heme proteins, SERRS employing thiolprotected Ag nanoparticles and TERS measurements may be preferable to SERRS measurements with citrate-reduced Ag nanoparticles. The TERS technique has recently received much attention as a promising analytical technique with nanoscale spatial resolution. In this study, an unrecognized advantage of the TERS technique is revealed.

2. EXPERIMENTAL SECTION 2.1. Absorption and Resonance Raman Spectral Measurements of Myoglobin and Cytochrome c. Mb (equine skeletal muscle) and Cyt c (bovine heart) were purchased from Sigma Chemical Co. and used as purchased. 10330

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Figure 1. Normalized absorption spectra of (a) met-Mb and (b, c) deoxy-Mb solutions at pH ∼ (a) 7.0, (b) 8.1, and (c) 7.0.

3. RESULTS AND DISCUSSION 3.1. Resonance Raman and SERRS Spectra of Myoglobin. Figure 1 shows the absorption spectra of aqueous solutions of Mb (0.1 mM) (a) before and (b, c) after the addition of sodium borohydride. Upon the addition of sodium borohydride, the absorption maximum of the γ (Soret) band shifted from 410 to 417 nm, and two characteristic peaks due to the α and β bands appeared in the 500−600 nm region. These spectral changes indicate that met-Mb was reduced to deoxyMb with the addition of sodium borohydride.41,42 In addition, it is noted that deoxy-Mb is not oxidized by the change of pH value from 8.1 to 7.0 as shown in Figure 1b,c. Figure 2a,b shows the resonance Raman spectra of met-Mb and deoxy-Mb. In the resonance Raman spectra of heme proteins, the position of the v4 band (1350−1370 cm−1, a breathing mode of four pyrrole rings) is known to be altered depending on the oxidation state of the heme moiety.9 In the present case, the resonance Raman spectrum of met-Mb acquired in aqueous solution shows an oxidation state marker band at 1370 cm−1 (Figure 2a). After the addition of sodium borohydride, this band shifts to 1354 cm−1 (Figure 2b), which indicates reduction of met-Mb to deoxy-Mb. Figure 2c shows the SERRS spectrum of met-Mb. The widths of the bands in the SERRS spectrum (Figure 2c) are larger than those in the corresponding resonance Raman spectrum (Figure 2a) because the Mb molecules adsorbed on the Ag surface are oriented randomly and the peak position of each molecule is slightly shifted.4−6 Compared with the conditions required for the resonance Raman measurements, the solution concentration and the incident laser intensity required for SERRS were

Figure 2. Normalized (a, b) resonance Raman (RR) spectra of (a) met-Mb and (b) deoxy-Mb. (c, d) Normalized SERRS spectra of (c) met-Mb and (d) deoxy-Mb with citrate-reduced Ag nanoparticles. (e) Normalized SERRS spectra of deoxy-Mb with thiol-protected Ag nanoparticles. The yellow band marks the region where the oxidation state marker band is located (1340−1400 cm−1 region).

reduced dramatically; the concentration was changed from 0.1 mM to about 66 nM, and the laser intensity was weakened from 50 mW to 0.5 mW. In spite of these changes, the SERRS spectra could be acquired with high signal-noise ratio. This high sensitivity is the distinguishing advantage of the SERRS measurement. One serious issue is noted in the SERRS analysis; the v4 band in the SERRS spectrum of deoxy-Mb (Figure 2d) occurs at 1370 cm−1 as is the case of that of met-Mb solution (Figure 2c). These results indicate that deoxy-Mb was oxidized to metMb during the SERRS measurement employing the citratereduced Ag nanoparticles. As previously mentioned, it is vital to obtain Raman spectra of biomolecules in their native oxidation state. Hence, the mechanism of oxidation during the SERRS measurement was contemplated with the aim of prevention. Though the pH of the reduced Mb solution (pH ∼ 8.1) was different from that of the citrate-reduced Ag nanoparticle colloid (∼7.0), the change in the oxidation state was not induced by the decrease in the pH value, as shown in Figure 1c. As described above, the Ag nanoparticles are catalytically active under irradiation with laser light of the LSPR wavelength.14 Thrall and co-workers reported the photooxidation of citrate by 10331

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Ag nanoparticles.15 Therefore, in this study, it is concluded that the citrate-reduced Ag nanoparticles work as photocatalysts for the oxidation of deoxy-Mb in the aqueous solution upon irradiation with the 514.5 nm laser light. Surface citrate on the Ag nanoparticle was oxidized, and the deoxy-Mb was then oxidized to met-Mb on contact with the bare Ag surfaces. Water might be reduced as the corresponding reduction reaction. Thus, to obtain the SERRS spectrum of deoxy-Mb without the effects of photooxidation, the measurements were acquired using thiol (6-mercaptohexanoic acid)-protected Ag nanoparticles. The alkanethiol is strongly adsorbed unto the Ag surface and forms a SAM membrane on the Ag nanoparticles.38,39 Thus, deoxy-Mb and Ag do not make direct contact, and the photooxidation of deoxy-Mb is prevented. In fact, in contrast to the spectrum of deoxy-Mb employing the citrate-reduced Ag nanoparticles, the position of the v4 band (Figure 2e) did not change (about 1358 cm−1), indicating that deoxy-Mb was not oxidized during the SERRS measurement. Thus, the SERRS spectra of deoxy-Mb could be obtained while preventing the photooxidation reaction by utilizing the thiolprotected Ag nanoparticles. 3.2. Resonance Raman and SERRS Spectra of Cytochrome c. The absorption spectrum (Figure 3) of Cyt

Figure 4. Normalized (a) resonance Raman (RR) and (b−d) SERRS spectra of reduced Cyt c. (b) Citrate-reduced, (c) 6-mercaptohexanoic acid-protected, and (d) 3-mercaptopropionic acid-protected Ag nanoparticles were used. The yellow band marks the region where the oxidation state marker band is observed (1340−1400 cm−1 region).

the distance between the Ag surface and the target molecule increases and the signal-noise ratio decreases upon incorporation of a thiol SAM membrane, it is vital to maintain the natural oxidation state of the molecule. Kitano and co-workers reported similar changes in the oxidation behavior of Cyt c based on the Ag surface conditions,36 but they did not discuss the details of the mechanism of the oxidation reaction. Herein, we have presented a systematic demonstration of the photooxidation of not only Cyt c but also Mb and Hb and successfully prevented this oxidation based on consideration of the mechanism. Furthermore, the usefulness of the TERS measurements is demonstrated. 3.3. TERS Spectra of Myoglobin and Cytochrome c. Figures 5a and 5b show the TERS spectra of deoxy-Mb and reduced Cyt c, respectively. The TERS spectral profiles are almost identical to the resonance Raman and SERRS spectra, which means that the molecules remained basically in the same forms during these measurements. It is also noted that a heat effect of the incident laser light for TERS should be larger than those for the resonance Raman and SERRS because the TERS experiments were done in air instead of the solution. In spite of this, the TERS spectra were almost the same with the resonance Raman and SERRS ones. These results mean that the heat effect of laser was not crucial and conformations of the molecules were not changed during the TERS measurements. Focusing on the oxidation state marker band, peaks are observed at 1357 and 1365 cm−1 in the spectra of deoxy-Mb

Figure 3. Normalized absorption spectrum of reduced Cyt c.

c indicated that Cyt c was in the reduced state (Fe2+), an aqueous solution of which was used for the ensuing experiments. The v4 band of Cyt c was located at 1365 cm−1 in the resonance Raman spectrum (Figure 4a). In comparison, the corresponding peak in the SERRS spectrum acquired with the citrate-reduced Ag nanoparticles occurred at 1375 cm−1 (Figure 4b). Thus, Cyt c was also oxidized in the presence of the citrate-reduced Ag nanoparticles and laser irradiation. The SERRS spectrum was thus acquired by using the thiol (6mercaptohexanoic acid)-protected Ag nanoparticles (Figure 4c), and the spectrum indicated that Cyt c remained unoxidized throughout the measurement. Even when Ag nanoparticles protected with another thiol with a shorter carbon chain (3mercaptopropionic acid) were utilized, the photooxidation reaction of Cyt c was suppressed (Figure 4d). From these results, it is clear that both Mb and Cyt c are oxidized during the SERRS measurements employing citricreduced Ag nanoparticles and that this oxidation can be prevented by adopting thiol-protected Ag nanoparticles. It was also confirmed that Hb was oxidized under the same experimental conditions. This oxidation reaction may occur for other redox proteins such as non-heme proteins and copper proteins. Thus, during SERRS measurements, the possibility of a photooxidation reaction must always be considered. Although 10332

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not affect the oxidation state of the molecules when there is sufficient separation between the tip and molecules, making it a powerful method for analysis of the state of single molecules. It must be noted, however, that there is a possibility that oxidation reactions may occur if the TERS tip makes direct contact with the evaluated materials or when metal nanoparticles are incorporated on a substrate to increase the signal intensity.

4. CONCLUSIONS In summary, the resonance Raman, SERRS, and TERS spectra of deoxy-Mb and reduced Cyt c were acquired, revealing the oxidation of the target molecules during SERRS measurements, by employing citrate-reduced Ag nanoparticles. Oxidation is attributed to the photocatalytic activity of Ag nanoparticles induced by irradiation with 514.5 nm laser light. This oxidation reaction can be prohibited by introducing a thiol SAM membrane on the Ag particle surface. In addition, the TERS technique facilitates the acquisition of spectra without changes in the oxidation states of Mb and Cyt c if the TERS tip is sufficiently separated from the sample. It is important to note that the oxidation state remains unchanged during the spectral measurement, not only for Mb and Cyt c but also for other redox materials. From this point of view, the TERS technique may be a valuable vibrational spectroscopic method for deeper understanding of the nature of redox proteins such as heme proteins.

Figure 5. Normalized TERS spectra of (a) deoxy-Mb and (b) reduced Cyt c.

and reduced Cyt c, respectively. Both spectra obviously demonstrate that the molecules remain in the unoxidized state during the TERS measurements. If the TERS tip was oxidized, the oxidation of molecules could be also prevented. However, spectra measured with an old TERS tip, which had been left for a long time and seemed to be oxidized, showed extremely low intensity and no defined peak. It means the effect of native oxidation of the TERS tip can be detected by the spectral changes. In addition, in the present experiments, the TERS tip was stored in a vacuum-sealed package until just before the TERS measurements. Therefore, the effects of the native oxidation of the TERS tip may be vanishingly small. In the present TERS instrument, the tip and substrate surface are separated by about 1−2 nm under tip-approaching conditions, which was fundamental to prevention of the oxidation reaction of the target molecules. It is noted here that the TERS spectra were measured in air, while the resonance Raman and SERRS ones were in solution. However, in the case of TERS, samples and TERS tip were separated only 1−2 nm, and thus, surface-adsorbed water forms a meniscus between them. The meniscus works as a reaction field as reported previously.43,44 In the present case, therefore, the oxidation reaction can proceed in the meniscus if it occurs as is the case with the measurements in the solution. Thus, it is reasonable to compare the TERS spectra with the resonance Raman and SERRS ones. Several groups reported the TERS measurements of reduced Cyt c22,29 and Hb nanocrystals,30 but TERS measurements of deoxy-Mb have not been reported. Deckert’s group reported that the oxidation state of Cyt c varied depending on its location in mitochondria.29 In that report, it was asserted that most of the Cyt c molecules were located in the intermembrane, and thus the TERS tip and the Cyt c molecules did not make direct contact. In another study,22 Zenobi and co-workers demonstrated that the TERS technique could detect the orientation of Cyt c on substrates at the singlemolecule level, which is not a feature of SERRS measurements. However, the possibility of the photooxidation of the target molecules during the measurements was not considered. From the present study, it is concluded that the TERS technique does

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Y.O.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2009−2013.

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REFERENCES

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