Research Article

Novel probe based on rhodamine B and quinoline as a naked-eye colorimetric sensor for dual sensing of heavy metals and hypochlorite as a biosensing and bioimaging agent

https://doi.org/10.21203/rs.3.rs-2345277/v1

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This work demonstrated the design and straightforward syntheses of several novel probe-based on rhodamine B and 2-mercaptoquinoline-3-carbaldehydes as a naked-eye colorimetric sensor, indicating a sensitive and selective recognition towards nickel (II) with a limit of detection 0.15 µmol mL^− 1 (0.01 mg L^− 1). Further, by employing the oxidation property of hypochlorite (OCl⁻), this novel sensor parallelly has been deployed to detect hypochlorite in laboratory conditions with a limit of detection of 0.19 µmol mL^− 1 and in living cells. Regarded to negligible cell toxicity toward mammalian cells, this novel sensor has the potential to determine these analytes in in-vivo investigation and foodstuff samples.

Naked-eye colorimetric sensor

rhodamine B

quinoline derivatives

heavy metals

nickel ions

hypochlorite

In the contemporary world, the issue of "prevention is better than cure" remains a highly contentious issue in therapeutic areas. One of the pivotal aspects of achieving this goal is the rapid detection of hazardous analysts in foods, water, and living organisms. With the sustainable improvement of the cosmopolitan metropolis, the severe problem of heavy metal pollution in the air, soil, and particularly in the water environment is cause for concern (Mohammed, Kapri et al. 2011, Raval, Shah et al. 2016, Briffa, Sinagra et al. 2020). Since the increase in water pollution could be considered an acute problem, rapid detection of heavy metal ion concentrations in drinking water is an effective remedy. One of these heavy metals that must be precisely taken into account is nickel. The transition series element nickel (Ni), which makes up around 3% of the earth's composition, is the 24th most common element on the earth. At a non-dangerous level, nickel has advantages for instance an activator of some enzyme reactions and participating in critical metabolic systems (Raval, Shah et al. 2016). However, the ingestion of nickel beyond acceptable levels induces the inhibition of oxidative enzyme activity, adverse effects on the lungs and kidneys, skin dermatitis, gastrointestinal distress, shortness of breath, and chest pain. It is really carcinogenic, and high levels of nickel cause the reduction of nitrogen and impaired growth. Based on World Health Organization (WHO), the maximum allowable level of nickel in potable water is 0.02 mg/L (Acheampong, Pereira et al. 2012). Hence, it is tremendously important to augment a swift and straightforward method to implement selective and sensitive recognition of nickel ions. Besides that, hypochlorite fulfills a key role in defending against the invasion of pathogens. Nevertheless, an excessive amount of hypochlorite preparation induces several diseases, namely kidney disease, arthritis, osteoarthritis, atherosclerosis, and cancer (Prokopowicz, Arce et al. 2010, Tang, Ding et al. 2015). Consequently, rapid recognition HOCl/OCl⁻ in vitro and in vivo is really imperative. Moreover, since hypochlorite is commonly deployed as a disinfection of drinking water and household bleach, a high concentration of hypochlorite must be damaging humans and animals, causing nose irritation or arousing eye and stomach discomfort. Owing to the destructive influence of hypochlorite, it is essential to swiftly detect and monitor OCl⁻ residues (Wang, Song et al. 2016, Shi, Huang et al. 2019, Li, Miao et al. 2020).

The sensor field as a privileged tool for clinical diagnostics has paved the way for developing earlier diagnoses and treatments. Most popular approaches for detecting nickel rely on electrochemical methods, atomic absorption spectroscopy, and inductive coupled plasma atomic emission spectrometry (ICP-AES) for declining the interference impacts (Şahin, Efeçınar et al. 2010, Mirabi, Rad et al. 2015, Dos Anjos, Alves et al. 2018). In spite of the great capability of the atomic techniques in selective detections, these methods have drawbacks such as their wrecking strategies, high cost as well as long-time analysis. In the sensor area, the rational design of the naked-eye colorimetric sensor as a swift and visual way culminates in detecting detrimental heavy metals and any imperative analysts (Khattab, Rehan et al. 2017, Aghayan, Mahmoudi et al. 2021). In this line, rhodamine B reveals a wealth of opportunities for chemosensor and biosensor applications, and several small molecules and macromolecules-centered rhodamine B have recently been introduced as an elevated sensor for mercury (Hong, Chen et al. 2019), zinc (Wechakorn, Suksen et al. 2016), copper (Mohammadnejad, Shiri et al. 2020), lead (Sunnapu, Kotla et al. 2015), iron (Chereddy, Suman et al. 2012), picric acid (Sivaraman, Vidya et al. 2014), and fluoride (Sivaraman and Chellappa 2013), Rhodamine-centered sensors universally are non-fluorescent and relatively colorless, notwithstanding they exhibit a color change to deep pink. When they are placed in an acidic solution, they demonstrate a strong fluorescence (Kim, Lee et al. 2008). This observation is owing to protonation at the carbonyl group, and following ring-opening of its spirolactam scaffold (Sivaraman, Anand et al. 2012). Metals ions could cause changing the color and fluorescence by assuming a role resembling that of the hydrogen ions in acidic solvents, providing that a suitable ligand is exhibited on the spirolactam ring (Milindanuth and Pisitsak 2018). As a result, in the present study, we made an effort to enhance a novel probe based on rhodamine B and quinoline as a naked-eye colorimetric sensor that could be efficiently and selectively deployed for detecting heavy metals, namely nickel and also hypochlorite. In addition to that, the chemodosimeter of the novel rhodamine-based sensor for analyte with high sensitivity showed a drastic color change from colorless to deep pink after the addition of the analyte (Parmar, Barad et al. 2013).

Reagents and instruments

The chemicals such as Nickel chloride, rhodamine B, and hydrazine were prepared by Aldrich. The solvents were analytical grade and purchased from Merck. Deionized water was used for the dilution of the solutions. The absorbance measurement was carried out by double-beam UV-Vis spectrophotometer using a 1cm quartz cell (Perkin-Elmer, Lambda 35, USA). The fluorescence measurement was carried out by a Pl spectrophotometer using a 1cm quartz cell (Cary Eclipse Agilent G980A). ¹H-NMR and ¹³CNMR spectra were recorded on Bruker AQS AVANCE 500, 400, and 300 MHz spectrometers respectively, using TMS as an internal standard and DMSO-d6 and CDCl₃ as solvent.

General synthesis of sensor based on rhodamine B and quinoline

In a 50 ml flask, 1 mmol (0.203 g) of the 2-mercapto-6-methyl quinoline-3-carbaldehyde compound and 1 mmol (0.465 g) rhodamine B hydrazide under reflux conditions and nitrogen atmosphere in methanol were mixed by a magnetic stirrer. The progress of the reaction was monitored by TLC in the mixture of n-hexane and ethyl acetate in a ratio of 6:4, the reaction was completed after 24 hours. The resulting precipitates were purified by washing with methanol and the final product 7a was obtained as a yellow precipitate with a yield of 85%.

After facile synthesis of rhodamine hydrazide and 2-mercaptoquinoline-3-carbaldehydes based on previous reports [25], we commenced our work by the addition of rhodamine hydrazide with 2-mercaptoquinoline-3-carbaldehydes in MeOH under refluxing conditions yields the novel rhodamine-based sensor with appropriate yield (Scheme 1). Next, the synthetic rhodamine-based sensor was characterized by FTIR, ¹H-NMR, and ¹³C-NMR.

The interaction between a synthesized rhodamine-based sensor and several metal ions (Fig. 1a) was investigated using the same settings. It was clear that the addition of an extensive diversity of environmentally metal ions namely Cu²⁺, Pb²⁺, Zr²⁺, Zn²⁺, Ag⁺, Al³⁺, Hg²⁺, Co²⁺, Mn²⁺, Cr³⁺, Sn²⁺, Fe³⁺, Cd²⁺, Al³⁺, Pd³⁺, Ni²⁺ did not have as a quite much effect on the sensor as Ni²⁺ (Fig. 1b). The outcome supported the high selectivity of the novel sensor which is considered a pivotal characteristic of an ion-selective chemosensor. Figure 1c depicts the UV-visible absorption spectra of this new sensor in the absence and presence of Ni²⁺. Ni²⁺ could chelate with the sensor, causing a ring-opening reaction of the sensor moiety, and then coordinate with carbonyl oxygen, nitrogen atoms of two imine groups, and quinoline nitrogen when added to the sensor solution. It resulted in a blue shift in the absorption spectrum, the formation of a new band at 564 nm, and a steady rise in absorbance with increasing Ni²⁺ concentration. Consequently, the sensor solution changed from yellow to deep pink.

For investigation of the interaction of Ni²⁺ with novel rhodamine and quinoline-based sensor, various concentrations of this element n were added to the solution and absorbance spectra were recorded up to 30 min. By increasing the concentration of Ni, the new peak at 560 nm was observed, and the intensity increased steadily. The absorption spectra are illustrated in Fig. 2a. As a matter of fact, the intensity of the absorbance at 560 nm pertaining to the addition of Ni²⁺ concentration drastically, and the calibration curve was plotted based on the absorbance increasing at 560 nm in the presence of Ni²⁺ (Fig. 2b) in the range of 0.1 to 0.8 × 10^− 5 with a detection limit of 0.15. The color of the solution swiftly changed from yellow to deep pink which can be rapidly diagnosed by the naked eye. A color gradient from yellow to deep pink was obviously perceived upon increasing the concentration of nickel which permitted a semiquantitative detection of the analyte in water by swift and facile visual analysis (Fig. 2c). Evidently, the color intensity became stronger with increasing the concentration of Ni²⁺.

Moreover, the role of functional groups in this naked-eye colorimetric sensor was perused. Accordingly, in addition to 2-mercaptoquinoline-3-carbaldehydes, 2-chloroquinoline-3-carbaldehyde and 2-hydroxyquinoline-3-carbaldehyde have been deployed in this sensory system. As expected, on account of the appropriate affinity of mercaptans with such metals, a rhodamine-based sensor prepared by 2-mercaptoquinoline-3-carbaldehydes revealed elevated interaction with nickel in comparison with hydroxy and chloro derivatives. (Fig. 3) Furthermore, different 2-mercaptoquinoline-3-carbaldehydes were used in sensor scaffolds and subsequently the interaction of sensor-encompassing substitutions namely methyl, methoxy or chlorine with Ni²⁺ were investigated. The outcome depicted the elevated tendency of the sensor prepared by 2-mercapto-6-methylquinoline-3-carbaldehyde to the analyte than the other derivatives (Fig. 4).

Detection of hypochlorite

This rhodamine B-based sensor had essentially no absorbance in the absence of ClO⁻; however, an increase in ClO⁻ enhanced the maximum absorption at 570 nm. (Fig. 5a), which approved that the interaction between the sensor and ClO⁻ resulted in the ring-opening of the spirocycle of the rhodamine hydrazide segment. To study the interaction of the sensor by ClO⁻, the fluorescence properties of the synthesized component were recorded and shown in Fig. 5. The fluorescent titration profiles of the sensor (5 × 10^− 5) with ClO⁻ (2–14 10^− 5 M) are illustrated in Fig. 5b. As the concentration of ClO⁻ was titrated into the sensor, there was a noticeable fluorescence amplification at 410 nm. The ring-opening rhodamine acid caused by the interaction of the probe rhodamine B-based sensor with ClO⁻ is another possible explanation for the outcomes. Additionally, the probe rhodamine B-based sensor exhibits remarkable linearity between fluorescence intensity and ClO⁻ concentration across the range of 0.05 to 0.9 10^− 5 M. The detection limit was calculated at 0.68 µM, which also demonstrated a highly sensitive characteristic. This outcome suggests that the probe may be used to quantitatively detect HOCl/ClO⁻.

The novel probe function in living cells

The yeast cells of Saccharomyces cerevisiae ATCC9763 were treated with two different concentrations (10^− 4 and 10^− 5 M) of the probe. The probe was not observed inside yeast cells by fluorescence microscopy. To determine the function of the novel probe in mammalian cells and its cell viability and cytotoxicity, the breast cancer MCF7 cells were treated with two concentrations (10^− 4 and 10^− 5 M) of the probe. Images of fluorescence microscopy show that the probe has entered the cells. Therefore, a probe rhodamine B-based sensor can be used for fluorescence microscopy imaging of MCF7 cells (Fig. 6a).

As shown in Fig. 6b, the cell viability and cell toxicity were obtained at 97.59% and 2.4%, and 75.16% and 24.83% for 10^− 5 and 10^− 4 M concentration respectively after 24 h via MTT assay. The results show that the cell viability decreases when the concentration of the probe increases, but this change is not very significant, and the probe has no cytotoxicity for MCF7 cells.

Here, we have devised a highly efficient novel probe based on rhodamine B and 2-mercaptoquinoline-3-carbaldehydes for dual sensing heavy metals and hypochlorite. The sensor depicted a significant color change from colorless to pink and fluorescence improvement upon the addition of nickel and ClO⁻ ions. The sensor moreover had a low limit of detection (0.15 µmol mL^− 1 for nickel and 0.19 µmol mL^− 1 for ClO⁻ ions). Considering negligible toxicity toward mammalian cells and the successful application of this novel sensor as an imaging agent for endogenous hypochlorite in living cells, this sensor-centered rhodamine could promisingly be used in industrial applications and foodstuff samples.

Acknowledgments

We are grateful for financial support provided by Alzahra University, the Iran National Science Foundation (INSF), and Tishk International University.

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No competing interests reported.

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