A turn-on fluorescence sensor for the highly selective detection of Al3+ based on diarylethene and its application on test strips

A novel turn-on fluorescent sensor for Al3+ based on photochromic diarylethene with a 2-hydroxybenzhydrazide unit has been successfully designed and synthesized. The photochromic and fluorescent characteristics were studied methodically in methanol under irradiation using UV/vis light and induced by Al3+/EDTA. This fluorescent sensor was highly selective toward Al3+ with an obvious fluorescent color change from dark blue to blue. The Job's plot and mass spectrometry (MS) analysis indicate a binding stoichiometry of 1 : 1 between the fluorescent sensor and Al3+. Moreover, a test strip containing this fluorescent sensor was prepared to allow for the easy detection of Al3+ in water. Finally, a logic circuit was designed using four input signals (In1: UV; In2: vis; In3: Al3+; In4: EDTA) and one output signal.


Introduction
Aluminum is the most abundant metal element in the earth's crust, most of it exists as a compound, such as aluminum oxide, aluminum hydroxide, and potassium sulfate and a signicant amount exists as Al 3+ in natural waters and in many biological tissues. 1,2 Furthermore, aluminum plays a very important role in the daily life of humans, 3,4 for example, aluminum products are widely used as food additives, cooking utensils, aluminum-based pharmaceuticals, in the automotive and aeronautic transport industry, and so forth. 5 With increased research, more and more studies have conrmed that excess Al 3+ is quite toxic to biological systems, 6 for example, Al 3+ toxicity may be related to Alzheimer's and Parkinson's diseases. 7,8 On the one hand, there is an increased risk of a large amount of free Al 3+ being released because of acid rain dropping onto the soil and causing the release of aluminum from the soil. It is believed that 40% of acidic soil in the world contains a high concentration of Al 3+ , which inuences plant growth. 9,10 On the other hand, this could lead to an increase of Al 3+ in our lives because of the widespread use of aluminum compounds in water treatment, cooking utensils, food additives and so forth. Al 3+ can spread to the tissues of humans and animals and eventually accumulates in the bones. 11,12 A high concentration of Al 3+ gives rise to serious bone diseases, such as myopathy, microcytic hypochromic anemia, dialysis dementia, encephalopathy, neuronal myopathy and can even lead to central nervous system damage. 13,14 The World Health Organization recommends a daily intake of approximately 3-10 mg. 15,16 Therefore, it is important to be able to detect the amount of Al 3+ in the environment owing to the risks to human health. To date, many conventional methods have been used to detect Al 3+ , such as atomic absorption spectroscopy 17 and inductively coupled plasma emission spectroscopy 18 for example. However, they require expensive instruments, complicated operating procedures, and high operating costs. Compared with the traditional methods, uorescent chemical sensors have many advantages, such as a simple operation, low cost and high sensitivity and they have attracted wide ranging attention. [19][20][21] In the past few years, large numbers of photoresponsive compounds have been reported for the detection of ions. [22][23][24][25] Among the photoresponsive materials that have been reported, photochromic diarylethenes are considered to be the most promising photo-switchable molecules on account of their prominent thermal stability, excellent fatigue resistance, and rapid response. [26][27][28] Diarylethene compounds can be functionalized as uorescence sensors as their uorescence can be reversibly adjusted by alternating ultraviolet light and visible light. In addition, it is well-known that 2-hydroxybenzhydrazide can react with the aldehyde group of diarylethene to form a Schiff base group, which is one of the most attractive and effective functional groups owing to their easy preparation and affluent bonding sites (N and O atoms). [29][30][31][32][33] Taking consideration of these aspects, we have designed and synthesized a diarylethene derivative 1O, as a target uorescent sensor that can forcefully bind metal ions through the introduction of a Schiff base moiety. [34][35][36] Therefore, a new diarylethene derivative 1O, containing a 2hydroxybenzhydrazide Schiff base unit was designed and synthesized as a uorescent sensor for detecting Al 3+ with a high selective and with sensitive characteristics. The structure of 1O was characterized using 1 H NMR, 13 C NMR, and infrared spectroscopy (IR), and the results are displayed in the ESI (Fig. S1-S3 †). The multifunctional uorescent switching characteristics induced by Al 3+ /EDTA and UV/vis light were systematically investigated. The photochromism of the diarylethene is shown in Scheme 1 and the analytical performance for the detection of Al 3+ is compared with other reported sensors in Table 1. Compared with these reported uorescent sensors, the diarylethenes uorescent sensor (1O) has a specic recognition ability for Al 3+ and the sensing process is not affected by interference from other metal ions. In addition, 1O exhibited multi-control uorescence switching behaviors in the presence of Al 3+ and lights. Moreover, sensor 1O has been successfully applied as a test paper and for the construction of a logic gate.

General procedures and materials
All solvents used were of analytical grade and were used without further purication. However, the solvents used in the Scheme 1 Photochromism of diarylethene 1O.

Synthesis of 1O
The synthetic route to the diarylethene (1O) is shown in Scheme 2. Compound 2 was prepared using the method previously reported in the literature. 42 The experimental characterization data and details of the procedures used to prepare 1O are detailed below. In a 50 mL ask, compound 2 (0.49 g, 1.03 mmol) and compound 3 (0.18 g, 1.20 mmol) were dissolved in absolute ethanol (10.0 mL) and reuxed for 5 h, then cooled to room temperature and concentrated under reduced pressure. The resulting solid was recrystallized from ethanol to obtain 1O (0.36 g, 0.62 mmol) as a pale yellow solid in a 60% yield. Mp 460-461 K; 1

Photochromic and uorescent properties of 1O
The photochromic and uorescence properties of 1O were measured in methanol (2.0 Â 10 À5 mol L À1 ). As shown in Fig. 1a, the maximum absorption of 1O was observed at 346 nm Scheme 2 Synthetic route to diarylethene 1O. (3 max ¼ 5.5 Â 10 4 L mol À1 cm À1 ) in methanol, which resulted from a p-p* transition, 43 at the same time the solution was colorless. When irradiated with 297 nm UV light, the absorption band at 346 nm decreased and a new absorption band centered at 541 nm appeared. This was accompanied by the solution changing from colorless to purple, owing to the formation of the closed-ring isomer 1C. Conversely, when irradiated with visible light (l > 500 nm), the absorption spectra of the closed-ring state 1C returns completely to the initial state 1O, and the solution changes from purple to colorless at the same time.
When excited at 365 nm light, the uorescence emission peak of 1O appeared at 452 nm in methanol. The absolute uorescence quantum yield of 1O was determined to be 0.004. Upon irradiating with 297 nm UV light, the emission intensity decreased, owing to the occurrence of the photocyclization reaction and the generation of the closed-ring isomer of 1C.  When the photostationary state (PSS) was reached the uorescence intensity of 1O was quenched to ca. 59% in methanol. The residual uorescence cannot be decreased any further due to the incomplete cyclization reaction and the existence of the open-ring isomers which have a parallel conformation. 44 At this time, the uorescence emission cannot be observed by the naked eye. By using visible light (l > 500 nm) to irradiate the solution of 1C, the uorescence intensity was fully restored to that of the open-ring 1O (Fig. 1b). Fig. 2 shows the absorbance spectra changes of 1O induced by Al 3+ /EDTA and UV/vis light in a methanol solution (2.0 Â 10 À5 mol L À1 ). As shown in Fig. 2a, when 5.0 equivalents of Al 3+ (0.1 mol L À1 ) was gradually added to the methanol solution of 1O, the maximum absorption peak at 345 nm decreased, the absorbance spectra gradually red-shied and a new absorption band appeared that was centered at 366 nm owing to the formation of the 1O-Al 3+ (1O 0 ) complexes. Upon addition of Al 3+ to the solution of 1O, the color of the solution did not change signicantly.

Absorption spectrum changes induced by Al 3+ /EDTA and UV/vis light
As shown in Fig. 2b, 1O 0 also underwent photochromism under UV/vis light irradiation. When irradiated with UV light at 297 nm, the color of the solution of 1O 0 changed from colorless to purple and a new absorption band centered at 542 nm emerged owing to the formation of the closed-ring isomer 1C 0 . Similarly, under visible light (l > 500 nm) irradiation, the absorption band centered at 542 nm disappeared completely and the system returned to the 1O 0 state, the color of the solution changed from purple to colorless. The maximum absorption peak of 1C was observed at 347 nm. When Al 3+ was added to the solution of 1C, the absorption peak at 347 nm red-shied to  Paper 368 nm and increased slightly. At the same time, the color of the solution changed from purple to light purple owing to the formation of 1C 0 . The absorption spectra returned to the initial state of 1C when as excess of EDTA (0.1 mol L À1 ) was added to the solution of 1C 0 (Fig. S5 †). This indicated that a reversible transformation between 1C and 1C 0 could be induced using Al 3+ and EDTA.

Fluorescence response to metal ions
The uorescence intensity of 1O in a methanol solution (2.0 Â 10 À5 mol L À1 ) changes following induction using Al 3+ /EDTA and UV/vis light. As shown in Fig. 3a, the emission intensity of 1O at 448 nm gradually increased when Al 3+ was increased from 0 to 5.0 equivalents, followed by a plateau upon further addition. At this time, a new compound 1O 0 , was formed, the uorescence color of the solution changed from dark blue to blue. Compared with 1O, the uorescence of 1O 0 was enhanced by 18-fold at the plateau. The absolute uorescence quantum yield of 1O 0 was determined to be 0.029. The uorescence intensity of 1O was restored when excess EDTA was gradually added. This is due to the occurrence of a complexation-dissociation reaction between Al 3+ and EDTA. Under UV irradiation at 297 nm, the uorescence intensity of 1O 0 dramatically declined with a uorescence color change from blue to dark blue owing to the formation of the closed-ring isomer 1C 0 , and the uorescent relative intensity decreased from 5289 to 2393. Moreover, the emission intensity of 1C 0 returned to that of 1O 0 upon irradiation with an appropriate wavelength of visible light (l > 500 nm) (Fig. 3b). As shown in Fig. 3c, a uorescence titration of 1C using Al 3+ was performed in methanol. When 2.0 equivalents of   Al 3+ was added to 1C, the emission intensity reached the maximum value at 448 nm. Compared with 1C, the uorescence of 1C 0 was enhanced 17-fold at the plateau. When the excess EDTA was added, the uorescence intensity returned to the initial state of 1C. It was shown that 1C and 1C 0 could be transformed into each other.
The uorescence response of 1O, which was induced by the addition of different metal ions such as Al 3+ , K + , Ca 2+ , Hg 2+ , Sr 2+ , Cd 2+ , Zn 2+ , Mg 2+ , Mn 2+ , Ba 2+ , Ni 2+ , Co 2+ , Pb 2+ , Sn 2+ , Cu 2+ , Cr 3+ , and Fe 3+ is shown in Fig. 4. It can be seen that the uorescence spectra of 1O was not obviously changed, except for the addition of Al 3+ . As shown in Fig. 4a, when various metal ions (4.0 mL, 0.1 mol L À1 ) were added to the methanol solution (2.0 Â 10 À5 mol L À1 ) containing 1O, only Al 3+ caused a drastic uorescence enhancement at 449 nm. At the same time, the uorescent color of 1O changed from dark blue to blue (Fig. 4c). The increase in the uorescence intensity could be attributed to the chelating enhanced uorescence (CHEF). In addition, the stable chelation of 1O with Al 3+ inhibited the isomerization of C]N. 45,46 As shown in Fig. 4b, the uorescence intensity of Al 3+ was much higher than that of the other metal ions. Thus, 1O could be used as a highly selective uorescent sensor for Al 3+ recognition.  Table 2: In1 (UV); In2 (vis); In3 (Al 3+ ); and In4 (EDTA).
In order to calculate the binding ratio between 1O and Al 3+ , a Job's plot was performed by uorescence titration according to the method previously reported. 47 It can easily be seen that the emission intensity of complexes 1O-Al 3+ approached the maximum value when the molar fraction of [1O]/([1O] + [Al 3+ ]) was about 0.5, indicating that 1O was bound to Al 3+ with a binding stoichiometry of 1 : 1 (Fig. 5).
A 1 H NMR titration experiment was performed in CD 2 Cl 2 to further investigate the binding mode between 1O and Al 3+ . As shown in Fig. 6, the signal peak Ha at 11.71 ppm belongs to the protons of the hydroxyl (-OH), and the signal peak Hb at 9.41 ppm corresponds to the protons of the Schiff unit (-CH]N). With the addition of Al 3+ , the signal peak Ha became wider and weaker, indicating that a bond between the hydroxyl group and Al 3+ was formed. In addition, the signal peak Hb shied from 9.41 to 9.52 ppm, showing the formation of a bond between the Schiff unit (-CH]N) and Al 3+ . The above results indicated that the O of the hydroxyl group and the N(-CH]N) on the Schiff base are the optimal binding sites. To further conrm the binding mechanism of 1O and Al 3+ , electrospray ionization-mass spectrometry (ESI-MS) experiments were performed as shown in Fig. S4. † An ESI-MS peak for 1O at 604.2 m/z was observed and assigned to [1O-H + ] À (calcd 604.1). When excess amounts of Al 3+ were added, a new ESI-MS peak at 754.0 m/z was observed due to the formation of complexes, this was assigned to [1O + Al 3+ + 2NO 3 À À 2H] À (calcd 754.1). This result further conrmed that the formation of a 1 : 1 complex between 1O and Al 3+ . In addition, the association constant (K a ) for the complexation of 1O with Al 3+ was calculated to be 4.72 Â 10 4 L mol À1 (R ¼ 0.9913) using the Hildebrand-Benesi equation 48 (Fig. S6 †). According to the reported method, 49 the detection limit (LOD) of 1O for Al 3+ was calculated to be 1.24 Â 10 À5 mol L À1 (Fig. S7 †).
In order to further determine the selectivity of 1O to Al 3+ in methanol solution, competitive experiments were performed. As shown in Fig. 7, the uorescence intensity showed no obvious changes upon adding Al 3+ (10.0 equiv.) to a solution of 1O in the presence of various metal ions (10.0 equiv.) except for Fe 3+ , indicating that 1O has a good selectivity for Al 3+ .

Application on test strips
In order to make testing of Al 3+ more convenient on site, a test strip coated with 1O was made. In this experiment, a Whatman lter paper was immersed in a methanol solution (1.0 Â 10 À3 mol L À1 ) of 1O and dried at room temperature. Then, the drying test strip was immersed in a solution of various metal ions, and the uorescence color of the test strip was observed under UV light. As shown in Fig. 8, only the test strip immersed in the Al 3+ solution showed a distinct color change (strong uorescence emission). Moreover, an increase in the Al 3+ concentration gave a more pronounced uorescence effect (Fig. S8 †). Therefore, this method can be used to detect Al 3+ more conveniently.

Application in logic circuits
As described above, the photochromic properties of diarylethene 1O could be effectively modulated by stimulation with UV/vis light, and Al 3+ /EDTA. The dual-controlled photoswitching behavior of diarylethene 1O is shown in Fig. 9a. Based on these characteristics, a combinational logic circuit consisting of four input signals and one output signal had been constructed, the input signals included In1 (297 nm UV light), In2 (l > 500 nm visible light), In3 (Al 3+ ), and In4 (EDTA) and the output signal was a change in the uorescence intensity at 448 nm (Fig. 9b). The input signal in the logic circuit was 'on' or 'off', corresponding to the different Boolean values of '1' or '0'. The emission intensity of diarylethene at 452 nm was considered to be the original value, and the uorescence intensity of 1O was signicantly enhanced aer the addition of Al 3+ . When the emission intensity at 448 nm was 18 times greater than the original value, the output signal could be regarded as an 'on' state with a Boolean value of '1'; otherwise, it was treated as an 'off' state with a Boolean value of '0'. Under the stimulation of different conditions, diarylethene 1O demonstrated an on-offon uorescence switching behavior. Therefore, each input from the four strings would give a corresponding output signal. For example, if the input string is '1, 0, 0 and 0', the corresponding input signals ln1, ln2, ln3 and ln4 are 'on, off, off and off' respectively, under these conditions, diarylethene 1O would be converted to 1C by stimulating with 297 nm light, and the uorescence emission intensity would be reduced. As a result, the corresponding output signal was 'off', and the output digit was '0'. Similarly, the same on-off-on signal would occur under the stimulation of the other conditions of uorescence switching and this combinational logic circuit was composed of all possible logical strings, as shown in Table 2.

Conclusions
In conclusion, a novel diarylethene derivative with a 2-hydroxybenzoic acid hydrazide unit was designed and synthesized, and showed high selectivity and sensitivity for Al 3+ in methanol solution. It showed multiple responses when induced using UV/  vis light and Al 3+ /EDTA. When Al 3+ was added to a methanol solution of 1O, the uorescent color changed from dark blue to blue. This diarylethene derivative could be used as a uorescent sensor to "recognize" Al 3+ . Moreover, a test strip with a sensor function was successfully prepared and a logic circuit was constructed on the basis of the unimolecular platform. This work provides a useful strategy for the development of chemical sensors, the monitoring of Al 3+ in the environment and the potential applications of molecular logic circuits.

Conflicts of interest
There are no conicts to declare.