Nickel Nanowires Combined with Surface-Enhanced Raman

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Nickel Nanowires Combined with Surface-Enhanced Raman Spectroscopy: Application in Label-Free Detection of Cytochrome c-Mediated Apoptosis Haijing Zhang, Yiming Kou, Junbo Li, Lei Chen, Zhu Mao, Xiao Xia Han, Bing Zhao, and Yukihiro Ozaki Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04204 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 20, 2018

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Analytical Chemistry

Nickel Nanowires Combined with Surface-Enhanced Raman Spectroscopy: Application in Label-Free Detection of Cytochrome cMediated Apoptosis Haijing Zhang, † Yiming Kou, ‡ Junbo Li, † Lei Chen,€ Zhu Mao, † Xiao Xia Han,*, † Bing Zhao,† and Yukihiro Ozaki§ †

State Key Laboratory of Supramolecular Structure and Materials, Institute of Theoretical Chemistry, Jilin University, Changchun 130012, P. R. China. ‡ National Engineering Laboratory for AIDS Vaccine, School of Life Science, Jilin University, Changchun 130012, P. R. China € Key Laboratory of Preparation and Applications of Environmental Friendly Materials, Ministry of Education, Jilin Normal University, Changchun 130103, P. R. China § Department of Chemistry, School of Science and Technology, Kwansei Gakuin University, Sanda, Hyogo 669-1337, Japan Supporting Information Placeholder ABSTRACT: Intrinsic properties of nickel have enabled

its wide applications as an effective catalyst. In this study, nickel nanowires (Ni NWs) as electron donors for oxidized cytochrome c (Cyt c) are investigated, which are NW diameter, temperature, and pH value-dependent. The reductive and magnetic properties facilitate the Ni NWs to rapidly and conveniently reduce Cyt c in complicated biological samples. Moreover, we find that the Ni NWs combined with resonance Raman spectroscopy have specificity towards Cyt c detection in real biological samples, which is successfully used to distinguish the redox state of the released Cyt c from isolated mitochondria in apoptotic Hela cells. Moreover, rapid labelfree Cyt c quantification can be achieved by surfaceenhanced Raman spectroscopy with a limit of detection of 1 nM and long concentration linear range (1nM˗1μM). The proposed Ni NWs-based reduction approach will significantly simplify the traditional biological methods and has great potential in the application of Cyt c-related apoptotic studies.

The nickel (Ni) well known as a low-cost catalyst has several key properties including oxidative addition and ready access to multiple oxidation states, which allowed the development of a broad range of innovative reactions.1-3 On the other hand, Ni nanomaterials as an active substrate have been applied in the research field of surface-enhanced Raman scattering (SERS), and their enhancement ability is found to be dependent on surface morphology due to the electromagnetic mechenism.4,5 Moreover, in the case of a study on pyrazine adsorbed

on nickel electrodes, the obtained intensity-potential profile was interpreted by the charge-transfer mechanism and thus Ni support was believed to be capable of communicating charges with pyrazine, but such charge transfer depends on laser wavelengths6 and so far the adsorbed molecules are limited to several organic small molecules, which is an obstacle for the practical applications of these transition metals. Cytochrome c (Cyt c) is an essential component of the respiratory chain, and it transports electrons undergoing oxidation and reduction7. Additionally, Cyt c is also involved in initiation of apoptosis, including binding into cardiolipin and release from mitochondria to the cytoplasm.8,9 Cyt c is free within the intermembrane of mitochondria during the release process and in the cytoplasm after release. It will be of great significance for early apoptotic cell detection if a label-free approach is developed to directly probe Cyt c release in real time. Traditional detection method for determination of the released Cyt c from mitochondria is Western blotting. 10,11 In recent years, several novel approaches have been developed such as fluorescence-based strategies 12,13 with highly selectivity and sensitivity. However, almost all these methods require time-consuming procedures for the preparation of specific aptamers or antibodies in order to capture Cyt c based on biorecognition. Moreover, none of these methods can distinguish the redox states of the released Cyt c. Noted that SERS can be used to monitor the redox state and conformation of Cyt c within living mitochondria14. However, it has never been used to determine redox states of the released Cyt c and quali-

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fy them outside mitochondria, which is very important biomarker for apoptosis. In this study, we examine the reduction activity of Ni NWs and their applications in the exploration of Cyt cmediated apoptosis. The dependence of the reductive property on the diameter size of the nanowires, temperature and pH condition during the reaction process is investigated by resonance Raman spectroscopy. Moreover, the selectivity of the as-prepared Ni NWs for Cyt c are examined. Furthermore, the Ni NWs are employed for quantifying the released Cyt c and determination of its redox states from isolated mitochondria in apoptotic HeLa cells. Ni nanowires were prepared according to a dropping method15. We found that the diameters of the Ni NWs were tunable by the heating temperature during the synthetic procedure. As shown in the transmission electron microscope (TEM) images of Figure 1, the average diameter of the as-prepared Ni NWs decreased (from 300, 250, 200, 100 to 60 nm) with the increasing heating temperature (from 30, 60, 80, 120 to 140 °C). The Ni NWs with rough surfaces would facilitate direct contact of the proteins. Moreover, the lattice distances of 0.21 and 0.18 nm are observed in the high-resolution TEM (HRTEM) images (Figure 1F), which correspond to the crystal planes of (111) and (200) of Ni, respectively. The purity of the Ni NWs is further confirmed by the X-ray diffraction (XRD). As shown in Figure 1 G, the three reflection peaks of the Ni NWs are all well-matched with the crystal planes of (111), (200), and (220) of the reference Ni. The magnetic property of the Ni NWs is examined. Note that the highest value of the saturation magnetization is observed in the Ni NWs heated at 120 °C (trace g in Figure 1H) rather than that heated at 140 °C. Accordingly, the Ni NWs prepared at 120 °C are chosen for the following experiments. An obvious color change (from dark red to light red) was observed by naked eyes when the Ni NWs were added into the oxidized Cyt c (Fe3+Cyt c) as seen in the inset of Figure 2A. The UV-Vis spectrum of the oxidized Cyt c with the Ni NWs displays the typical Soret band at 414 nm and two Q bands at 520 and 549 nm (Figure 2 b), confirming the reduction of the Fe3+Cyt c. Raman spectroscopy can provide detailed structural information about heme proteins16. Cyt c absorbs UV-Vis lights mainly in the Soret and Q-band regions at wavelengths of around 410 nm and 530 nm, which allow investigating Cyt c conformations by resonance Raman (RR) scattering using violet and green excitation lasers.17,18 Here, resonance Raman spectra were recorded with a 532 nm laser excitation and a Raman fingerprint of the native reduced Cyt c is observed (Figure 2 c), consistent with that of the Cyt c reduced by a commonly-used reducing agent (sodium dithionite, Figure S1). All these results demonstrate the reduction activity of the Ni NWs for the oxidized Cyt c. As shown in Figure 2B, besides the shifted v4 bands at 1364 cm-1

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Figure 1. TEM images of the Ni NWs prepared at 140 °C (A), 120 °C (B), 80 °C (C), 60 °C (D) and (E) 30 °C; (F) HRTEM images of the Ni NWs prepared at 120 °C; (G) XRD patterns of the Ni NWs prepared at 140 °C (a), 120 °C (b), 80 °C (c), 60 °C (d) and (e) 30 °C; (H) magnetic hysteresis curves of the Ni NWs prepared at 140 °C (g), 120 °C (h) and 80 °C (f).

and the v10 band at 1622 cm-1, the relative intensity of the two bands at 1129 and 1168 cm-1 is much higher in the reduced Cyt c (Figure 2 c) than that in the oxidized form c (Figure 2 d), and accordingly the reductive activity of the Ni NWs can be probed by the relative intensity of the two bands. To further investigate the reductive activity of the Ni NWs, we examined the nanowire diameter, temperatureand pH-dependent RR spectra of the reduced Cyt c by the Ni NWs prepared at 120 °C. Here, as shown in Figure 2C the two bands at 1168 cm-1 from the capillary (used as an external standard) and 1129 cm-1 assigned to the symmetric vibrational mode of the half-ring of the porphyrin are used to probe the reduction reactions.19 The reductive activity was found to be the nanowire diameter-dependent as seen in Figure 2D. The relative intensity of the two bands remarkably decreased with the increase of the nanowire diameter, indicating that the smaller diameter of the Ni NWs is, the faster reduction reaction will be. Moreover, we found the reduction activity is also affected by the temperature and pH values during the reduction reactions (Figure S2). To explore the reduction selectivity of the Ni NWs for heme proteins in vitro, UV-Vis spectra of four other heme proteins, cytochrome b5 (Cyt b5), hemoglobin (Hb), myoglobin (Mb) and horseradish peroxidase (HRP), after reacting with the Ni NWs are recorded as shown in Figure S3. Noted that the reduced Soret band only

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Figure 2. (A) UV-Vis spectra of the Fe3+Cyt c before (a) and after (b) adding the Ni NWs, the inset photo shows the corresponding color change of the Cyt c solution; RR spectra of the Fe3+Cyt c before (d) and after (c) adding the Ni NWs；(B) resonance Raman spectra of the Fe3+Cyt c before (d) and after (c) reduced by the Ni NWs; (C) RR spectra of (e) the PBS buffer (pH=7.3) in capillary as a control and (f) the reduced Cyt c by the Ni NWs; intensity ratio of the bands at 1129 cm-1(I1129) to 1168 cm-1 versus (D) the average diameters of the Ni NWs, the error bars indicate the standard deviations with 3-5 measurements. The concentration of the Cyt c is 200 µM with1 mg Ni NWs.

appeared in the case of the oxidized Cyt c, and all other four heme proteins remained their oxidized states. Thus the reduction activity of the Ni NWs is specific for Cyt c, which is of great importance for selectively reducing Cyt c in a complicated biological sample (e.g., like protein extracts). The specific electron donor property of the Ni NWs for Cyt c probably originates from the higher reduction potential of Cyt c than other heme proteins. Moreover, according to the energy levels of the Ni NWs and oxidized Cyt c, a very small energy barrier height of allows direct electron transfer from the Ni the Fe3+Cyt c (Figure S4).20 The reduction reaction can be further confirmed by using a Ni2+ chromogenic reagent to detect the reaction product (Figure S3B). In our study, the Ni NWs can be used directly in the isolated mitochondria solution. After the reduction reaction the Ni NWs are collected by an external magnet and the supernatants are removed and kept in capillaries for Raman measurements. Here, the Cyt c can be detected within one minute including the reduction reaction procedure. Here the Hela cells were firstly incubated with actinomycin D for 1 h, and the mitochondria were subsequently isolated. The released Cyt c thus can be detected by RR spectrometry after the addition of Ni NWs (the diagram in Figure 3). Although the bands at 750 and 1583 cm-1 appear in both the oxidized and reduced Cyt c as shown in Figure 2B, the Raman signal of the reduced state of Cyt c is stronger owing to the larger Raman cross-section than that of the oxidized state.21,22 As shown in Figure 3A, the released Cyt c is almost unde-

tectable (trace blank) but after the reduction by the Ni NWs, typical Raman bands at 750 and 1583 cm-1 assigned to the vibrational mode ν15 and ν2 23 of the Cyt c appear, indicating the existence of the oxidized Cyt c in the isolated mitochondria solution. A calibration curve is plotted based on Fe2+Cyt c concentration-dependent intensity of the Raman band at 750 cm-1 (Figure S5), according to which the concentration of the total released Cyt c in the isolated mitochondria form apoptotic Hela cells is calculated to be about 2 µM. The redox states of the Cyt c can be evaluated by Figure S6. Here, the mitochondria solution without actinomycin D pretreatment was used as a control (Figure S7), in which the Cyt c was undetectable even with the aid of the reductive Ni NWs. Additionally, the oxidized Cyt c (2 µM) can indeed be detected in pure protein solution by UV-Vis absorption, but it is almost undetectable in the isolated mitochondria solution due to the background interference. Moreover, the limit of detection (LOD) of the proposed approach for the reduced Cyt c can be further improved as low as 1 nM with the aid of silver nanoparticles (Ag NPs), which display high Raman signal enhancement. As shown in Figure 3 B, the SERS spectra of the reduced Cyt c display broadened shifted bands and variations in relative intensities compared with its resonance Raman spectrum (Figure 2 B), which is attributed to the interactions between the heme and the Ag. Such interaction involves the photo-induced electron transfer from the reduced Cyt c to Ag. 20 Moreover, the linear range of the approach is as wide as 1 nM˗0.1 μM (Figured 3C), and the control spectrum is shown in Figure S8. All the results demonstrate that the proposed SERS-based method exhibits a comparable LOD, much shorter detection time and wider concentration linear range than those of current related methods.22 In summary, the reduction activity of the Ni NWs for oxidized Cyt c was investigated in detail. The asprepared Ni NWs with magnetic property were capable of selectively reducing Cyt c among diverse heme proteins. More importantly, the Ni NWs were successfully used for rapid Cyt c reduction and determination of the released Cyt c from mitochondria in apoptotic Hela cells. It is noted that the redox states of the released Cyt c can easily be determined with the aid of the Ni NWs, and label-free highly sensitive Cyt c quantification can be achieved with the Ag NPs by SERS spectroscopy. Without the Ni, it will be impossible for SERS to determine the Cyt c redox states outside mitochondria. Moreover, the high sensitivity is also attributed to the combination of Ni and Ag (Figure S9). The reduction reaction can complete within one minute and the Ni NWs can be conveniently collected and separated by an external magnet, which display great potential in the applications of Cyt c function investigation in apoptosis.


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Figure 3. Schematic diagram of in situ detection of the Cyt c release from mitochondria in actinomycin D-induced apoptotic Hela cells by RR/SERS spectroscopy; (A) RR spectra of the Cyt c in isolated mitochondria solution before (black) and after (red) reduction by the Ni NWs, and RR spectrum of the reduced Cyt c (blue, 12 µM); (B) SERS spectra of reduced Cyt c at concentrations of 1 µM (dark cyan) and 1nM (orange), respectively; (C) Cyt c concentrationdependent relative SERS intensities of the band at 1374 cm-1 (the Si band at 520 cm-1 was used as an external standard), the error bars indicate the standard deviations with 3-5 measurements. Supporting Information Experimental details; a supporting table for Raman and SERS band assignments; temperature and pH-dependent reductive activity and selectivity of the Ni NWs; Fermi energy of the Ni NWs; Cyt c concentration-dependent Raman intensities; Raman and SERS spectra of living mitochondria. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation (Grant Nos. 21773079, 21773080, 21711540292 and 21503021) of China. REFERENCES (1) Tasker, S. Z.; Standley, E. A.; Jamison, T. F. Nature 2014, 509, 299-309. (2) Rosen, B. M.; Quasdorf, K. W.; Wilson, D. A.; Zhang, N.; Resmerita, A. M.; Garg, N. K.; Percec, V. Chem. Rev. 2011, 111, 1346-1416. (3) Garcia, J. J.; Brunkan, N. M.; Jones, W. D. J. Am. Chem. Soc. 2002, 124, 9547-9555. (4) Ren, B.; Huang, Q. J.; Cai, W. B.; Mao, B. W.; Liu, F. M.; Tian, Z. Q. J. Electroanal. Chem. 1996, 415, 175-178. (5) Tian, Z. Q.; Ren, B.; Wu, D. Y. J. Phys. Chem. B 2002, 106, 9463-9483. (6) Huang, Q. j.; Yao, J. L.; Mao, B. W.; Gu, R. A.; Tian, Z. Q. Chem. Phys. Lett. 1997, 271, 101-106. (7) Ma, X.; Zhang, L.-H.; Wang, L.-R.; Xue, X.; Sun, J.-H.; Wu, Y.; Zou, G.; Wu, X.; Wang, P. C.; Wamer, W. G.; Yin, J.-J.; Zheng, K.; Liang, X.-J. ACS Nano 2012, 6, 10486-10496.

(8) Bradley, J. M.; Silkstone, G.; Wilson, M. T.; Cheesman, M. R.; Butt, J. N. J. Am. Chem. Soc. 2011, 133, 19676-19679. (9) Kagan, V. E.; Tyurin, V. A.; Jiang, J.; Tyurina, Y. Y.; Ritov, V. B.; Amoscato, A. A.; Osipov, A. N.; Belikova, N. A.; Kapralov, A. A.; Kini, V. Nat. Chem. Biol. 2005, 1, 223-232. (10) Ott, M.; Robertson, J. D.; Gogvadze, V.; Zhivotovsky, B.; Orrenius, S. P. Natl. Acad. Sci. USA 2002, 99, 1259-1263. (11) Borutaite, V.; Budriunaite, A.; Morkuniene, R.; Brown, G. C. BBA - Mol. Basis Dis. 2001, 1537, 101-109. (12) Shamsipur, M.; Molaabasi, F.; Hosseinkhani, S.; Rahmati, F. Ana. Chem. 2016, 88, 2188-2197. (13) Chen, T.-T.; Tian, X.; Liu, C.-L.; Ge, J.; Chu, X.; Li, Y. J. Am. Chem. Soc. 2015, 137, 982-989. (14) Brazhe, N. A.; Evlyukhin, A. B.; Goodilin, E. A.; Semenova, A. A.; Novikov, S. M.; Bozhevolnyi, S. I.; Chichkov, B. N.; Sarycheva, A. S.; Baizhumanov, A. A.; Nikelshparg, E. I. Scientific Reports 2015, 5, 13793. (15) Zhang, J.; Xiang, W.; Liu, Y.; Hu, M.; Zhao, K. Nanoscale Res. Lett. 2016, 11, 118. (16) Han, X. X.; Schmidt, A. M.; Marten, G.; Fischer, A.; Weidinger, I. M.; Hildebrandt, P. ACS Nano 2013, 7, 32123220. (17) Susanne Döpner; Peter Hildebrandt; And, F. I. R.; Mauk, A. G. J. Am. Chem. Soc. 1998, 120, 11246-11255. (18) Murgida, D. H.; Hildebrandt, P. Chem. Soc. Rev 2008, 37, 937-945. (19) Hu, S.; Morris, I. K.; Singh, J. P.; Smith, K. M.; Spiro, T. G. J. Am. Chem. Soc. 1993, 115, 12446-12458. (20) Li, J.; Cheng, W.; Wang, X.; Zhang, H.; Jin, J.; Ji, W.; Han, X. X.; Zhao, B. Chem.–Eur. J. 2017, 23, 9034 –9038. (21) Han, X. X.; Köhler, C.; Kozuch, J.; Kuhlmann, U.; Paasche, L.; Sivanesan, A.; Weidinger, I. M.; Hildebrandt, P. Small 2013, 9, 4175-4181. (22) Sivanesan, A.; Ly, K. H.; Adamkiewicz, W.; Stiba, K.; Leimkühler, S.; Weidinger, I. M. J. Phys. Chem. C 2013, 117, 11866-11872. (23) Okada, M.; Smith, N. I.; Palonpon, A. F.; Endo, H.; Kawata, S.; Sodeoka, M.; Fujita, K. P. Natl. Acad. Sci. USA 2012, 109, 28-32.


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Nickel Nanowires Combined with Surface-Enhanced Raman

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