Silver Decahedral Nanoparticles-Enhanced Fluorescence Resonance

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Silver decahedral nanoparticles-Enhanced Fluorescence Resonance Energy Transfer sensor for Specific Cell Imaging Hui Li, Hongting Hu, and Danke Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5045274 • Publication Date (Web): 12 Mar 2015 Downloaded from http://pubs.acs.org on March 19, 2015

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Analytical Chemistry

Silver decahedral nanoparticles-Enhanced Fluorescence Resonance Energy Transfer Sensor for Specific Cell Imaging Hui Li, Hongting Hu, Danke Xu† †

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, China

Corresponding Author Tel/Fax (+) 00862583595835 E-mail: [email protected]

Abstract: We report on a silver decahedral nanoparticles (Ag10NPs)-based FRET (Fluorescence Resonance Energy Transfer) sensor for target cell imaging. Fluorophores-functionalized aptamers (Sgc8-FITC) were bound with Ag10NPs via the SH group on the aptamer to form Ag10-Sgc8-FITC. Then, quencher-carrying strands (BHQ-1) were hybridized with Sgc8-FITC to form Ag10NPs-based FRET sensor (Ag10-Sgc8-F/Q). The sensor interacted with membrane protein tyrosine kinase-7 (PTK-7) on CCRF-CEM cell surface to attain fluorescence imaging of CCRF-CEM cells. The addition of CCRF-CEM cells resulted in many sensors binding with cells membrane and the displacement of BHQ-1, thus disrupting the FRET effect and the enhanced fluorescence intensity of FITC. It was found that Ag10NPs largely enhanced the fluorescence intensity of FITC. The results also showed that the Ag10NPs-based FRET sensor (Ag10-Sgc8-F/Q) was not only superior to bare FRET sensor (Sgc8-F/Q) and sensor Ag-Sgc8-F/Q, but also highly sensitive and specific for CCRF-CEM cells imaging.

Keywords: Silver decahedral nanoparticles; Aptamers; Fluorescence resonance energy transfer; Enhanced fluorescence imaging

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Introduction Sensitive and specific analysis of cancer cells is significant for early diagnosis of cancer and investigation of cancer metastasis. Fluorescence resonance energy transfer (FRET) imaging of cancer cells is a powerful approach because of its low background, high sensitivity and multiplexing potential. The process where an excited state donor gives its excitation energy to a proximal ground state acceptor via nonradiative transfer is called FRET1. FRET-based methods are widely used in living cell imaging. Wang and co-workers developed an activatable aptamer sensor which was based on FRET between FAM and BHQ-1 for cell imaging2. For living cells, some biologically significant molecules such as mRNA3, glycans4, mitochondrial hydrogen peroxide(H2O2)5, biothiol6, 7were also imaged by FRET-based methods with high sensitivity. Nevertheless, signal amplification for FREF-based methods to attain higher sensitivity in cell imaging have not been investigated yet. Silver nanomaterials were reported to enhance the FRET efficiency and result in increased quantum yield and emission intensity of fluorophores8-11. We have developed a sensitive silver-enhanced FRET sensor for protein detection12. Silver-enhanced FRET sensors might have the potential to enhance the sensitivity of FRET imaging of cells. Therefore, we firstly attempted to establish a silver-enhanced FRET sensor for cell imaging with high sensitivity and specificity. Silver nanomaterials had metal-enhanced fluorescence (MEF) on nearby fluorophores and the emission intensity of fluorophores could be enhanced by MEF effect13, 14. MEF effect was highly related with shape15 and size16 of nanoparticles as well as distance between silver and fluorophores17. The parameters such as size and distance were investigated in our previous works for MEF study17, 18. Here, to attain optimal MEF effect, two shapes of nanoparticles: silver spherical nanoparticles (AgNPs) and silver decahedral nanoparticles (Ag10NPs) were synthesized. The MEF of AgNPs and Ag10NPs on fluorescein isothiocyanate (FITC) was investigated to show the superiority of Ag10NPs. For cell imaging, target recognition is important. Aptamers are one kind of recognition molecules for specific binding with corresponding receptors on the membrane of cells19-21. Aptamers were screened from cell-SELEX (systematic evolution of ligands by exponential enrichment) process22,

23

, and aptamer Sgc8 was selected by Tan’s group to

specifically recognize membrane protein tyrosine kinase-7 (PTK-7) on CCRF-CEM (CCL-119, T-cell line, human acute lymphoblastic leukemia) cell surface24. Compared to antibodies, aptamers


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are generally more stable and can be synthesized or modified easily23. Furthermore, the sulfydryl (SH) at 5’ end of aptamers facilitates the high efficient binding between aptamers and silver nanomaterials25-28. We prepared a silver-enhanced FRET sensor (Ag10-Sgc8-F/Q) for enhanced cell imaging. The procedure of silver decahedral nanoparticles (Ag10NPs)-based FRET sensor for target cell imaging was shown in Scheme 1. The sensor interacted with the membrane protein PTK-7 on CCRF-CEM cell surface to attain “off”-“enhanced on” fluorescence imaging of CCRF-CEM cells. It was found that Ag10NPs largely enhanced the fluorescence intensity of FITC and the average size of them was smaller than that of spherical silver nanoparticles (AgNPs) with the same MEF effect. The results also showed that the Ag10NPs-based FRET sensor (Ag10-Sgc8-F/Q) was not only superior to bare FRET sensor (Sgc8-F/Q) and AgNPs-based FRET sensor (Ag-Sgc8-F/Q), but also highly sensitive and specific.

Methods Materials and reagents: Silver nitrate (AgNO3), sodium borohydride (NaBH4), sodium L-ascorbate, polyvinylpyrrolidone (PVP, MW=40000), L-arginine were purchased from Sigma Aldrich (St, Louis, MO, USA). Trisodium citrate was purchased from Sinopharm Chemical Reagent Co., Ltd.. Na2S2O3 and K3[Fe(CN)6] were purchased from Nanjing Chemical Reagent Co., Ltd.. CCRF-CEM (CCL-119, T-cell line, human acute lymphoblastic leukemia) and Ramos (CRL-1596, B-cell line, human Burkitt's lymphoma) were cultured in RPMI 1640 medium (ATCC) with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) and penicillin (80U/mL)-streptomycin (0.08mg/mL) (KeyGEN Biotech, Nanjing, China) at 37℃ under a 5% CO2 atmosphere. Cells were washed with 1×PBS (Phosphate Buffered Saline, Gibco®, Scotland, UK ). PBSM (1×PBS+ 5 mM MgCl2), mPEG-SH (MW=5000, Yarebio, shanghai, China), MTT cell proliferation and cytotoxicity detection kit (KeyGEN Biotech; Nanjing, China), Dil for cell membrane staining (KeyGEN Biotech; Nanjing, China), Glass bottom cell culture dish (Φ15mm, Nest Biotechnology Co., Ltd, China). The sequences of used oligonucleotides were as follows: Sgc8-FITC(45bases):5’SH-(CH2)6-AAAAATCTAACTGCTGCGCCGCCGGGAAAATACTGTA CGGTTAGA-FITC,Sgc8-FITC(51bases):5’SH-(CH2)6-AAAAAAAAAAATCTAACTGCTGCG CCGCCGGGAAAATACTGTACGGTTAGA-FITC, Sgc8-FITC(57bases):5’SH-(CH2)6-AAAA



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AAAAAAAAAAAAATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-FITC, Sgc8-FITC(63bases):5’SH-(CH2)6-AAAAAAAAAAAAAAAAAAAAAAATCTAACTGCTGCG CCGCCGGGAAAATACTGTACGGTTAGA-FITC, Control-FITC:5’SH-(CH2)6-AAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATACTGTACGGTTAGA-FITC, BHQ-1:BHQ-1-TCTAACCGTACA.

Apparatus. Led spot light (blue, 3W, 5W, Changzhou, China) was used to prepare silver decahedral nanoparticles. The ultraviolet-visible (UV-vis) spectra and fluorescence intensity (excitation wavelength was λex=470 nm and emission wavelength was λem=520 nm) were measured on BioTek (Synergy H1, USA). Transmission electron microscope (TEM) (JEM-200CX, Japan) was used for collecting TEM images. Atomic absorption spectroscopy(180-80, Hitachi, Japan) was used to detect the concentration of silver. Flow cytometry (FC 500, Beckman Coulter, USA) was used to take fluorescence intensity of cells. Confocal scanning laser microscopy (CSLM) (TCS SP5, Leica, Germany) was used to take cell images.

Preparation of Silver decahedral nanoparticles (Ag10NPs). Trisodium citrate (2 mL, 50 mM), PVP (2 mL, 6%), L-arginine (0.2 mL, 5 mM), and AgNO3 (0.8 mL, 5 mM) were added to 6 mL deionized water followed by the addition of fresh NaBH4 (0.32 mL, 0.1 M) to make seeds, then the seeds were exposed to blue light (3 W) for 10 h to prepare Ag10NPs-1. Trisodium citrate (1.5 mL, 50 mM), PVP (1.5 mL, 6%), L-arginine (0.15 mL, 10 mM) and AgNO3 (0.6 mL, 10 mM) were added to 21 mL deionized water followed by the addition of fresh NaBH4 (0.24 mL, 0.1 M) to make seeds which were then exposed to blue light (5 W) for 10 h to prepare Ag10NPs-2. 10 mL seeds of Ag10NPs-2 and 10 mL Ag10NPs-2 were mixed and then exposed to blue light (5 W) for 10 h to prepare Ag10NPs-3. The absorption spectra were measured using BioTek spectrometer.

Metal-enhanced fluorescence of Ag10NPs. Ag10-Sgc8-FITC was prepared by mixing 1 mL of Ag10NPs with mPEG-SH (50 µL, 10 µM), Sgc8-FITC (57 bp) (50 µL, 10 µM) and NaCl (64 µL, 2 M) for 2 h. After standing for 12 h, the solution was centrifuged for 3 times, and the precipitate was re-dispersed by PBSM. Ag10-Sgc8-FITC were respectively mixed with PBS, NK (the mixture


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of 100 mM Na2S2O3 and 10 mM K3[Fe(CN)6]), and the fluorescence spectra were taken by BioTek （λex=470 nm). Different lengths of Sgc8-FITC (45 bp, 51 bp, 57 bp, 63 bp) were used to prepare Ag10-Sgc8-FITC, and the changes of fluorescence intensity（λex=470 nm, λem=520 nm）with NK and without NK were taken by BioTek.

Ag10NPs-enhanced FRET sensor. Ag10-Sgc8-F/Q sensor was prepared by mixing 1mL of Ag10-Sgc8-FITC with BHQ-1 (50 µL, 10 µM) for 1 hour. The solution was then centrifuged for 3 times, and the precipitate was re-dispersed by 100 µL PBSM. Flow cytometry was used to take fluorescence intensity of FITC in FL1-H channel. The length of Sgc8-FITC (45 bp, 51 bp, 57 bp, 63 bp), and the ratio of Sgc8-FITC to mPEG-SH were optimized to prepare Ag10-Sgc8-F/Q sensors. CCRF-CEM (2 × 105) and Ramos (2 × 105) cells were incubated with different Ag10-Sgc8-FITC sensors for 15 min. The cells were washed to remove medium and free sensors, and then dispersed in 1×PBS for flow cytometry detection.

Ag10NPs-enhanced FRET sensor for cell imaging. For localization of the sensor, Ag10-Sgc8-F/Q were incubated with CCRF-CEM (2× 105) cells for 2 h. After washing once with 1×PBS, Dil (5 µL, 100 µM) was added to incubate with cells for 20 min. After washing twice with 1×PBS, cells were dispersed in 1×PBS for imaging. For imaging cells, different amounts of CCRF-CEM cells (2.0× 105, 4.0× 104, 8.0× 103, 1.6× 103, 3.2× 102, 64) were incubated with Ag10-Sgc8-F/Q for 30 min. The cells were then washed and dispersed in 1×PBS for imaging.

Results and Discussion Characterization of nanoparticles and MEF effect. The FRET sensor (Ag10-Sgc8-F/Q) is composed of three parts: silver decahedral nanoparticles (Ag10NPs), the fluorophore-carrying aptamers (Sgc8-FITC) and the quencher (BHQ-1)-carrying strands. Sgc8-FITC was bound with Ag10NPs by self-assembly of SH at its 5’ end to form Ag10-Sgc8-FITC. Then, BHQ-1-carrying strands were hybridized with Sgc8 to form Ag10NPs-enhanced FRET sensor (Ag10-Sgc8-F/Q). The quencher BHQ-1 could quench the fluorescence of FITC thus lowering the background signal, and the sensor was in “off” state. When the target cells were added to the sensor, the BHQ-1-carrying strands were displaced from Ag10-Sgc8-F/Q resulting in enhanced fluorescence recovery of



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Ag10-Sgc8-FITC, and the sensor was in “enhanced-on” state. Ag10NPs enhanced the fluorescence intensity of Sgc8-FITC by two ways. Firstly, Sgc8-FITC molecules were enriched on the surface of Ag10NPs for multiple binding to target cells. Secondly, Ag10NPs could increase the fluorescence intensity of FITC via MEF effect. Spherical silver nanoparticles (AgNPs) were widely used to fabricate sensors for fluorescence-enhanced cell imaging and detection of DNA and proteins29. In this issue, we modified the method30 of synthesis and preparation of AgNPs to attain largest MEF effect on FITC when the distance between FITC and AgNPs was much longer than optimal lengths. The maximum absorption wavelength for AgNPs-1, AgNPs-2, AgNPs-3, AgNPs-4 and AgNPs-5 were 418 nm, 438 nm, 462 nm, 486 nm and 521 nm, respectively (Figure S1a), which indicated different optical property and different MEF effects on nearby fluorescent dyes. To demonstrate the MEF effect of AgNPs on Sgc8-FITC, Sgc8-FITC modified AgNPs (Ag-Sgc8-FITC) were prepared. NK solution (100 mM Na2S2O3 and 10 mM K3[Fe(CN)6]) was used to dissolve AgNPs into Ag+ but did not affect the fluorescence intensity of FITC. The MEF effect was equal to the ratio (F/F0) of fluorescence intensity with or without AgNPs. The data (Figure S1b) showed that the absorption peak of AgNPs disappeared with the addition of NK solution. Meanwhile, the fluorescence intensity of FITC was decreased, which was due to the disappearance of MEF effect. The MEF effect of AgNPs with different absorption peaks was shown in Figure S1c, demonstrating that AgNPs-4 had the highest MEF effect on FITC as AgNP-4 had a better overlap with FITC emission spectrum. AgNPs-5 aggregated slightly during the process of preparation of Ag-Sgc8-FITC, which caused a decreased MEF effect. Therefore, AgNPs-4 was chosen as a control for comparison with Ag10NPs. The concentration of AgNPs-4 was calculated to be 200.5 µg/mL by atomic absorption spectroscopy. Transmission electron microscope (TEM) was used to characterize the size and shape of AgNPs-4 (Figure S1d), indicating that the diameter of AgNP-4 was 89.4 nm±5.0 (N=12). Therefore, our data showed that AgNPs with the size of 89.4 nm had the largest MEF effect. As MEF effect was highly related with the shape of nanoparticles, we attempted to optimize the MEF effect by synthesizing nanoparticles with different shapes. Silver decahedral nanoparticles (Ag10NPs) with smaller sizes were prepared to substitute for AgNPs. Ag10NPs had excellent optical properties and were prepared according to the method with some


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modification31-33. The amount of reagents and light intensity were changed to prepare Ag10NPs with different absorption peak. Three kinds of Ag10NPs (Ag10NPs-1, Ag10NPs-2, Ag10NPs-3) with different absorption peaks were synthesized and their maximum absorption peaks were 478 nm, 500 nm, and 514 nm respectively (Figure S2a). For specific targeting, Sgc8-FITC was applied to bind with three kinds of Ag10NPs to form Ag10-Sgc8-FITC. A fast and effective method of binding aptamers to Ag10NPs was reported for protein detection in our previous work34. After modification with Sgc8-FITC, the maximum absorption peaks of three kinds of Ag10NPs shifted to 448 nm, 465 nm, and 482 nm, respectively (Figure 1a). Though Ag10NPs had excellent optical properties, their MEF effect on fluorescent dyes had not been reported yet. The MEF effects of three kinds of Ag10NPs on FITC were investigated and the data (Figure 1b) showed that three kinds of Ag10NPs were able to increase the fluorescence intensity of FITC. Ag10NPs-1 had the lowest F/F0. Ag10NPs-2 and Ag10NPs-3 had almost the same F/F0 resulted from AgNPs. However, the synthesis of Ag10NPs-2 was much easier than that of Ag10NPs-3. Therefore, Ag10NPs-2 was optimal for the next experiment. The shape of Ag10NPs-2 was shown in Figure S2b, and decahedral nanoparticles can be easily observed. The shape of Sgc8-FITC modified Ag10NPs-2 was still decahedral (Figure 1c) and the size was obviously smaller than that of AgNPs in Figure S1d. Sgc8-FITC with different numbers of bases was modified on the surface of Ag10NPs-2 to find out the relationship between distance and MEF effect. It was found that Sgc8-FITC with 51 bases owned highest increase in fluorescence intensity and Sgc8-FITC with 45 bases had the lowest increase (Figure 1d). In conclusion, Ag10NPs could still enhance fluorescence intensity of FITC when the distance between Ag10NPs and FITC was 45 bases, 51 bases, 57 bases and 63 bases.

Optimization of Ag10NPs-enhanced FRET sensor. A FRET sensor (Ag10-Sgc8-F/Q) was fabricated based on Ag10-Sgc8-FITC and fluorescence quencher BHQ-1 for enhanced cell imaging. Two parameters, the length of Sgc8-FITC, the ratio of Sgc8-FITC to mPEG-SH were carefully examined when Ag10-Sgc8-F/Q was prepared for target cell imaging. The Ag10-Sgc8-F/Q sensors with different length of Sgc8-FITC(45 bases, 51 bases, 57 bases, and 63 bases) were prepared and the quencher efficiency was 87.7%, 89.9%, 91.5% and 89.1%, respectively. As Sgc8 could specifically recognize CCRF-CEM cells and didn’t bind with Ramos. CCRF-CEM cells were chosen as target cells and Ramos cells were chosen as a control.



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The sensors with different lengths of Sgc8-FITC were cultured with CCRF-CEM and Ramos cells for 15 min. The selective binding affinity of sensors was monitored by flow cytometry, which showed 16.3-fold, 46.8-fold, 53.3-fold, and 34.1-fold fluorescence signal shift for CCRF-CEM cells (Figure 2a) and 4.2-fold, 6.4-fold, 5.4-fold, and 3.9-fold for Ramos cells (Figure 2b). The results indicated the high specificity of Ag10-Sgc8-F/Q for CCRF-CEM cells. Due to the largest fluorescence signal shift, Sgc8-FITC with 57 bases was chosen for the next experiments. The ratio of Sgc8-FITC to mPEG-SH was also optimized. mPEG-SH was beneficial to maintain the stability of Ag10-Sgc8-F/Q in cell culture and reduce nonspecific absorption of proteins on Ag10NPs’ surface. mPEG-SH was used to prevent the aggregation of Ag10NPs in buffers, such as PBSM and cell culture medium. The results showed 26.2-fold, 57.2-fold, 66.6-fold, 66.2-fold, and 68.2-fold fluorescence signal shift for CCRF-CEM cells (Figure 2c), and 6.9-fold, 10.9-fold, 11.5-fold, 12.8-fold, and 12.2-fold for Ramos cells (Figure 2d). The high specificity of Ag10-Sgc8-F/Q for CCRF-CEM cells was further demonstrated. The fluorescence signal shift rose with the ratio increasing and then reached a platform, hence the ratio of 1:1 (Sgc8-FITC: mPEG-SH=1:1) was chosen. The concentration of the sensor and the displacement time were optimized. Ag10-Sgc8-F/Q sensors with different concentrations were incubated with CCRF-CEM and Ramos cells. The concentration of Ag10-Sgc8-F/Q was calculated as 40.4 µg/mL by atomic absorption spectroscopy. The fluorescence intensity of CCRF-CEM increased with the increasing concentration of Ag10-Sgc8-F/Q. When the concentration was increased from 0.25 µg to 4.0 µg, an obvious enhanced fluorescence signal shift for CCRF-CEM cells was observed (Figure S3a) but just a little change for Ramos cells (Figure S3b). In addition, the incubation time of cells with Ag10-Sgc8-F/Q was also examined. The results showed that CCRF-CEM cells had an obvious fluorescence signal shift (Figure S3c) compared with Ramos cells (Figure S3d). Therefore, the sensor of Ag10-Sgc8-F/Q had good specificity for CCRF-CEM cells detection. The kinetics of the assay was investigated. The Ag10-Sgc8-F/Q sensors were incubated with CCRF-CEM cells from 15 min to 24 h (Figure 3). The fluorescence signal shift could be obviously observed in Figure 3a. The X-Mean of fluorescence intensity was used to show the relationship between fluorescence intensity and the incubation time (Figure 3b). The fluorescence intensity was increased with the incubation time, and the maximum fluorescence intensity was


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achieved when the incubation time was 2 h. With the continue increase of incubation time, the fluorescence intensity decreased. After 24 h incubation, the fluorescence intensity was very low. The fluorescence intensity of CCRF-CEM cells with Ag10-Sgc8-F/Q was quickly increased in 2 h and then slowly decreased with the increased time. There might be an equilibrium between cells and BHQ-1. BHQ-1 was firstly displaced by cells resulting in the gradual recovery of fluorescence (Ag10-Sgc8-FITC) and attaining maximum fluorescence intensity at 2 h. After that, BHQ-1 was gradually bound with Ag10-Sgc8-FITC to form Ag10-Sgc8-F/Q resulting in the slow decrease of fluorescence intensity.

The Specificity of Ag10NPs -enhanced FRET sensor for cell imaging. To show the superiority of Ag10-Sgc8-F/Q, some other sensors (Ag-Sgc8-F/Q, Sgc8-F/Q) were prepared as controls. Sgc8-F/Q were prepared by adding the mixture of Na2S2O3 and K3[Fe(CN)6] into Ag10-Sgc8-F/Q or Ag-Sgc8-F/Q. The results showed that the fluorescence intensity of Ag10-Sgc8-F/Q or Ag-Sgc8-F/Q was much higher than that of Sgc8-F/Q (Figure 4a). Therefore, AgNPs and Ag10NPs could increase the fluorescence intensity of FITC when Ag10-Sgc8-F/Q and Ag-Sgc8-F/Q interacted with target cells. For cell imaging, Ag-Sgc8-F/Q could not label cells totally but aggregated in binding buffer as shown in Figure S4, however, the aggregation of Ag-Sgc8-F/Q was not detectable by flow cytometry. When the concentration of Ag-Sgc8-F/Q sensor was lowered to 4.0 µg, they could be well dispersed on the cells and no aggregation was observed (Figure S4). Because of the large size of AgNPs, Ag-Sgc8-F/Q was more susceptible to environment. Therefore, Ag10-Sgc8-F/Q was more suitable for cell imaging. To further prove the specificity of Ag10-Sgc8-F/Q, another sensor Ag10-Control-F/Q which had a random sequence and would not bind with CCRF-CEM cells was prepared. The results showed that the fluorescence intensity caused by Ag10-Control-F/Q could be ignored compared to that caused by Ag10-Sgc8-F/Q (Figure 4b). The results from Figure 4b showed that Ag10-Sgc8-F/Q had excellent binding capacity and high specificity towards CCRF-CEM cells. Based on the optimal conditions, Ag10-Sgc8-F/Q was fabricated for enhanced cell imaging. To show the superiority of Ag10-Sgc8-F/Q, some control experiments were done and the results were shown in Figure 5. The fluorescence intensity of CCRF-CEM cells with Ag10-Sgc8-F/Q was much higher than that of Ramos cells. In addition, the fluorescence intensity from sensor Sgc8-F/Q or



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Ag10-Control-F/Q was extremely low (Figure 5). Hence, Ag10-Sgc8-F/Q had excellent specificity towards target CCRF-CEM cells.

Ag10NPs -enhanced FRET sensor for cell imaging. The incubation time of Ag10-Sgc8-F/Q with CCRF-CEM cells was investigated to find out the reaction characteristic of Ag10-Sgc8-F/Q for cell imaging. Ag10-Sgc8-F/Q was cultured with CCRF-CEM cells for different incubation time (15 min, 30 min, 60 min, 120 min, 4 h, and 12 h). Ag10-Sgc8-F/Q was well distributed on the cells and the outline of cells could be easily observed from 15 min to 120 min (Figure S5). The distribution of Ag10-Sgc8-F/Q on the cells became uneven and Ag10-Sgc8-F/Q aggregated on the cells after 4-h incubation (Figure S5). Therefore, the incubation time from 15 min- 2 h was chosen for the sensor to interact with CCRF-CEM cells. The location of Ag10-Sgc8-F/Q on the cell surface was demonstrated. Fluorescent dye Dil (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate) was widely used for binding phospholipid bilayer membranes of living cells and Dil labeling of cells membranes was nontoxic and highly effective. Fluorescent dye Dil (λex=543 nm) was used to label CCRF-CEM cell surface, and Ag10-Sgc8-F/Q (λex=488 nm) was used to bind with membrane protein PTK-7 on CCRF-CEM cell surface. Therefore, the fluorescent images of CCRF-CEM cells with Dil and FITC should be overlapped. The results showed that the fluorescent images of FITC were well overlapped with the images of Dil (Figure 6a,b,c). To show the detection ability of Ag10-Sgc8-F/Q for CCRF-CEM cells. Ag10-Sgc8-F/Q was incubated with the mixture of Dil-labeled Ramos cells (1×105) and CCRF-CEM cells (5×103). Ag10-Sgc8-F/Q bound with CCRF-CEM cells resulting in green images, while Dil-labeled Ramos cells resulting in red images. The green images of CCRF-CEM cells and red images of Ramos cells were showed in Figure 6d. The result showed that Ag10-Sgc8-F/Q could distinguish CCRF-CEM cells when a little amount of target cells were mixed with abundant control cells. The nonspecific absorption of Ag10-Sgc8-F/Q on Ramos could be ignored. To further prove the detection ability of Ag10-Sgc8-F/Q, different number (200000, 40000, 8000, 1600, 320, and 64) of CCRF-CEM cells were mixed with the sensor for 30 min. Fluorescent images of CCRF-CEM cells were shown in Figure 7, and the scale bar was 50 µm. When the cell number was 200000, the fluorescence intensity of single cell was low. With the decrease of cells


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number, the fluorescence intensity of single CCRF-CEM cells was increased. When the number of CCRF-CEM cells was decreased to 64, the cells were still able to be captured by Ag10-Sgc8-F/Q and observed with high fluorescence intensity. The results showed that Ag10-Sgc8-F/Q was sensitive and could be applied for enhanced cell imaging when cells concentration was very low. Compared with previous ones that also utilized the same cell-binding aptamer2,35,36, the Ag10-Sgc8-F/Q sensor had three advantages. Firstly, Ag10-Sgc8-F/Q had a relatively low signal-to-background ratio for cell imaging. Secondly, Ag10NPs work as a carrier for loading numerous Sgc8 for multiple binding with target cells. Thirdly, Ag10NPs could enhance the sensitivity of FRET sensor by MEF effect. Therefore, Ag10-Sgc8-F/Q was highly sensitive and specific for cell imaging. The in vitro cytotoxicity of Ag10-Sgc8-F/Q on CCRF-CEM cells was investigated. MTT assay was used to assess the viability of cells, showing that 4.0 µg of Ag10-Sgc8-F/Q was nontoxic to CCRF-CEM cells (Figure S6).

Conclusion We firstly report on a Ag10NPs-enhanced FRET sensor (Ag10-Sgc8-F/Q) for target cell imaging and the sensor is highly sensitive and specific towards target CCRF-CEM cells. Ag10NPs are excellent nanoparticles for fabrication of MEF-based sensors because of its unique optical property. Ag10NPs increased emission intensity of FITC and enhanced the sensitivity of Ag10-Sgc8-F/Q. Ag10NPs is superior to AgNPs for fabricating silver-enhanced sensors when they owned the same MEF effect on FITC. In addition, Ag10NPs is nontoxic to CCRF-CEM cells. Ag10-Sgc8-F/Q was simple, inexpensive, and convenient for target cells imaging. In addition, the principle of this assay could be extended to other cancer cells imaging and analysis. Conflict of interest: The authors declare no competing financial interest.

Acknowledgment We thank Prof. Weihong Tan in Hunan University for providing CCRF-CEM and Ramos cells. We acknowledge the financial support of the National Basic Research Program of China (973



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Program, 2011CB911003), National Natural Foundation of China (21227009, 21175066, 21475060, 21405077, 21328504), Natural Science Foundation of Jiangsu Province (BK20140591), and the National Science Funds for Creative Research Groups (21121091).

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

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Scheme 1. Silver decahedral nanoparticles-enhanced fluorescence resonance energy transfer sensor for specific cell imaging.


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Figure 1. (a) UV-vis spectra of three kinds of Sgc8-FITC modified Ag10NPs, Ag10NPs-1, Ag10NPs-2, Ag10NPs-3. (b)The MEF effect (F/F0=fluorescence intensity with Ag10NPs/without Ag10NPs) of Ag10NPs on FITC. (c) TEM image of Ag10-Sgc8-FITC. (d) For Ag10-Sgc8-FITC, the F/F0 at different length of Sgc8-FITC.



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Figure 2. Fluorescence intensity of CCRF-CEM cells (a) and Ramos cells (b) with Ag10-Sgc8-F/Q sensor at different length of Sgc8-FITC for 15 min. Fluorescence intensity of CCRF-CEM cells (c) and Ramos cells (d) with Ag10-Sgc8-F/Q sensor at different ratios of Sgc8-FITC and mPEG-SH for 15 min .


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Figure 3. (a) Fluorescence intensity of CCRF-CEM (2 × 105) cells with Ag10-Sgc8-F/Q for different incubation time, (b) the relationship between fluorescence intensity (X-Mean) and incubation time.



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Figure 4. (a) Fluorescent intensity of CCRF-CEM cells with Ag-Sgc8-F/Q, Ag10-Sgc8-F/Q and Sgc8-F/Q. (b) Fluorescent intensity of CCRF-CEM cells with Ag10-Sgc8-F/Q and Ag10-Control-F/Q.


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Figure 5. Fluorescent and merged images of CCRF-CEM cells with Ag10-Sgc8-F/Q, Sgc8-F/Q and Ag10-Control-F/Q. Fluorescent and merged images of Ramos cells with Ag10-Sgc8-F/Q.



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Figure 6. (a) Fluorescent images of CCRF-CEM cells with Ag10-Sgc8-F/Q (green) and (b) with Dil (red). Overlap images of Ag10-Sgc8-F/Q and Dil on CCRF-CEM cells. (d) Fluorescent image of CCRF-CEM cells (green) and Ramos cells (red). Ramos (1× 106) cells were mixed with Dil (5 µL, 100 µM) for 20 min. After washing twice with 1× PBS, Ag10-Sgc8-F/Q was incubated with the mixture of Dil-labeled Ramos cells (1×105) and CCRF-CEM cells (5×103) for 30 min. The cells were then washed and dispersed in 1×PBS for imaging.


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Figure 7. Fluorescent images of CCRF-CEM cells. Different number (200000, 40000, 8000, 1600, 320, and 64) of CCRF-CEM cells were mixed with the sensor for 30 min.The cells were then washed and dispersed in 1×PBS for imaging.



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Table of Contents (for TOC only)


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Silver Decahedral Nanoparticles-Enhanced Fluorescence Resonance

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