A Single Nanoprobe for Ratiometric Imaging and Biosensing of

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A Single Nanoprobe for Ratiometric Imaging and Biosensing of Hypochlorite and Glutathione in Live Cells Using Surface-Enhanced Raman Scattering Weikang Wang, Limin Zhang, Li Li, and Yang Tian Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02081 • Publication Date (Web): 06 Sep 2016 Downloaded from http://pubs.acs.org on September 7, 2016

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A Single Nanoprobe for Ratiometric Imaging and Biosensing of Hypochlorite and Glutathione in Live Cells Using SurfaceEnhanced Raman Scattering Weikang Wang, Limin Zhang, Li Li and Yang Tian* Shanghai Key Laboratory of Green Chemistry and Chemical Processes, Department of Chemistry, East China Normal University, Dongchuan Road 500, Shanghai 200241, China. ABSTRACT: Hypochlorite (ClO-) and glutathione (GSH) have been reported to closely correlate with oxidative stress and related diseases, however the clear mechanism is still unknown, mainly owing to a lack of accurate analytical methods in live cells. Herein we create a novel surface-enhanced Raman scattering (SERS) nanoprobe, 4-Mercaptophenol (4-MP)functionalized gold flowers (AuF/MP), for imaging and biosensing of ClO - and GSH in RAW 264.7 macrophages cells upon oxidative stress. The SERS spectra of AuF/MP change with the reaction between ClO - and 4-MP on AuFs within 1 min, and then recover after reacted with GSH, resulting in the ratiometric detection of ClO - and GSH with high accuracy. The single SERS probe also shows high selectivity for ClO- and GSH detection against other reactive oxygen species and amino acids which may exist in biological system, as well as remarkable sensitivity ascribed to a larger amount of hot spots on AuFs. The significant analytical performance of the developed nanoprobe, together with good biocompatibility and high cell-permeability enables the present SERS probe imaging and real-time detection of ClO- and GSH in live cells upon oxidative stress.

Hypochlorite (ClO-), as one of the biologically important reactive oxygen species (ROS), is a powerful oxidant which functions as an antimicrobial agent in water treatment and living organisms.1-3 The endogenous ClO-, which is generated mainly in leukocytes by myeloperoxidase (MPO)-catalyzed peroxidation of chloride ions, is centrally connected to innate host defense, playing a vital role in killing a wide range of pathogens.4-6 However, abnormal overproduction of ClO- is considered to be associated with hepatic ischemia-reperfusion injury, lung injury, rheumatoid arthritis, and even cancer.7-10 On the other hand, glutathione (GSH), as the most abundant antioxidant low-molecule-mass thiol in vivo, plays a central role in combating oxidative stress and maintain redox homeostasis that is pivotal for cell growth and function.11,12 Moreover, it takes part in many pathological and physiological processes through direct interaction with free radicals or enzymes.13,14 Therefore, changes in the level of GSH contribute to various diseases such as AIDS, leukocyte loss, liver damage, cancer, and neurodegenerative diseases.11,12,15,16 Nevertheless, the biological activities of ClO- and GSH have not yet been fully established.

raphy (HPLC) and fluorescence method have been developed.17-24 Among them, fluorescence method is an attractive approach in terms of sensitivity, spatial and temporal resolution, and ease of use in live cells and tissues.25-27 However, it usually suffers from the photobleaching and phototoxicity. Surface-enhanced Raman spectroscopy (SERS) has become a powerful analytical technique to provide molecular fingerprint information of various biomolecules, even in live cells and tissues.28-33 Compared

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Scheme 1. Schematic illustration for working principle of the present single SERS nanoprobe for imaging and biosensing of ClO- and GSH. 1

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with other methods, it has remarkable advantages in biochemical applications, including ultrahigh sensitivity, high spectral resolution for multiplex detection, flexible excitation wavelengths, noninvasive to biological samples, particularly resistance to photobleaching and autofluorescence. These unique features make SERS an ideal technique for quantitative analysis and bioimaging in live cells.34-36 However, it is still a challenging work to directly probe inorganic molecules such as ClO- or GSH using SERS because of their small Raman scattering crosssections.

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(AA), glucose and all the amino acid (99%) were obtained from Sigma-Aldrich (USA). KH2PO4, K2HPO4.3H2O, CaCl2, CdCl2, MgCl2.6H2O, PbCl2, MnCl2.4H2O, NaNO3, NaCl, AgNO3, AlCl3.6H2O, ZnCl2, FeCl3, FeCl2.4H2O, NaClO4, NaNO2, CuCl2.2H2O, and NaOH were obtained from Sinopharm Chemical Reagent Co. Ltd (China). Ultrapure water (18.2 MΩ cm-1) produced with a Milli-Q gradient system (Millipore, Billerica, MA) was used in all experiments. All of the chemicals were used without further purification. In the selectivity test, alkyl peroxyl radical (ROO•) was obtained by thermolysis of AAPH in air-saturated solution at 310 K. Peroxynitrite (ONOO−) was chemically provided by H2O2 and NaNO2. Nitric oxide (NO) and nitroxyl (HNO) were obtained from the solution of S-nitroso-Nacetyl-DL-penicillamine and Angeli’s salt (Na2N2O3). Hydroxyl radical (•OH) was generated by the Fenton reaction (Fe2+/H2O2 = 1: 6). The first singlet oxygen (1O2) was produced by H2O2 with NaClO. Superoxide anion (O2•–) was derived from dissolved KO2 in the DMSO solution.

In this work, as illustrated in Scheme 1, we first designed and developed a single ratiometric SERS nanoprobe to determine both ClO- and GSH with high selectivity, accuracy, and sensitivity, and successfully applied in real-time monitoring the levels of ClO- and GSH in live cells. Firstly, gold flowers (AuFs) were synthesized as SERS material for amplifying the Raman signals by 6-fold compared to that of Au nanoparticles. Meanwhile, 4Mercaptophenol (4-MP) possessing high SERS responsiveness was designed and optimized as a specific element toward ClO-, and conjugated on the surface of AuFs to fabricate the SERS probe (AuF/MP). The peak located at 635 cm-1 (C-C-C bending, in-plane benzene ring deformation) in SERS spectra gradually increased with the increasing concentration of ClO-, while that observed at 1077 cm-1 (C-C stretching and aromatic ring breathing) decreased. More interestingly, the SERS spectra recovered after addition of GSH, demonstrating the SERS probe can also be employed to determination of GSH followed by that of ClO-. Furthermore, the peak obtained at 822 cm-1 (C-H out of plane bending) stayed almost constant with the changes of both ClO- and GSH, which can play as an inner-reference element to provide the ratiometric determination of ClO- and GSH with high accuracy. In addition, the single SERS probe for ClO- and GSH detection showed high selectivity against other ROS and amino acids which may exist in biological system. The significant analytical performance of this single SERS nanoprobe, as well as the unique properties of AuFs including good biocompatibility and long-term stability, established a direct and reliable platform for real-time quantification of ClOand GSH in live cells. To the best of our knowledge, it is the first report for SERS imaging and biosensing of ClOand GSH in live cells.

Synthesis of AuF/MP Nanoprobe. Gold Nanoflowers were synthesized according to the previous method with modification. First of all, the Au seeds were synthesized. A 50 mL sample of aqueous HAuCl4 (0.25 mM) was added in a 100 mL flask and brought to boiling under stirring, then 0.5 mL of 5% sodium citrate solution was added. The process of reaction got to an end until the colour of solution became wine red. For the growth of AuFs, 80 mL of HAuCl4 (0.25 mM) aqueous solution was added to a sample vial, followed by adjusting the pH to 11.4 by 1 M NaOH solution. The mixture of 640 μL of NH2OH.HCl solution (40 mM) and 6.4 mL of the Au seeds then was added into the above solution at 298 K. With mild stirring (100 rpm), the color of the solution turned blue green, indicating the formation of AuFs. The resulting AuFs were centrifuged at 6000 rpm for 5 min and re-suspended in 8 mL deionized water for the subsequent fabrication of the nanoprobes. As the solution stirring constantly, a freshly prepared 4MP solution (2 μM) was added to AuFs to produce a 1:4 4MP solution/AuFs ratio, which facilitated uniform distributions of the 4-MP on the gold particle surfaces. After 1 h, excess 4-MP was removed from the AuFs through three rounds of centrifugation (6000 rpm, 5 min) and AuFs were re-suspended in phosphate-buffered saline (PBS; 20 mM pH=7.4). The AuF/MP nanoprobes were obtained for analytical experiments.

EXPERIMENTAL SECTION

Apparatus and Measurements. All measurements were carried out at ambient temperature. The SERS measurements were performed on the Thermo Scientific DXR Raman Microscope. A 780 nm laser was used for all the measurements. For SERS measurements of solution samples, a 10× (NA 0.4) microscope objective with a working distance of 1.3 mm and spot focused laser was used. The laser power and acquisition time were 10 mW and 2 s, respectively. For SERS imaging of live cells, a 50× (NA 0.75) microscope objective with a working distance of 0.38 mm and spot focused laser was used. The laser power and ac-

Chemicals and Reagents. 4-Mercaptophenol (4-MP), hydroxylamine hydrochloride (ClO-), sodium citrate, sodium hypochlorite solution (NaClO, ~ 10%), glutathione (GSH), hydrogen peroxide (H2O2, 30%), Sodium nitrite, and 2, 2-azobis (2-methylpropionamidine)dihydrochloride (AAPH) were obtained from Aladdin Chemistry Co. Ltd (China). Gold(III) chloride trihydrate (HAuCl4.3H2O, 99%), potassium superoxide (KO2), dimethyl sulfoxide (DMSO), methyl thiazolyl tetrazolium (MTT), Phorbol myristate acetate (PMA), ascorbic acid 2

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Information). With the addition of ClO-, the SERS spectrum significantly changed: a new peak ascribed to C=O stretching appeared at 1619 cm-1, together with a little change of benzene stretching vibration in the presence of ClO- (Table S1 and Figure S2, Supporting Information). Meanwhile, as shown in Figure 1D (curve b), the peak located at 635 cm-1 ascribed to C-C-C bending gradually increased with the addition of ClO-, whereas that obtained at 1077 cm-1 attributed to aromatic ring breathing, clearly decreased. These results indicate the hydroxy benzene group of 4-MP was converted into quinonyl group in the presence of ClO-, which was confirmed by FT-IR spectroscopy. From FT-IR spectroscopic data (Figure S3, Supporting Information), we can see that after reacted with ClO-, clear C=O vibrational peak was observed at 1636 cm1 in the SERS spectrum of AuF/MP (curve b), while –OH peak located at 3350 cm-1 (curve a) disappeared. These data indicate that 4-MP converted into 4mercaptocyclohexa-2, 5-dienone (4-MC) after reacted with ClO-. Moreover, –OH peak recovered after the following addition of GSH and C=O peak vanished, revealing that 4-MC turned into 4-MP again after the reduction of GSH. The observation was further evident by NMR data (Figure S4, Supporting Information). Because 4-MP was confined onto the surface of AuFs through very stable AuS covalent bond, phenol was chosen as a simplified model molecule to investigate the reaction of AuF/MP nanosensor with ClO- and GSH, instead of 4-MP. From NMR data, it is clear that –OH group in phenol clearly turned into C=O group after reacted with ClO- and C=O group reduced back to –OH group with the addition of GSH. The SERS peak observed at 822 cm-1 (C-H out of plane bending) showed almost constant with the change of ClO-, and thus selected as an inner reference peak for built-in correction.

quisition time were 0.1 mW and 1 s. The gold nanoflowers (AuFs) were characterized using a field emission gun S4800 scanning electron microscope (SEM, Hitachi, Japan) and JEM-2100 transmission electron microscope (TEM, JEOL Ltd., Japan). UV-Vis spectra were obtained using a UH-5300 spectrophotometer (Hitachi, Japan). Infrared spectroscopic data were collected by a Nicolet iS10 infrared spectrometer (Thermo Fisher Scientific, USA). All pH measurements were performed using a pH-3c digital pH meter (Shanghai Lei Ci Device Works, Shanghai, China) with a combined glass-calomel electrode. Cellular Culture. RAW264.7 mouse macrophages were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) containing high glucose supplemented with 10% fetal bovine serum, 100 units mL−1 penicillin, and 100 µg mL−1 streptomycin. In the proliferative period, RAW264.7 cells were dispersed in 6 cm glass bottom dishes for later cell imaging and spectroscopic detection. Cells were incubated for 4 h with culture medium containing AuF/MP (0.42 mg mL−1). Then the culture medium was removed and cells were washed three times with PBS to remove free unbound samples. Finally, the dish was put into a small incubator (temperature = 310 K) on the microscope stage for cell imaging and spectroscopic detection.

RESULTS AND DISUCUSSION Characterization of AuF/MP Nanoprobe. As a starting point of our work, AuFs were synthesized by the seeding approach.37-39 From the typical TEM images (Figure 1A and 1B), we can see that the as-synthesized nanoflowers were quasi-spherical, consisting of a solid core with many short, irregular, and sharp branches. The average dimension of AuFs was ~ 60 ± 8 nm (Inset in Figure 1A). Energy dispersive spectrometer (EDS) elemental mapping (Inset in Figure 1B) shows AuFs were gold crystalline. The surface plasmon resonance (SPR) of AuFs was observed in the range of 480-850 nm with a peak at 610 nm (Figure 1C). Previous studies have reported that Au nanoparticles with diameter of ~ 60-80 nm have the highest efficiency for SERS using red (633 nm) or near-infrared excitation (780 nm) excitation, which are just suitable for detection of biological samples to avoid autofluorescence and phototoxicity.40-42 AuFs demonstrated more than 6-fold in SERS intensity that Au nanoparticles with the same concentration (Figure S1, Supporting Information), due to a larger amount of hot spots for localized near-field enhancement effects resulted from tips of AuFs, as well as greater surface area originated from the roughness of AuFs surface.

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Next, 4-MP with high SERS signal was designed and optimized as specific recognition element for ClO- and GSH, and conjugated onto the surface of AuFs through Au-S bond. As shown in Figure 1D, a distinct SERS spectrum was obtained for the AuF/MP, in which O-H out-of-plane bending and benzene stretching vibration were clearly observed at 511 cm-1, as well as 1581 cm-1 and 1596 cm-1, ascribed to the characteristic of 4-MP (Table S1, Supporting

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Figure 1. (A) TEM image of AuFs. Inset: Size distribution of AuFs. (B) TEM image of AuF Inset: EDS analysis of AuF. (C) UV-vis spectra of AuFs solution. (D) SERS spectra of AuF/MP (a) in the absence of ClO- and GSH, (b) after addition of ClO-, and (c) after addition of GSH followed by (b).

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probe toward determination of ClO- and GSH over other ROS, metal ions, amino acids, and other biological species which may exist in live cells, was systematically examined. As summarized in Figure 3A, the intensity ratio ((I635-I0)/ I822 or (I1077-I0)/I822, I0 means peak intensity at 635 cm-1 of AuF/MP and at 1077 cm-1 of AuF/MP-ClO- ) of the developed probe showed negligible responses for other ROS including ROO•, O2•–, ONOO-, NO, •OH, H2O2, 1O2 and HNO against determination of ClO-. Meanwhile, as demonstrated in Figure 3B, no obvious signals were observed for metal ions and other potential interferences. On the other hand, the selectivity test was also carried out for GSH determination (Figure 3C and 3D). Other amino acids demonstrated negligible interferences over GSH, while ROS, metal ions and other potential interferences showed almost no responses. For the competition test, the effects of all these potential interferences on the electrochemical response for ClO- and GSH were investigated in detail. Relatively little changes (< 4.7%) were observed. Furthermore, this AuF/MP probe demonstrated high selectivity against pH change from 4.0 to 9.7 (Figure S5, Supporting Information). All these results indicate high selectivity of the present single probe for determination of ClO- and GSH over ROS, pH, metal ions, amino acids, and other biological molecules.

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Figure 2. (A) SERS spectra of AuF/MP in 20 mM PBS (pH 7.4) in the presence of NaClO with various concentrations (a to l: 0, 1, 4.25, 12.75, 17.01, 25.51, 34.02, 42.52, 51.03, 59.53, 61.66 and 63.78 μM). (B) Plots of ratiometric peak intensities (I1077/I822 and I635/I822) versus different NaClO concentrations. (C) SERS spectra of AuF/MP-NaClO in PBS (20 mM, pH 7.4) in the presence of GSH with various concentrations (a to k: 0, 1.17, 4.11, 5.88, 8.23, 11.76, 15.29, 17.64, 19.99, 21.16 and 23.52 μM). (D) Plots of ratiometric peak intensities (I1077/I822 and I635/I822) versus different GSH concentrations. Each data point (B and D) represents the average value from three replicate SERS spectra. Error bars equal to the standard deviations.

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Ratiometric Responses of AuF/MP Nanoprobe to ClO- and GSH. As plotted in Figure 2A, with increasing concentration of ClO-, the ratiometric peak intensities of I635/I822 increases and I1077/I822 decreases. A good linear relationship was clearly observed between I635/I822 (and I1077/I822) and the concentration of ClO- in the range of 1– 63.78 μM (Figure 2B). The detection limit (3σ/k) for ClOwas achieved to be 0.40 μM (upon I635/I822) and 0.24 μM (upon I1077/I822). More interestingly, with the increasing concentration of GSH, the SERS spectrum (Figure 1D) almost recovered. As demonstrated in Figure 2C, the peak at 635 cm-1 gradually decreased, while that at 1077 cm-1 increased. The ratiometric peak intensity of I635/I822 (and I1077/I822) showed good linearity with the addition of GSH from 1.17 to 19.99 μM (Figure 2D). The detection limit was achieved to 0.14 μM (upon I1077/I822) and 0.38 μM (upon I635/I822), which fulfills the requirements for evaluating the level of GSH in live cells. The SERS spectra recovered after addition of GSH, revealing that the developed SERS probe can also be employed to determination of GSH followed by that of ClO-. It is the first time that the single nanoprobe can subsequently be utilized for GSH detection followed by accurate determination of ClO-.

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Figure 3. Selectivity and competition tests of various relevant analytes against ClO- and GSH. Red and black columns stand for the selectivity and competition tests of AuF/MP nanoprobe, respectively. (A) 1-9: ROO•, O2•–, ONOO-, NO, •OH, H O , 1O HNO, ClO-. The concentration is 100 μM ex2 2 2 cept H2O2 (500 μM) against ClO- (50 μM). (B) 1-18: Ca2+, Cd2+, Mg2+, Pb2+, Cu2+, Mn2+, Zn2+, Al3+, Ag+, Hg2+, NO2-, NO3-, ClO3-, ClO4-, Cl-, Fe2+, Fe3+, ClO-. Concentration of metal ions and related ions is 1 mM except Ag+ (50 μM) and Cl- (0.1 M) against ClO- (50 μM). (C) 1-20: Cys, Phe, Val, Trp, Leu, Arg, Thr, Ala, Gln, His, Tyr, Lys, Asp, Glu, Gly, Pro, Asn, Ser, Met, GSH. Concentration of amino acids is 100 μM against GSH (10 μM). (D) 1-7: AA 100 μM, glucose 5 mM, UA 100 μM, HAS 0.5 mg/mL, Na+ 1 mM, Mg2+ 1 mM, GSH 10 μM. Error bars equal to the standard deviations.

Selectivity, Competition and Stability of AuF/MP Nanoprobe for ClO- and GSH. The complexity of intracellular system presents a great challenge for ClO- and GSH detection not only in sensitivity and accuracy, but more importantly in selectivity. The selectivity of AuF/MP

In addition, the SERS spectrum for the AuF/MP probe had not obvious change after storage for 24 h, indicating the long-term stability for imaging and biosensing in live 4

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cells (Figure S6, Supporting Information). We also found that the AuF/MP probe could react with ClO- very rapidly, within 1 min (Figure S7, Supporting Information), with good cycle capability (Figure S8, Supporting Information). Accordingly, the present SERS nanoprobe demonstrated high sensitivity, selectivity, and accuracy, as well as quick response and long-term stability for determination of ClO- and GSH. Thus, the remarkable analytical performance of this developed probe, together with the unique properties of AuFs such as easy to enter into live cells and good biocompatibility, established a new approach for A1 BF

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intensity of I635/I822 increases (Figure 4B2) and that of I1077/I822 decreases (Figure 4C2) after cells were induced by PMA (incubation for 30 min). Then, the SERS mapping recovered after cells were incubated with GSH for 30 min (Figure 4B3 and 4C3). Meanwhile, the SERS spectra were recorded from the live cells stimulated by PMA and then incubated with GSH. I635/I822 turned into stronger with increasing stimulation time by PMA, while I1077/I822 correspondingly decreases (Figure S10, Supporting Information). The ClO- concentration was found to be enhanced by 4-fold after stimulated by PMA for 30 min (Figure S10, Supporting Information), confirming that PMA may induce oxidative stress and generate ClO-. Meanwhile, the SERS spectra obtained in live cells also recovered (Figure S10, Supporting Information) similar to those observed in SERS imaging after addition of GSH, suggesting GSH could eliminate ClO- and recover the oxidative stress of live cells. In addition, we also use fluorescence method to confirm the results determined by the pre-

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In summary, we have developed a single SERS nanoprobe for real-time quantification of ClO- and GSH in live cells, by designing and synthesizing AuF/MP nanoprobe with a larger amount of hot spots for localized near-field enhancement effects, as well as selective recognition of ClO- with high SERS signal. The nanoprobe demonstrated high performance in sensitivity, accuracy, and selectivity against other ROS, RNS, metal ions, and amino acids. The remarkable analytical performance of this nanoprobe, combined with its unique properties such as quick response, good biocompatibility, and easy to enter into live cells, has successfully established a reliable approach for real-time imaging and biosensing of ClO- and GSH in RAW264.7 macrophages cells upon oxidative stress. The simplicity in development of this unique single nanoprobe should make it find broad applications in biochemical investigations related to oxidative stress. The present work has also provided a methodology for designing and constructing the single nanoprobe for other ROS, amino acid, proteins, and so on, which play the important role in biological and pathological processes.

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real-time monitoring ClO- and GSH in live cells. Figure 4. (A1-A3) Bright-field, dark-field, and overlay images of RAW264.7 cells after 4 h incubated with AuF/MP nanoprobes. (B1-B3 and C1-C3) SERS imaging of AuF/MP nanoprobes in RAW264.7 cells: (B1 and C1) in the absence of ClOand GSH; (B2 and C2) induced by PMA; (B3, C3) after incubation with GSH for 30 min followed by stimulation of PMA. (B represents I635/I822 and C represents I1077/I822).

SERS Imaging of Intracellular ClO- and GSH. For further biological application of AuF/MP nanoprobe in live cells, MTT (MTT=3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide) assays were carried out with RAW264.7 mouse macrophages. Cell variability still maintained above 90 % after the probe with the concentration up to 3.36 mg mL-1 incubated with cells for 48 h (Figure S9, Supporting Information). Thus, the present probe can be considered to be low toxic and biocompatible for detection of ClO- and GSH in live cells. Then, we applied the AuF/MP probe for bioimaging and biosensing of ClO- and GSH in RAW264.7 macrophages cells. The overlay image (Figure 4A3) of bright-field image (Figure 4A1) and dark-field image (Figure 4A2) shows that the nanoprobes were taken up into the cells through the process of endocytosis and mainly distributed in the cytoplasm region. Phorbol myristate acetate (PMA) was employed to generate ClO- in live cells, because it is the activator of protein kinase C and can induce the oxidative stress.43 From SERS mapping of AuF/MP nanoprobes in RAW264.7 cells, we can obviously see that the ratiometric

ASSOCIATED CONTENT Supporting Information Experimental section; SERS bands assignment; Comparison of AuFs and AuNPs for enhancing Raman signals; SERS spectra of benzene stretching vibration and C=O stretching of AuF/MP; FT-IR spectra; 1H NMR and 13C NMR spectra; pH dependence; Time dependence; Response time; Cycling capability; MTT assay; SERS detection of ClO- and GSH in live cells. This material is available free of charge via the Internet at http:// pubs. acs. org/. 5

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AUTHOR INFORMATION Corresponding Author *Phone: + 86 21 54341041; fax: +86 21 54341041; e-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Support from the NSFC (21305104 and 21505045), the NSFC for Distinguished Young Scholars (21325521 for Y. T.), the Program of the Shanghai Subject Chief Scientist (15XD1501600), and the Pujiang Program (15PJ1401800) is gratefully acknowledged.

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