A ratiometric fluorescence probe for monitoring cyanide ion in live cells

Accepted Manuscript Title: A ratiometric fluorescence probe for monitoring cyanide ion in live cells Author: Jianbin Chao Zhiqing Li Yongbin Zhang Fangjun Huo Caixia Yin Hongbo Tong Yuhong Liu PII: DOI: Reference:

S0925-4005(16)30033-8 http://dx.doi.org/doi:10.1016/j.snb.2016.01.033 SNB 19542

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

16-11-2015 30-12-2015 8-1-2016

Please cite this article as: Jianbin Chao, Zhiqing Li, Yongbin Zhang, Fangjun Huo, Caixia Yin, Hongbo Tong, Yuhong Liu, A ratiometric fluorescence probe for monitoring cyanide ion in live cells, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.01.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A Ratiometric Fluorescence Probe for Monitoring Cyanide Ion in Live Cells

Jianbin Chaoa*, Zhiqing Lia,b, Yongbin Zhanga, Fangjun Huoa, Caixia Yinc, Hongbo Tongb, Yuhong Liua,b

a

Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, P.R.

China b

School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006,

China c

Institute of Molecular Science, Shanxi University, Taiyuan, 030006, China

*

Corresponding author.

Jianbin Chao Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, P. R. China Tel.: ＋86-351-701 7838; Fax: ＋86-351-701 6048. E-mail address: [email protected] (Jianbin-Chao)

1

Graphical abstract

In this paper, a ratiometric emission fluorescent probe 1,1-dimethyl-3-ethyl-2-(2-(N-ethyl-carbazol-3-yl)-vinyl)-1H-benzo[e]indolium iodide (Probe) is facilely synthesized via the ethylene bridging of N-ethylcarbazole-3-formaldehyde and 1,1,2-trimethyl-3-ethyl-1H-benz[e]indolium iodide, which is a useful fluorescent probe for monitoring cyanide ion at extremely low concentrations (1-2 μM), quantitatively. The detection of cyanide is performed via the nucleophilic attack of cyanide toward the benzo[e]indolium group of the probe, resulting in a prominent fluorescence ratiometric change and a color change. The titration experiments for CN- show that it exhibits a large Stokes shift (92 nm), high selectivity, excellent sensitivity and good stability for CN- sensing in DMSO. The detection limit of probe for CN- is found to be 0.23 μM. Furthermore, laser scanning confocal microscopy of U251 cells demonstrates that this probe has excellent cell membrane permeability and might be used to monitor the minor CNconcentration in biological system

2

Highlights 1. A ratiometric fluorescent probe (Probe) is facilely synthesized via the ethylene bridging of N-ethylcarbazole-3-formaldehyde and 1,1,2-trimethyl-3-ethyl-1H-benz[e]indolium iodide. 2. It is a useful fluorescent probe for monitoring cyanide ion at very low concentrations (1-2 μM), quantitatively. 3. The detection limit of probe for CN- is found to be 0.23 μM. 4. It exhibits a large Stokes shift, good selectivity, sensitivity and stability. 5. Fluorescent CN- imaging in living cells by this probe is further successfully applied.

3

Abstract:

A

ratiometric

emission

fluorescent

probe

1,1-dimethyl-3-ethyl-2-(2-(N-ethyl-carbazol-3-yl)-vinyl)-1H-benzo[e]indolium iodide (Probe)

is

facilely

synthesized

via

the

ethylene

bridging

of

N-ethylcarbazole-3-formaldehyde and 1,1,2-trimethyl-3-ethyl-1H-benz[e]indolium iodide, which is a useful fluorescent probe for monitoring cyanide ion at extremely low concentrations, quantitatively. The detection of cyanide is performed via the nucleophilic attack of cyanide toward the benzo[e]indolium group of the probe, resulting in a prominent fluorescence ratiometric change and a color change. The titration experiments for CN- show that it exhibits a large Stokes shift (92 nm), high selectivity, excellent sensitivity and good stability for CN- sensing in DMSO system. The detection limit of Probe for CN- is found to be 0.23 μM. Furthermore, the probe has excellent cell membrane permeability and is applied successfully to rapidly detect CN- in living cells.

Keywords: Ratiometric fluorescence, Cyanide-sensing, Nucleophilic addition, Detection limit, Bioimaging

4

1. Introduction Because of the importance of anions in biological, environmental, and industrial processes, the development of optical chemosensors for anions has recently been an area of great interest [1-9]. Among various anions, cyanide ion is of concern because it is an extremely toxic anion and can directly lead to the death of organisms. However, cyanide salts are still used as industrial materials in plastics production, gold mining, and other fields [10-13]. According to the World Health Organization (WHO), the permissible level of cyanide in drinking water is 1.9 μM [14]. Therefore, the selective and sensitive detection of cyanides is recently attracting a considerable interest for the environmental protection as well as the human healthcare [15-20]. The conventional detection methods include titrimetric, mass spectrometric, and spectrophotometric determinations as well as electrochemical, flow injection, quartz crystal monitor, ion chromatography, and headspace gas chromatography analyses [21-30]. These detection methods suffer from a number of disadvantages such as requiring large sample sizes, long analysis times, high detection limits, and poor precision, largely due to interferences. The focus of our current research is to provide a single analytical sensor that combines the advantages found in many of the previously mentioned cyanide studies [21-30]. Among the less conventional methods devised for sensing cyanide ions, those which utilize chemical reactions that produce fluorometric and colorimetric responses have proven to be the most convenient owing to their simplicity, high sensitivity and inexpensive nature [31]. Therefore, strategies to design these types of chemosensors have attracted significant attention over the past few years, including the formation of cyanide complexes with transition metals [32-34], boron derivatives [35-37] and CdSe quantum dots [38], hydrogen-bonding interactions [39], deprotonation [40], displacement method [41-43], single-electron transfer reaction [44] and nucleophilic addition reactions [45-51]. We are especially interested in nucleophilic reaction-based receptors, because this type of sensing system takes advantage of the particular feature of the cyanide ion: its nucleophilic character, and enables the recognition system with some characteristic features such

5

as analyte-specific response and little competition, which are highly desirable features for an efficient recognition and detection system. As a versatile probe technique, elicitation of fluorescence has the advantages of high sensitivity, low cost, easy detection, and suitability as a diagnostic tool for biological concerns. However, development of ratiometric fluorescence probes for intracellular CN- measurement is still limited by the design of dual-emission organic fluorophores which should be specific and sensitive to CN-. Actually, the ratiometric sensor has attracted significant attention as an alternative to first-generation intensity probes due to its sensitivity and the built-in correction for avoiding environmental effects. In the present work, we report a novel ratiometric fluorescence probe (Figure 1) for

monitoring

cyanide

N-ethylcarbazole-3-formaldehyde

ion

via

as

an

the

ethylene

electron

donor

bridging (D)

of and

1,1,2-trimethyl-3-ethyl-1H-benz[e]indolium iodide as an electron acceptor (A). The D-π-A structure type is frequently adopted as fluorophore to construct intramolecular charge transfer (ICT) fluorescent probes displaying a large Stokes shift [52-54]. Here, CN- is expected to be detectable by nucleophilic attack toward the carbon atom of the C=N group, which is activated by the strong electron-withdrawing feature of the positively-charged benzo[e]indolium fragments and the N-ethylcarbazole group, and two well-separated emission peaks before and after the CN- addition could be obtained, as well as an obvious color change. Notably, this probe was found to show high selectivity and sensitivity for CN-, and also importantly, it is able to detect CN- at extremely low concentrations, quantitatively. In addition, it shows a large Stokes shift (92 nm), which is highly desirable for a fluorescent probe to achieve reliable and sensitive fluorescent detection. Moreover, fluorescent CN- imaging in living cells by this probe was also successfully applied.

2. Experimental section 2.1 Materials and Methods Probe was synthesized in our own laboratory. Unless otherwise stated, all other reagents were purchased from commercial suppliers and used without further 6

purification. Reactions were carried out on the magnetic stirrers and their reaction process were monitored on thin layer chromatography (TLC). Absorption spectra were measured with a UV-757CRT spectrophotometer (Shanghai Precision & Scientific Instrument Co., Shanghai, China) in a 4.5 mL (1 cm in diameter) cuvette with 2mL solution. Fluorescence measurements were conducted on a Hitachi F-7000 fluorescence spectrophotometer. NMR spectra were recorded on a Bruker instrument (AVANCE ‫ ׀׀׀‬HD) with TMS as the internal standard in DMSO-d6 of 600 MHz for 1H NMR and 150 MHz for 13C NMR, respectively. Mass spectra were acquired with an Agilent Accurate-Mass-Q-TOF MS 6520 system equipped with an electrospray ionization (ESI) source (Agilent, USA). Cell imaging was performed in a FV1000 confocal laser scanning microscope (Olympus Co., Ltd. Japan) with a 200× objective lens. A crimson single crystal of Probe was mounted on a glass fiber for data collection. Cell constants and an orientation matrix for data collection were obtained by least-squares refinement of diffraction data using a Bruker SMART APEX CCD automatic diffractometer. Data were collected at 296 K using Mo Kα radiation (α= 0.71073 Å) and the w-scan technique, and corrected for the Lorentz and polarization effects (SADABS). The structures were solved by direct methods (SHELX97), and subsequent difference Fourier maps were inspected and then refined in F2 using a full-matrix least-squares procedure and anisotropic displacement parameters [55]. 2.2

Synthesis

of

1,1-dimethyl-3-ethyl-2-(2-(N-ethyl-carbazol-3-yl)-vinyl)-1H-benzo[e]indolium iodide (Probe) N-ethylcarbazole-3-formaldehyde

(0.223

g,

1

mmol)

and

1,1,2-trimethyl-3-ethyl-1H-benz [e]indolium iodide (0.365 g, 1 mmol) were heated in dry ethanol (10 mL) at reflux for 4 h with vigorous stir. The reaction was cooled to room temperature and the precipitate was collected by filtration, washed with diethyl ether, and dried in vacuo. Probe was obtained as a crimson solid in 59.6% yield (0.34 g). As depicted in Figure 1, Probe was easily synthesized in a one-step reaction, and the purification was simple. The structure of Probe was fully characterized by NMR and Single Crystal Diffraction (Figure 2). 1H NMR (DMSO-D6, δ/ppm): 9.142 (s, 1H), 7

8.797 (d, J =16.2 Hz, 1H), 8.463 (d, 2H), 8.312 (d, 2H), 8.233 (d, J =7.8 Hz, 1H), 8.129 (d, J =8.4 Hz, 1H), 7.885 (d, J = 9 Hz, 1H), 7.835 (m, 1H), 7.770 (m, 3H), 7.603 (m, 1H), 7.399 (m, 1H), 4.875 (q, J= 6.6 Hz, 2H), 4.580 (q, J = 6.6 Hz, 2H), 2.095 (s, 6H), 1.568 (t, J = 6.6 Hz, 3H), 1.399 (t, J = 6.6 Hz, 3H).

13

C NMR (DMSO-D6,

δ/ppm): 181.86, 155.28, 131.48 130.52, 129.24, 128.85, 127.48, 127.38, 125.76, 123.50, 121.25, 121.04, 113.43, 110.78, 110.74, 108.58, 56.49, 53.92, 42.30, 40.52, 38.01, 26.32, 14.35. Crystal data for C32H31N2I: crystal size: 0.35 × 0.33 × 0.30, monoclinic, space group C2/c. a = 32.0137 (10) Å, b = 8.8744 (3) Å, c = 20.6922 (7) Å, β = 107.502 (1)°, V = 5606.6 (3) Å3, Z = 8, T = 296 K, θmax = 25.05°. Final residual for 326 parameters and 4955 reflections with I > 2σ (I): R1 = 0.0764, wR2 = 0.1729 and GOF = 1.060. HRMS (ESI) calcd. for [M]+ 443.2482, found 443.2469 (Figure S1). Inset Figure 1 and Figure 2 2.3 General UV-vis and fluorescence spectra measurements CN- solution was prepared by dissolving potassium cyanide in deionized water. Aqueous anions solutions were also prepared using deionized water. Stock solution of Probe (1.0 mM) was prepared in DMSO and the solution for spectroscopic determination was obtained by diluting the stock solution to 10 μM in DMSO medium. Spectral data were recorded after each addition. The excitation wavelength was 340 nm. The excitation and emission slit width were 2.5 nm and 5 nm, respectively. The resulting solution was shaken well and kept at room temperature for 10 min before recording its absorption and fluorescence spectra.

2.4 Cell culture and fluorescence imaging U251 cells were cultured in DMEM containing 10% FBS (Fetal Bovine Serum) and 1% antibiotic-antimycotic solution at 37 ℃ in a 5% CO2/95% air incubator. One day before imaging, the cells were plated on 35 mm diameter round glass Petri dishes and allowed to adhere for 24 h. Subsequently, the cells were incubated with Probe (10 μM) dissolved in DMSO for 5 min at 37 °C and then washed with pre-warmed PBS (pH=7.4) three times. Using excitation wavelengths at 405 and 488nm, respectively, 8

the images of the red channel were obtained in the 425-500 nm detection range, and the images of the blue channel were collected in the 520-620 nm range. In order to detect CN- in living cells, Cells were stimulated with CN- (3 μM) in its culture dish, and then fluorescence microscopic images were acquired over 30 s.

3. Results and discussion 3.1 Proposed mechanism The visible color and strong fluorescence of Probe was due to their outstanding ICT effect from the electron-donor carbazole group to the electron-acceptor benzoindole moiety. As illustrated in Figure 1, the reaction mechanism could be reasonably explained by the nucleophilic addition reaction of cyanide anion with the polarized C=N bond of the benzo[e]indolium group. As a result, the π-conjugation between benzo[e]indolium moiety and carbazole group was blocked. 1H NMR analysis was also carried out to demonstrate the proposed addition mechanism. As anticipated, after addition of potassium cyanide powder into the solution of Probe, the nucleophilic attack of CN- toward the positively-charged benzo[e]indolium group weakened its electron-withdrawing character and led all the 1H NMR signals up-field shifted (Figure 3). It can be seen that the proton signal (H-a, at δ 2.095) of two methyl groups was shifted up-field and divided into two single signals (H-a1, at δ 1.794 and H-a2, at δ 1.359), which became non-equivalent after formation of Probe-CN. Moreover, the proton signals (H-b, at δ 4.580 and H-c, at δ 1.399) of ethyl group connected with N+ were dramatically shifted up-field to δ 3.399 and δ 1.312, respectively. The vinyl protons at δ 8.797 (H-d) and δ 7.770 (H-e) were upfield shifted to δ 6.589 (H-d) and δ 7.207 (H-e) upon cyanide addition at room temperature due to the breaking of the conjugation. This observation clearly indicated that the cyanide anion was added to the benzo[e]indolium group (Figure 3). In addition, the formation of the Probe-CN adduct was further confirmed by mass spectrometry analysis (ESIMS), the peak at m/z 470.2570 (calcd. = 470.2590) corresponding to [probe-CN + H]+ was clearly observed (Figure S1). Inset Figure 3 9

3.2 The solvent dependence and the kinetic study The solvent of a system is often considered as a significant influencing factor on interactions. The fluorescent spectral response of Probe (10 μM) in the absence and presence of CN- (2 μM) at different ratio values between DMSO and H2O was evaluated as shown in Figure S2. From Figure S2, we could find that Probe was stable and displayed the best response for CN- in DMSO. So, in the subsequent UV-vis and fluorescence experiments, DMSO was selected as a testing system to investigate the spectral response of Probe to CN-. Time dependent fluorescence arising from the interaction of Probe (10 μM) with CN- (1 μM) was investigated by monitoring changes in fluorescence at 440 and 595 nm (I440/I595) as a function of time, suggesting that the reaction could be completed within 10 min in the experimental conditions (Figure S3). Kinetics measurement of Probe (10 μM) reaction with CN- (100 μM) under the pseudo-first-order conditions gave an observed rate constant of Kobs = 0.0041 s-1, t1/2 = 168.24 s (Figure S4). Thus, the detection was delayed for 10 min to ensure the reaction between Probe and CNaccomplished completely.

3.3 Investigation of Spectral Properties of Probe The absorption spectra of Probe (10 μM) after addition of various amounts of CN- (0-23 μM) were shown in Figure 4. Probe exhibited an absorption at 503 nm, which was attributed to the large π-conjugation. Upon the addition of CN-, the absorption peak at 503 nm was gradually attenuated and a broad band from around 250 to 300 nm was increased, suggesting that the π-conjugation was interrupted due to the nucleophilic attack of CN-, which also induced the obvious color change from crimson to colorless (inset of Figure 4); The fluorescence response of Probe (10 μM) toward CN- (0-2 μM) in DMSO was investigated as demonstrated in Figure 5. Initially, the Probe solution exhibited an intense emission peak centered at 595 nm (Absmax = 503 nm) with a large Stokes shift of 92 nm. The large Stokes shift could help to reduce the excitation interference. But the addition of CN- resulted in a gradual decrease in emission at 595 nm and an increase in emission at 440 nm. As a 10

consequence, an obvious color change in the solution from crimson to blue occurred, which could be observed easily under a handheld UV lamp (inset of Figure 5a). Meanwhile, a clear isosbestic point was also noted at 565 nm, indicating the formation of the Probe-CN adduct. Then the fluorescence quantum yield of Probe was Ф = 0.12 relative to rhodamine B (Ф = 0.69 in MeOH [56]). After the complete reaction with CN-, the Quantum yield of Probe-CN complex was Ф = 0.17 relative to quinine (Ф = 0.58 in 0.1 M H2SO4 [57]). A titration profile of the ratio of the emission intensities at 440 and 595 nm (I440/I595) to the CN- concentrations from 0 to 2 μM could be observed (Figure 5b). The ratio value was enhanced with the addition of CN- and became constant when the amount of CN- added reached 0.2 equiv (2 μM). To our knowledge, the notable fluorescent spectral change caused by 0.025 equiv of CN(0.25 μM) and obtaining the maximal spectral signal with 0.2 equiv of CN- (2 μM) were impressive and unprecedented compared with those of many reported CNprobes. Moreover, as plotted in the inset of Figure 5c, the signal ratio I440/I595 showed a good linearity with CN- concentration in the range of 1-2 μM [Y = 28.8X – 16.9 (R2 = 0.9902)]. The detection limit was calculated to be 0.23 μM (S/N = 3), which was below the WHO detection level. It was also worth mentioning that the shift of the two emission wavelengths was very large (△F = 155 nm), which contributed to the accurate measurement of the intensities of the two emission peaks, as well as resulted in a huge ratiometric value. These findings suggested that Probe was indeed a sensitive detector and quantitative monitor of CN- ions in DMSO media. Inset Figure 4 and Figure 5 An important feature of Probe was that it had special selectivity for a kind of analyte over other substance. In order to study its special recognition ability, we investigated the fluorescence behavior of Probe (10 μM) in the presence of various typical potential interfering analytes, such as F-, Cl-, Br-, I-, CO32-, HCO3-, NO3-, SO42-, AcO-, CNS-, HSO3-, SO32-, S2O32-, S2-, HS-, Cys, Hcy and GSH (1 equiv., respectively). Remarkably, as exhibited in Figure S5, unperturbed effect on the intensity ratio (I440/I595) of Probe was observed for all these typical analytes, compared with that obtained for CN-. Moreover, competitive assays indicated that the 11

coexistence of all these typical anions (100 equiv., respectively) and biothiols (Hcy and GSH: 5 equiv., Cys: 50 equiv., respectively) had little impact on the ability of Probe to detect CN--ions (Figure 6). These experiments manifested that Probe had high selectivity and strong anti-jamming capacity for CN- determination against kinds of analytes. Inset Figure 6 3.4 Bioimaging and biosensing of CN- in live cells Encouraged by the aforementioned results, we further investigated the applicability of Probe for intracellular CN- detection and imaging with U251 cells. Dynamic fluorescence imaging was performed to observe the reaction process between Probe and CN- in real time. We used the confocal laser scanning microscope to directly visualize the fluorescence change of U251cells incubated with Probe under a certain concentration of CN- (3 μM) conditions in its culture dish over time (0, 10 s, 30 s) (Figure 7). The red fluorescence (520-620 nm) and blue fluorescence (425-500 nm) were captured by 488 and 405 nm laser, respectively, and the fluorescence intensity was calculated by use of commercial software. It was obvious that the U251 cells displayed bright red fluorescence (Figure 7a) and very weak blue fluorescence (Figure 7d) emission, initially. In just thirty seconds, the red fluorescence intensity decreased sharply (Figure 7a-c); by contrast, the blue fluorescence intensity increased pronouncedly (Figure 7d-f). These cell experiments showed excellent cell-membrane permeability of Probe, and it could thus be used to mark CN- in living cells. Inset Figure 7

4. Conclusion In conclusion, a ratiometric probe for CN- is facilely synthesized via the ethylene bridging

of

N-ethylcarbazole-3-formaldehyde

1,1,2-trimethyl-3-ethyl-1H-benz[e]indolium

iodide.

The

nucleophilic

and addition

reaction between CN- and Probe blocks the π-conjugation of this fluorophore and results in blue shift both in absorption (218 nm) and emission spectra (155 nm). Upon the addition of CN-, Probe exhibits a remarkable emission ratio (I440/I595) 12

enhancement and responds linearly to minor CN- of 1-2 μM. Probe also shares some other desired properties, such as large Stokes shift, which can reduce the excitation interference; as well as high selectivity and excellent sensitivity and good stability, all of which are favorable for intracellular CN- imaging. Application of Probe to CNimaging in live U251 cells is also achieved successfully, indicating that the probe has good cell membrane permeability and can be used to rapidly detect CN- in living cells. Most importantly, Probe represents a new type of fluorescent probe for CN- with ratiometric emission property for the low detection limit of 0.23 μM which can be used to noninvasively measure CN- in biological systems. Therefore, the probe proposed here has a great potential application for real-time monitoring and quantification of the extremely toxic CN- found in biological and environmental samples.

Acknowledgements The work was supported by the National Natural Science Foundation of China (No. 21472118), the Shanxi Province Science Foundation for Youths (Nos. 2012021009-4 and 2013011011-1), the Shanxi Province Foundation for Returnee (No. 2012-007), the Taiyuan Technology star special (No. 12024703), the Program for the Top Young and Middle-aged Innovative Talents of Higher Learning Institutions of Shanxi (TYMIT, No. 2013802), talents Support Program of Shanxi Province (No. 2014401) and Shanxi Province Outstanding Youth Fund (No. 2014021002), the Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (20111001), and the Shanxi Province Foundation for College Students of Science and Technology Innovation projects (No.2013328).

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Biographies Jianbin Chao is a professor in the Research Institute of Applied Chemistry at Shanxi University major in organic chemistry. His current research interest is supramolecular chemistry. Zhiqing Li is studying her masters in the School of Chemistry and Chemical Engineering at Shanxi University. She received her B.Sc. in chemistry at Shanxi Normal University in 2014. Yongbin Zhang is an associate professor in the Department of Organic Chemistry, Research Institute of Applied Chemistry at Shanxi University. His current research interest is synthesis chemistry. Fangjun Huo is an Assoiate Professor in Research Institute of Applied Chemistry at Shanxi University major in organic chemistry. His current research interests are sensors, supramolecular chemistry. Caixia Yin is a Professor in Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science at Shanxi University major in inorganic chemistry. She current research interests are molecular recognition, sensors chemistry. Hongbo Tong is an associate Professor in the Research Institute of Applied Chemistry at Shanxi University major in organic chemistry. His current research interest is organometallic chemistry. Yuhong Liu is studying her masters in the School of Chemistry and Chemical Engineering at Shanxi University. She received her B.Sc. in chemistry at Yuncheng University in 2013.

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Figure captions Figure 1 Synthesis of Probe and schematic diagram of Probe for CN- detection. Figure 2 Crystal structure of Probe. Figure 3 1H NMR spectra of Probe in DMSO-d6 before (a) and after (b) the addition of KCN (Portion 1H NMR spectra). Figure 4 Absorption spectra of Probe (10 μM) upon the addition of CN- in DMSO. CN- concentrations were varied in the following order: 0, 1.5, 3, 4.5, 6, 7.5, 9, 10.5, 12, 13.5, 15, 16.5, 18, 20, 23 μM. Inset: Photograph of Probe solution (10 μM) in the absence or presence of CN- (25 μM). Figure 5 Fluorescence spectra of Probe (10 μM) upon the addition of CN- in DMSO. CN- concentrations were varied in the following order: 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2 μM. λex = 340 nm. Slits: 2.5 nm/5 nm. Inset a: Photograph of Probe solution (10 μM) in the absence or presence of CN- (2 μM) under a UV light. Inset b: The plot of I440/I595 versus the concentration of CN- (0-2 μM). Inset c: the linear relation for concentration of CN- in the range of 1-2 μM. Figure 6 Fluorescence responses of Probe (10 μM) in DMSO toward the competing anions (100 equiv. respectively) and biothiols (Hcy and GSH: 5 equiv., Cys: 50 equiv., respectively). The red bars represented the addition of 1.8 μM CN- to the probe solution. The black bars displayed the subsequent addition of different competing analytes to the fluorescent probe. λex = 340 nm. Slits: 2.5 nm/5 nm. Figure 7 Dynamic fluorescence imaging of U251 cells incubated with Probe (10 μM) under a certain concentration of CN- (3 μM) conditions in its culture dish. (a-c) Image in red channel (520-620 nm); (d-f) Image in blue channel (425-500 nm); (g) bright-field image of panel a; The excitation wavelengths were 488 and 405 nm for red and blue fluorescence, respectively.

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Figure 5

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Figure 6

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Figure 7

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