Distinguishing Folate-Receptor-Positive Cells from Folate-Receptor

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Distinguishing Folate-Receptor-Positive Cells from Folate-ReceptorNegative Cells Using a Fluorescence Off−On Nanoprobe Duan Feng, Yanchao Song, Wen Shi, Xiaohua Li, and Huimin Ma* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China S Supporting Information *

ABSTRACT: Based on the high affinity of folic acid (FA) for folate receptor (FR) that is overexpressed on the surface of many human cancer cells, we have developed a simple fluorescence nanoprobe (1) with multiple capability (fluorescence off−on response and cell-targeting ability) for imaging of FR-positive cells by covalently linking both FA and Rhodamine B (RB) to graphene oxide (GO) through disulfide bonds. The nanoprobe shows a weak fluorescence due to the electron transfer from GO to RB. However, the specific binding of FA to FR-positive cells leads to the internalization of the nanoprobe into the cells. As a result, the disulfide bonds of 1 are cleaved by intracellular glutathione, causing the release of the RB moiety from GO and thereby the generation of fluorescence. Compared to most of the reported fluorescence always-on nanoprobes for imaging FR-positive cells, the present fluorescence off-on nanoprobe can not only produce a high signal/ background ratio but also avoid the false positive results often caused by nonspecific adsorption of the always-on nanoprobes on the surface of nontarget cells. Notably, the proposed off−on nanoprobe has been demonstrated to distinguish the cells with different expression levels of FR by culturing and analyzing different cell mixtures (Hela/NIH-3T3 and Hela/MCF-7 cells). Moreover, the nanoprobe is capable of discriminating FR-positive from FR-negative cells even with similar morphology. This method is simple and selective for fluorescence imaging of FR-positive cells. fluorochrome, and its fluorescence signal is insusceptible to biological environments,12−14 which is desired for avoiding false positive results. Second, GO not only has good biocompatibility,15,16 but also is an efficient quencher for organic fluorochromes.17−23 Third, FA is a typical cell-targeting agent, because of its high affinity for folate receptor (FR) that is known to be overexpressed on the surface of many human cancer cells.24−27 Moreover, the level of FR appears to rise as the stage of the cancer increases, whereas, in normal cells, FR is only minimally distributed.28,29 This difference between cancer and normal cells allows to target cancer cells through detecting FR.3−9 On the other hand, FR can capture FA from the extracellular milieu and transport it inside the cell via an receptor-mediated endocytosis.30,31 The above facts, together with the known finding that disulfide bonds can be cleaved by glutathione (GSH; an intracellular predominant thiol),32 activate us to design a fluorescence nanoprobe with multiple capability (off−on response and targeting ability) for imaging of FR-positive cells. To the best of our knowledge, such an attempt has not been reported. Scheme 1 depicts our nanoprobe (1) and its fluorescence off−on response to FR-positive cells. The nanoprobe can be fabricated by treating GO with dichlorosulfoxide and cystamine successively, and then functionalizing the amino-coated GO

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evelopment of selective and sensitive methods for imaging cancer cells is of great importance for tumor diagnosis and therapy. Conventional methods such as computed tomography, magnetic resonance imaging, and radionuclide imaging, which are mainly suited for mature tissues, play a key role in this respect. For molecular or cellular imaging, however, fluorescence spectroscopy has a remarkable advantage, because of its high sensitivity and spatiotemporal resolution.1,2 Recently, nanomaterial-based fluorescence imaging has attracted much attention, which can be attributed to the fact that it is relatively convenient to endow a single nanoprobe with multiple capability. So far, several fluorescent nanoprobes have been elegantly designed for cancer cell imaging, for instance, by using quantum dots, gold nanoparticles, polymer, silicon, or carbon nanomaterials.3−10 Nevertheless, fluorescence signal from most of these systems is in an always-on state, which is not favorable to affording high signal/background ratio.11 Moreover, nonspecific adsorption of a fluorescence always-on nanoprobe on the surface of nontarget cells can lead to a false positive signal. In contrast, fluorescence off−on nanoprobes, whose fluorescence response is activated only upon binding to target cells, can overcome these problems, but such nanoprobes are still rare for imaging of cancer cells.3 Herein, we have developed a fluorescence off−on nanoprobe for this purpose by linking both Rhodamine B (RB) and folic acid (FA) to graphene oxide (GO) via disulfide bonds. In the nanoprobe, RB, GO, and FA are chosen as a signaling moiety, a quencher, and a cell-targeting moiety, respectively, based on the following considerations. First, RB is an excellent © 2013 American Chemical Society

Received: May 7, 2013 Accepted: June 10, 2013 Published: June 10, 2013 6530

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Preparation of FA-NHS. Similar to the reported procedure, 8 FA-NHS was prepared by introducing Nhydroxysuccinimide to FA. In brief, FA (5 g) was dissolved in 100 mL of DMSO containing 2.5 mL of triethylamine. To the resulting solution, N-hydroxysuccinimide (2.6 g) and dicyclohexylcarbodiimide (4.7 g) were added successively, and the reaction solution was stirred overnight at room temperature. The byproduct, dicyclohexylurea, was removed by filtration. The DMSO solution was then concentrated by evaporation under reduced pressure, followed by addition of ethyl ether (20 mL). The resulting precipitate, FA-NHS, was washed three times with ethyl ether, dried under vacuum, and stored as a yellow powder. Fabrication of Nanoprobe 1. GO was prepared and characterized following the reported procedure.33 The obtained GO was then treated by dispersing in water, followed by centrifugation at 14 000 rpm for 30 min. The supernatant was collected and lyophilized. The GO (5 mg) from the supernatant was suspended in N,N-dimethylformamide (1 mL) and refluxed with dichlorosulfoxide (5 mL) at 80 °C for 24 h, which could lead to the chloroformylation of carboxyl groups on GO. The reaction mixture was centrifugated, and the supernatant was decanted. The residue was washed three times with anhydrous tetrahydrofuran, followed by drying in vacuum at room temperature. The obtained chloroformylated GO (GO−COCl) was dispersed in dichloromethane, and then added dropwise to the dichloromethane solution of cystamine that was freshly prepared by neutralizing cystamine dihydrochloride (16 mg) with 4 M NaOH and then extracting with dichloromethane. The reaction mixture was stirred at room temperature for 4 h. After centrifugation, the supernatant was discarded and the residue was washed three times with dichloromethane. Then, the residue was dried under N2 gas, yielding the amino-coated GO (GO−CO−NH−CH2−CH2− S−S−CH2−CH2−NH2; abbreviated as GO−S−S−NH2). To the suspension of GO−S−S−NH2 in 0.2 M Na2CO3 (pH 9.0), Rhodamine B isothiocyanate (1.0 mg) and the above prepared FA-NHS (1.0 mg) were added. After stirring for 24 h at room temperature, the reaction mixture was centrifuged, and the residue was further purified by washing several times with water until the supernatant was almost colorless. The purified product (FA−CH 2 −CH 2 −S−S−CH 2 −CH 2 −NH−CO−GO−CO− NH−CH2−CH2−S−S−CH2−CH2−RB; abbreviated as nanoprobe 1) could be dispersed in water for use. The same procedure was applied to preparing other reference/control probes. (Note that the preparation of GO−S−S−RB and GO− S−S−FA is through the reaction of GO−S−S−NH2 with Rhodamine B isothiocyanate and FA-NHS, respectively; reference nanoprobe FA−CH 2 −CH 2 −CH 2 −CH 2 −CH 2 − CH 2 −NH−CO−GO−CO−NH−CH 2 −CH 2 −CH 2 −CH 2 − CH2−CH2−RB, abbreviated as FA−C−C−GO−C−C−RB, was obtained by replacing cystamine with hexamethylene diamine.) Calculation of the Amounts of RB and FA Conjugated to Nanoprobe 1. The amounts of RB and FA conjugated to per gram of GO in nanoprobe 1 were calculated based on the additivity of absorbance of RB and FA, which produce the characteristic absorption bands at 550 and 350 nm, respectively. The following equation set is used to calculate the conjugated amounts of RB and FA:

Scheme 1. Fluorescence Off−On Response of Nanoprobe 1 to FR-Positive Cell

with both Rhodamine B isothiocyanate and FA-NHS (reaction product of FA with N-hydroxysuccinimide8). The nanoprobe itself should have no or weak fluorescence due to the quenching effect of GO on the fluorochrome of RB. As can be seen from Scheme 1, however, the high affinity of FA for FR leads to the specific binding of the nanoprobe to the FRpositive cell, followed by the internalization of 1 into the cell through the receptor-mediated endocytosis. As a result, the disulfide bonds of the probe are cleaved by intracellular GSH, causing the release of the RB moiety from GO and thereby the generation of fluorescence.

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EXPERIMENTAL SECTION Reagents. Graphite flake-325 mesh, GSH, cysteine (Cys) and homocysteine (Hcy) were purchased from Alfa Aesar. Rhodamine B isothiocyanate, cystamine dihydrochloride, bovine serum albumin, and human serum albumin were obtained from Sigma−Aldrich. N-Ethylmaleimide was purchased from Acros Organics. CaCl2, NaCl, KCl, MgCl2, Na 2HPO 4, NaH 2 PO4 , glucose, Vitamin B, Vitamin C, glutamine, serine, and arginine were obtained from Beijing Chemicals, Ltd. N-Hydroxysuccinimide, FA and dimethylsulfoxide (DMSO) were obtained from J&K Chemical, Ltd. Dulbecco’s modified eagle media (DMEM), fetal bovine serum (FBS), penicillin (100 μg/mL), and streptomycin (100 μg/mL) were obtained from Invitrogen Co. A phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4) solution was obtained from Invitrogen Co. Ultrapure water (over 18 MΩ·cm) from a Milli-Q reference system (Millipore) was used throughout. Other reagents employed were all of analytical grade and were used without further purification. Apparatus. A Hitachi Model F-2500 spectrofluorimeter was used for fluorescence measurements. UV−vis spectra were recorded in 1 cm quartz cells with a Model TU-1900 spectrophotometer (Beijing, PRC). Atomic force microscopy (AFM) images were recorded with a Nanoscope IIIa instrument (Digital Instruments) operating in tapping mode, and the samples were prepared by depositing a diluted water dispersion of GO on a freshly cleaved mica surface and then allowing it to dry under ambient conditions. Fluorescence imaging experiments were performed on a Model FV 1000IX81 confocal laser scanning microscope (Olympus) with FV5LAMAR for excitation at 515 nm and a variable bandpass emission filter set to 550−650 nm through a 100 × 1.4 NA objective. Optical sections were acquired at 0.8 μm.

A350nm = εFA350 × C FA + εRB350 × C RB 6531

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tration) of GSH were first examined in PBS (pH 7.4) at 37 °C. As shown in Figure S4 in the Supporting Information, 1 mM GSH leads to a large fluorescence increase, suggesting that GSH at intracellular levels can efficiently cleave the disulfide bonds of 1, turning on the fluorescence signal. In contrast, GSH at extracellular levels causes a small enhancement in fluorescence, which means that nanoprobe 1 only generates a low background signal. This is rather desirable for sensitive imaging. To optimize the reaction conditions, effects of pH and time were studied on the fluorescence of 1 reacting with GSH (see Figure S5 in the Supporting Information). The results showed that the nanoprobe can function in the physiological pH range of pH 6.5−8.5 (see Figure S5A in the Supporting Information), and its fluorescence signal increases with the reaction time but reaches a plateau within ∼2.5 h (Figure S5B in the Supporting Information). For the purpose of reproducibility, a reaction time of 3 h was employed in our experiments. Moreover, the fluorescence of nanoprobe 1 without GSH (control experiment) hardly changes during the same period of time, which suggests that the system is stable. To confirm that the increase in fluorescence intensity was due to the cleavage of disulfide bond by a thiol, the responses of nanoprobe 1 to some common intracellular biothiols, such as homocysteine and cysteine at a concentration of 1 mM, were also investigated under the same conditions. As shown in Figure S6 in the Supporting Information, both homocysteine and cysteine induce the enhancement of fluorescence intensity of nanoprobe 1 as in the case of GSH, indicating that the enhanced fluorescence indeed arises from the action of thiols. The different increases in fluorescence may be ascribed to the different reactivity of the thiols.39 It should be pointed out that intracellular concentrations of homocysteine and cysteine (μM levels)36 are much lower than that of GSH (mM levels), suggesting that the cleavage reaction of 1 mainly depends on intracellular GSH. Moreover, a reference nanoprobe, FA−C−C−GO−C−C− RB, in which the disulfide bond (S−S) in nanoprobe 1 was replaced by alkane bond (C−C), was synthesized and examined to further prove that the fluorescence enhancement was caused by the disulfide bond cleavage other than any other mechanism. As depicted in Figure 1, reaction of FA−C−C−GO−C−C−RB with GSH under the same conditions produces a rather small increase in fluorescence. Other two reference nanoprobes, GO−S−S−RB and GO−S−S−FA that were functionalized with RB and FA through the disulfide bond linkage, respectively, were also synthesized. In the presence of GSH, GO−S−S−RB generates a large fluorescence enhancement, whereas GO−S−S−FA does not. The above findings clearly support that the fluorescence increase is due to the cleavage of disulfide bond, and RB acts as a signal unit. Potential interfering substances, such as metal ions, glucose, nonthiol amino acids, vitamins, human serum albumin, and bovine serum albumin, were examined in parallel under the same conditions to assess the selectivity of nanoprobe 1 for GSH. The results showed that GSH reacted with 1 producing the strongest fluorescence compared to the other substances (see Figure 2). The fluorescence enhanced by GSH is at least 5 times higher than those by the other substances tested. This indicates that the constructed nanoprobe possesses a high selectivity for GSH, which is attributed to the specific cleavage of the disulfide bond by a thiol.

A550nm = εFA550 × C FA + εRB550 × C RB

where A is the absorbance of nanoprobe 1 at different wavelengths; ε and C are the molar absorptivity and the concentration of a related substance, respectively. All absorbance measurements were performed in a quartz cell with an optical length of 1 cm. Fluorescence Response of Nanoprobe 1 to GSH. Unless otherwise stated, fluorescence responses of 1 (100 μg/ mL) to GSH with different concentrations at λex = 520 nm were performed in 0.1 M PBS (pH 7.4) at 37 °C for 3 h. Cell Imaging. Hela, MCF-7 and NIH-3T3 cells used in this study were cultured in DMEM containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin−streptomycin at 37 °C in a 5% CO2 incubator. For fluorescence imaging, the adherent cells grown on glass-bottom culture dishes (MatTek Co.) were incubated with nanoprobe 1 (100 μg/mL) in 1 mL of culture media at 37 °C for 3 h. Then, cell imaging was carried out after washing the cells thoroughly with 0.1 M PBS solution (pH 7.4). For fluorescence imaging of a cell mixture of Hela and NIH3T3 cells, Hela cells were first mixed with NIH-3T3 cells in an almost-equal concentration of ∼106 cells/mL. After coculturing for 12 h, the cell mixture was then incubated with nanoprobe 1 (100 μg/mL) at 37 °C for 3 h. For fluorescence imaging of a cell mixture of Hela and MCF-7 cells (the two types of cells have similar morphology), Hela cells were first stained by Hoechst 33324 (6 μg/mL) at 37 °C for 1 h, and then washed three times with FBS-free DMEM, followed by the same co-culturing and fluorescence imaging operations as described above.

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RESULTS AND DISCUSSION Preparation and Spectroscopic Properties of 1. To construct the nanoprobe, GO was first prepared following the known procedure,33 and characterized by atomic force microscopy (AFM). As shown in Figure S1A in the Supporting Information, the thickness of GO is ∼1.0 nm and its lateral dimension can be as large as ∼600 nm. Considering the fact that the smaller-sized nanomaterials are easier to enter the cells and distribute uniformly,13 the above GO was further treated by dispersion in water and then centrifugation under 14 000 rpm for 30 min. The smaller size of GO, which can be obtained by lyophilizing the supernatant, was collected for use. As shown in Figure S1B in the Supporting Information, the lateral dimension of the as-obtained GO is not more than 200 nm. Then, the above two sizes of GO were incubated with Hela cells for 3 h under the same conditions for differential interference contrast (DIC) imaging analysis, which reveals that the larger size of GO aggregates occasionally on the surface of the cells as black dots (see Figure S2 in the Supporting Information). Similar phenomena can be found in previous literature.34,35 This may be attributed to the difficult entrance of the larger size of GO into cells. Therefore, the smaller size of GO was used to prepare the nanoprobe in our study, and the amounts of RB and FA conjugated to per gram of GO in nanoprobe 1 were calculated to be ∼9.1 and 22 μmol, respectively, based on the additivity of absorbance from each component (see Figure S3 in the Supporting Information). It is known that the concentration of intracellular GSH is between 1 mM and 10 mM, whereas that of extracellular GSH is between 1 μM and 10 μM.36−38 Thus, the fluorescence responses of nanoprobe 1 to 1 mM (the lowest intracellular concentration) and 10 μM (the highest extracellular concen6532

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cancer cell membrane and can effectively deliver the species bearing FA into the cells via the receptor-mediated endocytosis. It should be pointed out that the fluorescence of nanoprobes in cells, like the 1-loaded Hela cells here, is usually not as uniform as that of small molecular probes. To verify whether the entrance of 1 into the cells is via the receptor-mediated endocytosis, different competition experiments were conducted (see Figure S8 in the Supporting Information). On one hand, GO−S−S−RB (a reference nanoprobe) was incubated with Hela cells under the same conditions, and the as-treated Hela cells only show a weak fluorescence, with respect to the 1-loaded cells (compare Figures S8a and S8c in the Supporting Information), indicating that GO−S−S−RB lacking the targeting moiety of FA is difficult to enter the cells. On the other hand, Hela cells pretreated with saturated free FA and then incubated with nanoprobe 1 also display a weak fluorescence (see Figure S8b in the Supporting Information), because free FA can bind to FR, thus decreasing the FR availability on the cell membrane and the internalization of 1. Both of the above results support that nanoprobe 1 is internalized into Hela cells through the receptor-mediated endocytosis, and nanoprobe 1 conjugated with FA possesses the capability for targeting FR-positive cells. To validate that the enhanced fluorescence in cells arises from the cleavage of disulfide bond by GSH, the uncleavable FA−C−C−GO−C−C−RB and the cleavable 1 were further compared for imaging of Hela cells. As shown in Figure S9 in the Supporting Information, the fluorescence signal of the Hela cells incubated with FA−C−C−GO−C−C−RB (lacking disulfide bond) remains off (see Figure S9a in the Supporting Information), but that of the 1-loaded Hela cells is turned on (Figure S9d in the Supporting Information). In addition, further research was conducted by pretreating Hela cells with different concentrations of N-ethylmaleimide, a thiol blocking reagent.36 As shown in Figure S9b in the Supporting Information, Hela cells treated with 1 μM of N-ethylmaleimide produce faint fluorescence, and this fluorescence can be further decreased (Figure S9c in the Supporting Information) by a higher concentration of N-ethylmaleimide (10 μM). These findings further verify that the fluorescence enhancement of nanoprobe 1 in Hela cells is due to the thiol-induced disulfide bond cleavage and the release of the RB moiety from GO. Distinguishing FR-Positive from FR-Negative Cells by Nanoprobe 1. To demonstrate the applicability of 1 for selectively targeting FR-positive cells, a comparative study was made with both FR-positive cells (Hela cells) and FR-negative cells (MCF-7 and NIH-3T3 cells). As depicted in Figure 3, Hela cells exhibit much stronger fluorescence than MCF-7 or NIH-3T3 cells, and the relative pixel intensity from Hela cells is ∼6.7 and 16.4 times higher than that from MCF-7 and NIH3T3 cells, respectively, clearly indicating that 1 can serve as a cell-targeting fluorescence nanoprobe to distinguish the cells with different expression levels of FR. The potential of nanoprobe 1 for distinguishing cancer cells from normal cells was examined by culturing and analyzing a model cell mixture of NIH-3T3 and Hela cells. These two types of cells are chosen because of their identifiable morphology.8 As depicted in Figure 4A, Hela cells show bright fluorescence, whereas NIH-3T3 cells produce negligible fluorescence. This difference in fluorescence obviously results from the fact that NIH-3T3 cells lack FR, while Hela cells overexpress FR, thereby suggesting that nanoprobe 1 is suited for simple discrimination of FR-positive cancer cells from normal cells.

Figure 1. Fluorescence responses of GO−S−S−FA, FA−C−C−GO− C−C−RB, GO−S−S−RB, and nanoprobe 1 (each 100 μg/mL) to 1 mM GSH. ΔF is the difference of fluorescence intensity of a probe with and without GSH. The data represent the mean ± standard deviations (n = 3). Stronger fluorescence of reference probe GO−S− S−RB than 1 is due to its higher content (23 μmol) of RB modified on per gram of GO. λex/em = 520/575 nm.

Figure 2. Fluorescence responses of nanoprobe 1 (100 μg/mL) to various substances: KCl (150 mM), CaCl2 (1 mM), MgCl2 (2.5 mM), glucose (10 mM), glutamine (1 mM), serine (1 mM), arginine (1 mM), vitamin B (1 mM), vitamin C (1 mM), human serum albumin (HSA, 100 nM), bovine serum albumin (BSA, 100 nM), and GSH (1 mM). The data represent the mean ± standard deviation (n = 3). λex/em = 520/575 nm.

Receptor-Mediated Endocytosis of Nanoprobe 1. The uptake of nanoprobe 1 by cells was investigated by confocal laser scanning microscopy, and Hela cells were used as model cancer cells because they overexpress FR.8 As shown in Figure S7 in the Supporting Information, with the increase of incubation time from 0 to 6 h, the fluorescence of Hela cells gradually increases, and after 3 h this increase becomes insignificant. This suggests that nanoprobe 1 can enter Hela cells and its disulfide bond is almost cleaved by intracellular GSH in 3 h, which is consistent with the result of Figure S5B in the Supporting Information in vitro. In other words, the uptake of nanoprobe 1 by Hela cells is a fast process. On the other hand, the 1-loaded Hela cells show bright fluorescence on both the membrane and the cytoplasm, but not on the nucleus, which is also rich in GSH. This indicates that nanoprobe 1 can reside in the membrane and enter the cytoplasm rather than the nucleus, which accords with the fact that FR mainly exists in the 6533

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Figure 3. (A) Confocal fluorescence images of different cells: (a) NIH-3T3, (b) MCF-7, and (c) Hela cells. The cells were incubated with nanoprobe 1 (100 μg/mL) at 37 °C for 3 h. The DIC images of the corresponding samples are shown at the bottom. Scale bar = 20 μm. (B) Relative pixel intensity obtained from the corresponding fluorescence images of panel (A) with ImageJ software. The pixel intensity of at least 2 cells was averaged, and the pixel intensity from Hela cells is defined as 1.0.

Figure 4. Fluorescence images of different cell mixtures. (A) The cell mixture of NIH-3T3 and Hela cells incubated (a) without (control) and (b) with nanoprobe 1 (100 μg/mL) for 3 h. The DIC images of the corresponding samples are shown in images c and d, where the white arrows indicate NIH-3T3 cells. (B) The cell mixture of MCF-7 and Hoechst 33324-stained Hela cells was incubated without nanoprobe 1 (images a and b; controls) and with nanoprobe 1 (100 μg/mL) (images d and e) for 3 h. Images a and d were obtained at λex = 405 nm; images b and e were obtained at λex = 515 nm. The DIC images of the corresponding samples are shown on the right (images c and f). The yellow arrows indicate Hela cells and red arrows indicate MCF-7 cells. Scale bars = 20 μm.

Differentiation of tumor cells with similar morphology by common optical microscopes is usually difficult, and solving this problem is very helpful for cancer diagnosis. Toward this end, the capability of nanoprobe 1 to distinguish FR-positive from FR-negative cancer cells with similar morphology was also evaluated. In this experiment, another cell mixture of MCF-7 and Hela cells, which have similar morphology but different expression levels of FR, was cultured and analyzed. In order to conveniently identify the two types of cells in cell imaging, Hela cells were pretreated with Hoechst 33324 (a fluorescent dye for nuclei; λex = 405 nm) for 30 min, and then co-cultured with MCF-7 cells for 12 h. The as-obtained cell mixture was incubated with nanoprobe 1 for 3 h and then subjected to fluorescence imaging. As shown in Figure 4B, Hoechst 33324stained Hela cells show strong fluorescence in their nuclei at λex = 405 nm, whereas MCF-7 cells do not (images a and d). This staining treatment of only Hela cells, together with the DIC results (images c and f), enables us to easily identify the two types of cells that have similar morphology. Images b and e in Figure 4B are from the cell mixture that was treated without and with nanoprobe 1. As is seen, both types of cells themselves (control) hardly produce fluorescence at λex = 515 nm (image b), showing a low background signal. After incubation with 1, however, Hela cells exhibit intense fluorescence, but MCF-7 cells scarcely generate fluorescence (image e). These observations indicate that the proposed fluorescence nanoprobe is capable of distinguishing FR-positive from FR-negative cells.

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CONCLUSIONS In summary, we have developed a cell-targeting fluorescence off−on nanoprobe, which can be used to distinguish the cells with different expression levels of FR. This applicability has been confirmed by culturing and analyzing different cell mixtures (Hela/NIH-3T3 and Hela/MCF-7 cells). Moreover, the nanoprobe is capable of discriminating FR-positive from FR-negative cells with similar morphology, and this capability has been proved by the staining experiments of nuclei. We expect that the proposed nanoprobe may be of potential for cancer diagnostic studies.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-10-62554673. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 6534

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ACKNOWLEDGMENTS We are grateful to the financial support from the NSF of China (Nos. 21275147 and 20935005), the Ministry of Science and Technology (No. 2011CB935800), and the Chinese Academy of Sciences (No. KJCX2-EW-N06-01).

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dx.doi.org/10.1021/ac401377n | Anal. Chem. 2013, 85, 6530−6535