Xanthone based Pb²⁺ selective turn on fluorescent probe for living cell staining

Abstract

A xanthone based Pb²⁺ selective turn-on fluorescent probe (L) was designed, synthesized, and characterized by different spectroscopic techniques. Binding of L to Pb²⁺ induced significant change in the absorption and/fluorescence properties. Other common cations, viz. Na⁺, K⁺, Ca²⁺, Mg²⁺, Ag⁺, Mn²⁺, Hg²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Cr³⁺ did not interfere. The limit of detection of the method was 1.8 × 10⁻⁷ M. L can detect intra-cellular Pb²⁺ in living cells.

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Pb²⁺, one of the most toxic heavy cations is believed to be responsible for memory loss, anemia, muscle paralysis, and mental retardation, particularly for children.^1,2 Based on the toxicity of Pb²⁺ ion, the permissible limit set by the US Environmental Protection Agency (EPA) and Bureau of Indian Standards (BIS) is 0.015 mg L⁻¹ and 0.1 mg L⁻¹, respectively. As this metal has been used for a long time in batteries, gasoline, pigments, and drinking water distribution systems,³ it is widely distributed in the environment. Several methods are available for trace level determination of Pb²⁺, viz. atomic absorption spectrometry, ion-selective electrodes and inductively coupled plasma mass spectrometry.⁴ While these methods allow the low level determination of Pb²⁺ as required by the World Health Organization (15 ppb),⁵ they require expensive instrumentation and tedious sample pre-treatment. Nowadays, fluorescence spectroscopy is exhaustively used for the determination of Pb²⁺ because of its high sensitivity, simplicity, rapidity and low operational cost. An efficient fluorescent probe changes its emission intensity and/or spectral behaviour upon selective binding with the analyte.^6,7 The xanthone nucleus which is present in numerous natural products, exhibits a wide range of interesting biological and pharmacological activities, e.g. antimicrobial,^8,9 antioxidant,^8,9 antimalarial,^8,9 anticancer¹⁰ and anti-HIV activity.¹¹ Presently, we have been engaged in the development of small fluorescent probes for the selective determination of toxic cations and anions.¹² Moreover, the fluorescence property of the xanthone nucleus has encouraged us to design a highly selective, facile and inexpensive synthesis method for its derivative as a fluorescent indicator (L) for Pb²⁺. Recently, several chemosensors based on the fluorometric and/or colorimetric determination of Pb²⁺ have been reported.^13–19 However, most of them have required tedious synthetic protocols involving multistep reactions. We have synthesized L in a single step from commercially available starting materials in a short time with high yield. L is less expensive than other reported fluorophores.

Absorption and emission spectra are recorded with a Shimadzu Multi Spec 1501 absorption spectrophotometer and Hitachi F-4500 fluorescence spectrophotometer, respectively. Stock solutions of L (100 μM) and tested metal ions (1000 μM) are prepared in DMSO and H₂O respectively. All the experiments have been performed in DMSO–H₂O (2 : 1, v/v). Scheme 1 shows the synthesis of L. It is characterized by MALDI-TOF MS, FTIR and ¹H NMR spectroscopic studies (Fig. S1, S2 and S3†).

Scheme 1 Synthesis of L.

Fig. 1 illustrates the changes in the UV-Vis spectra of L (100 μM) in the presence of Pb²⁺. Upon gradual addition of Pb²⁺ to the solution of L, the intensity of a new broad shoulder at 405 nm gradually increased. The inset shows the linear range.

Fig. 1 Changes of the UV-Vis spectra of L (1 μM) with externally added Pb²⁺ (0, 10, 20, 30, 40, 50 μM).

The effect of different metal ions (100 μM) viz. Na⁺, K⁺, Ca²⁺, Mg²⁺, Ag⁺, Mn²⁺, Hg²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Cr³⁺ and Pb²⁺ on the fluorescence properties of L (1 μM, λ_em = 510 nm, λ_ex = 405 nm) is presented in Fig. 2. While Pb²⁺ enhances the emission intensity of L to a significant extent, two other cations viz. Cu²⁺ and Fe³⁺ quench the emission intensity to a very small extent and the remaining cations have no effect.

Fig. 2 Effect of different cations (100 μM) on the emission spectra of L (1 μM). Other metal ions: Na⁺, K⁺, Ca²⁺, Mg²⁺, Ag⁺, Mn²⁺, Hg²⁺, Cd²⁺ (λ_em = 510 nm, λ_ex, 405 nm).

The very weak emission intensity of L increases gradually up to a maximum of 9 fold upon the addition of Pb²⁺ (Fig. 3). The linear region of the plot of fluorescence intensity vs. externally added Pb²⁺ concentration (inset, Fig. 3) can be used for the determination of unknown Pb²⁺ concentration. The enhancement of fluorescence is ascribed as the chelation-enhanced fluorescence (CHEF) effect that reduces the intra-molecular charge transfer (ICT) process in L. The possibility of other types of binding of L with Pb²⁺ is ruled out on the basis of the ¹H NMR of the [L–Pb²⁺] complex (discussed later) with the justification that the lone pair on carbonyl oxygen (sp²) is less available than the ether oxygen (sp³) for binding to Pb²⁺ and carbonyl oxygen is stabilized through intra-molecular hydrogen bonding as shown in Fig. 4.

Fig. 3 Emission spectra of L (1 μM) in the presence of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 and 200 μM Pb²⁺. Inset: plot of emission intensity of Lvs. [Pb²⁺].

Fig. 4 Proposed binding mode of L for Pb²⁺.

The limit of detection (LOD) of Pb²⁺ was found to be 1.8 × 10⁻⁷ M (Fig. S4†). The binding constant of L with Pb²⁺ was estimated using the Benesi–Hildebrand equation,²⁰ (F_∞ − F₀)/(F_x − F₀) = 1 + 1/K_a[C]ⁿ, where F₀, F_x and F_∞ are the emission intensities of L in the absence of Pb²⁺, at an intermediate [Pb²⁺] and at a concentration of complete interaction, respectively. While, K_a is the binding constant, C is the concentration of Pb²⁺ and n is the number of Pb²⁺ bound per L (here, n = 1/2). The value of K_a was 0.125 × 10⁴ M^−1/2 with R² = 0.96 (Fig. S5†). While Job's plot indicates 2 : 1 (L : Pb²⁺, mole ratio) stoichiometry (Fig. S6†) of the [L–Pb²⁺] complex, the mass spectrum of the [L–Pb²⁺] complex (Fig. S7†) also supports the composition. FTIR of L shows three OH stretching frequencies, viz. 3200.36 cm⁻¹ (hydrogen bonded, broad), 3494.3 and 3390.72 cm⁻¹. Formation of the L–Pb²⁺ complex shifts the OH stretching frequency from 3494.3 to 3501.98 cm⁻¹, keeping the other two OH stretching frequencies unaltered (Fig. S8†). Selectivity of L for Pb²⁺ has been tested and it was found that Cu²⁺ and Fe³⁺ interfere to a negligible extent (Fig. S9†), the interference was eliminated completely using thiocyanate as a masking agent. Thiocyanate has no adverse effect on the emission intensity of the [L–Pb²⁺] system.

¹H NMR titration of L with Pb²⁺ was performed in DMSO-d₆ (Fig. 5). One –OH (H_b) is shifted downfield due to coordination of its lone pair to Pb²⁺ while no significant change to the chemical shift values for the other two –OH protons indicate their non-involvement in the coordination of Pb²⁺.

Fig. 5 Changes in the ¹H NMR spectra of L with addition of Pb(NO₃)₂ in DMSO-d₆: (A) free L; (B) L with 0.5 equivalent of Pb²⁺ (C) L with 1 equivalent of Pb²⁺.

Fig. 6 clearly indicates that Pb²⁺ contaminated cells emit green fluorescence in presence of L under fluorescence microscope. Thus L will be useful for determination of Pb²⁺ toxicity in living cells.

Fig. 6 Fluorescence microscopic images of (A) Candida albicans cells; (B) Candida cells incubated with Pb²⁺; (C) Candida cells incubated with Pb²⁺ followed by addition of L; (D) Pollen cells; (E) Pollen cells incubated with Pb²⁺; (F) Pollen cells incubated with Pb²⁺ followed by addition of L. Bright field images of (G) Candida albicans cells incubated with Pb²⁺ followed by addition of L; (H) Candida cells incubated with Pb²⁺; (I) Pollen cells incubated with Pb²⁺ followed by addition of L; (J) Pollen cells incubated with Pb²⁺.

We have successfully synthesized and characterized a biologically important xanthone based inexpensive turn on fluorescent probe for the selective determination of Pb²⁺. Fluorescence enhancement is attributed to the Pb²⁺ assisted CHEF process and inhibition of ICT. L has a fair binding ability to Pb²⁺ with an association constant (K_a) of 0.125 × 10⁴ M^−1/2. The LOD of the probe was 1.8 × 10⁻⁷ M. The probe is very efficient for the detection of intra-cellular Pb²⁺. Table S1†demonstrates that the present probe is very competitive and cheaper than the available Pb²⁺ selective sensors for living cell imaging.

Acknowledgements

Authors sincerely thank the UGC-DAE-KOLKATA center for financial support. A. Sahana and S. Lohar are thankful to CSIR, New Delhi for providing fellowships. Authors thank the Indian Institute of Chemical Biology (IICB), Kolkata for extending the mass spectrometer facilities. We sincerely acknowledge the University Science Instrumentation Center (USIC), Burdwan University for providing the fluorescence microscope facility.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c2ay25935d

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