A Highly Selective and Sensitive Fluorescent Chemosensor for Detecting Al³⁺ Ion in Aqueous Solution and Plant Systems

Abstract

The solubilized form of aluminum, Al³⁺, is present under acid soil conditions and toxic to both animals and plants. Detecting and quantifying Al³⁺ is vital for both chemistry and biology. A new Schiff-based fluorescent turn-on sensor (probe L) for the selective detection of the Al³⁺ ion was synthesized by coupling 2-hydroxy-1-naphthaldehyde and 2-aminoisoindoline-1,3-dione, and the structure was characterized by nuclear magnetic resonance spectra. The probe L exhibited an excellent selective and sensitive response to the Al³⁺ ion over other metal ions in DMSO-H₂O (1:9 v/v). Fluorescence quantification revealed that probe L was promising for the detection and accumulation of Al³⁺. Treating rice seedlings with Al³⁺ at 25–200 μM inhibited their growth. Al³⁺ treatment produced reactive oxygen species in rice roots. Practical applications of the fluorescent probe for the quantification of Al³⁺ in water samples and rice seedlings are demonstrated. Detecting the Al³⁺ ion with the probe L is easy and a potential alternative to existing analytical methods. The method can be used for detecting the Al³⁺ content of aqueous solution and plant systems. The novel fluorescent probe L has good potential for monitoring Al³⁺ content in the environment and biological systems.

1. Introduction

Aluminum (Al) is the third most abundant element in the earth’s crust. With acidic rain and industrial pollution, a trivalent cation of Al³⁺ is solubilized and widely exists in the environment. Approximately 40 to 50% of the world’s arable soils are acidic, and the toxicity of Al is considered an important limiting factor of plant productivity on acidic soils [1,2,3]. Excessive Al content in contaminated soil leads to a decrease in soil microbial content and hampers plant growth [4]. Additionally, Al is widespread in food additives, pharmaceuticals, and storage/cooking utensils. Al³⁺ ions, the solubilized form of Al, enhance the peroxidation of phospholipids and proteins in cell membranes. Al³⁺ ions can move in the human body via food and water sources. Heavy metal intake by human populations through the food chain damages the nervous system and can induce Alzheimers and Parkinsons disease and amyotrophic lateral sclerosis [5,6,7,8].

Al in its ionic form, Al³⁺, is found in most animal and plant tissues and in natural water. Detecting and quantifying Al³⁺ is crucial for controlling its concentration in the biosphere and its direct impact on human health. Various kinds of sensors have been reported for selective Al³⁺ detection [9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26]. However, these sensors are limited by their complicated synthesis, low water solubility, and lack of sensitivity. For practical applications, a water-soluble sensor for detecting Al³⁺ is still in great demand. Recently, several fluorescent probes have been synthesized for sensing Al³⁺ in cells [27,28,29,30]. The use of fluorescent probes for biosensing and bioimaging the ion in animal cells has also been reported. However, these fluorescent probes have not yet been examined for their application in plant systems. The detection of metal ion by fluorescent probes in plant tissues has always been problematic because of interference from the intrinsic fluorescence of the cell wall and secondary metabolites such as tannins, alkaloids, and polyphenols [31,32].

In this study, we designed a fluorescent sensor, probe L (Scheme 1), that showed a selective colorimetric change toward Al³⁺ among the other tested metal ions. To demonstrate the potential of probe L for biological detection, we first analyzed the effect of Al³⁺ exposure on root elongation in rice (Oryza sativa L. cv. TN67). The toxicity and accumulation of Al³⁺ were evaluated in rice seedlings and we used probe L to quantify the Al³⁺ level in rice seedlings. The toxicity and accumulation of Al³⁺ inhibited cell elongation and cell division, which led to the stunted growth of roots. Al³⁺ treatment may decrease the uptake of water and nutrients and negatively affect plant growth.

2. Materials and Methods

2.1. Synthesis of Probe L

The synthesis of probe L was based on the following method (Scheme 1). 2-hydroxy-1-naphthaldehyde (0.278 g, 1.61 mmol) was added to a solution of 2-aminoisoindoline-1,3-dione (0.303 g, 1.87 mmol) in dry ethanol (15 mL) with two drops of acetic acid. The reaction mixture was refluxed overnight and then the solvent was removed under reduced pressure. The resulting solid was washed with cooled EtOH repeatedly to give probe L (0.469 g, 80%) as a yellow solid; melting point: 234.0 °C; ¹H NMR (300 MHz, DMSO-d₆) $\mathsf{\delta }$ 11.95 (s, 1H), 10.07 (s, 1H), 8.62 (d, 1H, J = 4.4 Hz), 8.05 (d, 1H, J = 4.4 Hz), 7.99–7.90 (m, 5H), 7.66–7.61 (m, 1H), 7.47–7.42 (m, 1H), 7.28 (d, 1H, J = 4.5 Hz); ¹³C NMR (75 MHz, DMSO-d₆) $\mathsf{\delta }$ 164.33, 159.50, 159.26, 134.99, 131.77, 130.16, 129.04, 128.44, 127.99,123.94, 123.54, 122.28, 118.67, 108.61; HRMS(EI): calculated for C₁₉H₁₂N₂O₃(M + H), m/z 316.0848; resulting m/z 316.0839. (Figures S1–S3).

2.2. Plant Materials and Growth Conditions

Rice plants (Oryza sativa L. cv. TN67) were grown as described in [33] with minor modification. The seeds were incubated at 37 °C for 3 d in darkness. Ten germinated seeds with coleoptiles reaching 2 mm in length were transferred to a Petri dish (9 cm in diameter) with a filter paper. The rice seedlings were incubated with deionized water supplemented with Al(ClO₄)₃ (Sigma-Aldrich, St. Louis, MO, USA) from 25 to 200 μM, treated with Al³⁺ solution (10 mL in each Petri dish) for 10 d, then subcultured with fresh solution for another 10 d. Control seedlings were incubated without Al³⁺. The rice seedlings were analyzed for root elongation and reactive oxygen species (ROS) production. For measuring dry weight and accumulation of Al³⁺, samples were dried by incubation at 37 °C for 7 d. Data were obtained from at least three biological replicates.

2.3. Use of Probe L to Determine Al Concentration in Rice

Roots of 20-day-old rice seedlings were rinsed thoroughly with deionized water and oven-dried for 2 d at 37 °C. Ten rice seedlings were ground into fine powder and dissolved with 24× deionized water. The extract was mixed with an equal volume of chloroform and then centrifuged at 2000 g for 2 min. Al³⁺ content was determined in the supernatant (270 μL) by supplementation with the fluorescence probe L (30 μL) dissolved in 10% DMSO. Samples of 400 μM probe L were incubated at 2 °C for 20 min. The Al³⁺-induced specific fluorescence was determined by fluorescence spectrometry (FP-8300, JASCO, Germany) at 375 nm excitation. Data are mean values from three independent biological replicates.

2.4. In Situ Detection of Reactive Oxygen Species (ROS)

Rice roots were treated with various concentrations of Al(ClO₄)₃ for 14 d. H₂O₂ levels were detected by treating roots with 3,3’-diamninobenzidine (DAB). To detect H₂O₂, root samples were placed in a solution [10 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 3.8] of 1 mg mL⁻¹ DAB overnight in darkness. Root samples were rinsed briefly with deionized water. Staining was observed by stereomicroscopy.

3. Results and Discussion

3.1. Absorption and Fluorescence Studies of Probe L Toward Various Metal Ions

We used by UV/vis and fluorescence measurements to investigate the chemosensor behavior of probe L with the following 16 metal ions (as perchlorate salts): Li⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Mn²⁺, Hg²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Pb²⁺, Cd²⁺, Zn²⁺, and Al³⁺ in DMSO-H₂O (1:9 v/v). The solution of probe L itself showed two absorption bands at 320 nm and 370 nm, respectively (Figure 1). The addition of Fe³⁺ and Fe²⁺ to the solution enhanced the absorption band. In the presence of Cu²⁺, the absorption spectrum of probe L was red-shifted, accompanied by a striking color change from dark green to black, which could be seen under UV light (Figure 2). In the presence of Al³⁺, the intensity of the absorption spectrum of probe L was decreased, accompanied by a striking color change from dark green to blue, which could be detected under UV light (Figure 2). These changes in absorption bands indicated a partial interaction between the ions and probe L. As compared with the weak emission band of probe L alone, the addition of Al³⁺ generated an additional fluorescent-enhanced emission band at 472 nm (Figure 3). The enhanced efficiency at 472 nm was 11-fold greater than the control without Al³⁺ (Figure S4). The fluorescent enhancement may be attributed to the formation of a rigid system after binding with Al³⁺.

3.2. Fluorescence Titration and Binding Studies

To further investigate the chemosensing properties of probe L, probe L was fluorescence-titrated with Al³⁺. The changes in fluorescence spectra for probe L as a function of Al³⁺ content are shown in Figure 4. With increasing amounts of Al³⁺ added to a solution of probe L in DMSO-H₂O (1:9 v/v), the emission band at 530 nm gradually increased, and concomitantly a new emission band appeared that peaked at 472 nm. A Job plot indicated a 1:1 stoichiometric complexation of probe L with Al³⁺ (Figure S5). Additionally, the formation of a 1:1 complex between probe L and Al³⁺ was confirmed by the appearance of a peak at m/z 393, assignable to [probe L + Al³⁺ + 3H₂O − 4H⁺] in the ESI/MS (Figure S6). From the fluorescent titration profiles, the association constant for probe L-Al³⁺ in DMSO-H₂O (1:9 v/v) was determined as 3 × 10⁷ M⁻¹ by a Hill plot (Figure S7). By using the abovementioned fluorescence titration results, the detection limit for Al³⁺ was estimated as 1 ppb.

3.3. Application in Water

In order to determine the optimal pH condition of the probe L in the water, we examined the effect of pH on the emission bands of the probe L–Al³⁺ complex in DMSO-H₂O (1:9 v/v) solution. The fluorescence intensity showed a significant emission band (Figure S8) accompanied by a marked color change from dark green to light blue (Figure S9), which could be detected under UV light in the pH range 5–10. Thus, probe L could be applied for the analysis of environmental aqueous samples with a relatively wide pH range.

The practical application of probe L (40 μM) for selective sensing of Al³⁺ (5 equiv.) in different sources of water (DMSO: H₂O = 1 : 9 v/v) was demonstrated (Figure 5). Al(ClO₄)₃ was first dissolved in water of the above source, followed by the addition of probe L to each water sample. All water samples containing Al³⁺ showed a clear color change from dark green to light blue. The result indicated that probe L can detect Al³⁺ in different water sources.

Testing paper detection is an inexpensive approach to detect the presence of an analyte. Toward this goal, we first coated filter paper with probe L by soaking in a DMSO-H₂O (1:9 v/v) solution followed by air drying. Al³⁺ at different concentrations was added to this probe L-coated test paper: the color of the paper changed from dark green to light blue instantly (Figure 6), caused by the interaction of probe L and Al³⁺, therefore implying that the test paper specifically recognized Al³⁺. The detection limit of Al³⁺ with this probe L-coated test paper was 1.0 × 10⁻⁵ M.

3.4. Effect of Al³⁺ Stress on Root Elongation and Oxidative Burst in Rice

The inhibition of root growth is the primary response of the plant exposed to heavy metals. We analyzed the effect of Al³⁺ exposure on root elongation to evaluate Al³⁺ toxicity in rice seedlings. After exposing rice seedlings to Al³⁺ for 14 d, root elongation was measured. At 25 and 50 μM, Al³⁺ significantly reduced root elongation as compared with control conditions (Figure 7A). The root elongation decreased with increasing Al³⁺ concentration. At 100 μM Al³⁺, root elongation was reduced to about 50% of the control conditions. At 200 μM Al³⁺, root growth was completely inhibited. Al³⁺ at 200 μM inhibited root elongation by approximately 80%. We determined production of ROS such as H₂O₂ by treating roots with DAB. Treatment with 25 and 50 μM Al³⁺ induced more ROS production compared with control plants (Figure 7B). The ROS level was greater with 100 and 200 μM Al³⁺ than in controls (Figure 7B). Accordingly, the dry weight of roots was significantly decreased with 100 and 200 μM Al³⁺ (Figure 7C). It was reported that treatment of 200 μM perchlorate had subtle effects on root length and root weight of rice plants [34]. With treatment of 400 μM perchlorate, most of the rice varieties exhibited noticeable toxicity symptoms such as wilting, abscission, and even necrosis [34]. In our study, root weight was markedly inhibited by treatment with 25 μM Al(ClO₄)₃ (Figure 7C). It is suggested that Al³⁺ resulted in the heavy metal stress in the rice plants. Thus, Al³⁺-induced ROS production may inhibit growth in rice seedlings (Figure 8B,C). Thereafter, we treated rice seedlings with 25–200 μM Al³⁺ for the quantification of Al³⁺ by the colorimetric fluorescent probe.

3.5. Practical Applications of Probe L for Quantifying Al Content in Rice Seedlings

With the excellent response of probe L to Al³⁺ in aqueous solution, we investigated its ability to detect Al³⁺ content in rice seedlings. The intensity of blue fluorescence was increased with increasing Al³⁺ solution standard from 25 to 200 μM (Figure 8A). Extracts of Al³⁺-treated rice seedlings were incubated with the fluorescent probe. Extracts with 50 μM Al³⁺ showed mild fluorescence. The maximal fluorescence intensity was observed with 200 μM Al³⁺ (Figure 8B). The intensity of fluorescence was further quantified by fluorescence spectroscopy. Fluorescence emission spectra were measured at 495 nm (Figure 8C). Fluorescence intensity was increased with increasing Al³⁺ concentration from 25 to 100 μM (Figure 8D). The highest levels of Al³⁺ occurred with 200 μM Al³⁺. The stationary phase of Al³⁺ accumulation in rice seedlings may be due to reaching its saturation level (Figure 8D). Therefore, Al³⁺ accumulation in rice was associated with Al³⁺ concentration, with greater accumulation at 200 μM Al³⁺.

An Al³⁺ standard curve was constructed by linear regression with the Al³⁺ calibration standards in control seedling extracts (Figure 8E). The standard curve showed an acceptable regression line with concentration range 0 to 300 μM (Figure 8F). We then determined Al³⁺ content in 25, 50, 100, and 200 μM-treated samples. The level of heavy metals detected in rice seedlings was 98, 200, 353, and 446 μM, respectively. The differences in Al³⁺ toxicity (Figure 7) may be related to the accumulation in rice (Figure 8). This result agrees with the ratiometric fluorescence changes observed in aqueous solutions. Therefore, the fluorescence probe can be used for the quantification of Al³⁺ content in plants.

4. Conclusions

We developed a new fluorescent probe, probe L, for detecting Al³⁺ content. The probe exhibited rapid, highly selective and sensitive detection of Al³⁺ content, with distinct changes in fluorescent emission. Fluorescent quantification demonstrated that probe L could be used to detect plant samples with heavy metal Al³⁺ pollution. This study provides a promising fluorescent probe for the detection and accumulation of Al³⁺ that could be an easy potential alternative to existing analytical methods for investigating Al³⁺ content in plants.