Novel integrated sensing system of calixarene and rhodamine molecules for selective colorimetric and fluorometric detection of Hg²⁺ ions in living cells

Highlights

• Three novel rhodamine based calixarene fluorescent probes (5-7) were synthesized for highly selective detection of Hg²⁺ ions.

• The probe (7) could quantitatively detect Hg²⁺ ions with a low detection limit of 3.4 nM.

• Cytotoxicity and cell imagine studies against MCF-7 and MIA PaCa-2 cell lines were performed.

Abstract

Three novel and facile calixarene derivatives (5, 6 and 7), which were appended with four rhodamine units at the upper rim of calixarene skeleton, were firstly prepared and evaluated for selective detection of metal ions in solution. Receptors (5) and (7) indicated immediate turn on fluorescence output toward Hg²⁺ ions over other most competitive metal ions with the ultralow detection limits, indicating their high efficiency and reliability. The binding response to Hg²⁺ ions in solution was also observed through a chromogenic change (from colorless to pale pink). Furthermore, in vitro and bio-imaging studies with MCF-7 or MIA PaCa-2 cell lines were also performed to investigate the use of receptors in biological systems in order to monitor of mercury ions. Results showed that new receptors (5) or (7) were cell permeable and suitable for real-time imaging of Hg²⁺ in living cells (MCF-7) or (MIA PaCa-2) without any damage to healthy cell lines (HEK 293).

Introduction

Mercury is one of the most toxic heavy metals, and can be easily transport via cell membranes in the tissues such as blood-brain, gastrointestinal tissues, barrier skin and red cell membrane through dissolving in lipid bilayer [1,2]. Even at low concentration, mercury ions cause primary problem for living beings [3,4]. Consequently, selective recognizing and rapid tracking of mercury ions in physiological processes are therefore of paramount importance in order to avoid its harmful effects to living population. Hereby, different techniques such as voltammetry, atomic-absorption spectroscopy (AAS), and inductively coupled plasma emission spectrometry (ICP-AES) are still being investigated for this purpose [[5], [6], [7], [8], [9]]. Among techniques, there is an increasing interest on fluorescent based methods. Therefore, design and synthesis of fluorescent sensors have been intensively investigated in parallel with this method in recent years. Up till now, a number of fluorescence based methods including the utilization of synthetic or commercial ionophores, such as azine, hydroxyquinolines, cyclens or cyclams, diazatetrathia crown ethers and calixarenes for Hg²⁺ ions have been reported in the literature [[10], [11], [12], [13], [14], [15], [16], [17]]. Calixarene based fluorescent sensors are one of the most popular and favored supramolecular structure due to their readily modification and functionalization by ionophore or fluorophore units at lower or upper rim of the calix skeleton [[18], [19], [20]]. However, there are limited number of studies reporting on the application of calixarenes modified with fluorogenic groups as sensing probe for Hg²⁺. In these studies, while lower rim of the calixarene skeleton (phenolic oxygen units) is usually modified with functional groups, only some of them showed a strong fluorescence response toward Hg²⁺ ions with lower selectivity and detection limit. In addition, no scientific results on calixarenes modified with strong fluorophore units as rhodamine are reported till now. Rhodamine is a xanthene derivative and a subset of the triarylmethane dyes. Rhodamine molecule as a fluorophore group has some photophysical advantages such as large absorption coefficient, long emission and absorption wavelengths, and high quantum yield. Therefore, there is an increasing interest on design, synthesis and application of rhodamine-based fluorescent sensors due to its particular structure [21,22].

In this study, we modified the upper rim of calixarene skeleton with rhodamine units for the first time by the reaction of para chloromethylated (2), chlorosulfonated (3), and/or formylated (4) calixarene and aminated rhodamine molecule (Rh-A). Therefore, we found three different rhodamine based calixarene molecules (5, 6 and 7) containing an amine, sulfonamide, and azomethine linker, respectively.

Herein, we have investigated the sensor applications of three novel rhodamine based calixarene molecules as a selective fluorescence probe toward Hg²⁺ ions in solution. Moreover, cytotoxicity experiments were conducted to determine the usage of these molecules as an Hg²⁺ imaging sensor in healthy cells.