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Gold nanocluster-based fluorescence biosensor for targeted imaging in cancer cells and ratiometr

Biosensors and Bioelectronics 65 (2015) 183 –190

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Biosensors and Bioelectronics
journal homepage: www.elsevier.com/locate/bios

Gold nanocluster-based ?uorescence biosensor for targeted imaging in cancer cells and ratiometric determination of intracellular pH
Changqin Ding a, Yang Tian a,b,n
a b

Department of Chemistry, Tongji University, Siping Road 1239, Shanghai 200092, People′s Republic of China Department of Chemistry, East China Normal University, North Zhongshan Road 3663, Shanghai 200062, People′s Republic of China

art ic l e i nf o
Article history: Received 25 July 2014 Received in revised form 30 September 2014 Accepted 13 October 2014 Available online 18 October 2014 Keywords: Au nanoclusters Ratiometric ?uorescence biosensor pH determination Targeted imaging Cancer cells Folate receptor

a b s t r a c t
The dysregulated pH is working as a mark of cancer. It is a challenge for developing a biosensor for targeted imaging in cancer cells and monitoring of intracellular pH. Here, a ratiometric ?uorescence biosensor for pH determination was developed with targeted imaging into folate acceptor (FR)-rich cancer cells at the same time. AuNCs protected by bovine serum albumin (BSA) worked as reference ?uorophore and ?uorescein-isothiocyanate (FITC) acted as the response signal for pH. For targeted imaging of cancer cells, the AuNCs were simultaneously conjugated with folic acid (FA). The developed ratiometric biosensor can monitor pH with a wide linear range from 6.0–7.8 with a pKa at 6.84. Under every different pH condition, the probe showed high selectivity over various metal ions and amino acids with its ?uorescence ratio stayed almost constant ( o 5%). It also showed good cyclic accuracy when pH switched between 6.0 and 8.0, as well as low cytotoxicity. The AuNC-based inorganic–organic nanohybrid biosensor showed good cell-permeability, low cytotoxicity, and long-term photostability. Accordingly, the pH biosensor was employed to gain targeted imaging in FR ? ve Hela cells with FR ? ve lung carcinoma cells A549 as comparison, and achieved to monitor the pH changes in Hela cells. & 2014 Elsevier B.V. All rights reserved.

1. Introduction Intracellular pH (pHi) plays a critical role in the physiological and pathological processes (Roos and Boron, 1981). The regulation of pHi is essential for most cellular processes, including receptormediated signal transduction, calcium regulation, ion transport, cell volume regulation, vesicle traf?cking, cellular metabolism, cell membrane polarity and so on (Golovina and Blaustein, 1997; Loiselle and Casey, 2003). Abnormal pHi shows great relationship with human physiology and pathophysiology diseases such as cancer, Alzheimer's disease, and cardiopulmonary problems (Izumi et al., 2003; Lagadic-Gossmann et al., 1999; Tang et al., 2007). Recent years, the detection of pHi value in cancer cells draws more attention, as the dysregulated pH is working as a mark of cancer (Zhang et al., 2010). It has been reported that the intracellular pH is higher than extracellular pH in cancers tumor, which may help to promote the proliferation, migration, invasion of cancer tumors and some other cancer processions (Webb et al., 2011). On this point, monitoring pH changes in cancer cells is critically important

n Corresponding author at: Department of Chemistry, Tongji University, Siping Road 1239, Shanghai 200092, People's Republic of China. Fax: ? 86 21 62237105. E-mail address: ytian@chem.ecnu.edu.cn (Y. Tian).

for studying cellular functions and gaining a better understanding of physiological processes. Up to now, pH sensitive microelectrodes, nuclear magnetic resonance, UV–vis absorption spectroscopy, and ?uorescence spectroscopy have been reported for the detection of pH in vivo (Zhang et al., 2010). Among these, ?uorescence probe provides a powerful tool to assess pH in vivo and in vitro with technical and practical advantages such as high sensitivity, subcellular resolution and easy operation (Li et al., 2014; Patterson et al., 1997). In our previous work, a two-photon “turn-on” ?uorescence probe has been designed based on carbon dots for monitoring pH changes in live cells and tissues (Kong et al., 2012). Unfortunately, this kind of “turn-on” ?uorescence probe, as well as those reported “turn-off” ?uorescent probes for pH remains two limitations: one is lack of accuracy in quantitative determination, because the ?uorescence intensity changes with the variation of pH and the ?uorescent probe unevenly distributes in live cells; another is their lack selectivity in recognition of different cells, because they could be took up by various kinds of cells without speci?c recognition. To solve these two problems, we designed a ratiometric ?uorescent biosensor with targeted marker for imaging and biosensing of pH in cancer cells. Ratiometric ?uorescence measurements can eliminate the in?uence of variations in the local probe concentration and distribution, thus enhancing the accuracy of measurements (Du et al., 2013; Fu et al., 2013; Gao et al., 2014; Miao et al., 2013;

http://dx.doi.org/10.1016/j.bios.2014.10.034 0956-5663/& 2014 Elsevier B.V. All rights reserved.


C. Ding, Y. Tian / Biosensors and Bioelectronics 65 (2015) 183 –190

Fig. 1. (A) Working principle of the developed FA-FITC@AuNC ?uorescent bisensor for pH detection and cancer cell-targeted imaging and (B) Reaction scheme of FITC with H ? and OH ? .

Zhu et al., 2012). Silica nanoparticals and polymer gel coating with different dyes and quantum dots (QDs) were widely used as the reference ?uorescences for construction of raiometric ?uorescence probes (Dennis et al., 2012; Peng et al., 2010; Tsou et al., 2014). But these probes are dif?cult to enter into live cells because of their big sizes and some of them have high potential toxicity due to their heavy metal components. More recently, AuNCs have been reported as new type of luminescent nanomaterials for catalysis, sensors, and bioimaging because of their good biocompatibility, high electrocatalytic activity, and unique optical property (Kong et al., 2011; Liu et al., 2014; Sperling et al., 2008; Xie et al., 2009; Xue et al., 2012; Zhuang et al., 2014). AuNCs protected by BSA showed good photoluminescence properties with a large Stokes shift (?150 nm), good biocompatibility, and low cytotoxicity due to its small size (central core o 5 nm) and BSA shell. In this article, we developed a AuNC-based ?uorescence biosensor for targeted imaging of cancer cells and ratiometric ?uorescence detection of intracellular pH simultaneously. As shown in Fig. 1A, BSA-protected AuNCs were employed as reference signal and conjugated with FITC to form a ratiometric ?uorescence probe FITC@AuNC for pH detection. Here, FITC acted as the speci?c recognition element for H ? with ?uorescence emission centered at 516.5 nm. When pH increases (OH ? increases), the lactone ring of FITC molecular will open to form anion, causing strong ?uorescence emission; when pH decreased (H ? increases), the lactone ring will closed with weak ?uorescence (Fig. 1B), while the red ?uorescence ascribed to AuNCs at 625 nm remained constant. On the other hand, FA was also hybrid on FITC@AuNC surface to form FA-FITC@AuNC ?uorescent probe for targeted imaging of cancer cells. FA transports physiologically across the plasma membrane

by using either reduced folate carrier or FR. The latter is frequently overexpressed in cancer cells, enabling FA be used as a good target-receptor for FR ? ve cancer cells (Lu and Low, 2002; Wang et al., 2013). The FA-FITC@AuNC biosensor can monitor pH gradients in a pH range of 6.0–7.8 with high selectivity, good sensitivity, and high cyclic accuracy. Meanwhile, AuNC-based ?uorescent biosensor showed low-cytotoxicity and long-term stability against light illumination. Moreover, it also exhibited the ability to achieve target-imaging of FR ? ve cancer, indicating an acidi?cation process and its recovery when these nanoparticles entering into cancer cells.

2. Material and methods 2.1. Reagents and chemicals Gold (III) chloride trihydrate (HAuCl4 ? 3H2O, 99%), dimethyl sulfoxide (DMSO), nhydroxysuccinimide (NHS), and methyl thiazolyl tetrazolium (MTT) were purchased from Sigma-Aldrich. Fluorescein-isothiocyanate (FITC), N, N′-Dicyclohexylcarbodiimide (DCC), folic acid (FA), and ouabain octahydrate were obtained from Aladdin Chemistry Co. Ltd. Metal salts, bovine serum albumin (BSA), amino acids, glucose, diethylether, and triethylamine were obtained from Sinopharm Chemical Reagent Co. Ltd. Solutions of metal ions were all prepared from their chloride salts. Dialysis tube (MWCO: 3500) and Sephadex G50 were obtained from Ebioeasy Corporation and Biosharp Corporation separately. Cell culture media and supplements were supplied by Invitrogen Corporation. All chemicals from commercial sources were used as

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received without further puri?cation. All aqueous solutions were prepared with Milli-Q water (18.2 MΩ cm ? 1). 2.2. Synthesis of AuNCs, FITC@AuNC, and FA-FITC@AuNC probes BSA-stabilized AuNCs were synthesized according to a green bio-mineralization method (Xie et al., 2009) with a little modulation. The combination of FITC and AuNCs was achieved through the reaction between the isothiocyanate group of FITC and the amino group on the surface of AuNCs to form FITC@AuNC and stored at 4 °C. For preparation of FA-FITC@AuNC probe, AuNCs were ?rstly conjugated with FA by using coupling reagents DCC and NHS (Lee and Low, 1994). In a typical experiment, FA was reacted overnight with 500 mg DCC and 300 mg NHS at room temperature. After ?ltration, the reaction product FA@AuNC was precipitated and washed three times with diethylether, dried under vacuum, and stored as yellow powder. Then FA@AuNC solution was mixed with the solution of NHS-folate in DMSO with appropriate proportion in dark for another 2 h. Followed, FITC in EtOH was added and the reactant was stirred in dark at room temperature for another 12 h. Finally, the nanohybrid probe was dialyzed in PBS (0.05 M, pH ? 7.4) for 8 h by using dialysis tube, puri?ed through Sephadex G50 column and stored at 4 °C.

3. Results and discussion 3.1. Characterization of AuNCs and FA-FITC@AuNC The as-prepared AuNCs showed mono-dispersed with average size of ?5 nm, as shown in Fig. 2A. A further observation revealed that the AuNC was consistent with ?111? spacing of Au (Fig. 1B), which was con?rmed by XRD data with a diffraction peak at 38.56° (JCPSD no. 04-0784, Fig. 2B). After combined with FA and FITC, such diffraction peak shifted to 35.32°, indicating the increase of Au lattice from ?2.06 ? to ?2.24 ?. The change was also evident by XPS results. As demonstrated in Fig. 2C, the Au 4f XPS spectrum of AuNCs displayed a dominant component of Au°: 83.5 eV (Au 4f7/2) and 87.3 eV (Au 4f5/2), while Au 4f XPS spectrum of FA-FITC@AuNC showed two main peaks at 84.1 eV (Au 4f7/2) and 87.7 eV (Au 4f5/2), typically ascribed to Au ? (Guével et al., 2011; Wei et al., 2010). FT-IR spectrum of AuNCs (Fig. 2D, curve a) exhibited four characteristic peaks located at 3450 cm ? 1 (VO–H), 1260 cm ? 1 (VC–H), 1662 cm ? 1 (VCQO), and 1540 cm ? 1 (VN–H). The presence of these hydrophilic groups including ?NH2, ?COOH and/ or ?OH imparts AuNCs water-solubility. On the other hand, FT-IR spectrum for FITC (Fig. 2D, curve b) shows four characteristic peaks located at 3420 cm ? 1, 2040 cm ? 1, 1620 cm ? 1, and





Fig. 2. Characterization of the ?uorescent biosensor. (A) TEM and HRTEM (inset) images of AuNCs; (B) X-ray diffraction patterns of (a) AuNCs and (b) FA-FITC@AuNC; (C) XPS data of (a) AuNCs and (b) FA-FITC@AuNC; (D) FT-IR spectra of (a) AuNCs, (b) FITC, (c) FA and (d) FA-FITC@AuNC.


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absorption peaks at 280 nm and 350 nm indicated the combination of BSA and FA (Stella et al., 2000). Furthermore, UV–Vis absorption spectrum showed a new absorption shoulder around 355 nm after the combination of FA (Fig. S2B, SI), which also indicates the successful conjugation of FA, FITC and AuNCs. 3.2. Analytical performance of FA-FITC@AuNC biosensor for pH determination The standard ?uorescence pH titration was performed in PBS at the excitation of 488 nm, as shown in Fig. 3B. In this ratiometric biosensor, the organic molecule FITC was used as response signal for determination of pH, while AuNC served as reference signal because of its good stability under different pH (Fig. S3, SI). With the increasing pH of the buffer solution, the green emission from FITC continuously increased, while the red ?uorescence ascribed to AuNCs stayed constant. As a result, FGreen/FRed, the ratio of the integrated intensities at 510–550 nm (FGreen) and 580–680 nm (FRed), gradually increased with the increasing pH. The signal ratio showed good linearity with pH in the range of 6.0–7.8 with a pKa at 6.84, as plotted in the inset of Fig. 3B. As the 2δ of the pH titration experiments is 0.099, suggesting that the FA-FITC@AuNC could detect pH change with a level of ?0.1. Much importantly, the complexity of intracellular system presents a great challenge for biosensors in requirement of selectivity and stability. The selectivity experiments were carried out by monitoring the FGreen/FRed ratio of the ?uorescent biosensor in the presence of metal ions and amino acids which may coexist in living system. Various metal ions such as aboundant cellular cations (1 mM for K ? , Na ? , Ca2 ? , Mg2 ? ) and trace metal cations in organisms (10 μM for Co2 ? , Cd2 ? , Cu2 ? , Fe2 ? , Fe3 ? , Mn2 ? , Ni2 ? , Zn2 ? ) were tested under three different pH conditions (pH 6.34, 7.25, and 8.09 respectively). As shown in Fig. 4A, under the same pH conditions, there is no obvious difference in the FGreen/FRed ratio in the presence of various cations ( o 5%) compared to that in different pH situations. Similarly, several typical amino acids and glucose (10 μM) were examined under three different pH conditions. It could be seen that the FGreen/FRed ratio of ?uorescent biosensor coexisted with various amino acids and glucose stayed nearly unchanged under the same pH condition, while shifted with the changes of pH (Fig. 4B). The results indicate the high selectivity of the developed ratiometric probe for determination of pH in the complicated live cells. The photostability of FA-FITC@AuNC biosensor was also investigated under different pH conditions (pH 6.19, 7.42, and 8.06) and summarized in Fig. 4C. After being exposed to a ?uorescence spectrophotometer (λex ? 488 nm) equipped with a 90 W Xenon lamp for 2 h, no obvious changes ( o 5%) were observed for the FGreen/FRed ratio of the biosensor under every pH condition, suggesting the good photostability of this AuNC-based inorganic–organic ?uorescent biosensor. Fig. 4D showed the good ?uorescence reversibility responses of FA-FITC@AuNC biosensor in the ratio of integrated intensities (FGreen/FRed) in a PBS solution when pH was switched between 6.0 and 8.0 for three cycles. Besides, the relationship between the ?uorescence ratio (FGreen/FRed) and reaction time showed that such probe could quickly response to pH changes within 30 s (Fig. S4, SI). These experiments indicated that the dual-emission FA-FITC@AuNC ?uorescent probe had high selectivity, long-term stability, good reversibility and quick response for pH determination in biological system. 3.3. Cytotoxicity. The possibility of FA-FITC@AuNC probe for molecular receptortargeted optical detection of cancer was tested in three types of cancer cell lines with different levels of FR expression: FR ? ve Hela


Fig. 3. Fluorescent determination of pH. (A) Fluorescence spectra of (a) AuNCs, (b) FITC, and (c) FA-FITC@AuNC under 488 nm excitation; (B) Fluorescence spectra of the ratiometric biosensor to various pH titration. Inset: Plot of FGreen/FRed as a function of the pH (5.0–9.0) (excited at 488 nm, FGreen: 510–550 nm, FRed: 580– 680 nm).

1380 cm ? 1, which are ascribed to the vibration of O–H, NQCQS, CQO and C?O?C respectively. Meanwhile, ?ve peaks located at 3420 cm ? 1, 1700 cm ? 1, 1600 cm ? 1, 1480 cm ? 1, and 1190 cm ? 1 were clearly observed for FT-IR spectrum of FA, corresponding to the vibration of VO–H, VCQO, δN–N, VC–N and VC–O respectively. After FITC and FA were conjugated on AuNCs surface, the appeared peaks at 1650 cm ? 1 (Amide I VCQO) and 1459 cm ? 1 (Amide III VC–N), and the disappeared peak at 2040 cm ? 1 (VNQCQS) in the IR spectra (Fig. 2D, curve d) indicated the successful attachment of FA and FITC on AuNCs by covalent combining with BSA. The ?uorescence emission of AuNCs was observed to be centered at 640 nm under excitation at 488 nm, as shown in Fig. 3A (curve a). Using rhodamine B as a standard, the ?uorescence quantum yield (QY) of AuNCs was estimated to be ?9% (Fig. S1, Supplementary Information (SI)). However, after combined with FITC and FA, such emission shifted to 625 nm. Meanwhile, the emission peak of FITC shifted from 515 nm to 516.5 nm upon excitation of 488 nm (Fig. 3A, curve b). The successful preparation of FA-FITC@AuNC was also proved by UV–Vis spectrum when the asprepared probe was puri?ed through Sephadex G50 column, in which FA-FITC@AuNC ?owed out ?rstly (Fig. S2A, I, SI) and then FA-FITC–BSA followed (Fig. S2A, II, SI). The chromatogram was obtained by detecting the intensity of ?uorescence emission (λex ? 488 nm) and UV–Vis absorbance at the same time. The intensities of ?uorescent emissions at 516.5 nm and 625 nm presented the existences of FITC and AuNCs, respectively. UV–Vis

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Fig. 4. Selectivity, photostability, and reproducibility test. (A and B) Fluorescence responses of FA-FITC@AuNC under different pH of 6.34, 7.25, and 8.09 respectively, (A) toward various metal ions (1 mM for Na ? , K ? , Ca2 ? , and Mg2 ? ; 10 μM for other cations); and (B) toward various amino acids (10 μM for cysteine, valine, histidine, isoleucine, leucine, lysine, methionine, serine, threonine, and glutamic acid) and 10 μM glucose. (C) Photostability of FA-FITC@AuNC i under different pH of 6.19, 7.42, and 8.06 respectively for 2 h. Error bars represent standard error measurements (S.E.M.) of three parallel experiments. (D) Fluorescence reversibility responses of FA-FITC@AuNC between pH 6.0 and 8.0.

cells, FR ? ve lung carcinoma cells A549, and FR ? ve human kidney cells 293 T. To develop further biological imaging applications, the long-term cellular toxicity of both ?uorescent probes were determined by standard MTT assays. In the presence of FITC@AuNC probes with concentration from 5 to 100 μg mL ? 1, the cellular viabilities of Hela cells were estimated to be greater than 85% and 80% after incubation for 24 and 48 h (Fig. S5A, SI), while those of A549 cells were greater than 80% and 75% (Fig. S5B, SI). Meanwhile, the cellular viabilities of cells treated with FA-FITC@AuNC probes for 24 and 48 h were up to 80% and 75% in Hela cells, as well as 85% and 90% in A549 cells. Besides, non-cancer FR ? ve human kidney 293 T survived much more with FA-FITC@AuNC probe ( 4 88% for 24 h, 4 93% for 48 h) than with FITC@AuNC probe ( 4 93% for 24 h, 4 75% for 48 h) under the same treatment concentration (Fig. S5C, SI). These results indicated that both FITC@AuNC and FA-FITC@AuNC probes is generally low-toxic for cellular imaging, possibly because of good biocompatibility of the surrounded biomolecule BSA. 3.4. FR-targeted imaging and cellular uptake of FA-FITC@AuNC The FR-targeted optical images of FA-FITC@AuNC and FITC@AuNC were obtained by using FR ? ve Hela cells and FR ? ve lung

carcinoma cells A549. Fig. 5 presents the overlap images of ?uorescent channel FRed (580 nm–680 nm) and bright ?eld of FR ? ve A549 cells and FR ? ve Hela cells, which were treated with FA-FITC@AuNC and FITC@AuNC. Hoechst 33342 staining was carried out to differentiate the nucleus from cytosol. It demonstrated that AuNCs probes were dispersed mainly near the nucleus of cells (Fig. S6, SI). The relative difference in the uptake of probes was measured by collecting the ?uorescence of FRed (Fig. 5C). There is no obvious difference between the consuming amount of FITC@AuNC probes into FR ? ve Hela cells (Fig. 5A1) and FR ? ve A549 cells (Fig. 5B1) after incubation of 2 h. It appeared to be no signi?cant staining or cellular uptake responding to FA-mediated process in the intracellular uptake process of FITC@AuNC probes. However, FR ? ve Hela cells took nearly three times of FA-FITC@AuNC probes (Fig. 5A2) as much as of FITC@AuNC probes (Fig. 5A1). As compared, FR ? ve A549 cells and 239 T cells took up less FA-FITC@AuNC than Hela cells did during the same culture time (Fig. S8, SI), which was corresponded with the lack of folate receptor on its cytomembrane. Furthermore, when Hela cells were pretreated with FA for 2 h, the uptake of FA-FITC@AuNC into Hela cells signi?cantly decreased (Fig. 5A3). These results clearly suggest that FA-FITC@AuNC were speci?cally taken up by Hela cells via a process corresponding to the FR on the surface of cells, and gathered near


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Fig. 5. Fluorescent microscopic images showing interaction of FA-FITC@AuNC and FITC@AuNC with (A) Hela cells and (B) A549 cells: (A1) FR ? ve Hela and (B1) FR ? ve A549 cells with FITC@AuNC, (A2) FR ? ve Hela and (B2) FR ? ve A549 with FA-FITC@AuNC of incubation for 2 h at 37 °C, and FR ? ve Hela cells incubated with FA-FITC@AuNC for 2 h after treated (A3) with FA for 2 h, (A4) at 4 °C, and (A5) 10 mM NaN3 for 2 h. (C) Cellular internalization amount of FA-FITC@AuNC and FITC@AuNC in different types of cell lines under various treatments. Error bar: standard error measurements (S.E.M.). Scale bar: 50 μm.

nucleus with the ?uorescence intensity remaining intact. When cells were cultured with probes at 4 °C (Fig. 5A4) or pretreated with NaN3 (Fig. 5A5) for 2 h to block the energy-dependent endocytosis, the consuming amount of probes were identically decreased (Fig. 5C). These results indicate that the uptake of FAFITC@AuNC probes were taken-up into Hela cells mainly via a pathway with folic-receptor involved and energy dependent. It also indicated the maintenance of the probes and its potential application for pH detection in the intracellular regions. Next, the process of cellular uptake of FA-FITC@AuNC probes into Hela cells were monitored by culturing Hela cells in serumfree media with 50 μg mL ? 1 FA-FITC@AuNC probes at 37 °C for 0.5 h, 1 h, 2 h, 4 h, and 24 h, respectively. The ?uorescence intensities from 510 to 550 nm (FGreen) and from 580 to 680 nm (FRed) were collected to indicate pH values in the cellular uptake process as well as the consuming amount of probes in cells. Fig. S7 (SI) showed that after 2 h incubation, the consuming of probes into cells reached maximum. Then, the amount of probes in cells

decreased rapidly and almost disappeared after 24 h incubation (Fig. S7A-E, SI). It indicates a quick metabolism as well as the low cytotoxicity of such probes in cells. Besides, it could be noticed that the probes were ?rstly gathered near cytomembrane (Fig. S7B, SI), then transferred near nuclear (Fig. S7D, SI). In this process, the emission of FGreen from FITC increased gradually with the color of the ?uorescent merged images turned from false red (Fig. S7B, SI) to yellow (Fig. S7D, SI). It suggests that the pH of the environment of probes increased among the probe moving from near-cytomembrane to near-nuclear region. Though the precise mechanism of FR transport of FA into cells remained unsolved, it is clear that folate conjugates were taken up nondestructively by mammalian cells via receptor-mediated endocytosis (Lu and Low, 2002; Rothberg et al., 1990a, 1990b; Turek et al., 1993). Such folate conjugates were observed to internalize endosomes after they bond to FR on the cancer cell surface (Turek et al., 1993; Varma and Mayor, 1998; Wu et al., 1997). It has also been reported that folate conjugate-containing endosomes have

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Fig. 6. (A–C) Confocal ?uorescence images of (A) FGreen (510–550 nm), (B) FRed (580–680 nm), and (C) overlapped ?uorescence and bright ?eld image of Hela cells incubated with FA-FITC@AuNC for 1 h excited by 488 nm; (D) Fluorescence emission scan from Hela cells with FA-FITC@AuNC probes. (E–G) Na ? –H ? exchange dependent pseudo color images (FGreen/FRed) of Hela cells with FA-FITC@AuNC probes stimulated by ouabain. (E) Hela cells incubated in Cl ? -free Ringer's solutions containing 0.1 mM ouabain for 45 min, followed by (F) treated in Na ? -free Ringer's solutions for another 5 min, and (G) adding 100 mM Na ? for another 5 min. (H) Bar graph representing FGreen/FRed. (I– L) Na ? –H ? exchange dependent pseudo color images (FGreen/FRed) of Hela cells with FA-FITC@AuNC probes stimulated by NH4Cl: (I) Hela cells are incubated in Cl ? -containing Ringer's solutions with 30 mM NH4Cl for 4 min, followed by (J) washing NH4Cl from the solution, and (K) adding Na ? -containing Ringer's solutions for another 2 min; (L) Bar graph representing FGreen/FRed. Values are the mean ratios generated from the intensities from ?ve randomly selected ?elds. Error bars represent standard error measurements (S.E.M.). Scale bar: 50 μm.

been shown to have pH values between 4.3 and 6.9 due to a process called endosome acidi?cation (Lee et al., 1996). As a targeted-introducer, FA acted a similar role in the uptake of FA-FITC@AuNC probe for the detection of intracellular pH. As a result, FAFITC@AuNC probe entered FR ? ve Hela cells speci?cally via a pathway corresponding to its rich folate receptors on the surface. Such process was energy-depended, and could be blocked by low temperature or the presence of NaN3. After being taken into the cells, the two emissions (FGreen and FRed) of probe acted differently when it transferred from the regions near cytomembrane to gather near the nuclear. In this transmission, the average ratio of two emission channel increased gradually, indicating an increase in the pH of the intracellular environment. 3.5. Bioimaging and biosensing of pH in cancer cells According to the ?uorescence images (Fig. 6A and B) and their overlay images with bright-?eld image (Fig. 6C), it was clear that the probe had good cell-permeability. The ?uorescence scan in Hela cells treated with FA-FITC@AuNC probes (Fig. 6D) con?rmed the present probes maintained the dual-emission in cellular environment, with two emissions centered at 520 nm and 640 nm. The emissions in cells had a little red-shift compared to that in PBS due to the various cellular environment and deviations from different detectors (Zhuang et al., 2014).

Next, FA-FITC@AuNC biosensor was used in the application of intracellular pH detection. The Na ? –H ? exchange has been a common mechanism for regulating cytosolic pH (Paradiso et al., 1984). Upon excitation at 488 nm, the ratio images of Hela cells treated with FA-FITC@AuNC were collected from two separated channels. By using a “pseudo color image” technology, the ratio value of the gray value of green channel and red channel was coded into different colors: red for the ratio of 0.1 and blue for the ratio of 1.5. After Hela cells were incubated in Cl ? -free Ringer's solutions containing 0.1 mM ouabain for 45 min, the average emission ratio FGreen/FRed is 1.061 7 0.191 (Fig. 6E), similar to that in Hela cells incubated without ouabain treatment (Fig. 6H). Then, the ratio slightly decreased to 1.008 7 0.265 when the cells were continuously incubated with a Na ? -free Ringer's solutions for another 5 min (Fig. 6H). The pseudo color clearly changed from blue to red (Fig. 6F). Such change indicated a rapidly acidi?cation of cells under stimulate of the Na ? -free Ringer's solutions. After adding another 100 mM Na ? solution, such ratio increased back to 1.057 7 0.149 (Fig. 6G and 6 H), suggesting a recover of pH in the intracellular environment. In another set, Hela cells with FA-FITC@AuNC were incubated in a Cl ? -containing Ringer's solution for 30 min, then treated with 30 mM NH4Cl for 4 min (Fig. 6I). The acid load was achieved by washing the NH4Cl from the solutions (Fig. 6J). After another stimulate by Na ? , cells would come alkalinized to a higher pH, with


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the pseudo-color image turned into blue (Fig. 6K). Such changes was monitored by using a mode of time scan (Fig. 6L). It could be noticed that after the remove of NH4Cl, the ratio value of FGreen and FRed channels decreased to 0.975 7 0.040, indicate the pH in cells decreased to ?6.5. As the addition of Na ? , the ratio value of two channels increased to 1.622 7 0.066 ?rstly, then recovered to 1.495 7 0.059. It showed that such stimulate will cause a sudden increase of pH in cells, then recovered after ?4 min. These initial experiments in Hela cells demonstrated that FA-FITC@AuNC probe could be used to observe the changes of pH in live cells through the ?uorescence ratio of different emission channels, suggesting the great potential of this dual-emission probe for further fundamental biology researches.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi: http://dx.doi.org/10.1016/j.bios.2014.10. 034

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4. Conclusions To conclude, a ratiometric ?uorescence biosensor for pH detection, FA-FITC@AuNC, has been developed successfully for speci?c bioimaging and biosensing in cancer cells at the same time. In this self-calibration biosensor, ?uorescent AuNCs work as reference signal, FITC plays the role for pH response and FA acts as speci?c recognition element for cancer cells target. The developed biosensor has been used to monitor pH gradients in a pH range of 6.0–7.8 with good sensitivity, high cyclic accuracy, and short response time. It also exhibits high selectivity over various metal ions and biological species. Furthermore, the AuNC-based inorganic-organic biosensor shows good biocompatibility and longterm stability against light illumination. As a result, the target imaging have been testi?ed by using FR ? ve Hela cells, FR ? ve A549 cells and FR ? ve 293 T cells, and achieved the biosensing of pH in Hela cells. It has been also con?rmed an acidi?cation process and its recovery in the transversion of FA conjugates entering into cancer cells. This investigation has provided a methodology to designing the ratiometric ?uorescent biosensor with targeted molecules, by conjugating different function units, such as targeted and detector molecules on one kind of speci?c nanomaterial. In addition, this work can be extended to construct future ratiometric ?uorescent biosensors for targeted imaging, drug deliver, or the detection of other biomolecules, such as metal ions, proteins, and other biological species.

Acknowledgments This work was ?nancially supported by the NSFC (21175098, and 21175044) and National Nature Science Fund for distinguished young scholars (21325521).