Exploration and application of a highly sensitive bis(salamo)-based fluorescent sensor for B4O72− in water-containing systems and living cells

A highly selective fluorescent sensor H4L based on a bis(salamo)-type compound with two N2O2 chelating moieties as ionophore was successfully developed. Sensor H4L was found to have excellent selectivity for B4O72− over many other anions (Br−, CI−, CN−, CO32−, HCO3−, H2PO4−, HSO4−, NO3−, OAc−, S2O3−, SCN−, SO42−, Hcy (homocysteine) and H2O2), and it exhibited an approximately 150-fold enhancement of the fluorescence response to B4O72− in Tris-HCl buffer (DMF/H2O = 9:1, v/v, pH = 7) solutions. Significantly, its fluorescence intensity was enhanced in a linear fashion with increasing concentrations of B4O72−. The detection limit of sensor H4L towards B4O72− was 8.61 × 10−7 M. The test strips could conveniently, efficiently and simply detect B4O72− ions in Tris-HCl buffer (DMF/H2O = 9:1, v/v, pH = 7) solutions. Furthermore, sensor H4L showed excellent membrane permeability in living cells, and it was successfully used to monitor intracellular B4O72− by confocal luminescence imaging.

could be of interest. Up until now, with the development of optical sensors for recognizing heavy and transition metal ions in living organisms [5][6][7][8][9][10][11][12][13][14][15] , intense efforts have been devoted to the design and synthesis of high sensitivity fluorescent sensors due to their low cost and rapid response as well as the easy operability of the fluorescent technique [16][17][18][19][20][21][22] . According to the relevant literature, the metal complexes of N 2 O 2 salen-type ligands and corresponding analogues could be used in catalysis 23,24 , nonlinear optical materials and magnetic materials [25][26][27][28][29][30][31][32][33][34] , supramolecular architecture 35,36 , ion recognition [37][38][39][40][41][42][43][44][45] , biological fields and so forth [46][47][48][49][50][51][52] . Today, studies on the participation of salamo-type compounds in ion recognition have yet to be explored [53][54][55][56][57][58][59][60][61][62][63] . Notably, compared with most of the known fluorescent probes for Zn 2+ , Cu 2+ , and CN − , there are relatively few reports on fluorescent probes for B 4  Effect of the pH on sensor H 4 L. In order to remove the interference by protons during the detection of B 4 O 7 2− and to find the optimal sensing conditions, further tested was performed in the pH range of 1 to 12. As shown in Fig. 1    2− may be attributed to that borates are hydrolyzed to form boric acid: Four coordinated organoboron compounds based on N,O-chelation are constructed mainly by structures 1, 2 and 3 as the ligand backbone (Fig. S4a). The weak fluorescence of sensor H 4 L was attributed to the lone pairs of electrons on the nitrogen atoms, which lead to intra-molecular photoinduced electron transfer (PET). Due to the lack of electronic properties, the Lewis bases such as the N atoms of the salamo moieties from the H 4 L unit coordinate to the B atoms, resulting in a unique electronic structure and optical properties after B atoms are incorporated into the conjugated system. Four coordinated organoboron compounds can produce strong fluorescence with the excitation of light 64 . On the other hand, sensor H 4 L exhibited a very weak fluorescence intensity due to the photoinduced electron transfer process from the hydroxy oxygen atom to amino groups. However, when sensor H 4 L was coordinated with a B 4 O 7 2− ion, the chelation-enhanced fluorescence process would be started, and the photoinduced electron transfer process would be inhibited at the same time (Fig. S4b). Hence, an obvious enhancement of the fluorescence intensity was observed.
Fluorescent titration was carried out to gain more insight into the recognition properties of sensor H 4 L as a B 4 O 7 2− probe. As shown in Fig. 3, (Fig. 5). In order to be applied in real life and to find the optimal sensing conditions, the fluorescence intensity of sensor H 4 L over a period of time in the presence of B 4 O 7 2− was determined in Tris-HCl buffer (DMF/H 2 O = 9:1, v/v, pH = 7) solutions. As shown in Fig. S6a, it was found that there were nearly no changes in the fluorescence intensity of H 4 L-B 4 O 7 2− over a period of time, suggesting that H 4 L-B 4 O 7 2− was very stable. Additionally, the fluorescence intensities at different temperatures were also determined. As shown in Fig. S6b, H 4 L exhibited satisfactory B 4 O 7 2− sensing abilities when the temperature was in the range of 0-90 °C. Therefore, it was demonstrated that sensor H 4 L could work in a short time and at room temperature, and it can be applied in real life.
Prior to the imaging experiments, the cytotoxicity of H 4 L at different concentrations (0-100 µM) was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in BHK-21 cells. The results after 48 h revealed that H 4 L exhibited almost no toxicity or low toxicity (Fig. 6).The ability of sensor H 4 L to detect B 4 O 7 2− in living cells was further studied by confocal luminescence imaging. As seen in Fig. 7, the BHK-21 cells incubated with sensor H 4 L (30 μM) alone for 30 min at 37 °C maintained a good shape and were viable, the solvent for the H 4 L concentrate is DMSO, and they also showed very good intracellular fluorescence. Interestingly, an enhanced intracellular fluorescence was detected in cells containing sensor H 4 L incubated with

Materials and General Methods
2-Hydroxy-3-methoxybenzaldehyde (99%), methyl trioctyl ammonium chloride (90%), pyridiniumchlorochromate (98%) and borontribromide (99.9%) were purchased from Alfa Aesar. Hydrobromic acid 33 wt% solution in acetic acid was purchased from J&K Scientific Ltd. The other reagents and solvents were analytical grade reagents from the Tianjin Chemical Reagent Factory and were used as received. Melting points were obtained by the use of a microscopic melting point apparatus made by the Beijing Taike Instrument Limited Company and were uncorrected. 1 H NMR spectra was determined by a German Bruker AVANCE DRX-400 spectrophotometer. All of the UV-vis and fluorescence spectroscopy experiments were recorded on Shimadzu UV-2550 and Perkin-Elmer LS-55 spectrometers, respectively.
Statistical analysis. Statistical methods used are detailed at each experiment individually.

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.