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Self-Assembly of Folate onto Polyethyleneimine Coated CdS/ZnS Quantum Dots for Targeted Turn-On Fluorescence Imaging of Folate Receptor Over-Expressed Cancer Cells Yi Zhang, Jing-Min Liu, and Xiu-Ping Yan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac3025653 • Publication Date (Web): 30 Nov 2012 Downloaded from http://pubs.acs.org on December 4, 2012

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Analytical Chemistry

Self-Assembly

of

Folate

onto

Polyethyleneimine

Coated

CdS/ZnS Quantum Dots for Targeted Turn-On Fluorescence Imaging of Folate Receptor Over-Expressed Cancer Cells Yi Zhang,†, ‡ Jing-Min Liu,† and Xiu-Ping Yan*,† †

State Key Laboratory of Medicinal Chemical Biology, and Research Center for

Analytical Sciences, College of Chemistry, Nankai University, Tianjin 300071, China ‡

Department of Medical Chemistry, College of Pharmacy, Tianjin Medical

University, 22 Qixiangtai Road, Tianjin 300070, China *Corresponding author. Fax: (86)22-23506075. E-mail: [email protected]

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ABSTRACT: Folate receptor (FR) can be over-expressed by a number of epithelial-derived tumors, but minimally expressed in normal tissues. As folic acid (FA) is a high affinity ligand to FR, and not produced endogenously, development of FA conjugated probes for targeted imaging FR over-expressed cancer cells is of significance for assessing cancer therapeutics and for better understanding the expression profile of FR in cancer. Here we report a novel turn-on fluorescence probe for imaging FR over-expressed cancer cells. The probe was easily fabricated via electrostatic self-assembly of FA and polyethyleneimine-coated CdS/ZnS quantum dots (PEI-CdS/ZnS QDs). The primary fluorescence of PEI-CdS/ZnS QDs turned off firstly upon the electrostatic adsorption of FA onto PEI-CdS/ZnS QDs based on electron transfer to produce negligible fluorescence background. The presence of FR expressed on the surface of cancer cells then made FA desorb from PEI-CdS/ZnS QDs due to specific and high affinity of FA to FR. As a result, the primary fluorescence of PEI-CdS/ZnS QDs adhering to the cells turned on due to the inhibition of electron transfer. The most important merits of the developed probe are its simplicity and the effective avoidance of the false positive results due to the simple electrostatic self-assembly of FA onto the surface of PEI-CdS/ZnS QDs and the involved fluorescence “off-on” mechanism. The probe was demonstrated to be sensitive and selective for targeted imaging of FR over-expressed cancer cells in turn-on mode.

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Folate

receptor

(FR),

a

folate-binding

protein

with

a

naturally

38-kDa

glycol-polypeptide, can be over-expressed by a number of epithelial-derived tumors, including malignancies of the ovary, mammary gland, lung, kidney, testis, prostate and hematopoietic cells of myelogenous origin, but minimally expressed in normal tissue.1-3 As folic acid (FA) is a high affinity ligand to FR and not produced endogenously, FA conjugated probes for the over-expression of FR in cancer cells have emerged rapidly in the filed of targeted imaging and therapeutic agents delivery.4-11 However, the necessity for covalent conjugation of FA onto reporters or carriers makes the preparation of these probes time- and labor-consuming for in vitro targeting. Quantum dots (QDs) as promising fluorescence probes, possess many advantages over conventional organic fluorophores, such as high photoluminescence efficiency, great photostability, size-dependent emission wavelength, and sharp emission profile.12-14 QDs have been widely explored as fluorescent markers or reporters for FR positive (FR+) cells, for example, by conjugating FA,7,15-18 FA-cyclodextrin,19 FA-lipid,20,21 FA-BSA,22 FA-gelatine,23 FA-poly(lactide)-VE-TPGS,24 FA-PAMAM25 and FA-PEG-PAMAM26 onto QDs to target FR+ tumor cells. In the above mentioned applications, functionalized QDs could probe, illuminate or even penetrate the targeting cells. However, several hours are always needed for FR+ cells to endocytose the modified QDs to show obvious difference from negative control.4-10 Furthermore, the above modified QDs are always bright, thus it is compelled to wash the detecting objects for several times to avoid false positive phenomenon. In spite of washing for three times, the fluorescence of QDs on the surface of FR negative cells or background

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is still be noticeable due to unspecific endocytosis. For these reasons, an “on-off” or “off-on” switch system should be more suitable and reliable for the QDs based fluorescence probe.27-30 Herein we report a novel fluorescence turn-on probe for targeted imaging FR over-expressed cancer cells based on the self-assembly of FA and polyethyleneimine (PEI)-coated CdS/ZnS QDs (PEI-CdS/ZnS QDs). The primary fluorescence of PEI-CdS/ZnS QDs turns off firstly upon the electrostatic adsorption of FA onto PEI-CdS/ZnS QDs based on electron transfer to produce a probe with negligible fluorescent background. The competition of FR expressed on the surface of cancer cells enables FA desorption from PEI-CdS/ZnS QDs. Thus, the electron transfer between FA and PEI-CdS/ZnS QDs is inhibited, and the primary fluorescence of PEI-CdS/ZnS QDs adhering to the cells via electrostatic interaction between PEI and phospholipids on cell membrane is recovered. In this way, FR over-expressed cells are illuminated. The principle and application of the developed fluorescence turn-on probe for imaging FR over-expressed cancer cells are illustrated in Scheme 1. The most important merits of this probe are its simplicity and the effective avoidance of the false positive results due to the simple electrostatic self-assembly of FA onto the surface of PEI-CdS/ZnS QDs and the fluorescence “off-on” mechanism involved.

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Scheme 1. Schematic Illustration for the Developed Fluorescence Turn-on Probe for Imaging FR Over-expressed Cancer Cells

EXPERIMENTAL SECTION Chemicals and Materials. All chemicals used were of at least analytical grade. Ultrapure water (18.2 MΩ cm) obtained from a WaterPro water purification system (Labconco Corporation, Kansas City, MO, USA) was used throughout. CdCl2⋅2.5H2O, ZnSO4⋅7H2O,

Na2S⋅9H2O,

tris(hydroxymethyl)aminomethane,

boric

acid,

Na2HPO4⋅12H2O, NaH2PO4⋅2H2O, HCl, NaOH, KCl and NaCl were from Guangfu Fine Chemical Research Institute (Tianjin, China). PEI (branched, M.W. 10000, 99%) and FA was from Alfa Aesar (Tianjin, China). All the amino acids, ascorbic acid, glucose, citric acid and oxalic acid were from Beijing Newprobe Biotechnology Co. Ltd. (Beijing, China). Dialysis membrane tubing with a molecular weight cutoff of 12000-14000 (Dingguo Biotech Co. Ltd., Beijing, China) was used for dialysis experiments. 4’,6-diamidino-2-phenylindole (DAPI) and folate receptor 2 (FR2, human) as a typical FR were from Sigma-Aldrich (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 medium, trypsin and

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FBS of Invitrogen were purchased from Life Tech. Co. (Carlsbad, CA, USA). The phosphate buffer solution (PBS) (0.01 M) was made up of 8.0 g NaCl, 0.2 g KCl, 3.63 g Na2HPO4·12H2O and 0.24 g KH2PO4 in 1 L ultrapure water and adjusted to a desired pH for direct use. Instrumentation. The morphology of dialyzed QDs was characterized on a JEOL 100 CXII transmission electron microscope (TEM) operating at a 200 kV accelerating voltage. Absorption spectra were recorded on a Shimadzu UV-3600 UV-vis-NIR spectrophotometer. Fourier transform-infrared (FT-IR) spectra were recorded with a Nicolet MAGNA-560 FT-IR spectrometer. The steady-state fluorescence and resonance light scattering (RLS) experiments were performed on a Hitachi FL-4500 spectrofluorometer equipped with a plotter unit and a quartz cell (1 cm × 1 cm). In all the tests of steady-state fluorescence, λex of 350 nm, delay of 0 s, slit (ex/em) of 5 nm/10 nm, PMT voltage of 700 V and response of 0.5 s were adopted. The concentration of cadumium was determined with Thermo Elemental X Series inductively coupled plasma mass spectrometer (ICPMS). The quantum yield of the QDs was measured on an Edinburgh FLS920 spectrometer with an integrating sphere attachment under excitation of 360 nm. The fluorescent decay lifetime of the QDs was measured on a PTI spectrometer under excitation of 334 nm laser. The fluorescence imaging was taken on a Nicon ECLIPSE TE 2000-U inverted fluorescence microscope system equipped with a motorized stage and recorded with a high-definition cooled color matrix CCD (DS Fi1c, 5-Megapixel, Nikon, Japan). Determination of the zeta potential (ζ) of PEI (2 mg L-1), FA (20 µM), PEI-CdS/ZnS (500× dilution of the stock

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solution), and FA-assembled PEI-CdS/ZnS (20 µM of FA and 500× dilution of the stock solution) in PBS solution at pH 7.4 was performed on a Malvern Zetasizer 3000HSa (He-Ne laser, λ=632.8 nm). The X-ray photoelectron spectroscopy (XPS) measurements were carried out on a 5000 VersaProbe spectrometer (ULVAC-PHI, Japan) fitted with a monochromatic Al Kα X-ray source (1486.7 eV). Synthesis and Purification of PEI-CdS/ZnS QDs. The PEI-CdS/ZnS QDs were synthesized according to a modified procedure.31, 32 Briefly, 280 mg of PEI, 1 mmol of CdCl2, and 0.5 mmol of Na2S were dissolved in 47.5 mL of ultrapure water in a 100-mL flask. After the mixture was heated to 60 oC for 15 min, ZnSO4 (2.5 ml, 0.08 M) solution was then dropwise added and the temperature was maintained at 60 oC for 20 min. The mixture was then cooled down to room temperature, and dialyzed using the dialysis membrane tubing with a molecular weight cutoff of 12000-14000 against 250 mL PBS (0.01 M, pH 7.4) for 1 h six times to remove all the free PEI (M.W. 10000). The Cd2+ and Cd-PEI complex in the dialysate were monitored by ICPMS and UV-vis spectrophotometry, respectively (Figure S1 in the Supporting Information). After dialysis, the purified QDs were diluted to 100 mL with the PBS (0.01 M, pH 7.4) as the PEI-CdS/ZnS QDs stock solution for further application. For characterization, the purified QDs were droped onto supporting film for TEM measurement, or evaporated to dry powder for FT-IR measurement. Fabrication of FA Assembled PEI-CdS/ZnS QDs. 2 µmol of FA was added into 2 mL of the PEI-CdS/ZnS QDs stock solution. The mixture was set for 10 min and dialyzed against PBS (0.01 M, pH 7.4) for 30 min twice to remove excessive FA. The

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purified solution of the FA assembled PEI-CdS/ZnS QDs was diluted to 4 mL with PBS (0.01 M, pH 7.4) for further experiments. Cell Culture. The Hela human cancer of cervix cell line (#CCL-2), HepG2 human hepatic carcinoma cell line (#HB-8065) and A549 human lung carcinoma cell line (#CCL-185) were obtained from American Type Culture Collection. The Hela and HepG2 cells were cultured in Corning-culture dishes in DMEM medium with 10% FBS, while A549 cells in RPMI-1640 medium with 10% FBS at 37 oC and 5 % CO2 to 70-80 % confluency. The cells were transferred into 6-well plates, cultured for 24 h, washed with 5 mL of PBS (0.01 M, pH 7.4) twice to ensure no dead cells and dissociative FA left, then kept in 2 mL of PBS (0.01 M, pH 7.4) prior to imaging experiments. Cell Imaging. Before imaging, the amount of FA assembled PEI-CdS/ZnS QDs and the incubation time were optimized by measuring the fluorescence recovery induced by cells on a Hitachi FL-4500 spectrofluorometer. In a typical imaging test, 0.1 mL of the FA assembled PEI-CdS/ZnS QDs was added into each well of the cells. Fluorescence imaging under blue light excitation was carried out after 10-min incubation and no washing steps were needed. A 20× objective was used to collect the fluorescence (4× for the negative control photoes) and the excitation light intensity was set up as maximum. The aperture stop was 70% of NA and the fluorescence shutter totally opened. The intensity of fluorescence source, the sensitivity of the camera, and the time of exposure were always the same for each picture. Furthermore, in order to illustrate the proportion of targeted imaging, the cell nuclei were further stained by DAPI. After the 10-min incubation with the QDs, the cells were washed by PBS (pH 7.4) twice, fixed by cold

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paraformaldehyde (4%) for 20 min, washed by PBS, stained by DAPI (0.1 mg L-1) for 10 min, washed by PBS, and finally kept in PBS for imaging. RESULTS AND DISCUSSION Characterization of PEI-CdS/ZnS QDs. The TEM image reveals spherical PEI-CdS/ZnS QDs with the diameter of 41.2 ± 5.5 nm (Inset of Figure 1A). The PEI-CdS/ZnS QDs shows the absorption spectra with a characteristic absorption shoulder peak at ~350 nm and the fluorescence emission spectra with a maximum emission peak at ~500 nm (Figure 1B). Comparison of the XRD patterns of PEI-CdS and PEI-CdS/ZnS QDs shows a shift of the peaks in CdS/ZnS pattern, especially the (111) peaks toward larger angles (Figure 1C), revealing the transformation of a CdS lattice to a ZnS lattice during the formation of the ZnS shell, and confirming the formation of the CdS/ZnS heterostructure. In addition, the significantly broad peaks indicate the formation of Zn and Cd sulfide crystals of reduced sizes down to the nanoscale. FT-IR spectra of the resultant PEI-CdS/ZnS QDs in Figure 1D show six characteristic absorption bands of PEI (3420 cm−1 for N–H stretch, 2930 and 2870 cm−1 for C-H stretch, 640 cm−1 for N–H bend, 1460 cm-1 for C-H bend and 1110 cm−1 for C–N stretch), confirming the capping of PEI on the surface of CdS/ZnS QDs. In addition, both shape changes and wavelength shift in peaks of N–H stretch and N–H bend indicate a strong interaction between amino-groups of PEI and Cd or Zn. The N1s XPS peak corresponding to N-Cd/N-Zn with binding energy of 399.2 eV (Figure 1E and 1F) also proves the chemical bond between PEI and Cd or Zn. The zeta potentials for PEI, FA, PEI-CdS/ZnS, and FA-assembled PEI-CdS/ZnS were +21.8 mV, -43.9 mV, 9



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+24.7 mV, and +3.23 mV, respectively, implying the combination between PEI and QDs and also the adsorption of FA onto PEI-CdS/ZnS QDs.

Figure 1. (A) TEM image and (B) UV-vis absorption spectra and fluorescence emission spectra of PEI-CdS/ZnS QDs, (C) XRD patterns of PEI-CdS and PEI-CdS/ZnS QDs, (D) FT-IR spectra of PEI and PEI-CdS/ZnS QDs, (E) typical survey spectra and (F) the N1s spectra peak fitting from XPS data. R2 of the peak fitting is 0.9996. The fluorescence stability of PEI-CdS/ZnS QDs was studied against both pH and storage time. The PEI-CdS/ZnS QDs gave maximal emission at pH 7.0-7.6 in PBS (0.01

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M) (Figure 2A). Moreover, the fluorescence of PEI-CdS/ZnS QDs in PBS (0.01 M, pH 7.4) was stable without shift in the emission maximum during one month storage at ambient temperature and in room-light, indicating no self-aggregation and photo-oxidization of the PEI-CdS/ZnS QDs (Figure 2B). In addition, the PEI-CdS/ZnS QDs were also stable in several distinct buffers such as borate buffer and Tris-HCl buffer (Figure S2 in the Supporting Information). Compared with most aqueous QDs capped with thiols,33-36 the as-prepared PEI-CdS/ZnS QDs show superior stability at room temperature and against illumination.

Figure 2. Fluorescence intensity of PEI-CdS/ZnS QDs in PBS (0.01 M) at different pH (A) and stored in PBS (0.01 M, pH 7.4) for various periods (B).

Assembly of FA onto PEI-CdS/ZnS QDs. To fabricate FA self-assembled PEI-CdS/ZnS QDs as a fluorescence probe for targeting FR, the interaction between FA and PEI-CdS/ZnS QDs was studied. PEI, a kind of polyelectrolyte with multiple amino groups on each polymer chain (Figure 3A), is water soluble, and part of its amino groups can bind to the QDs strongly and confine the QDs all through the growth period, while other amino groups are protonated on the surface of the protected QDs and enable 11



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QDs to interact with other substances.37 As the −NH2 groups in PEI (pKb = 3.0-5.0) are protonated and the −COOH groups in FA (pKa = 3.55) are dissociated at pH 7.4 in PBS (Figure 3B), electrostatic interaction between the −NH3+ of PEI and the −COO− of FA is expected.

Figure 3. The molecular structure of PEI (A) and FA (B). (C) Fluorescence emission spectra of PEI-CdS/ZnS QDs at different concentration of FA. (D) The time course of the fluorescence of PEI-CdS/ZnS QDs in the absence and presence of 2.0 µM FA. (E) F0-F (quenched fluorescence intensity) vs. the concentration of FA; inset: Stern–Volmer plots (F0/F) for FA concentration dependence of the fluorescence intensity of the PEI-CdS/ZnS QDs (in the range of 0-5.0 µM FA).

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Addition of FA to the solution of PEI-CdS/ZnS QDs in PBS (pH 7.4) resulted in significant quenching of the fluorescence of the QDs in conjunction with an obvious blue shift in the emission maximum (Figure 3C). Temporal change of the quenched fluorescence intensity of the PEI-CdS/ZnS QDs in the presence of 2.0 µM FA was also monitored for 27 min. Maximal and stable quenched fluorescence intensity was observed after 10-min interaction (Figure 3D). Figure 3E shows the FA concentration dependent quenching of the fluorescence of the PEI-CdS/ZnS QDs at 500 nm. To understand the mechanism of the fluorescence quenching of PEI-CdS/ZnS QDs by FA, the absorption and the fluorescence emission spectra of PEI-CdS/ZnS QDs and FA were studied. Owing to no overlap between the fluorescence emission of PEI-CdS/ZnS QDs and the absorbance of FA (Figure 1B; Figure S3 in the Supporting Information), fluorescence resonance energy transfer from PEI-CdS/ZnS QDs to FA is excluded. Meanwhile, the absorbance of 20 µM FA at 350 nm (the excited wavelength of PEI-CdS/ZnS QDs) is very weak, and the fluorescence of 20 µM FA is also negligible at 500 nm. As surface functionalized QDs are both excellent electron donors and acceptors, the fluorescence of the QDs can be quenched by electron acceptors or donors.38, 39 The fluorescence quenching did not follow the Stern−Volmer equation (F0/F = 1 + KsvC, where C is the concentration of the added FA, F0 and F is the fluorescence intensity in the absence of FA and at the concentration of the added FA respectively, Ksv is the Stern–Volmer constant). Plotting F0/F versus the FA concentration yields an upward curve (Figure 3E inset), which suggests both dynamic quenching and static

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quenching were operative, and hence the formation of FA assembled PEI-CdS/ZnS QDs.40 To further explore the mechanism of the fluorescence quenching, the quantum yield, the fluorescent decay lifetime, the FT-IR and RLS spectra of PEI-CdS/ZnS QDs in the absence or prescence of FA were also examined. The RLS spectra of PEI-CdS/ZnS QDs were examined in the absence and presence of 2.0 µM FA to distinguish the binding modes between FA and PEI-CdS/ZnS QDs (Figure S4A in the Supporting Information). The change of the RLS intensity of PEI-CdS/ZnS QDs in the presence of FA suggests the relatively stable interaction or adsorption of FA onto QDs.41, 42 Fluorescence time profile was also recorded to examine the mechanism for the fluorescence-quenching (Figure S4B in the Supporting Information). A significant decrease in the average fluorescence lifetime from 83.9 to 67.0 ns and even to 53.3 ns was observed after the addition of 1.0 and 10 µM FA, indicating that the addition of FA affected the electron-hole state of PEI-CdS/ZnS QDs and resulted in an acceleration of the recombination of excitons, because fluorescence is sensitive to both electron and hole dynamics.43 Meanwhile, the quantum yield of PEI-CdS/ZnS QDs decreased when FA was added (Table S1 in the Supporting Information), which also proves that the fluorescence reduction originated from the quenching effect rather than inter-filtering effect, for the absorption of FA was deduced.44 In addition, the FA assembly to PEI-CdS/ZnS QDs was also verified by the characterized peaks in the FT-IR spectrum of samples (Figure S5 in the Supporting Information).

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FA Assembled PEI-CdS/ZnS QDs as Turn-On Fluorescence Probe for FR. Owing to the high affinity of FA to FR, FA more strongly interacts with FR than PEI on the surface of QDs. As a result of competitive binding of FR, FA would desorb from the surface of PEI-CdS/ZnS QDs and bind with FR, leading to the recovery of the fluorescence of the QDs. As shown in Figure 4A, the fluorescence of the FA assembled PEI-CdS/ZnS QDs increased with the concentration of FR. A linear relationship between the enhanced fluorescence intensity of the QDs and the concentration of added FR was found over the range from 0.2 to 1.2 µg mL-1 with a correlation coefficient R=0.993 (Figure 4B). The precision for nine replicate measurements of the FR at 0.2 µg mL-1 ranged from 1.8% to 2.1% and the detection limit for FR was 10 ng mL-1. The limit of detection for FR is comparable with that obtained from a DNA-based fluorescence sensor,9 but higher than that obtained from an electrochemical sensor.48

Figure 4. (A) Fluorescence “turn-on” of FA assembled PEI-CdS/ZnS QDs by FR. (B) Enhanced fluorescence intensity of the QDs at 500 nm (λex=350 nm) vs. the concentration of the added FA from 0.2 to 1.2 µg mL-1 showing a linear fitting with a correlation coefficient R=0.993.

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To evaluate the selectivity of the FA assembled PEI-CdS/ZnS QDs for FR, we studied the fluorescent response of the PEI-CdS/ZnS QDs and the FA-assembled PEI-CdS/ZnS QDs to Na+ (2.0 mM), K+ (2.0 mM), Mg2+ (2.0 mM), Ca2+ (2.0 mM), Zn2+ (2.0 mM), glucose (0.4 mM), oxalic acid (0.4 mM), urea (0.4 mM), ascorbic acid (0.4 mM), citric acid (0.4 mM) and essential amino acids (2.0 mM) (Figure 5). While FA exhibited a significant quenching effect on the fluorescence intensity of the PEI-CdS/ZnS QDs, other co-existing molecules, such as ascorbic acid and glucose at 200-fold higher concentration of FA, metal ions and several amino acids even at 1000-fold higher concentration of FA, did not cause an obvious fluorescence change (less than 10%). The above results show that the FA assembled PEI-CdS/ZnS QDs have high selectivity for FR.

Figure 5. Effect of diverse coexisting substances on the fluorescence of FA assembled PEI-CdS/ZnS QDs in 0.01 M PBS. Red bars: 0, only the QDs; 1-5, the QDs plus Na+, K+, Mg2+, Ca2+, Zn2+ (2.0 mM), respectively; 6-10, the QDs plus glucose, oxalic acid, urea, ascorbic acid, citric acid (0.4 mM), respectively; 11-17, the QDs plus histidine, leucine, isoleucine, lysine, methionine, phenylalanine, threonine (2.0 mM), respectively. Blue bars: 0, the QDs plus FR (1.4 mg L-1); 1-17, the QDs plus FA plus each corresponding coexisting substance. All data were obtained after incubation at ambient temperature for 10 min. 16


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Figure 6. Fluorescence images of Hela (A and D), HepG2 (B and E) and A549 (C and F) cells incubated with the FA assembled PEI-CdS/ZnS QDs (A, B, C) and DAPI (D, E, F).

Targeted Imaging. To evaluate the feasibility of the prepared FA assembled PEI-CdS/ZnS QDs as a fluorescence turn-on probe for targeted imaging of FR expressed on cancer cells, the probe was used to detect the FR expression by Hela cells (a kind of human cervical carcinoma cell with a high growth rate and over-expression of FR), and HepG2 cells (a human liver carcinoma cell line with a high-expression of FR), with FR negative A549 cells (adenocarcinomic human alveolar basal epithelial cells) as controls. As shown in Figure 6, the brightness of fluorescence was in the order of Hela > HepG2 > A549 after an incubation with the probe. The results are consistent with the fact of over-expression of FR for Hela cells, high-expression of FR for HepG2 cells, and no obvious expression of FR for A549 cells.45-47 The FA-assembled QDs-imaged cells were also counted against the DAPI stained cells for the comparison of imaging efficiency. There were 74 in 86 (86%) Hela cells, 96 in 119 (80%) HepG2 cells, and 0 in 73 (0%) A549 cells imaged by the FA assembled PEI-CdS/ZnS QDs. The percentages of imaging also prove the ability of targeted imaging by the FA assembled

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PEI-CdS/ZnS QDs. As a negative control, these cells were incubated with PEI-CdS/ZnS QDs but without FA (Figure S6). Most of the cells were attached by the QDs (electrostatic interaction between PEI and cell surface), but there was no such an obvious difference between different cell lines. CONCLUSIONS We have prepared FA functionalized PEI-CdS/ZnS QDs as a sensitive and selective fluorescence probe for FA by the electrostatic self-assembly of FA and PEI-CdS/ZnS QDs. The probe has been demonstrated effective for targeted imaging of FR over-expressed cancer cells in “turn-on” mode based on an electron transfer inhibition assay.

Acknowledgment XPY appreciate the financial support from the National Basic Research Program of China (Grant 2011CB707703), the National Natural Science Foundation of China (Grants 21275079, 20935001), the Fundamental Research Funds for the Central Universities and the Tianjin Natural Science Foundation (Grant 10JCZDJC16300), and YZ appreciate the financial support from the China Postdoctoral Science Foundation (Grant 2011M500529). We greatly appreciate Prof. Yu Liu and Dr. Zhuo-Yi Gu (Nankai University) for their help in the measurement of the fluorescence quantum yields, Prof. Yan Li and Miss Ye Teng (Nankai University) for their help in the experiment of RLS, and Prof. Jie Yang (Tianjin Key Lab of Cellular and Molecular Immunology, College of

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Basic Medical Science, Tianjin Medical University, China) for her help in the experiments of cells culturing and imaging. SUPPORTING INFORMATION AVAILABLE 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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For TOC only

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ZnS

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