Intracellular Zinc Quantification by Fluorescence Imaging with a FRET

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Intracellular Zinc Quantification by Fluorescence Imaging with a FRET System Yang Shu, Na Zheng, An-Qi Zheng, Ting-Ting Guo, Yong-Liang Yu, and Jian-Hua Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00018 • Publication Date (Web): 22 Feb 2019 Downloaded from http://pubs.acs.org on February 26, 2019

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Analytical Chemistry

Intracellular Zinc Quantification by Fluorescence Imaging with a FRET System Yang Shu, Na Zheng, An-Qi Zheng, Ting-Ting Guo, Yong-Liang Yu*, Jian-Hua Wang Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Shenyang 110819, China

Keywords: Zinc, quantitative analysis, cell imaging, FRET, carbon dots, aptamer Abstract: Fluorescence imaging of cellular metals is widely reported. However, the quantification of intracellular metals with fluorescence imaging is so far not feasible and highly challenging. In this work, a ratiometric probe with two fluorescent labeled complementary DNA strains is designed for intracellular zinc quantification via fluorescence imaging, based on fluorescence resonance energy transfer (FRET) from carbon dots (CDs) to fluorescein (FAM). The donor CDs is modified with Zn2+ aptamer, while the receptor FAM is conjugated with the complementary DNA sequence to ensure selectivity. MCF-7 cells are cultured sequentially with Zn2+ (20, 40, 55, 70, 85, 100 μmol L-1) and CDs-FAM (100 μg mL-1), which are used for fluorescence imaging (at ex 405 nm, em 440-490 nm for CDs, em 500-550 nm for FAM) to provide a relative fluorescence ratio ((F-F0)/F0, F= ICDs/IFAM), followed by quantifying intracellular zinc with ICPMS. A linear correlation is achieved between the relative fluorescence ratio in fluorescence images and the intracellular zinc content derived by ICPMS, which facilitates intracellular zinc quantification via fluorescence imaging. It is especially useful for real-time tracing of intracellular zinc

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during cell culturing process or in vivo. Cellular uptake of Zn2+ by MCF-7 cells is further evaluated with this approach by culturing with 100 μmol L-1 Zn2+ for different times, and a maximum uptake of 60.5 fg per cell is observed at an incubation time of 60 min. This value is further well demonstrated by ICPMS detection.


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INTRODUCTION Zinc plays a very important role in the physiological process of organisms, e.g., regulation of enzyme and receptor functions as well as gene expression and transmission. The abnormal metabolism of Zn2+ is closely related to many diseases 1. In the recent years, a variety of high sensitive and selective detection methods have been developed for the sensing of Zn2+ 2-5. Fluorescence imaging in biological cells has become a hot topic due to its unique characteristic of noninvasive, real time, in situ, simple operation and fast response 6,7. Among the large number of fluorescence probes for cellular imaging of various metal species, carbon dots (CDs) have attracted extensive attentions due to their low cytotoxicity compared with metal quantum dots 10. One of the most favorable features of CDs is their long half life in the cell interior, which enables continuous and long term detection for fluorescence imaging 11. Thus CDs have been demonstrated to exhibit excellent performance on intracellular imaging of metal species, including zinc 12-16. However, there are obvious limitations for fluorescence imaging. It is worth mentioning that in the practices of fluorescence imaging for metals in biological cells, e.g., zinc, there is so far no attempt for the quantification of zinc in the cells. The hitherto conducted studies illustrate the relationship between fluorescence and the contents of zinc in the incubation/culture media, but usually provide no information on the exact concentrations of zinc in the living cells. Although the fluorescence intensity in the imaging is directly correlated with the intracellular zinc content, the quantitative analysis of intracellular zinc is still highly challenging due to the lack of suitable analytical protocols. Considering the fact that a lot of metal species existing in the interior of biological cells, it is highly necessary to develop selective fluorescent probes for



recognizing the cellular metals 17. For this purpose, aptamer, as a specific DNA sequence, is a suitable choice for providing the desired selectivity for the metal of interest 18,19. Zn2+ aptamer probes have been frequently screened and applied for the detection of zinc in aqueous medium, blood and biological cells 20,21. In the present work, we report a novel approach for the quantification of intracellular zinc by fluorescence imaging with Zn2+ aptamer modified CDs as fluorescent probe. The aptamer is base-paired with a complementary sequence with fluorescein (FAM) label. FRET occurs between the fluorescence donor CDs and the acceptor FAM. Intracellular zinc content in a batch of MCF-7 cells is firstly quantified with ICPMS, which is thereafter correlated with the fluorescence ratio of CDs to FAM in the image, which is in turn closely correlated with the intracellular zinc content. Therefore, a linear relationship between fluorescence ratio and intracellular zinc content is established, which facilitates quantitative analysis of zinc content in MCF-7 cells by fluorescence imaging.

EXPERIMENTAL SECTION Chemicals. The aptamer and its complementary strand are provided by Suzhou Jinweizhi Biotechnology Co., Ltd. (Suzhou, China). The sequence of the aptamer is 5 ́-NH2-(CH2)6-GCATCAGTTAGTCATTACGCTTACGGCGGCTCTATCCTAACTG ATATATTGTGAAGTCGTGTCCC-3 22, while that of the complementary strand is 5 ́-GCGTAATGACTAACTGATGC-FAM-3 ́. N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) are obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). The dialysis bag (MW 100-500, MW 7000) is received from Shanghai Yuanye Biotechnology Co., Ltd. (China). Citric acid, branched polyethyleneimine (PEI), NaOH, Na2HPO4·12H2O, KCl, NaCl, KH2PO4, ZnSO4 and anhydrous ethanol are achieved from Sinopharm Chemical


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Reagent Co., Ltd. (Shanghai, China). All reagents are of analytical reagent grade and deionized water of 18 MΩ cm is used throughout. Dulbecco’s modified medium (DMEM, high glucose), trypsin and fetal bovine serum are obtained from Thermo Scientific (Logan, Utah, USA). Penicillin/streptomycin is provided by Invitrogen, USA. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit is the product of Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Instrumentations. The shape and size of the CDs are characterized with a JEM-2100F transmission electron microscope (TEM, Japan Electron Optics Laboratory, Japan) and Bruker Dimension icon atomic force microscope (AFM, Brook, Germany). Fourier transform infrared (FT-IR) spectra are obtained on a Nicolet-6700 spectrophotometer (Thermo Fisher Scientific, USA) by using KBr pellets. UV-vis absorption spectra are recorded with a UH5300 spectrophotometer (Hitachi, Japan) with a 1.0 cm quartz cell. Fluorescence spectra are obtained with a F-7000 fluorescence spectrophotometer (Hitachi High-Technologies Corporation, Japan). The excitation wavelength at 310 nm is used for the fluorescence measurement (PTM voltage, 600 V; excitation slit, 5 nm; emission slit, 10 nm; scan speed, 1200 nm/min). The zeta potential is obtained by using a Zeta potential analyzer (British Malvern Instruments Ltd., UK). The quantum yields are recorded on a Quantaurus-QY Absolute Photoluminescence Quantum Yield Measurement System (Hamamatsu Photonics, Japan). X-ray photoelectron spectroscopy (XPS) spectra are conducted with an ESCALAB 250 surface analysis system (Thermo Instruments Inc, USA). Fluorescence images of MCF-7 cells are obtained using an Fluoview FV1200 confocal microscope (Olympus, Japan). The cytotoxicity is recorded on an μQuantenzyme-labeled instrument (Bio-tek, USA). An Agilent 7500a inductively coupled plasma mass spectrometer (ICP-MS) (Agilent Technologies, USA) is used



for the quantification of zinc by detecting the isotope of 66Zn. Plasm gas: 15.00 L min-1, Aux gas: 1.00 L min-1, Carrier gas: 1.00 L min-1. Preparation of the Fluorescence Nanoprobe. CDs are prepared and modified with aptamer according to reported methods 23,24. 1.5000 g citric acid and 0.0900 g branched PEI are added into 8 mL of secondary deionized water in a 25 mL hydrothermal reactor. The mixture is transferred into an electric thermostatic drier and kept at 180°C for 4 h. After cooling down to room temperature, it is neutralized to pH 7.0 with 1 mol L-1 NaOH solution. The product is subjected to dialysis (MW 100-500) against secondary deionized water for 48 h to obtain CDs, and solid product is collected by vacuum drying. 5.0 mg CDs are activated using 0.0830 g EDC in a phosphate buffered saline (PBS) for 15 min at room temperature with magnetic stirring at 10 rpm. 0.1240 g NHS and 5 nmol aptamer are then added with further stirring for 12 h. The mixture is thereafter incubated at 4°C for 24 h. The aptamer modified CDs, i.e., CDs-aptamer, is achieved by dialyzed in a dialysis bag (MW 7000) until the dialysate exhibits no UV-vis absorption and fluorescent emission. 5 nmol aptamer complementary strand is then added to the above solution and incubated for 24 h at room temperature in the dark. A fluorescent probe conjugated by two fluorescent labeled complementary DNA strain (CDs-FAM) is finally obtained. For the purpose of testing cytotoxicity of CDs-FAM, MCF-7 cells are cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotics penicillin and maintained at 37°C with 5% CO2. After removal of the medium, the cells are further incubated with fresh medium containing CDs or CDs-FAM (2-500 μg mL-1) for 20 h. 10 μL of MTT solution (5 mg mL-1) is then added to each well and incubated for 4 h, and 100 μL of Formazan lysate is introduced into each well to dissolve the formed crystals. The absorbance is measured at 570 nm with a Polarstar microplate reader.


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CDs-FAM Probe for Zinc Sensing in Aqueous Medium. 5, 10, 20, 40, 60, 80, 100 μmol L-1 of Zn2+ is added into 40 μL of CDs-FAM aqueous solution (100μg mL-1). The reaction mixtures are filled to 300 l with PBS. The fluorescence ratio of CDs at λex/λem=310/440 nm (ICDs) to that of FAM at λex/λem=310/520 nm (IFAM) is derived, denoted as F= ICDs/IFAM. The relative fluorescence ratio, i.e., (F-F0)/F0, is calculated as the function of Zn2+ concentration, where F and F0 are derived in the presence and absence of Zn2+, respectively. Intracellular Zinc Quantification with Fluorescence Imaging by Correlating to ICPMS Detection. For the purpose of providing suitable level of intracellular zinc for imaging and ICPMS detection, the cells are subject to incubation with certain level of zinc-containing medium and CDs-FAM. The order of incubation by Zn2+ and CDs-FAM or vice versa is vital for providing reliable results. After careful scrutiny, the following order is followed in the ensuing studies. MCF-7 cells are incubated in 24-well plates in the culture medium with 0-100 μmol L-1 Zn2+ for 0, 0.5, 1 and 2 h, followed by washing with PBS and further incubated with CDs-FAM (100 μg mL-1) for 2 h to label the cells. For fluorescence cell imaging, 4% of paraformaldehyde is thereafter added and further incubated for 15 min to fix the cells. After washing with PBS buffer for three times, fluorescence images are taken on a confocal microscope (ex at 405 nm, em at 440-490 nm for CDs, em at 500-550 nm for FAM) and a relative fluorescence ratio (F-F0)/F0 is derived. For intracellular zinc quantification with ICPMS, the above cells are thereafter detached by 0.25% trypsin-EDTA followed by re-suspending in 1.0 mL of PBS buffer solution at pH 7.4. Finally, the cell density (cell number mL−1) is evaluated by using a



hemocytometer. The cell suspension is then added into 2 mL of freshly prepared aqua regia (HCl/HNO3 3:1). Considering that hydrochloric acid may seriously interfere with zinc quantification by ICPMS, the mixture is heated to dryness and diluted with 3 mL of deionized water. The total content of zinc is then quantified by ICPMS, and the amount of zinc in a single cell is calculated. The correlation of ICPMS detection (intracellular zinc content: fg/cell) with fluorescence imaging (fluorescence density: pixel) is performed by following SPSS statistics analysis, to derive a linear correlation (calibration curve) between the relative fluorescence ratio in fluorescence cell images and the intracellular zinc content obtained by ICPMS, and thus to facilitate intracellular zinc quantification via fluorescence imaging.

RESULTS AND DISCUSSION Characterizations of the Probe. TEM and AFM images of CDs are shown in Figure 1 with insets to illustrate the particle size distribution and the particle height profile. It is seen that CDs are well monodispersed spherical, with uniform particle size and exhibit an average diameter of 4.0±1.0 nm. In addition, the experimental results demonstrated virtually no difference for either TEM or AFM images of CDs-FAM with respect to those achieved for the bare CDs. FT-IR spectrum of CDs (Figure 1C) shows absorption bands at 3221 cm-1 and 2980 cm-1 corresponding to stretching vibrations of O-H and N-H, respectively. The absorptions at 1660 cm-1 and 1603 cm-1 are attributed to the stretching vibrations of C=O and C-N, respectively, while those at 1296 cm-1 and 1121 cm-1 arise from the bending of O-H and the stretching of C-O. After modification with aptamer, significant enhancements on the absorptions at 1257 cm-1 and 2935 cm-1 are observed,


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corresponding to the bending vibration of O-H and the stretching vibration of N-H, respectively. The enhancement on the absorption at 1645 cm-1 is due to the stretching vibration of N-C=O. These results well prove the conjugation of aptamer on CDs.

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Figure 1. (A) TEM image of CDs with the inset of size distribution. (B) AFM image of CDs with the inset of height profile along the line. (C) FT-IR spectra of CDs and CDs-aptamer. (D) UV-vis absorption spectra of CDs, aptamer and CDs-aptamer within a range of 300-450 nm.

UV-vis absorption spectra of CDs and CDs-aptamer are given in Figure 1D. CDs have a typical absorption at 350nm due to n-π* transition of C=O bonds, and the aptamer shows a typical absorption of DNA at 260 nm. CDs-aptamer exhibits absorptions at both 260 nm and 350 nm. In addition, the conjugation of negatively charged aptamer fragment results in a variation of zeta potential from -3.37 mV to -7.27 mV. The above observations further confirm the grafting of aptamer onto CDs.



The cytotoxicity of CDs-FAM is tested. The viability of MCF-7 cells incubated with CDs, CDs-FAM for 20 h is determined by MTT assay (Figure S1). At a concentration of 100 μg mL-1 for both CDs and CDs-FAM, the cell viabilities are kept at>95%. This illustrates favorable biocompatibility for CDs and CDs-FAM. FRET in CDs-FAM for Fluorescence Imaging and Zinc Sensing. Fluorescence imaging strategy with CDs-FAM as probe is shown in Scheme S1. After CDs-aptamer is connected to the complementary strand, the two DNA sequences hybridize to form a stable double-stranded structure. The photoluminescence from CDs is received by FAM, thus FRET occurs between them. Zn2+ interacts with aptamer and the specific binding results in an increase of the distance between CDs and FAM, and therefore ceasing the FRET process. 40004000 4000

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Figure 2. (A) The excitation and emission fluorescence spectra of CDs-aptamer and FAM. (B) Emission spectra of CDs-aptamer, CDs-FAM, and CDs-FAM in the presence of 100 μmol L-1 Zn2+.

Figure 2A shows that the maxima of excitation and emission wavelengths for CDs-aptamer and FAM are ex/em=350/440 nm and 470/520 nm, respectively. There is obvious overlap between the emission spectrum of CDs-aptamer and the excitation spectrum of FAM, which proves that FRET can occur between them at an


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appropriate distance. Figure 2B indicates effective quenching on the fluorescence emission at 440 nm for CDs-aptamer in the presence of the complementary strand, and the enhancement on the fluorescence emission at 520 nm for FAM. In the presence of Zn2+, the fluorescence intensity of the CDs-aptamer recovers and FRET is weakened due to the dissociation of the DNA double chain. The CDs-FAM probe is capable of sensing zinc in aqueous medium, by using a ratiometric fluorescence approach, that is, based on the calibration between the relative fluorescence ratio (F-F0)/F0 and zinc content. F is the fluorescence ratio of ICDs/IFAM (at ex 405 nm, em 440-490 nm for the donor CDs, em 500-550 nm for the receptor FAM), while F0 is the fluorescence ratio for the control system at identical conditions. Figure S2 indicated that the fluorescent response of the probe with Zn2+ is a very fast process, and the fluorescence ratio (F-F0)/F0 keeps constant for at least 5 h. It is also illustrated that a neutral medium is preferential for the sensing system, probably due to the effect of pH on the base pairing of DNA fragment and the electrostatic interaction between the negatively charged phosphate group and Zn2+ 25,26. For further studies, pH value of the sensing system is fixed at pH 7.4, which is very close to physiological conditions. Considering that the binding of Zn2+ to the aptamer is associated with spatial configuration of the DNA fragment, a favorable temperature of 40°C is used. 5, 10, 20, 40, 60, 80, 100 μmol L-1 of Zn2+ is added to CDs, CDs-FAM aqueous solutions, respectively, and the fluorescence spectra are measured. CDs are taken as control to show a single emission at ex/em 310/440 nm (Figure S3A). The increase of Zn2+concentration within 5-100 μmol L-1 results in a very limited variation of the fluorescence ratio (I-I0)/I0 for the CDs, i.e., from 0.03 to 0.08 (Figure S3B). As for the CDs-FAM nanoprobe, the fluorescence ratio (F-F0)/F0 increases significantly and is



linearly correlated to the concentrations of Zn2+ within a range of 5-100 μmolL-1 (Figure 3B), giving rise to a regression equation of Y=0.081+2.470×10-3X, along with a correlation coefficient 0.994, and a limit of detection (LOD) of 1.54 μmol L-1. This observation indicated that the CDs-FAM probe significantly improves the sensitivity for zinc detection with respect to the bare CDs. This result is obviously advantageous over other nanoparticle based sensing procedures and it is favorable for intracellular zinc quantification.

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Figure 3. (A) Fluorescence spectra of CDs-FAM respond to different concentrations of Zn2+ in the aqueous medium. (B) Linear relationship between the fluorescence ratio of (F-F0)/F0 and the Zn2+ concentrations in the aqueous medium. λex 310 nm; CDs-FAM: 13 μg mL-1; Zn2+: 5, 10, 20, 40, 60, 80, 100 μmol L-1; Incubation time, 2 min; pH 7; 40℃.

Figure 4A shows the responses of CDs-FAM probe for 50 μmol L-1 of different metal ions including Zn2+, Fe2+, Al3+, Ba2+, Na+, Mg2+, Pb2+, Ca2+, Co2+, Ni2+, Fe3+, K+, Mn2+, Cu2+. It is obvious that the nanoprobe exhibits selective response to Zn2+. A few nitrogen doped CDs have been previously applied for the sensing of Fe3+, Cu2+ 27-29. The surface functional groups, e.g.,–COOH, –OH, –NH2, contribute to the specific recognition. In the present case, although CDs-FAM has been modified with Zn2+


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aptamer, the other functional groups may still interact with Fe3+, Cu2+and cause a slight response. However, this response causes virtually no effect on the quantification of zinc. Figure 4B further indicates minimum effect of a number of coexisting substances at a concentration level of 5 mmol L-1, e.g., serine, glycine, valine, phenylalanine, leucine, histidine, Mg2+, K+, Ba2+, Na+, Fe2+ and Ca2+, on the quantification of zinc. These observations well demonstrated the favorable selectivity of the present CDs-FAM probe for intracellular zinc imaging and quantification.

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Figure 4. (A) Selective response of CDs-FAM fluorescent probe to some metal ions at the concentration level of 50 μmol L-1. (B) The minimum effects from various coexisting substances on the quantification of Zn2+. The concentrations of Zn2+ and the coexisting substances are 50 μmol L-1 and 5 mmol L-1, respectively.

The Correlation of Fluorescence Imaging with ICPMS Detection for Intracellular Zinc Quantification. MCF-7 cells are pretreated with zinc reinforced culture media to vary the intracellular content of zinc, followed by incubation with CDs-FAM to incorporate the fluorescent probe into the cell interior. Figure 5A illustrated that with the increase of Zn2+ concentration in the culture medium within 20-100 μmol L-1, an obvious enhancement on the blue fluorescence of CDs (ICDs) and



a decrement on the green fluorescence of FAM (ICDs) are observed. The fluorescence intensities, i.e., ICDs and IFAM, are measured by using Image J software. The fluorescence ratio, F=ICDs/IFAM, is employed for the purpose of improving the sensitivity for zinc detection, and meanwhile for eliminating the interference of intrinsic fluorescence attributed to the cells. F0 is the fluorescence ratio obtained with those cells without treatment by Zn2+ reinforced culture medium. Figure 5B indicated an obvious increment on the relative fluorescence ratio with the increase of Zn2+ concentration. Control

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Figure 5. (A) Confocal microscopy images of Zn2+ in live MCF-7 cells after incubation with 0, 20, 40, 55, 70, 85, 100 μmol L-1 of Zn2+ for 60 min, at ex 405 nm, em 440-490 nm for blue channel, em 500-550 nm for green channel. (B) The variation of relative fluorescence ratio according to the fluorescence images in Figure 5A analyzed by Image J software.

The amount of intracellular zinc after incubation with culture media reinforced with Zn2+ at 20-100 μmol L-1 is thereafter quantified by ICPMS. As shown in Figure 6A, the endogenous zinc content in the cell is approximately 22.8 fg cell-1. By increasing the concentration of Zn2+ in the culture medium, a significant increment of


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intracellular zinc content is observed. The trend of variation for intracellular zinc content is closely correlative with the fluorescence density in the fluorescence images. It is interesting to see that when comparing the relative fluorescence ratio derived from the fluorescence imaging method with the intracellular zinc content per cell achieved with detection by ICPMS, a linear correlation is obtained giving rise to a Pearson correlation coefficient of 0.998. This clearly indicated the feasibility of using the CDs-FAM probe based fluorescence imaging method for the direct quantification of intracellular Zn2+ by correlating with ICPMS detection. The dependence of the relative fluorescence ratio in the fluorescence images upon the intracellular zinc content per cell as achieved by ICPMS detection is shown in Figure 6B. A linear calibration is derived between the fluorescence ratio (F-F0)/F0 and the intracellular zinc content, with a linear range of 34.7-60.3 fg cell-1. The linear regression equation is (F-F0)/F0 = -0.343 + 0.00966mZn with a similar correlation coefficient as that derived from the SPSS statistics analysis. 60

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Figure 6. (A) Intracellular zinc content in MCF-7 cells measured by ICPMS after incubation with 0, 20, 40, 55, 70, 85, 100 μmol L-1 of Zn2+ for 60 min. (B) Linear correlation between the relative fluorescence ratio achieved by fluorescence imaging and the intracellular zinc content per cell obtained by ICPMS detection.



The real time variation of intracellular zinc level in MCF-7 cells with incubation time is investigated with the above mentioned fluorescence imaging method. Figure 7 indicated that the relative fluorescence ratio reaches a maximum after incubating with 100 μmol L-1 Zn2+ for 60 min, while it remains virtually unchanged by further increasing the incubation time to 120 min. The intracellular Zn2+ at various incubation time is calculated by using the above established linear calibration. It is seen that a maximum intracellular zinc level of 60.5 fg cell-1 is achieved after incubation of MCF-7 cells with 100 μmol L-1 Zn2+ for 60 min. For the purpose of further verifying the accuracy of the fluorescence imaging method, a parallel detection of the intracellular zinc content is performed by ICPMS. Table 1 illustrates the results by CDs-FAM probe based fluorescence imaging method with respect to those achieved by ICPMS detection, where favorable agreements were obtained with a Pearson correlation coefficient of 0.971. Control

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0.0

Control 20 Control 20

40 55 70 85 120 100 40 60 80 100 Time (min)

Figure 7. (A) Confocal microscopy images of Zn2+ in live MCF-7 cells after incubation with 100 μmol L-1 Zn2+ for different times. ex at 405 nm, em at 440-490 nm for blue channel, em at 500-550 nm for green channel. (B) The variation of relative fluorescence ratio derived from the CDs-FAM probe based fluorescence imaging with the incubation time.


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Table 1. Intracellular zinc content in MCF-7 cells as obtained by the established fluorescence imaging approach with respect to those derived by ICP-MS detection. The cells are incubated with 100 μmol L-1 Zn2+ for 0, 20, 40, 60, 80, 100 and 120 min. Incubation time/min

By this method (fg cell-1)

By ICPMS (fg cell-1)

20

49.2±1.1

47.3±0.4

40

55.9±1.6

54.1±0.7

60

63.2±1.2

60.5±1.5

80

62.8±1.6

61.8±0.3

100

61.8±2.9

62.5±1.0

120

63.8±1.7

60.4±1.3

CONCLUSIONS In the present work, the CDs-FAM based FRET system is used for fluorescence imaging of zinc in MCF-7 cells. By correlating the relative fluorescence ratio from the in vitro fluorescence images of MCF-7 cells with the intracellular content of zinc obtained by ICPMS, a linear calibration is derived between them. This facilitates direct quantification of intracellular zinc by fluorescence imaging of live cells. The Zn2+ aptamer incorporating on CDs improves the selective response of the fluorescent probe to zinc, while FRET is used to improve the sensitivity of detection. It should be emphasized that the present approach is especially useful for real-time tracing of intracellular zinc during cell culturing or zinc quantification in vivo.



ASSOCIATED CONTENT *Supporting Information Viability of MCF-7 cells after incubation with various concentrations of CDs (A) and CDs-FAM (B). Schematic illustration of the FRET process based on fluorescent nanoprobe CDs-FAM for fluorescence imaging of Zn2+. The effect of incubation time, pH value and temperature on the detection of zinc with CDs-FAM probe. Fluorescence spectra of CDs respond to different concentrations of Zn2+ in the aqueous medium; Linear relationship between the fluorescent ratio of (I-I0)/I0 and the Zn2+ concentrations in the aqueous medium.

AUTHOR INFORMATION *Corresponding author. E-mail address: [email protected] (Yu) Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work is financially supported by the Natural Science Foundation of China (Nos. 21575020, 21727811, 21675019), Fundamental Research Funds for the Central Universities (Nos. N170505002, N170504017, N170507001).


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For TOC only 100 μM 0.3 0.3

(F-F0)/F0

Blue channel

20 μM

Green channel

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.2 0.2

0.1 0.1

0.0 0.0

32 32

40 40

48

56 56

Zn content (fg cell-1)

ICP-MS


64

Intracellular Zinc Quantification by Fluorescence Imaging with a FRET

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