A Fluorescence Ratiometric Probe for Detection of Cyanide in Water Sample and Living Cells

In the present work, Compound 1 has been synthesized as a novel fluorescence ratiometric probe for CN. Upon treatment with CN, Probe 1 exhibited a fluorescence ratiometric response, with the emission wavelength shift from 570 nm to 608 nm. When 90 μM CN was introduced, the emission ratios (I570/I608) of the probe changed dramatically from 0.52156 to 4.21472. The detection limit was also measured to be 0.24 μM (S/N = 3). In addition, Probe 1 had a selective response to CN, while other anions caused nearly no interference. The sensing reaction product of Probe 1 with CN was characterized by H NMR spectra and ESI Mass spectrometry. Furthermore, Probe 1 has been successfully applied to detect CN in natural water samples. The fluorescence imaging experiments in living cells also demonstrated that Probe 1 could monitor CN in biological samples.


Introduction
Anion recognition has received intense attention due to its important role in an extensive range of environmental, clinical, chemical and biological applications [1].Cyanide (CN − ) is one of the most important anions, and it has been widely used in various industrial fields such as gold mining, electroplating, metallurgy, synthetic fibers and resins [2].But unfortunately, the cyanide is extremely detrimental to the living organism; it can inhibit the cellular respiration upon interacting strongly with the heme unit at the active site of cytochrome a 3 [3].Uptake of the toxic cyanide could occur through absorption by lungs, exposure to skin, and also from contaminated food and polluted drinking water [4][5][6].Therefore, it is very important to develop an efficient method to detect cyanide concentration in natural water sample and biological sample.
Among various methods for measurement of CN − , the fluorescence method based on fluorescent probe is more attractive due to its desirable features including high sen-sitivity, simplicity, and potential for in vivo imaging [7].Accordingly, in the past decade, a large number of fluorescent probes for detection of CN − have been reported in the literature [8].Whereas many of them only utilized the changes in emission intensity as detecting signals.A major limitation of the intensity-based fluorescent probe is that the signal output could be interfered by the factors such as environmental conditions, probe distribution, and instrumental efficiency [9,10].By contrast, a ratiometric measurement, employing the ratio of two emissions at different wavelengths as the detecting signal, could provide a built-in correction for the above mentioned factors and thus allow more accurate analysis [11,12].However, there are only very few fluorescence ratiometric probes that have been applied to monitor CN − concentration in water samples or biological samples [13][14][15][16][17][18].
Encouraged by these considerations, we developed Compound 1 as a novel fluorescence ratiometric probe for CN − in this work.Upon treatment with CN − , the probe showed ratiometric response.In addition, the probe has been successfully applied to detection of CN − level in natural water samples and living cells.

Reagents and Apparatus
Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification.Solvents were purified by standard methods prior to use.Twice-distilled water was used throughout all experiments.
Mass spectra were recorded on a LXQ Spectrometer (Thermo Scientific) operating on ESI. 1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance 400 spectrometer operating at 400 MHz and 100 MHz respectively.Elemental (C, H, N) analysis were carried out using Flash EA 1112 analyzer.Electronic absorption spectra were obtained on a SHIMADZU UV-2450 spectrometer.Fluorescence spectra were measured on a Photon Technology International (PTI) Quantamaster fluorometer with 3 nm excitation and emission slit widths.Cells imaging were performed with an inverted fluorescence microscope (Carl Zeiss, Axio Observer A1).All pH measurements were performed with a pH-3c digital pH-meter (Shanghai ShengCi Device Works, Shanghai, China) with a combined glass-calomel electrode.

Synthesis of Compound 3
Fresh distilled DMF (6.5 mL) was added dropwise to POCl 3 (6.5 mL) at 20˚C -50˚C with N 2 atmosphere and stirred for 30 minutes to yield a red solution.This solution was added to a solution of 7-diethylaminocoumarin (4.50 g, 20.7 mmol) in 30 mL DMF to allow a scarlet suspension.The mixture was stirred at 60˚C overnight and then poured into 300 mL of ice water.NaOH solution was added to adjust the pH = 5.0 of the mixture to yield large amount of precipitate.The crude product was filtered, thoroughly washed with water, dried and recrystallized in absolute ethanol to give 3 (3.67 g, yield 72.3%). 1

Optical Response to CN −
The sensing properties of Probe 1 in response to CN − were investigated in 20 mM potassium phosphate buffer/ CH 3 CN (v/v 1: 4, pH 7.4) at room temperature.As shown in Figure 1(a), in the absence of CN − , Probe 1 displayed fluorescence emission centered at 570 nm.However, when increasing concentrations of CN − were introduced, the emission at 570 nm gradually decreased.Concomitantly, a new emission centered at 608 nm appeared and increased, with a well-defined isoemission point at 588 nm.The changes in fluorescence emission spectra also elicited an obvious variation in emission color.With the addition of CN − , the fluorescence color of Probe 1 changed from green to red (Figure 1(a)).Therefore, Probe 1 can be used as a nake eye indicator for CN − .In addition, the emission ratio (I 608 /I 570 ) of Probe 1 response to CN − displayed a large increase from 0.52156 to 4.21472 after 90 μM CN − added (8-fold enhancement) (Figure 2).The emission ratios (I 608 /I 570 ) also showed a good linearity with CN − concentration in the range of 0 -30 μM (Figure 1(b)), indicating the probe can be potentially used to quantitatively detection of CN − .The detection limit for CN − was estimated to be 0.24 μM (S/N = 3) according to a reported procedure [19].The low detection limit together with the large emission ratio enhancement demonstrates that Probe 1 is highly sensitive to CN − .The absorption spectra of Probe 1 in the nm.

Response Time and Effect of pH
The kinetic studies of Probe 1 in the absence or presence of CN − was investigated by fluorescence spectra.As displayed in Figure 5, in the absence of CN − , almost no variation in emission intensity (at 608 nm) of Probe 1 was found, implying that Probe 1 was stable in the assay condition.However, upon addition of CN − (90 μM), a dramatic enhancement in emission intensity at 608 nm was observed, denoting the rapid reaction of Probe 1 with CN − .And the emission intensity reached a plateau after 30 min reaction.The responses of Probe 1 toward CN − at different pH conditions were also conducted (Figure 6).Probe 1 can be employed to detect CN − in the pH range of 5.5 -9.5, and function properly at physiological pH.Thus, Probe 1 can be potentially utilized to detect CN − in biological samples.

Reaction Products of Probe 1 with CN −
In order to investigate the reaction product of Probe 1 with CN − , the product of the Probe 1 with CN − was isolated and subjected to 1 H NMR characterization.As shown in Figure 7, the resonance signal of hydrogen (H d ) at 4-position of the coumarin ring was completely disappeared in the isolated product of Probe 1 with CN − .This observation clearly indicated that the hydrogen at the 4-position of the coumarin ring was substituted by CN − and formed a 1-CN adduct.Moreover, the formation of 1-CN adduct was further confirmed by ESI Mass spectrometry, where a major peak at m/z 373.35 is assigned to [1-CN+H] + (Figure 8).Thus, we proposed a possible reaction mechanism as shown in

Detection of Cyanide in Natural Water Samples
The water resource may be contaminated by CN − from the industrial waste.According to the World Health Organization, the maximum acceptable level of cyanide in drinking water is 1.9 μM [20].Thus it is high importance to monitor the level of CN − in water samples.The crude water samples were obtained from Yangtzi River, pond water and tap water, and were filtered through microfilmtration membrane before use.After the probe being treated with the water samples, ratiometic values (I 608 /I 570 ) were determined.The CN − concentration in these water samples was not detected.Next, the water samples were spiked with standard CN − solutions and then analyzed with Probe 1, the results are shown in Table 1.PROBE 1 was able to measure the concentrations of spiked CN − with good recovery.

Fluorescence Imaging in Living Cells
To study the utility of Probe 1 detecting CN − in biological sample, the Probe 1 was applied for fluorescence imaging in living cells.The pancreatic cancer cells was incubated with Probe 1 (1 μM) for 30 min at 37˚C.After washing with PBS buffer three times, the cells were used

Conclusion
A fluorescence ratiometric probe, Compound 1, for CN − has been constructed.Upon treatment with CN − , the fluorescence of Probe 1 exhibited red shift from 570 nm   to 608 nm, with the emission ratio (I 608 /I 570 ) changing dramatically from 0.52156 to 4.21472.Probe 1 also had a selective response towards CN − , while other anions gave almost no influence on Probe 1 detection of CN − .Furthermore, Probe 1 has been successfully applied to detection of CN − in natural water samples and living cells.

Scheme 1 .
Scheme 1.(a) the sensing reaction of Probe 1 with CN − ; (b) the synthetic procedure of Probe 1.

Figure 4 , 3 HSO 3 H
Probe 1 response to other anions was also investigated.The anions such as F − , Cl − , Br − , I − , PO  HCO  , 3 NO  , SCN − exerted no visible effect on the fluorescence ratios (I 608 / I 570 ) of Probe 1. Obviously, large fluorescence ratio change was only observed for Probe 1 treated with CN − .Moreover, the ratiometric responses of Probe 1 toward CN − in the presence of other anions were examined.Most of other anions gave nearly no influence on Probe 1 detection of CN − (Figure 4).These results demonstrated that Probe 1 had selective response towards CN − .

Figure 9 .
It included a nucleophilic addition reaction of the CN − with the coumarin ring, and subsequent an elimination reaction.The specific nucleophilic addition reaction renders Probe 1 selective response to CN − .

a
Relative standard deviations were calculated based on three times of measurement.

Figure 10 .
Figure 10.Fluorescence image of the pancreatic cancer cells stained with Probe 1 (1 μm) for 30 min with emission at 530 ± 10 nm a) and emission at 610 ± 10 nm b); fluorescence image of the pancreatic cancer cells pre-treated with CN − (60 μm) for 10 min, and then stained with Probe 1 (1 μm) for 30 min with emission at 530 ± 10 nm c) and emission at 610 ± 10 nm d).