An Anthracene and Indole-based Fluorescent Probe for the Detection of Chromium(III) Ions in Real Water Samples

A novel fluorescent probe possessing anthracene with an indole unit was designed and synthesized to detect chromium(III) ions (Cr3+) with high sensitivity and selectivity. The probe was synthesized in one step by mixing two commercially available chemicals, 2-aminoanthracene and Indole-5-carboxaldehyde. The probe molecule (ANT–In) demonstrates distinct properties, for instance, “turn-on” fluorescence response, high sensitivity and selectivity in less than one minute, and low detection limit (0.2 µM) via hydrolysis of the C = N bond. Additionally, the probe ANT–In was successfully used to identify the presence of chromium(III) ions in real water samples.


Introduction
Chromium(III) ions are essential for biological, chemical, and environmental systems [1]. It is a necessary trace element in the human diet for the "glucose tolerance factor" to function correctly [1]. Chromium(III) ions are essential in the metabolism of lipids, carbohydrates, proteins, and nucleic acids in living organisms because they activate some enzymes and stabilize some proteins and nucleic acids [2]. Thus, the National Research Council strongly advises ingesting 50-200 µg d − 1 of chromium(III) ion daily [3,4]. Chromium(III) ion shortage in the diet can affect the metabolism of glucose and lipids [5] and causes an increase in risk factors for diabetes [6], cardiovascular disease [7], and nervous system diseases [8]. Nevertheless, high chromium(III) ion concentration can negatively affect enzymatic activities and DNA damage [9,10]. Chromium(III) ions can also be harmful during industrial activities, including manufacturing steelworks chromate, tanning, and chrome pigment [11][12][13]. Therefore, chromium(III) ion detection with reliable, effective, and practical methods is in high demand.
Herein, we developed an anthracene and indole-based fluorescent probe (ANT-In) to detect chromium(III) ions. ANT-In demonstrates privileged properties such as operability in aqueous mediums, fast turn-on response, excellent sensitivity and selectivity, and applicability in real water samples.

General Methods
All reagents were purchased from commercial suppliers (Aldrich and Merck) and used without further purification. 1 H NMR and 13 C NMR were measured on a Varian VNMRJ 600 Nuclear Magnetic Resonance Spectrometer. Mass analyses were conducted with Thermo Q Exactive Orbitrap device. Fluorescence emission spectra were obtained using the Varian Cary Eclipse Fluorescence spectrophotometer.

Preparation of UV-vis and Emission Measurement Solutions
The stock solution of probe molecule ANT-In (1 mM) was prepared in CH 3 CN, and stock solutions of metal ion salts (20 mM) were prepared in triple distilled deionized water. The metal ions solution was added to the probe solution (2 mL) using a micropipette during the measurements. For fluorescence measurements, samples were contained in 10.0 mm path length quartz cuvettes (2.0 mL volume). Upon excitation at 400 nm, the emission spectra were integrated over the range 410 to 700 nm (Both excitation and emission slit width 5 nm / 5 nm). All measurements were conducted in triplicate at least.

Synthesis of ANT-In
2-aminoanthracene (100.0 mg, 0.517 mmol) and Indole-5-carboxaldehyde (75 mg, 0.517 mmol) were mixed in 10 mL ethanol in the presence of catalytic amount (2-3 drops) of acetic acid (AcOH). The solution mixture was refluxed for 6 h under the nitrogen atmosphere. The obtained solid was filtered and recrystallized in an EtOH-CH 2 Cl 2 mixture (3:1 v/v) to get the desired product of 121 mg ANT-In as a dark green solid (74%) (Scheme 1). 1

Preparation of Real Water Samples
The recovery experiments were performed to determine the Cr 3+ ions in drinking water, and tap water. Drinking and tap water samples were collected from the district of Gebze in Kocaeli Province in Turkey. First, different Cr 3+ ions concentrations were spiked into the actual water samples and detected with ANT-In based on fluorescence measurements. Next, the Cr 3+ concentrations were calculated using a linear regression equation by spiked water samples' fluorescent response (λem = 500 nm). The experiments were repeated three times to get an average value of the detected Cr 3+ concentrations. Then, the recovery percentages were calculated to evaluate the degree of deviation of the detected value compared to the amount of added Cr 3+ .

Results and Discussions
The synthesis route of ANT-In has been shown in Scheme 1. The probe molecule was synthesized via a facile reaction of 2-aminoanthracene and Indole-5-carboxaldehyde (Scheme 1). As specified in the Supporting Information (SI), the probe's chemical identity confirmed by 1 H NMR, 13 C NMR and HRMS techniques (SI).
Firstly, we determined the ideal sensing medium for Cr 3+ detection. Since ANT-In is not entirely soluble in aqueous solutions, a suitable organic co-solvent is required to raise the probe's solubility. CH 3 CN: H 2 O (7:3 v/v) was an effective system among different solvent combinations. To rule out any pH changes, we evaluated the effect of pH variations on the fluorescence intensity of the sensing medium, demonstrating that ANT-In was pH insensitive (pH 6.0-12.0) and pH adjustment of the sensing media did not affect ANT-In's ability to detect Cr 3+ (Fig. S1). In addition, the probe can detect Cr 3+ with a quite wide range of pH values from pH 6-10. Hence, HEPES buffer was used to set the pH of the sensing medium to pH = 7.0.
The free ANT-In was fluorescence off mode because of photoinduced electron transfer (PET), in which the lone pair electrons of the nitrogen atom are transferred to the anthracene unit. Upon adding Cr 3+ to the ANT-In, a new emission peak emerged at 500 nm. The saturation point was obtained when 4 eq Cr 3+ were introduced with a 26-fold enhancement (Fig. 1). Whereas the fluorescence response was rapid (1 < min), and complete saturation took 5 min (Fig. S2). We calculated the detection limit as 0,2 µM based on a signal-to-noise ratio of 3 (Fig. S3). The sensitivity of ANT-In to other possible metal species, including Cr 3+ , Fe 3+ , Al 3+ , Na + , Li + , Ag + , Ca 2+ , Mg 2+ , Ba 2+ , Pd 2+ , Hg 2+ , Cu 2+ , Zn 2+ , Pb 2+ , Ni 2+ , Cd 2+ , Co 2+ , Ce 3+ , Cr 6+ was investigated under same conditions. Fortunately, all other metal ions did not result in any fluorescence change except for Al 3+ and Fe 3+ , but their intensities were lower than that indicated by Cr 3+ , even if their concentration (10µM) were much more than Cr 3+ (2µM) concentration (Fig. 2a). Meanwhile, we investigated the interference of other metal ions for Cr 3+ . The interference experiments revealed that ANT-In could detect Cr 3+ in mixtures of other metal ions without difficulty (Fig. 2b).
The HRMS technique was used to gain a better insight into the sensing mechanism. Using HRMS analysis of the probe solution (ANT-In + Cr 3+ ), a main molecular ion peak at m/z 194.09509 indicated the exact molecular weight of 2-aminoanthracene. As depicted in Scheme 2, Cr 3+ readily coordinate with the nitrogen atom of the C = N unit and the nitrogen atom in the indole part. Afterwards, nucleophilic  addition of a water molecule leads to hydrolysis in forming highly emissive starting material 2-aminoanthracene. In order to provide support for the postulated sensing mechanism, NMR analysis was performed. The NMR results revealed that initially, there was no signal at 9.87 ppm (Fig. 3a); however, after treating the ANT-In solution with Cr 3+ ions, a new aldehyde proton, belonging to Indole-5-carboxaldehyde, appeared at 9.87 ppm (Fig. 3b). The conclusion that can be drawn from this finding is that a Cr 3+ ion-mediated hydrolysis process occurred.
Encouraged by the probe's high sensitivity and selectivity for Cr 3+ , we performed the practical application in real water samples. First, tap and drinking water samples were obtained and used in experiments without further purification. Subsequently, a specific amount of Cr 3+ was spiked in the water samples (17.5 µM). To minimize the matrix effect, we developed independent calibration curves for each water sample (Fig. S4), and recovery values were calculated 101.8% and 98.4% for drinking water and tap water, respectively (Table 1). In light of obtained results, our probe ANT-In (10 µM) could determine the amount of Cr 3+ , and it was practicable and reliable for Cr 3+ measurement in real water samples.

Conclusion
In this article, we designed and synthesized a simple fluorescence probe ANT-In for detecting Cr 3+ in an aqueous medium. The ANT-In exhibited remarkable selectivity, short response time (less than 1 min), and low detection limit. In addition, the designed probe successfully detected Cr 3+ -spiked drinking and tap water samples.