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Highly selective and sensitive surface enhanced Raman scattering nanosensors for detection of hydrogen peroxide in living cells
Biosensors and Bioelectronics 77 (2016) 292–298
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Highly selective and sensitive surface enhanced Raman scattering nanosensors for detection of hydrogen peroxide in living cells Lu-Lu Qu n, Ying-Ya Liu, Sai-Huan He, Jia-Qing Chen, Yuan Liang, Hai-Tao Li n School of Chemistry and Chemical Engineering, Jiangsu Normal University, Xuzhou, Jiangsu 221116, China
art ic l e i nf o
a b s t r a c t
Article history: Received 14 July 2015 Received in revised form 15 September 2015 Accepted 18 September 2015
Determination of hydrogen peroxide (H2O2) with high sensitivity and selectivity in living cells is a challenge for evaluating the diverse roles of H2O2 in the physiological and pathological processes. In this work, we present novel surface enhanced Raman scattering (SERS) nanosensors, 4-carboxyphenylboronic acid (4-CA) modified gold nanoparticles (Au NPs/4-CA), for sensing H2O2 in living cells. The nanosensors are based on that the H2O2-triggered oxidation reaction with the arylboronate on Au NPs would liberate the phenol, thus causing changes of the SERS spectra of the nanosensors. The results show the nanosensors feature higher selectivity for H2O2 over other reactive oxygen species, abundant competing cellular thiols and biologically relevant species, as well as excellent sensitivity with a low detection limit of 80 nM, which fulfills the requirements for detection of H2O2 in a biological system. In addition, the SERS nanosensors exhibit long term stability against time and pH, and high biocompatibility. More importantly, the presented nanosensors can be successfully used for monitoring changes of H2O2 levels within living biological samples upon oxidative stress, which opens up new opportunities to study its cellular biochemistry. & 2015 Elsevier B.V. All rights reserved.
Keywords: Surface enhanced Raman scattering Hydrogen peroxide Selective Sensitive Living cells
1. Introduction Hydrogen peroxide (H2O2), one of the major reactive oxygen species (ROS) in living organisms, has diverse physiological and pathological consequences (Marco et al., 2007). Emerging evidence supports a physiological role for H2O2 as a second messenger in cellular signal transduction (Reth, 2002). However, over accumulation of H2O2 causes oxidative stress which can induce damage to proteins, DNA, and lipids, and is associated with aging and severe human diseases including cardiovascular disorders, cancer, and neurodegenerative diseases (Toren and Nikki, 2000; Imlay et al., 1988). Therefore, a selective and accurate method to measure H2O2 is useful for underlying molecular mechanisms and elucidating the biological roles of H2O2. Over the past decades, several elegant methods have been developed for detection of H2O2, such as electron spin resonance (ESR) spectroscopy (Tetsuya et al., 2003), chemiluminescence (Hu et al., 2008), electrochemical sensing (Kafi et al., 2008) and chromatography (Toshimasa et al., 2003). Nevertheless, these methods often cause destruction of tissues or cells, which is generally conflicting with the in-situ monitoring of H2O2 in live biological samples (Lin et al., 2013; Xuan et al., 2012). By contrast, molecular n
Corresponding authors. E-mail addresses: [email protected] (L.-L. Qu), [email protected] (H.-T. Li).
http://dx.doi.org/10.1016/j.bios.2015.09.039 0956-5663/& 2015 Elsevier B.V. All rights reserved.
fluorescence imaging through staining with a H2O2-responsive fluorescent indicator offers an attractive approach for the in vivo detection of H2O2 owing to its high sensitivity and aptness for living cells (Lippert et al., 2011; Van de Bittner et al., 2010; Lee et al., 2007; Karton-Lifshin et al., 2011). However, the live-cell fluorescence imaging is usually limited severely by the photobleaching and phototoxicity induced by the excitation light (Magidson and Khodjakov, 2013). Surface enhanced Raman scattering (SERS), which can provide molecular vibrational information of the analytes, has been proved to be a powerful detection technique and extensively applied in various fields such as diagnosis, biosensing (Lee et al., 2014; Ye et al., 2014; Dong et al., 2015). The typical application of SERS is the direct probing of organic molecular systems which attached to metallic SERS substrates and had high Raman cross-sections (Li et al., 2010, 2011). In this scenario, the use of SERS for the direct sensing of inorganic species hit a snag due to their small Raman scattering cross-sections. To overcome this limitation, novel SERS nanosensors that combine metallic nanoparticles and specific organic Raman reporter molecules with both high Raman crosssection and recognizable ability to inorganic species have been designed (Wang et al., 2013; Yin et al., 2011; Zamarion et al., 2008). Especially, SERS signals of the Raman reporter molecule vary depending on the concentrations of the target inorganic species, which is the ideal criterion to determine the target inorganic species. For example, mobile and biocompatible pH nanosensors
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Scheme 1. Illustration of the Au NPs/4-CA nanosensors for sensing H2O2 in living cells.
have been designed by modifying 4-mercaptobenzoic acid on gold nanoaggregates (Kneipp et al., 2007; Kennedy et al., 2009). Redox potential SERS nanosensors have been developed by decorating a noble-metal nanoshell with redox-responsive small molecules (Auchinvole et al., 2012; Thomson et al., 2015). Also, we have fabricated a novel SERS nanosensor by functionalizing gold nanoparticles with oxidized cytochrome c for the selective and sensitive monitoring of intracellular superoxide anion radical (Qu et al., 2013). However, to the best of our knowledge, SERS nanosensors for selective and sensitive detection of the H2O2 have not yet been reported. With the idea of developing SERS as a new approach for rapid determination of H2O2 concentration under physiological conditions in mind, we embarked on a search to find a selective chemosensing agent for H2O2. Recent researches show that arylboronates could be oxidized by H2O2 selectively to provide phenols under mild conditions (Weinstain et al., 2014; Sun et al., 2013; Miller et al., 2005). Based on this reaction, we fabricated SERS nanosensors by modifying Au NPs with 4-CA (Au NPs/4-CA) for the real-time determination of H2O2 in living cells (Scheme 1). The Au NPs/4-CA nanosensors can rapidly and selectively respond to H2O2 in physiological solutions with tens of nanomolar sensitivity, and can be used for real-time SERS detection of H2O2 in living cells, demonstrating their practical functionality in complex biological systems.
2. Experimental 2.1. Reagents and materials All reagents were of analytical grade and used without further purification. Hydrogen peroxide (H2O2, 30 wt%), sodium hypochlorite ((NaClO), ferrous ammonium sulfate ((NH4)2 Fe(SO4)2 6H2O) and sodium chloride (NaCl) were bought from Aladdin Chemical Company (Shanghai, China). 4-Carboxyphenylboronic acid (4-CA, 97%), HAuCl4 3H2O, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), tertbutyl hydroperoxide (t-BuOOH), pyrogallic acid, phorbol myristate acetate (PMA), glutathione (GSH), tris(hydroxymethyl)aminomethane (Tris), L-cysteine (L-cys), S-nitroso-N-acetyl-dl-penicillamine (SNAP, the source of NO), 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) were obtained from Sigma-Aldrich (St. Louis, MO). All reactions using air- or moisture-sensitive reagents were performed in dried glassware under an atmosphere of dry N2. Deionized water (18 MΩ cm 1) used throughout the work was obtained from a Water Pro water purification system (Billerica, MA, USA). HeLa and normal human liver cells were purchased from the Chinese Academy of Sciences in Shanghai originally from American Type Culture Collection (Manassas, VA, USA). 2.2. Instruments Dynamic light scattering (DLS) measurements were performed using a Zetasizer Nano ZS (Malvern Instruments, Southborough, UK). Transmission electron microscopy (TEM) images of Au NPs were acquired from a JEM-1011 electron microscope (JEOL, Japan) with an accelerating voltage of 100 kV. HPLC–MS was implemented on a Hewlett Packard Series 1100 HPLC (column: Agilent Prep-C18, 5 μm, 4.6 250 mm). 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. An inverted microscope (Ti U, Nikon, Japan) with an external triple channel optical system was equipped with a 40 plan fluor objective (N.A. 0.75, Nikon, Japan). The use of dark-field condenser (N.A. 0.80–0.95, Nikon, Japan) with 100 W halogen tungsten lamp enables excitation light scattering from NPs. In the front of a filter turret (TI-FLC Epi-FL, Nikon, Japan), a 785 nm laser was used for Raman excitation. The spectrograph with a resolution of 2 cm 1 was equipped with a back-illuminated deep depletion CCD (PIXIS 400BR, Princeton Instruments, USA) which measures Raman spectra from 400 to 1800 cm 1 with a high sensitivity and fast acquisition rate. An automation controller system (MAC 6000, Ludl Electronic Products, Ltd., USA) was coupled to the fine adjustment knob of the microscope to precisely control the stage position in the z-direction with a resolution of 0.1 mm. 2.3. Fabrication of Au NPs/4-CA Au NPs were prepared by a modified citrate reduction method (Lee and Meisel, 1982). All the glass wares were immersed into aqua regia (HCl: HNO3 in a ratio of 3:1) over night and washed with plenty of deionized water for at least 5 min. 4.8 mL of
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1.00 wt% HAuCl4 was added into 100 mL deionized water at room temperature and rapidly heated to boil under vigorous stirring. Subsequently, 10 mL of 1.00 wt% trisodium citrate solution was added in the boiling solution, which resulted in a rapid color change from light yellow to black and then wine red within few minutes. The solution was kept boiling for an additional 10 min to ensure complete reduction of HAuCl4. After that, it was cooled to room temperature by removing the flask from the oil bath. The resulting colloidal solution was centrifuged at 5000 rpm for 5 min and then suspended in 100 mL deionized water for SERS nanosensors preparation. Prior to modification of Au NPs, a solution of cystamine (0.1 mM) was added to the Au NPs and the mixture was incubated for 2 h. After rinsing with water, a freshly prepared 4-CA solution (10 mM) containing EDC (40 mM) and NHS (10 mM) was added dropwise into Au NPs colloids (4-CA solution/colloid, volume ratio, 1:4) under continuous stirring, which increased the coupling efficiency of the amino group of cystamine and the carboxyl group of 4-CA and facilitated even uniform distributions of 4-CA on Au NPs surfaces (4-CA molecules on per Au NPs: about 1000, Text S1). After a 30 min incubation, the Au NPs solution was subjected to three rounds of centrifugation and resuspension in PBS (pH ¼7.4) to remove free 4-CA. 2.4. In vitro tests The experiments were performed at ambient temperature, and all H2O2 solutions were used within 30 min of preparation. H2O2 solutions (20 μL) at different concentrations were added into the prepared Au NPs/4-CA (180 μL), followed by SERS detection within 1 min. In addition, selectivity of Au NPs/4-CA nanosensors was examined by SERS responses. All assays were carried out in 20 mM PBS buffered (pH ¼7.4). Unless otherwise stated, stock solutions of selected biologically relevant reactive species were added to 4-CA (2.0 mM) modified Au NPs in PBS. SNAP (1 mM) in PBS buffer (100 mM, pH ¼7.4) was used to produce nitric oxide (NO) at room temperature. OH was prepared through the reaction of ferrous ammonium sulfate (0.1 mM) with H2O2 (1 mM). A pyrogallol (1 mM) autoxidation assay in Tris–HCl (50 mM, pH ¼ 8.0) was performed to generate O2 in a 25 °C water bath for 20 min. 1O2 was prepared based on the reaction of H2O2 with NaClO. ONOO was generated by KNO2 and H2O2 in HCl at 0 °C as previously reported (Kutala et al., 2008) and was frozen at less than 18 °C.
resultant of Au NPs/4-CA and H2O2 (10 μL) at different concentrations were added respectively, and incubated for 24 h. Sequentially, an MTT solution (5 μL, 5 mg/mL) was added into each well and incubated for another 4 h in the CO2 incubator. After removing the supernatant medium, the reaction was terminated by adding DMSO (150 μL) to dissolve the formazan crystals. The absorbance of the wells was measured on a microplate reader (Bio-Rad model 680) with a test wavelength of 570 nm. Cells incubated without Au NPs were used as a control.
3. Results and discussion 3.1. Characterization of Au NPs/4-CA nanosensors The size distribution and the dispersity of Au NPs/4-CA nanosensors were initially characterized by DLS measurements. As shown in Fig. S1, the average size of Au NPs/4-CA corresponds to 60 nm with a narrow size distribution. In addition, there was no obvious difference between the DLS spectrum of Au NPs and Au NPs/4-CA, indicating that the Au NPs were well dispersed after the surface functionalization of 4-CA. TEM characterization of the Au NPs/4-CA nanosensors further shows that they are spherical with a diameter of 60 nm (Fig. 1A), which is in close agreement with the result of DLS measurements. As 60–80 nm Au NPs are the most efficient for Raman enhancement at the near-infrared excitations (785 nm) which is known as a ‘clear window’ for optical detection, the nanosensors of Au NPs/4-CA could be suitable for the detection of biological samples (Krug et al., 1999). Furthermore, SERS response examination of Au NPs/4-CA showed that an obvious SERS spectrum can be obtained (black curve in Fig. 1B), meaning that 4-CA molecules were successfully functionalized on Au NPs by amide bonds since SERS is a surface selective technique and Au NPs have been washed with ultrapure water. In the SERS spectrum, the strong Raman bands at 821, 1011, 1031, 1178, 1270, 1562 cm 1
2.5. Cellular studies Cells were propagated in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin, and maintained in an incubator containing 95% air and 5% CO2 at 37 °C. The cells were plated into culture dishes (6 cm in diameter) and allowed to adhere for 24 h. The growth medium was removed and replaced by the culture medium containing Au NPs/4-CA. After incubation for 4 h, the cells were washed for three times with PBS to remove unbound samples and fixed on a microscopic glass slide to perform SERS studies. Then, PMA, a stimulator of cell respiratory burst for generating ROS, was added into the Au NPs/4CA-loaded cells with a final concentration of 400 ng/mL and incubated for an additional 4 h. The cells were washed three times with 0.20 M PBS buffer before imaging and SERS experiments. The biocompatibility of Au NPs/4-CA nanosensors in absence and presence of H2O2 was investigated by a MTT assay. The cells (1 105 cells/mL) were seeded onto 96-well microtiter plates to a total volume of 200 μL/well and maintained at 37 °C in a 5% CO2/95% air incubator for 12 h. Then, Au NPs/4-CA (10 μL) and the
Fig. 1. (A) Typical TEM image of Au NPs/4-CA nanosensors. (B) Raman response of Au NPs/4-CA in the absence and presence of H2O2. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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are attributed to the B–O symmetric stretching, B–O–H deformation, C–H in-plane deformation, B–O–H deformation, B–O symmetric stretching, C ¼C stretching vibrations, respectively (Table S1) (Piergies et al., 2013; Alver and Parlak, 2010; Kong et al., 2013). It also can be seen from Fig. 1B that, when H2O2 was introduced to Au NPs/4-CA, the SERS spectrum changed significantly, and bands at 1365, 1600 cm 1 attributing to in-plane O–H deformation, C ¼ C stretching (Hou and Fang, 2007) respectively, appeared in the new spectrum accompanying with the decrease of the peak at 1562 cm 1 (blue curve in Fig. 1B). These changes of characteristic SERS bands indicate the H2O2-mediated transformation of arylboronates to phenols on Au NPs. Specially, the significant SERS shift of C ¼C stretching vibration from 1562 to 1600 cm 1 may be due to the change in polarizability of the phenyl ring on Au NPs surface resulting from the transformation of boronic acid groups of 4-CA into hydroxyl groups (Kho et al., 2012; Guerrini et al., 2013), which could be used as spectral markers for monitoring the reaction of Au NPs/4-CA and H2O2. The H2O2-sensed reaction was further confirmed by HPLC–MS analysis (Fig. S2). A peak at m/z 138 well matches with the product C7H6O3 (calcd. 138.12 [M]) on mass spectrum from the reaction mixture of 4-CA and H2O2. This further confirms that Au NPs/4-CA nanosensors can be exactly responsive to the reaction of H2O2 thereby detecting H2O2 in solution. 3.2. Detection conditions of Au NPs/4-CA nanosensors SERS activity of Au NPs/4-CA nanosensors which mainly dominated by the amount of 4-CA molecules on Au NPs surfaces is of great significance for sensing biological samples. Thus, to acquire nanosensors for H2O2 with excellent SERS activity, we compared SERS spectra of nanosensors prepared by incubating the same concentration of Au NPs (about 2.5 nM) with the 4-CA solution at different concentrations. As displayed in Fig. S3, the intensities of the SERS spectra increase with 4-CA concentrations from 0.02 to 2.0 μM, and reach a plateau after 2.0 μM. The possible reason is that the coverage of 4-CA molecules at the “hot spot” sites of Au NPs capable of providing sufficiently large enhancement is complete, thus resulting in saturated SERS signals (Le Ru et al., 2006). Hence, 2.0 μM was chosen for the concentration of the 4-CA to assemble on Au NPs surfaces for maximizing SERS signals of Au NPs/4-CA nanosensors. Also, for biological applications, it is necessary for SERS nanosensors to function over a suitable pH range, particularly at a physiological pH value. Therefore, we investigate the influence of pH (5.8–8.0) on SERS responses of Au NPs/4-CA nanosensors, as depicted in Fig. S4. No distinct change could be observed in SERS spectra, illustrating Au NPs/4-CA nanosensors were pH-insensitive over a biologically relevant pH range and it is suitable to perform SERS detection in aqueous solution buffered to physiological pH (pH¼ 7.4). In addition, temporal SERS observation of Au NPs/4-CA nanosensors was carried out in the presence of H2O2 (Fig. S5). Upon treatment of H2O2, an obvious appearance of the 1600 cm 1 peak was observed, accompanied by a robust decrease of the 1562 cm 1 peak. Within 20 min of reaction, these peaks were tended to be constant, which indicated that the SERS nanosensors had a very fast response to H2O2 and the sensing reaction could be completed within 20 min. Thus, the nanosensors may be highly competent for the detection of H2O2 in biological samples, especially for the tracking of H2O2 concentration. 3.3. Stability and cytotoxicity of Au NPs/4-CA nanosensors An ideal biological nanosensor should be stable for a long period of time. Therefore, we characterize the stability of Au NPs/ 4-CA nanosensors by measuring their SERS signals (both frequency
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Fig. 2. Raman spectra of Au NPs/4-CA nanosensors after storage for different time in air. Inset: the Raman intensity of Au NPs/4-CA at 1562 cm 1 versus storage time.
and intensity) at different time under natural conditions, as shown in Fig. 2. After storage over 24 h, the responding SERS spectra of nanosensors did not change obviously, which could speculate that the Au NPs/4-CA did not aggregate seriously, illustrating their excellent stability in favor of sensing application in living cells. At the same time, Au NPs/4-CA nanosensors are allowed to be directly exposed to the surrounding circumstances, which is very important for biosensors and have great potential in the investigation of biological processes. For further SERS analysis of biological samples, the long-term cellular toxicity of Au NPs/4-CA nanosensors toward HeLa cells was assessed using the MTT assay (Fig. S6). In the presence of the nanosensors and the resultant of nanosensors and H2O2 with concentrations ranging from 1 nM to 10 nM, the cellular viabilities were estimated to be greater than 88% and 85% after incubation for 24 h, respectively. The results indicate that the Au NPs/4-CA nanosensors are generally low toxic for cellular detection, meaning that the Au NPs/4-CA nanosensors exhibit high biocompatibility and are suitable for use in biological applications. 3.4. Sensitivity and selectivity of Au NPs/4-CA nanosensors In order to evaluate the sensitivity of the Au NPs/4-CA nanosensors, different concentrations of H2O2 were tested. SERS spectra of 4-CA with different concentrations of H2O2 are shown in Fig. 3A. As the concentration of H2O2 increased, the ratiometric peak intensity of I1562/I1131 decreased, while simultaneously a new peak at 1600 cm 1 appeared and the ratiometric peak intensity of I1600 /I1131 increased obviously (Fig. 3). There is a linear relationship between the ratiometric peak intensities of I1562/I1131 (or I1600/I1131) and logarithmic concentration of H2O2 over the range of 0.1– 20 μM, with a detection limit of 0.08 μM (I1562/I1131) based on a signal-to-noise ratio of S/N ¼ 3. As the H2O2 concentration is approximately at micromolar levels in abnormal physiological condition such as oxidative stress-related diseases (Antunes and Cadenas, 2001; De Gracia Lux et al., 2012), thus, these Au NPs/4-CA nanosensors are suitable for the detection of H2O2 in biological samples. The selectivity of Au NPs/4-CA nanosensors toward H2O2 was also systematically investigated by recording SERS response of 4-CA in the presence of various biologically relevent analytes, which may coexist in the living system (Fig. 4). An obvious increase at the band ratio of I1600/I1131 was observed when Au NPs/4CA nanosensors were treated with H2O2 (11-fold over 20 min). In contrast, other biologically relevent analytes (OH, O2 , 1O2, ONOO , t-BuOOH, O3, NO, NaClO, GSH, L-cys) had little effect on the I1600/I1131 ratio. Taken together, these results establish that the
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Fig. 3. (A) Raman spectra of Au NPs/4-CA with varying H2O2 concentration: (a) 0.1 μM; (b) 0.2 μM; (c) 0.4 μM; (d) 1.0 μM; (e) 2.0 μM; (f) 4.0 μM; (g) 8.0 μM; and (h) 20 μM. (B) Plots of ratiometric peak intensities versus logarithmic H2O2 concentration based on I1562/I1131 (open squares) and I1600/I1131 (open circles). Each data point represents the average value from three SERS spectra. Error bars show the standard deviations.
Au NPs/4-CA nanosensor selectively reacts with physiological levels of H2O2 in a concentration-dependent manner. 3.5. Monitoring of H2O2 in living cells Encouraged by the promising results obtained in buffer, we explored whether Au NPs/4-CA could be used to detect H2O2 in the cellular environment. Considerable research demonstrates that tumor incidence is closely associated with reactive oxygen species (Kundu et al., 2009; Zorov et al., 2014). Thus, HeLa cells and normal human liver (HL-7702) cells were selected to investigate whether there were SERS spectral distinctions between tumor cells and normal cells. Uptake of Au NPs/4-CA was confirmed by the bright-field and dark-field microscope (DFM) images, as shown in Fig. 5A1,A2 and B1,B2. It can been seen, after an incubation of 4 h, numerous gold nanoparticles have been taken up into the cell through the process of endocytosis to form accumulations in late endosomes and lysosomes. They can be observed at the resolution of a light microscope, which enable us to localize the positions of Au NPs/4-CA nanosensors and record their SERS spectra more rapidly. In addition, the incorporation of Au NPs/4-CA did not bring a significant change to the cell morphology, which further demonstrated the excellent biocompatibility of Au NPs/4-CA. Fig. S7A-a, B-a showed Raman spectra of Au NPs/4-CA accumulated in cells. The spectra of Au NPs/4-CA in the HeLa cell and normal HL7702 cell did not change significantly at the typical bands of 1562 and 1600 cm 1, may be due to the generated H2O2 is not enough to react with Au NPs/4-CA. Then, a membrane permeable free radical initiator, PMA, which can cause oxidative damage to DNA, lipids, and proteins was added to induce oxidative stress and give rise to a variety of ROS species. After 4 h, SERS maps were generated using the intensity ratio of the 1600 cm 1 and the 1131 cm 1
Fig. 4. (A) Raman spectra of Au NPs/4-CA in absence (a) and presence of biologically relevant reactive analytes: (b) H2O2, (c) OH, (d) O2 , (e) 1O2, (f) ONOO , (g) t-BuOOH, (h) O3, (i) NO, (g) NaClO, (k) GSH, and (l) L-cys. (B) Histograms demonstrated Au NPs/4-CA and Au NPs/4-CA with the indicated biologically relevant reactive analytes based on I1600/I1131. Data shown are 0.5 mM for other biologically relevant species, 0.2 mM for GSH, and 0.1 mM for L-cys.
bands (Fig. 5A3,A4; 5B3,B4). The intensity of the 1600 cm 1 band appeared, demonstrating that the 4-CA on Au NPs is reduced via H2O2 generated under PMA stimulation. Meanwhile, Raman spectra of Au NPs/4-CA were collected from the HeLa and HL-7702 cells without stimulation of PMA, as displayed in curves d of Fig. S7A,B. Obviously, after equivalent time 4 h, there were no obvious changes in SERS spectra for non-stimulated HeLa cells and HL7702 cells, further indicating the generation of H2O2 under PMA stimulation. It is important to note that the observed intensity ratio of the 1600 cm 1 and the 1131 cm 1 bands in HeLa cells is with a higher degree than that of HL-7702 cells (Fig. 5C,D), due to that the H2O2 concentration in HeLa cells is higher than that in HL7702 cells. Using the I1600/I1131 ratio change from the exogenous H2O2 application experiment as a calibration curve, we calculated that under the aforementioned conditions, PMA stimulated HeLa cells and HL-7702 cells generate H2O2 at a rate of 1.08 nmol/104 cells/h and 0.85 nmol/104 cells/h, which is in agreement with previously reported measurements (Weinstain et al., 2014), indicating Au NPs/4-CA could be used as nanosensors for qualitative and quantitative detection of H2O2 in living cells.
4. Conclusions In summary, a novel SERS nanosensor has been developed for biosensing of H2O2 in living cells selectively and sensitively by functionalizing 4-CA on Au NPs (Au NPs/4-CA). The nanosensor is constructed based on the specific transformation of arylboronate into phenol in the presence of H2O2, which leads to SERS spectrum changes of Au NPs/4-CA. The present nanosensor exhibits high selectivity over other reactive oxygen species, abundant
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Fig. 5. Bright-field images of the HeLa cells (A1) and HL-7702 cells (B1) after 4 h incubation with Au NPs/4-CA. DFM images of the corresponding HeLa cells (A2) and HL-7702 cells (B2) containing Au NPs/4-CA. SERS mapping of Au NPs/4-CA after addition of PMA with 4 h for HeLa cells (A3) and HL-7702 cells (B3). Overlay images of SERS mapping and DFM images for HeLa cells (A4) and HL-7702 cells (B4). SERS spectra of Au NPs/4-CA from different position of SERS mapping of HeLa cells (C) and HL-7702 cells (D). Inset: Raman intensity of the nanosensors at the ratiometric peak I1600/I1131 and I1562/I1131 at different position of cells. The assay mixture consisted of cells (2 106 cells/mL), Au NPs/4-CA (2.0 nM), PMA (400 ng/mL) in 0.20 M PBS buffer (pH ¼ 7.4).
competing cellular thiols and biologically relevant species, as well as good sensitivity. Meanwhile, the Au NPs/4-CA nanosensor shows long-term stability against incubation time and pH, and high biocompatibility. More importantly, the hybridized nanosensors with high analytical performance could be successfully applied to the detection of H2O2 level in living cells under oxidative stress. The SERS nanosensors have been established to be an accurate and reliable approach for determination of H2O2 in a biological system, which may play critical roles in understanding the biological and pathological events.
Acknowledgments This research was supported by the National Natural Science Foundation of China (21505057, 21375051), the Natural Science Foundation of Jiangsu Province (BK20150227), the Natural Science Foundation of Jiangsu Normal University (14XLR011), and the Shanghai Key Laboratory of Functional Materials Chemistry (SKLFMC2014B01). Appendix A. Supplementary material Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.bios.2015.09.039.
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Highly selective and sensitive surface enhanced Raman scattering nanosensors for detection of hydrogen peroxide in living cells Lu-Lu Qu n, Ying-Ya Liu, Sai-Huan He, Jia-Qing Chen, Yuan Liang, Hai-Tao Li n School of Chemistry and Chemical Engineering, Jiangsu Normal University, Xuzhou, Jiangsu 221116, China
art ic l e i nf o
a b s t r a c t
Article history: Received 14 July 2015 Received in revised form 15 September 2015 Accepted 18 September 2015
Determination of hydrogen peroxide (H2O2) with high sensitivity and selectivity in living cells is a challenge for evaluating the diverse roles of H2O2 in the physiological and pathological processes. In this work, we present novel surface enhanced Raman scattering (SERS) nanosensors, 4-carboxyphenylboronic acid (4-CA) modified gold nanoparticles (Au NPs/4-CA), for sensing H2O2 in living cells. The nanosensors are based on that the H2O2-triggered oxidation reaction with the arylboronate on Au NPs would liberate the phenol, thus causing changes of the SERS spectra of the nanosensors. The results show the nanosensors feature higher selectivity for H2O2 over other reactive oxygen species, abundant competing cellular thiols and biologically relevant species, as well as excellent sensitivity with a low detection limit of 80 nM, which fulfills the requirements for detection of H2O2 in a biological system. In addition, the SERS nanosensors exhibit long term stability against time and pH, and high biocompatibility. More importantly, the presented nanosensors can be successfully used for monitoring changes of H2O2 levels within living biological samples upon oxidative stress, which opens up new opportunities to study its cellular biochemistry. & 2015 Elsevier B.V. All rights reserved.
Keywords: Surface enhanced Raman scattering Hydrogen peroxide Selective Sensitive Living cells
1. Introduction Hydrogen peroxide (H2O2), one of the major reactive oxygen species (ROS) in living organisms, has diverse physiological and pathological consequences (Marco et al., 2007). Emerging evidence supports a physiological role for H2O2 as a second messenger in cellular signal transduction (Reth, 2002). However, over accumulation of H2O2 causes oxidative stress which can induce damage to proteins, DNA, and lipids, and is associated with aging and severe human diseases including cardiovascular disorders, cancer, and neurodegenerative diseases (Toren and Nikki, 2000; Imlay et al., 1988). Therefore, a selective and accurate method to measure H2O2 is useful for underlying molecular mechanisms and elucidating the biological roles of H2O2. Over the past decades, several elegant methods have been developed for detection of H2O2, such as electron spin resonance (ESR) spectroscopy (Tetsuya et al., 2003), chemiluminescence (Hu et al., 2008), electrochemical sensing (Kafi et al., 2008) and chromatography (Toshimasa et al., 2003). Nevertheless, these methods often cause destruction of tissues or cells, which is generally conflicting with the in-situ monitoring of H2O2 in live biological samples (Lin et al., 2013; Xuan et al., 2012). By contrast, molecular n
Corresponding authors. E-mail addresses: [email protected] (L.-L. Qu), [email protected] (H.-T. Li).
http://dx.doi.org/10.1016/j.bios.2015.09.039 0956-5663/& 2015 Elsevier B.V. All rights reserved.
fluorescence imaging through staining with a H2O2-responsive fluorescent indicator offers an attractive approach for the in vivo detection of H2O2 owing to its high sensitivity and aptness for living cells (Lippert et al., 2011; Van de Bittner et al., 2010; Lee et al., 2007; Karton-Lifshin et al., 2011). However, the live-cell fluorescence imaging is usually limited severely by the photobleaching and phototoxicity induced by the excitation light (Magidson and Khodjakov, 2013). Surface enhanced Raman scattering (SERS), which can provide molecular vibrational information of the analytes, has been proved to be a powerful detection technique and extensively applied in various fields such as diagnosis, biosensing (Lee et al., 2014; Ye et al., 2014; Dong et al., 2015). The typical application of SERS is the direct probing of organic molecular systems which attached to metallic SERS substrates and had high Raman cross-sections (Li et al., 2010, 2011). In this scenario, the use of SERS for the direct sensing of inorganic species hit a snag due to their small Raman scattering cross-sections. To overcome this limitation, novel SERS nanosensors that combine metallic nanoparticles and specific organic Raman reporter molecules with both high Raman crosssection and recognizable ability to inorganic species have been designed (Wang et al., 2013; Yin et al., 2011; Zamarion et al., 2008). Especially, SERS signals of the Raman reporter molecule vary depending on the concentrations of the target inorganic species, which is the ideal criterion to determine the target inorganic species. For example, mobile and biocompatible pH nanosensors
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Scheme 1. Illustration of the Au NPs/4-CA nanosensors for sensing H2O2 in living cells.
have been designed by modifying 4-mercaptobenzoic acid on gold nanoaggregates (Kneipp et al., 2007; Kennedy et al., 2009). Redox potential SERS nanosensors have been developed by decorating a noble-metal nanoshell with redox-responsive small molecules (Auchinvole et al., 2012; Thomson et al., 2015). Also, we have fabricated a novel SERS nanosensor by functionalizing gold nanoparticles with oxidized cytochrome c for the selective and sensitive monitoring of intracellular superoxide anion radical (Qu et al., 2013). However, to the best of our knowledge, SERS nanosensors for selective and sensitive detection of the H2O2 have not yet been reported. With the idea of developing SERS as a new approach for rapid determination of H2O2 concentration under physiological conditions in mind, we embarked on a search to find a selective chemosensing agent for H2O2. Recent researches show that arylboronates could be oxidized by H2O2 selectively to provide phenols under mild conditions (Weinstain et al., 2014; Sun et al., 2013; Miller et al., 2005). Based on this reaction, we fabricated SERS nanosensors by modifying Au NPs with 4-CA (Au NPs/4-CA) for the real-time determination of H2O2 in living cells (Scheme 1). The Au NPs/4-CA nanosensors can rapidly and selectively respond to H2O2 in physiological solutions with tens of nanomolar sensitivity, and can be used for real-time SERS detection of H2O2 in living cells, demonstrating their practical functionality in complex biological systems.
2. Experimental 2.1. Reagents and materials All reagents were of analytical grade and used without further purification. Hydrogen peroxide (H2O2, 30 wt%), sodium hypochlorite ((NaClO), ferrous ammonium sulfate ((NH4)2 Fe(SO4)2 6H2O) and sodium chloride (NaCl) were bought from Aladdin Chemical Company (Shanghai, China). 4-Carboxyphenylboronic acid (4-CA, 97%), HAuCl4 3H2O, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), tertbutyl hydroperoxide (t-BuOOH), pyrogallic acid, phorbol myristate acetate (PMA), glutathione (GSH), tris(hydroxymethyl)aminomethane (Tris), L-cysteine (L-cys), S-nitroso-N-acetyl-dl-penicillamine (SNAP, the source of NO), 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) were obtained from Sigma-Aldrich (St. Louis, MO). All reactions using air- or moisture-sensitive reagents were performed in dried glassware under an atmosphere of dry N2. Deionized water (18 MΩ cm 1) used throughout the work was obtained from a Water Pro water purification system (Billerica, MA, USA). HeLa and normal human liver cells were purchased from the Chinese Academy of Sciences in Shanghai originally from American Type Culture Collection (Manassas, VA, USA). 2.2. Instruments Dynamic light scattering (DLS) measurements were performed using a Zetasizer Nano ZS (Malvern Instruments, Southborough, UK). Transmission electron microscopy (TEM) images of Au NPs were acquired from a JEM-1011 electron microscope (JEOL, Japan) with an accelerating voltage of 100 kV. HPLC–MS was implemented on a Hewlett Packard Series 1100 HPLC (column: Agilent Prep-C18, 5 μm, 4.6 250 mm). 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. An inverted microscope (Ti U, Nikon, Japan) with an external triple channel optical system was equipped with a 40 plan fluor objective (N.A. 0.75, Nikon, Japan). The use of dark-field condenser (N.A. 0.80–0.95, Nikon, Japan) with 100 W halogen tungsten lamp enables excitation light scattering from NPs. In the front of a filter turret (TI-FLC Epi-FL, Nikon, Japan), a 785 nm laser was used for Raman excitation. The spectrograph with a resolution of 2 cm 1 was equipped with a back-illuminated deep depletion CCD (PIXIS 400BR, Princeton Instruments, USA) which measures Raman spectra from 400 to 1800 cm 1 with a high sensitivity and fast acquisition rate. An automation controller system (MAC 6000, Ludl Electronic Products, Ltd., USA) was coupled to the fine adjustment knob of the microscope to precisely control the stage position in the z-direction with a resolution of 0.1 mm. 2.3. Fabrication of Au NPs/4-CA Au NPs were prepared by a modified citrate reduction method (Lee and Meisel, 1982). All the glass wares were immersed into aqua regia (HCl: HNO3 in a ratio of 3:1) over night and washed with plenty of deionized water for at least 5 min. 4.8 mL of
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1.00 wt% HAuCl4 was added into 100 mL deionized water at room temperature and rapidly heated to boil under vigorous stirring. Subsequently, 10 mL of 1.00 wt% trisodium citrate solution was added in the boiling solution, which resulted in a rapid color change from light yellow to black and then wine red within few minutes. The solution was kept boiling for an additional 10 min to ensure complete reduction of HAuCl4. After that, it was cooled to room temperature by removing the flask from the oil bath. The resulting colloidal solution was centrifuged at 5000 rpm for 5 min and then suspended in 100 mL deionized water for SERS nanosensors preparation. Prior to modification of Au NPs, a solution of cystamine (0.1 mM) was added to the Au NPs and the mixture was incubated for 2 h. After rinsing with water, a freshly prepared 4-CA solution (10 mM) containing EDC (40 mM) and NHS (10 mM) was added dropwise into Au NPs colloids (4-CA solution/colloid, volume ratio, 1:4) under continuous stirring, which increased the coupling efficiency of the amino group of cystamine and the carboxyl group of 4-CA and facilitated even uniform distributions of 4-CA on Au NPs surfaces (4-CA molecules on per Au NPs: about 1000, Text S1). After a 30 min incubation, the Au NPs solution was subjected to three rounds of centrifugation and resuspension in PBS (pH ¼7.4) to remove free 4-CA. 2.4. In vitro tests The experiments were performed at ambient temperature, and all H2O2 solutions were used within 30 min of preparation. H2O2 solutions (20 μL) at different concentrations were added into the prepared Au NPs/4-CA (180 μL), followed by SERS detection within 1 min. In addition, selectivity of Au NPs/4-CA nanosensors was examined by SERS responses. All assays were carried out in 20 mM PBS buffered (pH ¼7.4). Unless otherwise stated, stock solutions of selected biologically relevant reactive species were added to 4-CA (2.0 mM) modified Au NPs in PBS. SNAP (1 mM) in PBS buffer (100 mM, pH ¼7.4) was used to produce nitric oxide (NO) at room temperature. OH was prepared through the reaction of ferrous ammonium sulfate (0.1 mM) with H2O2 (1 mM). A pyrogallol (1 mM) autoxidation assay in Tris–HCl (50 mM, pH ¼ 8.0) was performed to generate O2 in a 25 °C water bath for 20 min. 1O2 was prepared based on the reaction of H2O2 with NaClO. ONOO was generated by KNO2 and H2O2 in HCl at 0 °C as previously reported (Kutala et al., 2008) and was frozen at less than 18 °C.
resultant of Au NPs/4-CA and H2O2 (10 μL) at different concentrations were added respectively, and incubated for 24 h. Sequentially, an MTT solution (5 μL, 5 mg/mL) was added into each well and incubated for another 4 h in the CO2 incubator. After removing the supernatant medium, the reaction was terminated by adding DMSO (150 μL) to dissolve the formazan crystals. The absorbance of the wells was measured on a microplate reader (Bio-Rad model 680) with a test wavelength of 570 nm. Cells incubated without Au NPs were used as a control.
3. Results and discussion 3.1. Characterization of Au NPs/4-CA nanosensors The size distribution and the dispersity of Au NPs/4-CA nanosensors were initially characterized by DLS measurements. As shown in Fig. S1, the average size of Au NPs/4-CA corresponds to 60 nm with a narrow size distribution. In addition, there was no obvious difference between the DLS spectrum of Au NPs and Au NPs/4-CA, indicating that the Au NPs were well dispersed after the surface functionalization of 4-CA. TEM characterization of the Au NPs/4-CA nanosensors further shows that they are spherical with a diameter of 60 nm (Fig. 1A), which is in close agreement with the result of DLS measurements. As 60–80 nm Au NPs are the most efficient for Raman enhancement at the near-infrared excitations (785 nm) which is known as a ‘clear window’ for optical detection, the nanosensors of Au NPs/4-CA could be suitable for the detection of biological samples (Krug et al., 1999). Furthermore, SERS response examination of Au NPs/4-CA showed that an obvious SERS spectrum can be obtained (black curve in Fig. 1B), meaning that 4-CA molecules were successfully functionalized on Au NPs by amide bonds since SERS is a surface selective technique and Au NPs have been washed with ultrapure water. In the SERS spectrum, the strong Raman bands at 821, 1011, 1031, 1178, 1270, 1562 cm 1
2.5. Cellular studies Cells were propagated in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin, and maintained in an incubator containing 95% air and 5% CO2 at 37 °C. The cells were plated into culture dishes (6 cm in diameter) and allowed to adhere for 24 h. The growth medium was removed and replaced by the culture medium containing Au NPs/4-CA. After incubation for 4 h, the cells were washed for three times with PBS to remove unbound samples and fixed on a microscopic glass slide to perform SERS studies. Then, PMA, a stimulator of cell respiratory burst for generating ROS, was added into the Au NPs/4CA-loaded cells with a final concentration of 400 ng/mL and incubated for an additional 4 h. The cells were washed three times with 0.20 M PBS buffer before imaging and SERS experiments. The biocompatibility of Au NPs/4-CA nanosensors in absence and presence of H2O2 was investigated by a MTT assay. The cells (1 105 cells/mL) were seeded onto 96-well microtiter plates to a total volume of 200 μL/well and maintained at 37 °C in a 5% CO2/95% air incubator for 12 h. Then, Au NPs/4-CA (10 μL) and the
Fig. 1. (A) Typical TEM image of Au NPs/4-CA nanosensors. (B) Raman response of Au NPs/4-CA in the absence and presence of H2O2. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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are attributed to the B–O symmetric stretching, B–O–H deformation, C–H in-plane deformation, B–O–H deformation, B–O symmetric stretching, C ¼C stretching vibrations, respectively (Table S1) (Piergies et al., 2013; Alver and Parlak, 2010; Kong et al., 2013). It also can be seen from Fig. 1B that, when H2O2 was introduced to Au NPs/4-CA, the SERS spectrum changed significantly, and bands at 1365, 1600 cm 1 attributing to in-plane O–H deformation, C ¼ C stretching (Hou and Fang, 2007) respectively, appeared in the new spectrum accompanying with the decrease of the peak at 1562 cm 1 (blue curve in Fig. 1B). These changes of characteristic SERS bands indicate the H2O2-mediated transformation of arylboronates to phenols on Au NPs. Specially, the significant SERS shift of C ¼C stretching vibration from 1562 to 1600 cm 1 may be due to the change in polarizability of the phenyl ring on Au NPs surface resulting from the transformation of boronic acid groups of 4-CA into hydroxyl groups (Kho et al., 2012; Guerrini et al., 2013), which could be used as spectral markers for monitoring the reaction of Au NPs/4-CA and H2O2. The H2O2-sensed reaction was further confirmed by HPLC–MS analysis (Fig. S2). A peak at m/z 138 well matches with the product C7H6O3 (calcd. 138.12 [M]) on mass spectrum from the reaction mixture of 4-CA and H2O2. This further confirms that Au NPs/4-CA nanosensors can be exactly responsive to the reaction of H2O2 thereby detecting H2O2 in solution. 3.2. Detection conditions of Au NPs/4-CA nanosensors SERS activity of Au NPs/4-CA nanosensors which mainly dominated by the amount of 4-CA molecules on Au NPs surfaces is of great significance for sensing biological samples. Thus, to acquire nanosensors for H2O2 with excellent SERS activity, we compared SERS spectra of nanosensors prepared by incubating the same concentration of Au NPs (about 2.5 nM) with the 4-CA solution at different concentrations. As displayed in Fig. S3, the intensities of the SERS spectra increase with 4-CA concentrations from 0.02 to 2.0 μM, and reach a plateau after 2.0 μM. The possible reason is that the coverage of 4-CA molecules at the “hot spot” sites of Au NPs capable of providing sufficiently large enhancement is complete, thus resulting in saturated SERS signals (Le Ru et al., 2006). Hence, 2.0 μM was chosen for the concentration of the 4-CA to assemble on Au NPs surfaces for maximizing SERS signals of Au NPs/4-CA nanosensors. Also, for biological applications, it is necessary for SERS nanosensors to function over a suitable pH range, particularly at a physiological pH value. Therefore, we investigate the influence of pH (5.8–8.0) on SERS responses of Au NPs/4-CA nanosensors, as depicted in Fig. S4. No distinct change could be observed in SERS spectra, illustrating Au NPs/4-CA nanosensors were pH-insensitive over a biologically relevant pH range and it is suitable to perform SERS detection in aqueous solution buffered to physiological pH (pH¼ 7.4). In addition, temporal SERS observation of Au NPs/4-CA nanosensors was carried out in the presence of H2O2 (Fig. S5). Upon treatment of H2O2, an obvious appearance of the 1600 cm 1 peak was observed, accompanied by a robust decrease of the 1562 cm 1 peak. Within 20 min of reaction, these peaks were tended to be constant, which indicated that the SERS nanosensors had a very fast response to H2O2 and the sensing reaction could be completed within 20 min. Thus, the nanosensors may be highly competent for the detection of H2O2 in biological samples, especially for the tracking of H2O2 concentration. 3.3. Stability and cytotoxicity of Au NPs/4-CA nanosensors An ideal biological nanosensor should be stable for a long period of time. Therefore, we characterize the stability of Au NPs/ 4-CA nanosensors by measuring their SERS signals (both frequency
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Fig. 2. Raman spectra of Au NPs/4-CA nanosensors after storage for different time in air. Inset: the Raman intensity of Au NPs/4-CA at 1562 cm 1 versus storage time.
and intensity) at different time under natural conditions, as shown in Fig. 2. After storage over 24 h, the responding SERS spectra of nanosensors did not change obviously, which could speculate that the Au NPs/4-CA did not aggregate seriously, illustrating their excellent stability in favor of sensing application in living cells. At the same time, Au NPs/4-CA nanosensors are allowed to be directly exposed to the surrounding circumstances, which is very important for biosensors and have great potential in the investigation of biological processes. For further SERS analysis of biological samples, the long-term cellular toxicity of Au NPs/4-CA nanosensors toward HeLa cells was assessed using the MTT assay (Fig. S6). In the presence of the nanosensors and the resultant of nanosensors and H2O2 with concentrations ranging from 1 nM to 10 nM, the cellular viabilities were estimated to be greater than 88% and 85% after incubation for 24 h, respectively. The results indicate that the Au NPs/4-CA nanosensors are generally low toxic for cellular detection, meaning that the Au NPs/4-CA nanosensors exhibit high biocompatibility and are suitable for use in biological applications. 3.4. Sensitivity and selectivity of Au NPs/4-CA nanosensors In order to evaluate the sensitivity of the Au NPs/4-CA nanosensors, different concentrations of H2O2 were tested. SERS spectra of 4-CA with different concentrations of H2O2 are shown in Fig. 3A. As the concentration of H2O2 increased, the ratiometric peak intensity of I1562/I1131 decreased, while simultaneously a new peak at 1600 cm 1 appeared and the ratiometric peak intensity of I1600 /I1131 increased obviously (Fig. 3). There is a linear relationship between the ratiometric peak intensities of I1562/I1131 (or I1600/I1131) and logarithmic concentration of H2O2 over the range of 0.1– 20 μM, with a detection limit of 0.08 μM (I1562/I1131) based on a signal-to-noise ratio of S/N ¼ 3. As the H2O2 concentration is approximately at micromolar levels in abnormal physiological condition such as oxidative stress-related diseases (Antunes and Cadenas, 2001; De Gracia Lux et al., 2012), thus, these Au NPs/4-CA nanosensors are suitable for the detection of H2O2 in biological samples. The selectivity of Au NPs/4-CA nanosensors toward H2O2 was also systematically investigated by recording SERS response of 4-CA in the presence of various biologically relevent analytes, which may coexist in the living system (Fig. 4). An obvious increase at the band ratio of I1600/I1131 was observed when Au NPs/4CA nanosensors were treated with H2O2 (11-fold over 20 min). In contrast, other biologically relevent analytes (OH, O2 , 1O2, ONOO , t-BuOOH, O3, NO, NaClO, GSH, L-cys) had little effect on the I1600/I1131 ratio. Taken together, these results establish that the
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Fig. 3. (A) Raman spectra of Au NPs/4-CA with varying H2O2 concentration: (a) 0.1 μM; (b) 0.2 μM; (c) 0.4 μM; (d) 1.0 μM; (e) 2.0 μM; (f) 4.0 μM; (g) 8.0 μM; and (h) 20 μM. (B) Plots of ratiometric peak intensities versus logarithmic H2O2 concentration based on I1562/I1131 (open squares) and I1600/I1131 (open circles). Each data point represents the average value from three SERS spectra. Error bars show the standard deviations.
Au NPs/4-CA nanosensor selectively reacts with physiological levels of H2O2 in a concentration-dependent manner. 3.5. Monitoring of H2O2 in living cells Encouraged by the promising results obtained in buffer, we explored whether Au NPs/4-CA could be used to detect H2O2 in the cellular environment. Considerable research demonstrates that tumor incidence is closely associated with reactive oxygen species (Kundu et al., 2009; Zorov et al., 2014). Thus, HeLa cells and normal human liver (HL-7702) cells were selected to investigate whether there were SERS spectral distinctions between tumor cells and normal cells. Uptake of Au NPs/4-CA was confirmed by the bright-field and dark-field microscope (DFM) images, as shown in Fig. 5A1,A2 and B1,B2. It can been seen, after an incubation of 4 h, numerous gold nanoparticles have been taken up into the cell through the process of endocytosis to form accumulations in late endosomes and lysosomes. They can be observed at the resolution of a light microscope, which enable us to localize the positions of Au NPs/4-CA nanosensors and record their SERS spectra more rapidly. In addition, the incorporation of Au NPs/4-CA did not bring a significant change to the cell morphology, which further demonstrated the excellent biocompatibility of Au NPs/4-CA. Fig. S7A-a, B-a showed Raman spectra of Au NPs/4-CA accumulated in cells. The spectra of Au NPs/4-CA in the HeLa cell and normal HL7702 cell did not change significantly at the typical bands of 1562 and 1600 cm 1, may be due to the generated H2O2 is not enough to react with Au NPs/4-CA. Then, a membrane permeable free radical initiator, PMA, which can cause oxidative damage to DNA, lipids, and proteins was added to induce oxidative stress and give rise to a variety of ROS species. After 4 h, SERS maps were generated using the intensity ratio of the 1600 cm 1 and the 1131 cm 1
Fig. 4. (A) Raman spectra of Au NPs/4-CA in absence (a) and presence of biologically relevant reactive analytes: (b) H2O2, (c) OH, (d) O2 , (e) 1O2, (f) ONOO , (g) t-BuOOH, (h) O3, (i) NO, (g) NaClO, (k) GSH, and (l) L-cys. (B) Histograms demonstrated Au NPs/4-CA and Au NPs/4-CA with the indicated biologically relevant reactive analytes based on I1600/I1131. Data shown are 0.5 mM for other biologically relevant species, 0.2 mM for GSH, and 0.1 mM for L-cys.
bands (Fig. 5A3,A4; 5B3,B4). The intensity of the 1600 cm 1 band appeared, demonstrating that the 4-CA on Au NPs is reduced via H2O2 generated under PMA stimulation. Meanwhile, Raman spectra of Au NPs/4-CA were collected from the HeLa and HL-7702 cells without stimulation of PMA, as displayed in curves d of Fig. S7A,B. Obviously, after equivalent time 4 h, there were no obvious changes in SERS spectra for non-stimulated HeLa cells and HL7702 cells, further indicating the generation of H2O2 under PMA stimulation. It is important to note that the observed intensity ratio of the 1600 cm 1 and the 1131 cm 1 bands in HeLa cells is with a higher degree than that of HL-7702 cells (Fig. 5C,D), due to that the H2O2 concentration in HeLa cells is higher than that in HL7702 cells. Using the I1600/I1131 ratio change from the exogenous H2O2 application experiment as a calibration curve, we calculated that under the aforementioned conditions, PMA stimulated HeLa cells and HL-7702 cells generate H2O2 at a rate of 1.08 nmol/104 cells/h and 0.85 nmol/104 cells/h, which is in agreement with previously reported measurements (Weinstain et al., 2014), indicating Au NPs/4-CA could be used as nanosensors for qualitative and quantitative detection of H2O2 in living cells.
4. Conclusions In summary, a novel SERS nanosensor has been developed for biosensing of H2O2 in living cells selectively and sensitively by functionalizing 4-CA on Au NPs (Au NPs/4-CA). The nanosensor is constructed based on the specific transformation of arylboronate into phenol in the presence of H2O2, which leads to SERS spectrum changes of Au NPs/4-CA. The present nanosensor exhibits high selectivity over other reactive oxygen species, abundant
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Fig. 5. Bright-field images of the HeLa cells (A1) and HL-7702 cells (B1) after 4 h incubation with Au NPs/4-CA. DFM images of the corresponding HeLa cells (A2) and HL-7702 cells (B2) containing Au NPs/4-CA. SERS mapping of Au NPs/4-CA after addition of PMA with 4 h for HeLa cells (A3) and HL-7702 cells (B3). Overlay images of SERS mapping and DFM images for HeLa cells (A4) and HL-7702 cells (B4). SERS spectra of Au NPs/4-CA from different position of SERS mapping of HeLa cells (C) and HL-7702 cells (D). Inset: Raman intensity of the nanosensors at the ratiometric peak I1600/I1131 and I1562/I1131 at different position of cells. The assay mixture consisted of cells (2 106 cells/mL), Au NPs/4-CA (2.0 nM), PMA (400 ng/mL) in 0.20 M PBS buffer (pH ¼ 7.4).
competing cellular thiols and biologically relevant species, as well as good sensitivity. Meanwhile, the Au NPs/4-CA nanosensor shows long-term stability against incubation time and pH, and high biocompatibility. More importantly, the hybridized nanosensors with high analytical performance could be successfully applied to the detection of H2O2 level in living cells under oxidative stress. The SERS nanosensors have been established to be an accurate and reliable approach for determination of H2O2 in a biological system, which may play critical roles in understanding the biological and pathological events.
Acknowledgments This research was supported by the National Natural Science Foundation of China (21505057, 21375051), the Natural Science Foundation of Jiangsu Province (BK20150227), the Natural Science Foundation of Jiangsu Normal University (14XLR011), and the Shanghai Key Laboratory of Functional Materials Chemistry (SKLFMC2014B01). Appendix A. Supplementary material Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.bios.2015.09.039.
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