Rhodamine-assisted fluorescent strategy for the sensitive and selective in-field mapping of environmental pollutant Hg(II) with potential bioimaging

Abstract

Mercury is a global pollutant that can damage human brain, central nervous system and endocrine moreover, other biological systems. Its real-time monitoring is a key challenge in the control of mercury emissions or pollution. In this study, a simple and efficient fluorescent sensor (PST) comprising of rhodamine B and 2-amino-5-bromopyridine was developed for the detection of environmental toxicant Hg²⁺. The newly synthesized sensor displayed a highly selective and sensitive “turn-on” response by the addition of Hg²⁺ among miscellaneous metal cations (Ag⁺, Al³⁺, Ba²⁺, Ca²⁺, Cd²⁺, Fe³⁺, Fe²⁺, K⁺, Li⁺, Mg²⁺, Mn²⁺, Cu²⁺, Pb²⁺, Ni²⁺, Na⁺ and Zn²⁺) in CH₃CN: H₂O (8:2 v/v) system. The 1:1 binding stoichiometry, limit of detection (LOD) 0.63 μM along with binding constant of 1.04 × 10⁵ M⁻¹ was calculated for the complex PST-Hg²⁺. The reversible nature of the complex was confirmed in the presence of ethylenediaminetetraacetate (EDTA). The significant enhancement of fluorometric and absorption response was attributed to the spirolactam ring opening via photo-induced electron transfer (PET) of xanthene moiety. This spirolactam ring opening was further verified by density functional theory, ¹H NMR and Fourier transform infrared spectroscopy (FT-IR). The real-time applicability of the sensor in environmental and biological fields was proved as detection kit on TLC strips and with the help of Laser confocal microscopy for live cell imaging by using HeLa cells. Conclusively, PST can be regarded as accurate ‘two in one’ kit for colorimetric recognition of Hg²⁺ in the environment and fluorescent cell imaging in live HeLa cells.

Introduction

Heavy metals play an important role in our daily life; therefore increasing attention has been given, not only because of physiological and biochemical functions but their adverse effects on human health [1], [2]. Among the heavy metal ions, mercury is regarded as a highly toxic even at very low concentration. Many kinds of microorganisms’ particularly sulfate-reducing bacteria are able to produce methylmercury from other forms of mercury. Methylmercury is a potent neurotoxin and can cause severe health problems including, myocardial infarction, Minamata and some kinds of autism by damaging the endocrine, immune and central nervous systems. Circulation of mercury through various channels such as water, air, food, etc. is alarming because it persists in the environment and subsequently accumulates in the food chain [3], [4].

Over the last several years, researchers and environmental engineers are continuously striving to develop new or upgrade the existing technologies to detect mercury in environmental and biological samples. Evidently, several techniques including atomic absorption spectroscopy, mass spectrometry, inductively coupled plasma-optical mass spectrometry (ICP-MS), high-performance liquid chromatography (HPLC), voltammetry and capillary electrophoresis have been attempted for the detection of Hg²⁺ [5], [6], [7]. Lack of large-scale implementation of aforementioned techniques is mainly due to labor-intensiveness, extreme operational conditions, and high equipment costs. These facts certainly demand the development of an efficient, infield and real-time methods for the detection of different heavy metal ions [7].

The detection of Hg²⁺ by fluorescence spectroscopy has become a lucrative and promising strategy because of high selectivity, sensitivity, real-time monitoring, economic and operational simplicity [8]. It has been observed that fluorescence-based sensing of heavy metal ions is nondestructive with high-outcome sensitive method for real calculations of spatial and specific analyte applications [9], [10], [11], [12]. Many fluorescent sensors with high sensitivity and selectivity have been proposed for the detection and quantification of Hg²⁺ [4], [9], [13], [14], [15], [16]. Among them, rhodamine-based sensors are the best candidates and are widely used for dual-responsive sensors through fluorometric and colorimetric signals [15], [17], [18]. A number of rhodamine-based chemical sensors have been successfully engineered such as; “off-on” type colorimetric sensors for the in-vivo and in vitro recognition of toxic metal ions. This “off-on” response is due to a particular structural transformation of rhodamine from ring closed (off) to ring-opened (on) equilibrium between spirolactam to amide moiety [19]. Furthermore, the longer emission wavelength of rhodamine fluorophore makes it suitable to serve as a reporting site for the analyte. In recent years, various groups [20], [21], [22], [23], [24], [25] have reported several rhodamine-based colorimetric and fluorometric sensors with selective response to Hg²⁺. However, the majority of the chemosensors contains a thiospirolactone rhodamine derivative. This thiospirolactone moiety serves as an ideal site for the construction of a selective and reversible sensor for Hg²⁺. Most of the reported fluorescent probes for the selective detection of Hg²⁺ are lacking at least one of the following parameters that include: various steps and cost involved in synthesis, sensitivity, and selectivity, lower limit of detection (LOD), intracellular detection of toxic metal ions and medium for detection, low binding affinity for metal ions and most importantly naked eye colorimetric detection.

Herein, we report a pyridine-rhodamine conjugate PST as an “off–on” colorimetric model for selective detection of Hg²⁺ in aqueous acetonitrile (8:2 v/v) media. The changes in the electronic spectral configuration of PST occurs through spirolactam ring opening mechanism upon addition of Hg²⁺ among miscellaneous metal cations. These spectral changes are accompanied by the visible color change from colorless to pink. Hence, the sensor PST is regarded as “bare-eye” detector for Hg²⁺. This colorimetric change is reversible upon the addition of EDTA^2-. To support the binding mode between the metal ion and PST, the density functional theory (DFT) calculations have also been performed which are in good agreement with the experimental findings. The dip strip and confocal laser microscope experiments evidenced the practical utility of PST as detection of Hg²⁺ in the environment and live cell imaging, respectively. The synthetic route of PST is shown in Scheme 1.