Single Probe for Imaging and Biosensing of pH ... - ACS Publications

Apr 21, 2015 - Jingfang Shangguan , Dinggeng He , Xiaoxiao He , Kemin Wang , Fengzhou Xu , Jinquan Liu , Jinlu Tang , Xue Yang , and Jin Huang...
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A Single Probe for Imaging and Biosensing of pH, Cu2+ ions, and pH/Cu2+ in live cells with Ratiometric Fluorescence Signals Yingying Han, Changqin Ding, Jie Zhou, and Yang Tian Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b00628 • Publication Date (Web): 21 Apr 2015 Downloaded from http://pubs.acs.org on April 28, 2015

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Analytical Chemistry

A Single Probe for Imaging and Biosensing of pH, Cu2+ ions, and pH/Cu2+ in live cells with Ratiometric Fluorescence Signals Yingying Han,† Changqin Ding,† Jie Zhou,† Yang Tian*,†‡ †

Department of Chemistry, Tongji University, Siping Road 1239, Shanghai 200092

‡

Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, North Zhongshan Road 3663, Shanghai 200062, P. R. China ABSTRACT: It is very essential to disentangle the complicated inter-relationship between pH and Cu in the signal transduction and homeostasis. To this end, reporters that can display distinct signals to pH and Cu are highly valuable. Unfortunately, there is still no report on the development of biosensors which can simultaneously respond to pH and Cu2+, to the best of our knowledge. In this work, we developed a single fluorescent probe, AuNC@FITC@DEAC (AuNC: gold cluster, FITC: fluorescein isothiocyanate, DEAC: 7-diethylaminocoumarin-3-carboxylic acid), for biosensing of pH, Cu2+, and pH/Cu2+ with different ratiometric fluorescent signals. Firstly, 2,2',2''-(2,2',2''-nitrilotris(ethane-2,1-diyl)tris((pyridin-2-ylmethyl)azanediyl))triethanethiol (TPAASH) was designed for specific recognition of Cu 2+, as well as for organic ligand to synthesize fluorescent AuNCs. Then, pH sensitive molecule – FITC emitting at 518 nm and inner reference molecule – DEAC with emission peak at 472 nm were simultaneously conjugated on the surface of AuNCs emitting at 722 nm, thus constructing a single fluorescent probe, AuNC@FITC@DEAC, to sensing pH, Cu2+, and pH/Cu2+ excited by 405 nm light. The developed probe exhibited high selectivity and accuracy for independent determination of pH and Cu 2+, against reactive oxygen species (ROS), other metal ions, amino acids, and even copper-containing proteins. The AuNC-based inorganic-organic probe with good cell-permeability and high biocompatibility was eventually applied in monitoring both pH and Cu2+, and in understanding the interplaying roles of Cu2+ and pH in live cells by ratiometric multicolor fluorescent imaging.

perature and pH.17 It is well known that intracellular pH plays a vital role in cell biology including receptormediated signal transduction, calcium regulation, ion transport and homeostasis.18-24 Thus, for understanding the mechanisms of Cu homeostasis at molecular level, it is very essential to disentangle the complicated interrelationship between Cu and pH in the signal transduction and homeostasis. For this regard, reporters that are able to display distinct signals to Cu and pH are highly valuable.

INTRODUCTION Metals are essential components of all live cells, and in many cases cells trigger and utilize dynamic metal movements for signaling purposes.1 Such processes have well been established for alkali and alkaline earth metals like sodium and calcium,2,3 but not for transition metals like copper (Cu). Copper is a redox-active metal that fluctuates between the oxidized (Cu2+) and reduced (Cu+) states, and is utilized in all domains of life.4,5 The ability of Cu to cycle between a stable Cu2+ and unstable Cu+ is used by cuproenzymes involved in redox reactions, e.g. Cu, Znsuperoxide dismutase, cytochrome oxidase, and tyrosinase.6 However, Cu is also a highly toxic metal element. The imbalance of Cu2+ and Cu+ can result in the formation of reactive oxygen species (ROS) and subsequent damage events, which are connected to a variety of neurodegenerative aliments, including Menkes and Wilson’s diseases, Alzheimer’s disease and familial amyotrophic lateral sclerosis.7-16 Over the past decades, many efforts have been paid on investigation of copper homeostasis from a single cell system such as bacteria and yeast to multicellular organisms. Recently, dynamic Cu movements have been found to be linked with oxygen, possibly as well as tem-

Up to now, a lot of elegant methods have been reported for determination of Cu, including atomic absorption spectrometry (AAS), inductively coupled plasma mass spectroscopy (ICP-MS), inductively coupled plasmaatomic emission spectrometry (ICP-AES), fluorescent sensors, and electrochemical approaches.25-29 Fluorescence sensing,30,31 combined with molecular imaging,32,33 provides a powerful methodology for studying cell biology in a noninvasive manner with high spatial and temporal resolution.34,35 In recent years, a number of well-designed fluorescent probes specific for either Cu2+ or pH have been reported.36-41 Our group is very interested in design and synthesis of inorganic-organic nanohybrid fluores-

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cent probes and have developed C-Dot-based fluorescent biosensors for monitoring of Cu2+ and pH in live cells.34,42,43 Since these fluorescent probes are specific to Cu2+ or pH, they are unable to independently respond to pH, Cu2+, and pH/Cu2+. One of the solutions to this problem is to simultaneously employ different types of fluorescent probes in a cell. But, the mixture of different fluorescent probes generated cross-talk, the different localization, and the different metabolisms, making the scenario very complicated. Thus, there is a pressing need to develop a single fluorescent probe which is able to respond to pH, Cu2+, and pH/Cu2+ with independent fluorescence signals for dissecting their interplaying roles in live cells.

containing proteins, and amino acids. Secondly, pH responsive molecule – FITC with emission peak at 518 nm and inner-reference molecule – DEAC emitting at 472 nm excited by 405 nm were simultaneously conjugated on the surface of AuNCs. Thus, a single fluorescent probe, AuNC@FITC@DEAC, was constructed to determine pH, Cu2+, and pH/Cu2+ using different ratiometric fluorescent signals with high accuracy. Eventually, the inorganicorganic single fluorescent probe was successfully used in simultaneously monitoring pH and Cu2+ in live cells by ratiometric multicolor fluorescent imaging. The developed probe is also capable of reporting the interplaying roles of Cu2+ and pH in signal transduction and upon oxidative stress in macrophages cells.

Here, we report on the rational design, synthesis, luminescent properties, and live cell imaging studies of AuNC@FITC@DEAC, the first single fluorescent probe, which can respond to pH, Cu2+, and pH/Cu2+ with different ratiometric fluorescent signals under one excitation wavelength, as demonstrated in Scheme 1. Three novel

EXPERIMENTAL SECTION Synthesis of TPAA and TPAASH. TPAA was prepared according to the previous procedure.44 1H NMR (400 MHz, CDCl3): δ (ppm) = 8.48 (dd, 3H), 7.58 (td, 3H), 7.31 (d, 3H), 7.12 (dd, 3H), 3.90 (s, 6H), 2.75 (t, 6H), 2.65(t, 6H); 13C NMR (100 MHz, CDCl3): δ (ppm) = 159.79, 149.07, 136.24, 121.99, 121.69, 55.00, 54.33, 47.22. Next, TPAASH was synthesized based on literature procedure 45 with modifications. 1H NMR (400 MHz, CDCl3): δ (ppm) = 8.51 (t, 3H), 7.65 (m, 3H), 7.50 (d, 3H), 7.16 (t, 3H), 3.74 (s, 6H), 2.702.78 (m, 6H), 2.56-2.63(m, 18H), 1.72(s, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm) = 159.67, 148.87, 136.52, 122.50, 121.81, 60.63, 59.19, 53.27, 52.58, 24.79. TOF MS EI +: calculated for [M+H]+ 600.2977, found 600.2971.

Cu2+

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Preparation of AuNCs and AuNC@FITC@DEAC. AuNCs were synthesized by a literature procedure with minor modifications.46 In a typical synthesis, 20 mg HAuCl4•3H2O (0.1 mmol) and 104 mg TPAASH (0.3 mmol) were dissolved in 17.5 mL mixed solvent (acetic acid:methanol = 1:6 v/v) and stirred for 30 min. After the brown solution became clear, 5 mL cold NaBH4 solution (0.1 M) was added into the reactant solution under rapid stirring and ice bath. The reactant solution was stirred continuously for another 3 h. After solvent evaporation, the raw product was dispersed in 15 mL ultra-high purity water and purified through dialysis tube (MWCO: 3500) in ultra-high purity water for 4 days. The as-prepared AuNCs capped with TPAASH was collected into a glass container with a tight lid and stored at 4°C.

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Cu2+

Scheme 1. Working Principle of the Developed AuNC@FITC@DEAC Fluorescent Probe for pH, Cu2+, and pH/Cu2+ Detection

To prepare AuNC@FITC@DEAC probe, FITC was firstly conjugated onto the surface of AuNCs. The AuNCs were added into a solution of FITC and cysteamine and stirred for 5 h to form AuNC@FITC@Cys. After purified through dialysis in ultrapure water, the as-prepared AuNC@FITC@Cys was mixed with DEAC activated by EDC and NHS and stirred overnight. Finally, the nanohybrid probe AuNC@FITC@DEAC was dialyzed in ultrapure water for 8 h by using dialysis tube (MWCO: 3500).

strategies were developed in the present work. First of all, the organic molecule TPAASH was designed and synthesized for specific recognition of Cu2+. Meanwhile, TPAASH was employed for organic ligand to further prepare luminescent gold clusters – AuNCs with emission peak at 722 nm upon the excitation at 405 nm. The fluorescence of AuNCs showed specific quenching with the addition of Cu2+, resulting in determination of Cu2+ with high selectivity against other metal ions, ROS, copper-

For the selectivity experiment, 1O2 was provided by the reaction of H2O2 (10 μM) with NaClO (10 μM). Hypochlorite anion (ClO−) was generated by NaClO (10 μM).

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Analytical Chemistry

Nitroxyl (HNO) and nitric oxide (NO) derived from Angeli’s salt and the solution of S-nitroso-N-acetyl-DLpenicillamine, respectively. Superoxide anion (O2•−) derived from dissolved KO2 (10 μM) in the DMSO solution. Peroxynitrite (ONOO−) was chemically provided by the reaction between NaNO2 (10 μM) and H2O2 (10 μM). (ROO•) was chemically generated by thermolysis of AAPH (10 μM) in air-saturated aqueous solution at 310 K.

Next, for the sake of constructing a single fluorescent probe to sense to pH, Cu2+, and pH/Cu2+, pH responsive molecule – FITC with an emission peak at 518 nm (curve b, Figure 1D) and inner-reference molecule – DEAC emitted with peak at 472 nm (curve c, Figure 1D) were simultaneously conjugated on the surface of AuNCs to form AuNC@FITC@DEAC probe. As demonstrated in Figure 1D (curve d), the AuNC@FITC@DEAC fluorescent probe showed three emission peaks at 465 nm, 525 nm, and 720 nm, respectively, upon one excitation wavelength at 405 nm. The emission peak of FITC red-shifted, while that of

Confocal Fluorescence Imaging and Biosensing in Hela Cells. One day before fluorescence imaging experiments, cells were plated on a 35-mm Petri dish with 14mm bottom well in culture media at 37°C. Then, the culture media were exchanged into PBS (pH=7.4) with 10 μM AuNC@FITC@DEAC probe and incubated for 1 h. After washed with PBS three times to remove the extracellular probe, the cell fluorescence image was obtained by using an Olympus FV1000 confocal laser scanning microscope equipped with an oil immersion 60 × objective at an excitation wavelength of 405 nm. The fluorescence emissions were collected simultaneously from the DEAC channel, FITC channel, and AuNCs channel in the range of 430−485 nm, 500−600 nm, and 655–755 nm, respectively. Then, exogenous Cu2+ was introduced directly into the Petri dish and the cells were incubated for 30 min to determine the Cu2+ changes in cells. The pH sensing was operated by monitoring the Na+/H+ exchange in cells. RESULTS AND DISCUSSION Design and synthesis of AuNCs and AuNC@FITC@DEAC fluorescent probes. As a starting point of the present study, TPAASH was designed for specific recognition of Cu2+ through the synthetic route as illustrated in Scheme S1. The structure of TPAASH was confirmed by 1H NMR, 13C NMR, HR-MS, and IR (Supporting Information, Figure S1-S5 and Figure S6A). Meanwhile, the organic molecule TPAASH was employed as ligand for chemical synthesis of AuNCs. The TEM image in Figure 1A shows that AuNCs well monodispersed in solution with an average diameter centered at 1.92 nm. From the high-resolution TEM given in the inset of Figure 1A, we can see that the AuNCs were composed of polycrystalline gold, which was also evident by X-ray diffraction pattern demonstrated in Figure 1B. As demonstrated in Figure 1C, X-ray photoelectron spectroscopic result for Au 4f7/2 can be deconvoluted into two components centered at Au1+ (curve b, 84.5 eV and 87.9 eV) and Au0 (curve c, 83.5 eV and 87.0 eV), respectively, which are in a good agreement with that previously reported AuNCs.47 A broad absorption band centered at 500 nm was observed for AuNCs protected by TPAASH, together with a shoulder peak at 650 nm (Supporting Information, Figure S7, curve a). As expected, strong fluorescence was observed at 722 nm for the as-prepared AuNCs upon excitation by 405 nm (Figure 1D, curve a). The quantum yield (QY) of the asprepared AuNCs was estimated to be 1.3% using Rhodamine B in ethanol as the reference (Supporting Information, Figure S8).

Figure 1. (A) Typical TEM image of AuNCs. The insets show the size distribution histogram (top) and the HRTEM image (bottom) of AuNCs. (B) XRD patterns of AuNCs. (C) X-ray photoelectron spectroscopy of AuNCs. (D)Fluorescence spectra of (a) AuNCs, (b) FITC, (c) DEAC, and (d) AuNC@FITC@DEAC (405 nm excitation). DEAC had a blue shift, after conjugated onto the surface of AuNCs. FT-IR was employed to track the modification processes (Supporting Information, Figure S6). Ratiometric responses of AuNC@FITC@DEAC to pH, Cu2+, and pH/Cu2+. The titration of the fluorescent probe AuNC@FITC@DEAC with pH or Cu2+ was first carried out to prove the working principle. As shown in Figure 2A, with the decreasing pH of the buffer solution, the green emission (Fgreen: 500-600 nm) from FITC continuously decreased, while no obvious variations were obtained at the blue fluorescence (Fblue: 430-485 nm) from DEAC and the red fluorescence (Fred: 655-755 nm) ascribed to AuNCs (Supporting Information, Figure S9A). The fluorescence signal pattern is blue-black-red, upon addition of H+, agreeing well with the illustration in Scheme 1. As a result, Fgreen/Fblue, the ratio of the integrat-

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ed intensities at 500-600 nm (Fgreen) and 430-485 nm (Fblue), gradually decreased with the decreasing pH. The fluorescent intensity ratio demonstrated good linearity with pH in the range of 6.08 - 9.09 with a pKa at 7.54, as summarized in the inset of Figure 2A.

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in Figure 3A, the blue fluorescence from DEAC stayed constant with the changes of both H+ and Cu2+ concentrations. However, the green fluorescence ascribed to FITC obviously dropped down as the decreasing pH in the range of 9.09 to 6.08, independent of Cu2+ changes from 2×10-7 to 1.1×10-5 M, as demonstrated in Figure 3B. Meanwhile, the red fluorescence emission from AuNCs gradually decreased with the rising concentration of Cu2+ from 2×10-7 to 1.1×10-5 M, and unaffected by the decreasing pH down to 6.08 (Figure 3C). Accordingly, the AuNC@FITC@DEAC probe exhibits a fluorescence signal pattern of blue-black-black with the addition of both H+ and Cu2+, in a good agreement with that given in Scheme 1.

Figure 2. (A) Fluorescence spectra of the ratiometric probe upon the exposure to different pH (405 nm excitation). Inset: The plot of Fgreen/Fblue as the function of pH. (B) Fluorescence spectra of the ratiometric probe upon the exposure to different amounts of CuCl2 (405 nm excitation). Inset: The plot of Fred/Fblue as the function of Cu2+.

Figure 3. Fluorescence spectra of the ratiometric probe upon the exposure to different pH and different concentrations of CuCl2. (A) Fluorescence spectra of DEAC; (B) Fluorescence spectra of FITC; (C) Fluorescence spectra of AuNCs. (405 nm excitation).

On the other hand, as demonstrated in Figure 2B, the red fluorescent emission ascribed to TPAASH-ligand AuNCs showed continuous quenching with the addition of Cu2+. By contrast, negligible changes were observed in the green emission from FITC (Supporting Information, Figure S9B) and blue emission from DEAC. Thus, the AuNC@FITC@DEAC probe exhibits a fluorescence signal pattern blue-green-black, upon addition of Cu2+, in good accordance with that illustrated in Scheme 1. The integrated fluorescence intensity ratio between red and blue channels Fred/Fblue gradually decreased with increasing concentration of Cu2+. As plotted in the inset of Figure 2B, the signal ratio Fred/Fblue shows a good linearity with Cu2+ concentration in the range of 2×10−7−1.1×10−5 M. The detection limit was calculated to be ∼97 nM (S/N = 3). The reaction time between AuNCs and Cu2+ was estimated within 10 min (Supporting Information, Figure S10). In addition, the average excited state lifetime of AuNCs with TPAASH ligand was measured to be 309.2 ns (2.0 ns (25%), 30.5 ns (26%), and 325.1 ns (48%), Supporting Information, Figure S11). The emission from the AuNCs probe may be attributed to ligand-to-metal-metal charge transfer (LMMCT) from the nitrogen atom in the ligands to the Au atoms, similar to those reported previously.48-50 Interestingly, the excited state lifetime showed negligible change after Cu2+ was added, suggesting the quenching mechanism of AuNCs with Cu2+ is possibly static quenching.51

For the selectivity test, the AuNC@FITC@DEAC probe was incubated with various reactive oxygen species (ROS) such as 1O2, ClO−, H2O2, HNO, NO, O2•−, ONOO−, and ROO•, metal ions including Cu+, Ni2+, Zn2+, Ca2+, Mg2+, K+, Na+, Fe2+, Fe3+, Co2+, Cd2+, Cr3+, Pb2+, Ag+, and Mn2+, as well as amino acids. No marked changes on Fgreen/Fblue ratio were obtained for ROS (Figure S12A), metal ions including Cu2+ (Figure S12C), and amino acids (Figure S12G). Meanwhile, ROS, other metal ions except Cu2+, and amino acids had negligible effects on Fred/Fblue ratio, as demonstrated in Figure S12D and B, and Figure S12H. More interestingly, selectivity of the AuNC@FITC@DEAC probe for determination of Cu2+ ions against Cu-containing proteins, such as cytochrome c oxidase (COX), Cu, Zn-superoxide dismutase (SOD), ceruloplasmin, and tyrosinase, was also examined. No obvious changes in fluorescent signal were observed for these Cu-containing proteins (Figure S12E). Meanwhile, Cu-containing proteins had no effects on the determination of Cu2+ by using the developed fluorescent probe (Figure S12F). Taken together, these results indicate the developed ratiometric probe can be employed to separately and simultaneously determine pH and aqueous Cu2+ with different emission channels. In addition, the AuNC-based inorganic-organic fluorescent probe also exhibits good photostability (Supporting Information, Figure S13). Therefore, the present probe is promising for ratiometric imaging and biosensing of pH and Cu2+ in the complicated live cells.

Then, in order to understand the relationship between pH and Cu2+ in live cells, we investigated the fluorescent responses of AuNC@FITC@DEAC probe with the simultaneous changes of H+ and Cu2+ concentrations. As shown

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Simultaneous multicolor imaging of Cu2+ and pH in live cells. For further cell imaging and biosensing, the long-term cellular toxicity of the AuNC@FITC@DEAC probe toward Hela cell lines was first examined by means of a standard MTT assay. The cellular viabilities were determined to be greater than 97% and 94% after the cells were incubated with the developed fluorescent probe for 24 and 48 h, respectively, at concentrations of 0.25, 1.0, 2.5, 5.0 and 10.0 μM (Supporting Information, Figure S14). The data demonstrate that the AuNC-based probe is generally low-toxic for cellular imaging. The observation was also evident by the results of flow cytometry experiments. Apoptosis assay of Hela cells after AuNC@FITC@DEAC probe treatment was conducted to evaluate the biocompatibility by flow cytometry (FACS) measurements (Supporting Information, Figure S15). Negligible differences were found between control cells and the cells treated with the present probe, revealing good biocompatibility of the AuNC@FITC@DEAC probe.

(Figure 4D), and color turned almost back (Figure 4Q). The result suggests a recover of pH in the intracellular environment. From the separated imaging of three channels, we can see that no marked variations were observed for fluorescence from blue channel (Figure 4J, 4N, and 4R). However the green fluorescence from FITC dye responsive to pH first quenched with the addition of Na+free Ringer’s solution (Figure 4O) and then turned bright after adding Na+ solution (Figure 4S). Surprisingly, the red fluorescence from AuNCs probe also changed with the pH variations, which should have no response to pH. As demonstrated in Figure 4P, the red fluorescence ascribed to Cu2+ response quenched with the decreasing pH

A

Then, we proceeded to evaluate the ability of the AuNC@FITC@DEAC probe for imaging in live cells. To this end, the present fluorescent probe was incubated with Hela cells for 30 min, then washed with Hank’s Balanced Salt Solution (HBSS) for 3 times. After the uptake, the fluorescence in Hela cells was monitored by confocal microscopy upon 405 nm excitation. Figure 4 demonstrates pseudocolored image of Hela cells with the AuNC@FITC@DEAC probe (Figure 4A), bright-field image (Figure 4B), and the overlay image of fluorescence and bright-field (Figure 4C) in Hela cells. The overlay image shows that the AuNC-based inorganic-organic probe had good cell-permeability and located in cytoplasm, The multicolor imaging of Hela cells treated with the AuNC@FITC@DEAC probe was collected from three separated channels: Fblue: 430−485 nm from DEAC dye (Figure 4E); Fgreen: 500−600 nm from FITC dye (Figure 4F); Fred: 655−755 nm from AuNCs probe (Figure 4G). The fluorescent imaging of control experiments for the present fluorescent probe incubated with Hela cells was shown in Figure 4A and 4E-4G.

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Figure 4. Fluorescence microscope images of Hela cells with AuNC@FITC@DEAC probes. (A) Overlapped three channels, (B) bright field image, and (C) overlapped fluorescence and bright field image. (E, F, and G) The images of blue, green, and red channels. (D and H) (D) Fgreen/Fblue and (H) Fred/Fblue in HeLa cells: (a) control experiment, (b) after induction by Cl--free Ringer’s solutions containing 0.1 mM ouabain, (c) Na+-free Ringer’s solution, and (d) adding 100 mM Na+ solution. (I, M, and Q) The images of Hela cells after treatment with (I) Cl--free Ringer’s solutions containing 0.1 mM ouabain for 45 min; (M) Na+-free Ringer’s solution for 5 min, and (Q) adding 100 mM Na+ solution. (J, K, and L) The images of blue, green, and red channel after treatment with Cl--free Ringer’s solutions containing 0.1 mM ouabain for 45 min; (N, O, and P) The images of blue, green, and red channel after treatment with Na+-free Ringer’s solution for 5 min; (R, S, and T) The images of blue, green, and red channel after adding 100 mM Na+ solution. Scale bar: 20 μm.

Next, the AuNC@FITC@DEAC probe was first employed in imaging and sensing of intracellular pH variations. The Na+-H+ exchange has been a usual mechanism for regulating cytosolic pH.52 After Hela cells were incubated in Cl--free Ringer’s solutions containing 0.1 mM ouabain for 45 min, the pseudocolored imaging shown in Figure 4I had no obvious change with that in Figure 4A. The average emission ratio Fgreen/Fblue is 0.885±0.013 (Figure 4D), similar to that in Hela cells incubated without ouabain treatment (Figure 4D). Then, the ratio slightly decreased to 0.855 ± 0.031 when the cells were continuously incubated with a Na+-free Ringer’s solution for another 5 min (Figure 4D). The color was clearly changed from green-yellow to light blue (Figure 4M). Such change indicated a rapidly acidification of cells under stimulate of the Na+-free Ringer’s solutions. Followed by adding 100 mM Na+ solution, the ratio increased to 0.949 ± 0.026

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Analytical Chemistry as the addition of Na+-free Ringer’s solution, and then recovered with the increasing pH after addition of Na+ solution (Figure 4T). The results suggest that intracellular pH decrease may induce the increasing concentration of intracellular Cu2+, and vice versa. The ratio changes of Cu2+ (Fred/Fblue) with pH variations were also summarized in Figure 4H. This is the first observation for pH-induced changes of Cu2+ concentration in live cells, although the mechanism is still unknown at the present stage. A

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normal condition, which agrees with that previously reported.53 Then, AuNC@FITC@DEAC probe was found to respond to the changes in the Cu2+ concentration: the pesudocolor changed from yellow-green to green-blue and blue with the addition of 5 μM and 10 μM Cu2+, respectively (Figure 5E and 5I). Accordingly, the Fred/Fblue ratio decreased to 0.208 ± 0.017 and 0.162 ± 0.004 (Figure 5N). From the separated images of three channels, it is obvious that the fluorescence from blue channel stayed almost constant (Figure 5B, 5F, and 5J). As similar to the titration results in solution, the red fluorescence from AuNCs gradually quenched with the increasing concentration of Cu2+ (Figure 5D, 5H, and 5L). However, the green fluorescence from FITC dye also unexpectedly quenched with the increasing concentration of Cu2+ (Figure 5C, 5G, and 5K), which should not respond to Cu2+. The pH changes with the concentration changes of Cu2+ were illustrated in Figure 5M. This means that the increasing concentration of Cu2+ in live cells may trigger the decreasing pH. This is also the first observation for Cu2+triggered intracellular pH decreasing in live cells. Thus, taken together, to the best of our knowledge, this represents the first report of simultaneous determination of pH and Cu2+ in multi-color imaging using a single fluorescent probe. The results suggest that the AuNC@FITC@DEAC probe has great potential in dissecting the interplaying roles of pH and Cu2+ in the complex interaction networks of the signal transduction.

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In summary, we have designed and synthesized TPAASH for specific recognition of Cu2+ and for ligands to prepare luminescent AuNCs. Then, we have developed AuNC-based single fluorescent probe AuNC@FITC@DEAC, which responds to pH, Cu2+, and pH/Cu2+ with different patterns, by conjugating pH responsive molecule – FITC and inner reference molecule – DEAC. The developed probe has demonstrated high selectivity, sensitivity, and accuracy with ratiometric fluorescent signals. Meanwhile, the AuNC-based probe with small size has also shown long-term stability, good cellpermeability, and low cytotoxicity. Accordingly, the AuNC@FITC@DEAC probe has been successfully applied in imaging and biosensing of pH and Cu2+, as well as used in ROS regulation of pH and Cu2+ changes in macrophages cells. Further optimization of this kind of probes may render new opportunities in understanding signal transduction and oxidative pathways. This investigation has provided a methodology to designing and synthesizing of inorganic-organic probes with multi-color and multiresponsive, extended to other metal ions, other types of biomolecules. The work is underway in our laboratory. We expect this type of probes may have broad applications in fundamental biology research.

10uM

2+ CCu Cu2+

Figure 5. Fluorescence microscope images of Hela cells with AuNC@FITC@DEAC probes. (A, E, and I) The pseudocolored images of Hela cells before (A) and after treatment with (E) 5 μM and (I) 10 μM CuCl2. (B, C, and D) The images of blue, green, and red channels before treatment with CuCl2; (F, G, and H) The images of blue, green, and red channels after treatment with 5 μM CuCl2; (J, K, and L) The images of blue, green, and red channels after treatment with 10 μM CuCl2. (M) Integrated fluorescence intensity from 500 to 600 nm over that from 430 to 485 nm in HeLa cells. (N) Integrated fluorescence intensity from 655 to 755nm over that from 430 to 485 nm in HeLa cells. Scale bar: 20 μm. On the other hand, we sought to assess whether AuNC@FITC@DEAC as a fluorescent probe could report changes in the Cu2+ level in live cells by ratiometric fluorescence imaging. For this regard, the fluorescence in live cells was monitored by confocal microscopy upon 405 nm excitation, after the fluorescent probe was uptaken in Hela cells for 30 min. From Figure 5A, we can see that the average emission ratio Fred/Fblue is 0.234 ± 0.004 (Figure 5N), revealing very low levels of available Cu2+ in the

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Supporting Information. Supplementary Experimental Section, Synthetic route for TPAA and TPAASH, NMR and MS Data of TPAA and TPAASH, FT-IR spectra, UV-vis absorption spectrum and photoemission spectrum of the luminescent Au NCs, Quantum yield, Fluorescence responses of Au NCs towards H+ ions and FITC towards Cu2+ ions, Reaction Time, Time-Resolved Fluorescence measurements, Selectivity for amino acids, Photostability, MTT Assay, and Apoptosis Assay. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION *Corresponding Author E-mail: [email protected] Fax: +86-21-62237105.

ACKNOWLEDGMENT This work is financially supported by the NSFC (21175098) and National Natural Science Fund for distinguished young scholars (21325521). East China Normal University is also greatly appreciated.

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Analytical Chemistry

Table of Contents

Cu2+

Cu2+ Cu2+

H+

H+

Cu2+

Cu2+ Cu2+

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