A “turn-on” ESIPT fluorescence probe of 2-(aminocarbonyl)phenylboronic acid for the selective detection of Cu(ii)

Herein, we report a highly selective fluorescent probe for the detection of Cu(ii). The detection mechanism relies on the Cu(ii)-catalyzed oxidative hydroxylation of 2-(aminocarbonyl)phenylboronic acid into salicylamide, thus recovering the excited-state intramolecular proton transfer (ESIPT) effect and inducing more than 35-fold fluorescence enhancement. The simple structure and readily available fluorescent probe give a novel method for quantitatively detecting Cu(ii) in the linear range of 0–22 μM, with a limit of detection down to 68 nM, and exhibiting high selectivity for Cu(ii) over 16 other metal ions.


Introduction
As one of the critical heavy metal elements, the level of copper ion content signicantly impacts the ecological environment and human health. Especially aer biological enrichment, excessive Cu(II) causes metabolic and neurological disorders, leading to rheumatoid arthritis, Wilson's syndrome, Parkinson's disease, and Alzheimer's disease. [1][2][3] Therefore, it is of great signicance to develop an efficient detection assay for Cu(II) in clinical diagnosis and disease prevention.
In recent years, powerful assays for Cu(II) detection have been developed, including titrimetry, 4 UV-Vis spectrophotometry, 5 atomic absorption spectrophotometry (AAS), 6 inductively coupled plasma-atomic emission spectroscopy (ICP-AES), 7 ion selective electrode (ISE), 8 and chemiluminescence. 9 Among various analysis methods, the uorescent probe is an appealing tool for Cu(II) detection owing to its simple operation, high sensitivity and in suit detection in living cells. [10][11][12] Nowadays, several uorescent probes for Cu(II) detection based on the sensing strategy of coordination or chemical reaction have been designed. However, based on the paramagnetic characteristics of Cu(II), the complex generated from the probe and Cu(II) ion usually leads to uorescent quenching, which gives false-positive results due to the lower sensitivity and anti-interference property. 13 Alternatively, reaction-based uorescence probes exhibit higher sensitivity and selectivity owing to the uorescent signal change related to the unique chemical reaction between analyte and probe molecular. [14][15][16] Gratifying examples of uorescence enhancement probes for Cu(II) have been reported based on distinct reactions, such as the Cu(II)induced hydrolysis, 17,18 ring-opening of the spirolactam, 19,20 C-O bond cleavage, [21][22][23] Cu(II)-mediated oxidation reaction of ophenylenediamine, [24][25][26] Cu(II)-catalyzed Heck coupling and click reaction. [27][28][29] Among these probes, the boronic ester/acid-based uorescence probe possesses excellent biocompatibility, which facilitates the in situ detection of intracellular Cu(II), and has been favored in recent years. 30 However, the borate groups mostly bind with the uorophore, such as coumarin, 31 uorescein, 32 resorun, 33 benzotriazole 34 by chemical modication, leading to challenges in synthetic procedure and high cost. Therefore, it is urgent to develop a new uorescence probe of Cu(II) using arylboronic acid with a simple structure and low cost.
Salicylamide, the simplest derivative of salicylic acid, has long been used as an anti-bacterial and anti-viral drug. 35,36 Notably, salicylamide has attracted considerable attention because of its excited-state intramolecular proton transfer (ESIPT) uorescent property, which was rst reported by Weller in the 1950s for salicylic acid. 37 In general, molecules can exhibit ESIPT uorescence if their structures incorporate an intramolecular hydrogen bonding interaction between a hydrogen bond donor (-OH and NH 2 ) and a hydrogen bond acceptor (]N-and C]O), and the molecular structure of salicylamide ts this feature. 38,39 Therefore, we envision that 2-(aminocarbonyl)phenylboronic acid could be utilized as a "turnon" uorescent probe for the detection of Cu(II), in which the boronic acid group acts as the recognition group of Cu(II) and hinders the ESIPT effect of the probe molecule. By employing the Cu(II) catalytic oxidative hydroxylation of the boronic acid group to the hydroxyl group, the ESIPT effect can be recovered owing to keto-enol tautomerism. The ESIPT process starts with the absorption of radiation from an enol cis conformer ( 1 E) and into the excited state ( 1 E*), this conformer undergoes ESIPT to form a keto tautomer ( 1 K*) giving rise to an emission, leading to the uorescent "turn on" (Scheme 1). Using widely distributed and cheap 2-(Aminocarbonyl)phenylboronic acid as a uorescence probe, we provide a concise and efficient assay for quantitatively determining Cu(II).

General instrumentation
Emission spectra were performed on a uorescence spectrometer (F-4600). Error limits were estimated: l (AE1 nm), s (AE10%), and 4 (AE10%). 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were recorded on a Bruker Avance AV400 spectrometer (Bruker, Billerica, MA, USA) unless otherwise noted. The chemical shis (d) were quoted in parts per million from tetramethylsilane for 1 H and DMSO-d 6 for 13 C spectroscopy. The pH values of the real water samples were recorded by applying B6337-02 benchtop pH/conductivity meters.

Fluorescence detection of Cu(II)
The experimental details of the uorescent detection condition optimization are provided in the ESI. † As for sensitivity testing, 2-(aminocarbonyl)phenylboronic acid (100 mM), Et 3 N (150 mM) and the various concentrations of Cu(II) (0-96 mM) were added in a cuvette sequentially. Aer the above-mixed solutions reacted for 2.5 h, the uorescence spectra were measured and recorded using a uorescence spectrophotometer.
The limit of detection (LOD) is calculated using the following formula: where s is the standard deviation of the blank signals (n = 3).
Scheme 1 Detection mechanism of the probe and the ESIPT process of salicylamide.

Fluorescence detection of Cu(II) in real water samples
Ganjiang River water was collected from the Ganjiang River, tap water was collected from a chemistry laboratory, and rainwater was collected from Ganzhou. Cu(II) was spiked into various water samples to obtain sample stock solutions with 5 mM of Cu(II). Real water samples were added to a sensing system containing 2-(aminocarbonyl)phenylboronic acid (100 mM) and Et 3 N (150 mM), resulting in a nal solution with 20 mM of Cu(II) in the DMF solution. Then, their uorescent spectra were measured and recorded by applying a uorescence spectrometer. All average values were obtained from three parallel experiments (n = 3).

Results and discussion
To verify the design hypothesis, the uorescence response of 2-(aminocarbonyl)phenylboronic acid to Cu(II) in various solvents was tested, as shown in Fig. 1a. The solutions of 2-(aminocarbonyl)phenylboronic acid alone exhibit weak uorescence emission above 400 nm, while the maximum uorescence enhancement could be observed in DMF upon the addition of Cu(II), indicating the relatively signicant solvent effect in the sensing system. The effect of each component in the sensing system was investigated via a series of control experiments. As shown in Fig. 1b, we found that no uorescence enhancement was produced regardless of discarding any of the components. Moreover, under ultraviolet irradiation at 365 nm, blue uorescence could be observed with the naked eye in the complete detection system (inset of Fig. 1b). To gain insight into the proposed reaction mechanism, we conrmed the structure of the nal uorescent product in the millimolar level detection reaction as salicylamide by employing 1 H NMR and 13 C NMR ( Fig. S1 and S2 †). The aforementioned results clearly demonstrate that the reaction between Cu(II) and 2-(aminocarbonyl) phenylboronic acid leads to the oxidative hydroxylation of the boronic acid moiety in the presence of Et 3 N, resulting in ESIPT uorescence.
Alkaline conditions proved to be favorable for the Cu(II)catalyzed nucleophilic substitution of boronic acid. [40][41][42] The effect of the triethylamine concentration on the assay system was then examined. As shown in Fig. S3, † the uorescence intensity at 410 nm dramatically increased along with the increasing concentration of Et 3 N (0-210 mM), and uorescent intensity enhanced up to 13 times. Thus, the suitable concentration of triethylamine was chosen as 150 mM. For further investigation, we optimized detection conditions, such as the H 2 O/DMF ratio and incubation time. It can be found that the uorescence intensity decreased when elevating the ratio of    (Fig.  S4 †). Because the stock solution of Cu(II) was prepared in deionized water, the test solution contained a smidgen of water.
Incubation time is an important indicator of the response capability of the probe, so we examined the alteration of uorescence at various incubation times. As shown in Fig. 2, no obvious uorescence enhancement occurred in the absence of Cu(II), exhibiting the excellent stability of the probe in this system. Aer adding Cu(II), the uorescence intensity of the sensing system increased gradually with an extended incubation time, reaching a steady state at 2.5 h. Consequently, we chose 2.5 h as the best incubation time for subsequent experiments.
To test sensitivity, uorescence spectra with different concentrations of Cu(II) were studied. When the concentration of Cu(II) was increased from 0 to 96 mM, the uorescence emission intensity increased signicantly and obtained the highest 35-fold uorescence enhancement (Fig. 3a). A linear equation of F 410 nm = 15.196 + 26.874 [Cu 2+ ] (R 2 = 0.9964) was obtained at a concentration range of 0-22 mM Cu(II), with a limit of detection (LOD) calculated as low as 68 nM based on LOD = 3s/k (where s is the standard deviation of blank measurement and k is the slope between the uorescence intensity versus Cu(II) concentration) (Fig. 3b). As shown in Table S1, † the proposed assay is comparable to other previously reported uorescence probes for monitoring Cu(II) (see Table S1 † for details) and is much lower than the limit of Cu in drinking water (∼20 mM) set by the U.S. Environmental Protection Agency. 43 Furthermore, we summarized the studies of Cu(II) sensors on applicability and analytical methods in LOD and linear range, as illustrated in Table 1. Compared with the reported results, our study exhibits higher sensitivity, lower detection limit, and wider linear range. Particularly, our analytical method showed some impressive advantages, such as operational simplicity, convenience and low cost.
High selectivity to detect objects over potentially competing species is an important indicator of the anti-interference capability of the probe. Competitive experiments on Cu(II) were carried out to evaluate the selectivity of the probe for Cu(II). As demonstrated in Fig. 4, only Cu(II) resulted in a remarkable uorescence enhancement at 410 nm, whereas a range of common metal ions, including alkali and alkali-earth metal ions (K + , Na + , Ca 2+ , Mg 2+ , Sr 2+ ), transition metal ions (Ag + , Zn 2+ ,  Mn 2+ , Co 2+ , Pd 2+ , Hg 2+ , Fe 3+ , Cr 3+ , Al 3+ ) and rare-earth metal ions (Pr 3+ , Ce 4+ ) merely caused slight enhancement in the uorescence, even when K + , Na + , Mg 2+ and Ca 2+ were added at micromolar levels (Fig. 4). The results demonstrate the specic oxidative hydroxylation of the arylboronic acid catalyzed by Cu(II) and indicate the capability of the probe in the selective detection of Cu(II). Finally, a sensing system was applied to detect Cu(II) in real water samples. Tap water, Ganjiang river water and rainwater were collected and pretreated based on previous studies. 48,49 When exogenous Cu(II) (20 mM) was added to these samples, the uorescence intensity increased signicantly. As shown in Table 2, the recovery rates are 94.50% and 95.50% with relative standard deviations of 0.93% and 0.76% for tap water and rainwater, respectively. These results suggest that the uorescence probe has potential applicability for Cu(II) monitoring in real water samples.

Conclusions
In this study, we reported a simple structure of arylboronic acid as a "turn-on" uorescence probe for the detection of Cu(II). The recognition mechanism is based on the Cu(II)-catalyzed oxidative hydroxylation of the arylboronic acid moiety to phenol, leading to the recovery of the ESIPT effect and switching on the uorescent. The probe exhibited excellent sensitivity (detection limit of 68 nM) and high selectivity for Cu(II) over 16 other metal ions. The probe is also reliable for determining exogenous Cu(II) in real water samples from Ganzhou, demonstrating its potential applicability for the detection of Cu(II) in real-world scenarios.

Conflicts of interest
The authors declare that they have no known competing nancial interests or personal relationships that could have inuenced the work reported in this paper.