Introduction

Studies relevant to the detection and quantification of heavy and transition-metal ions have been appealing due to their exceedingly hazardous impact on the environment and biological systems [1]. Among various toxic heavy metal ions, the mercury ion (Hg2+) is one of the most poisonous and endangering both human health and the environment [2] because it is released into environment by natural processes and human activities like water erosion, solid waste incineration, wind erosion, forest fires, oceanic and volcanic eruptions, fossil fuel combustion and industrial productions [3, 4]. Despite its toxicity, mercury compounds find variety of applications in industrial processes and products, including paints, electrical materials and batteries [5]. The presence of mercury even at very low concentrations can accumulate in the human body and produce many health issues, including foetal brain damage, significant cognitive and motor abnormalities and minamata sickness [6]. Furthermore, due to more soft nature of mercury ions, it has a strong binding affinity towards thiol groups in proteins, which causes cell dysfunction and numerous diseases including kidney disorder, abdominal pain and nausea. In addition, mercury has the power to create various hormonal imbalance and problems in thyroidal system by quickly entering through skin and respiratory cell membranes in living system [7,8,9]. Hence, the need for highly selective and sensitive determination of mercury(II) ions is vital.

In literature, many analytical methods were reported for recognition of mercury(II) ions including neutron activation analysis, plasmon-resonance rayleigh scattering spectroscopy (RSS), graphite furnace atomic absorption spectrometry (GF-AAS), anodic stripping voltammetry, cold vapor atomic absorption spectroscopy (CV-AAS), cold vapor atomic fluorescence spectrometry (CV-AFS), atomic absorption spectroscopy, inductively coupled plasma mass spectrometry and emission spectroscopy. Although these methods are well established, they are expensive, time-consuming and can require complex sample preparation [10,11,12,13,14,15]. Due to its low cost, simplicity, superior sensitivity, simple instrumentation, low detection limit, real-time response, and non-destructive nature, sensing by fluorescence technique is typically preferred over above discussed methods for quantifying Hg2+ [16,17,18,19,20,21,22].

Schiff base (-C = N-) based fluorophores have demonstrated excellent results among various chemosensors due to their ease of synthesis and ability to provide a suitable electronic/geometrical environment after complexation with metal ions. Generally Schiff base based sensors are designed using aromatic, poly-aromatic units or heterocyclic moieties with conjugated double bond systems. By incorporating fluorescence assisting groups in Schiff base structural framework fluorescence property can be achieved. In addition to this the nitrogen atom of -C = N- group is well known for binding mercury and resulting complex exhibits biological and photophysical application. Including several environmental and biological applications, the Schiff base based sensors and their complexes have demonstrated anticancer, antibacterial, analgesic, antifungal, anti-inflammatory, antiviral, antioxidant, antimalarial, antiglycation, and anti-ulcerogenic properties [23,24,25,26].

Several chemosensors containing sulphur and nitrogen donor atoms have been exploited for sensing of mercury(II) ions. Many of these sensors were developed through difficult synthetic routes and demonstrated poor selectivity towards Hg2+. The detection limit of these fluorophores for mercury(II) ions was found between 0.2 and 12.0 μM [27,28,29,30]. Additionally, the sulphur containing receptors cause cell malfunctions and subsequently leads to many diseases in the biological system [31, 32]. Thus, the designing and developing sensors with nitrogen biding sites for the selective recognition of soft mercury(II) ions might be an ideal choice. The simple synthesis of ratiometric fluorescent chemosensors typically involves pyrene units due to its peculiar signal of excimer-to-monomer emission. Due to its high chemical stability, unique photophysical properties and remarkable fluorescence detection, pyrene derivatives have been exploited regularly as a fluorescence probe [33,34,35,36,37,38,39,40].

For biological and environmental applications, there is still a need for highly selective and sensitive fluorescent sensor which can only respond to mercury(II) ions. Hence, it is crucial and highly prized to develop a very sensitive and selective fluorescent chemosensors for quantification of Hg2+ [41, 42].

Herein, we report synthesis and characterization of a novel sulphur free, highly selective and sensitive molecular sensor for mercury(II) ions detection based on pyrene as a backbone with limit of detection 9.0 × 10–8 M and its bio-imaging studies along with test strips practical applications. The following Table 1 shows comparison of reported mercury(II) sensor with present work.

Table 1 Comparison of fluorescent probes for Hg2+
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Experimental

Materials and Methods

The chemicals and reagents used in the experiments were all analytical or spectroscopic grade. The inorganic metal nitrate salts Mg(NO3)2.6H2O, Cd(NO3)2.4H2O, Hg(NO3)2.H2O, Co(NO3)2.6H2O, Ni(NO3)2.6H2O, Cu(NO3)2.3H2O, Zn(NO3)2.6H2O, Pb(NO3)2, Mn(NO3)2.4H2O, NaNO3, KNO3, Sr(NO3)2, Al(NO3)3.9H2O, Anhydrous FeCl3, CrCl3.6H2O, mercury(II) acetate, o-phenylene diamine (OPD) and 1-pyrene carboxaldehyde were purchased from Sigma Aldrich. The entire UV–Visible and fluorescence studies were done by taking Milli-Q water. The elemental analysis (C, H and N) was carried out using Thermoquest CHN analyzer. The IR spectra were obtained using a Nicolet 6700 FT-IR spectrometer. In DMSO-d6, 1H-NMR spectra were collected using a Jeol 400 MHz FTNMR spectrometer. Mass spectra were recorded using a Waters-USA Xevo G2-XS Qtof mass spectrometer. A Jasco-P-670 UV–Visible spectrophotometer was used to collect electronic spectra in 200–800 nm range. The Hitachi F-7000 Fluorescence Spectrophotometer was used to measure the fluorescence spectra.

Synthesis of Sensor PAPM (Scheme 1)

Scheme 1
scheme 1

Synthetic route of PAPM (L)

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To the methonolic solution of OPD (0.5 g, 4.62 mmol, in 10 mL), a solution of 1-pyrene carboxaldehyde (2.12 g, 9.25 mmol, in 20 mL MeOH) was added dropwise at room temperature with steady stirring. To this, acetic acid (0.5 mL) was added and reaction mixture was boiled under reflux for 16 h. The completion of the reaction was confirmed by TLC. The precipitated yellow compound was filtered, washed with methanol (25 mL) and dried in air. The product has been recrystallized from mixture of chloroform and methanol (1:1). Yield: 2.42 g, 98%. M.P. 225–227 °C. Elem. Anal. Calculated for C40H24N2: C, 90.20; H, 4.54; N, 5.26. Found: C, 90.17; H, 4.48; N, 5.21. 1H-NMR (400 MHz, DMSO-D6) δ 9.48 (s, 2H), 8.52 (d, J = 8.0 Hz, 2H), 8.44 (d, J = 8.1 Hz, 2H), 8.36—8.23 (m, 11H), 8.11 (t, J = 8.0 Hz, 3H), 7.81(s, 2H), 7.24(s, 2H). 13C-NMR (101 MHz, DMSO-D6) δ 152.27, 132.16, 131.43, 130.83, 129.10, 127.85, 127.26, 126.50, 126.16, 125.44, 125.01, 124.84, 124.22, 123.30, 122.31, 111.98. IR (KBr, cm−1) ν: 1587 (m. C = N), 3041 (Ar–H); ESI–MS: m/z 533.21 [(M + 1)+].

Synthesis of Mercury(II) Complex (Scheme 2)

Scheme 2
scheme 2

Synthetic route for synthesis of Mercury(II) Complex

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The synthesis of the mercury complex is induced by the dropwise addition of mercuric acetate (0.15 g, 0.41 mmol, in 10 mL MeCN) to the solution of PAPM (0.2 g, 0.37 mmol, in 15 mL MeCN) at room temperature with constant stirring. Upon addition of all the mercuric acetate, the reaction mixture turns to colorless and stirring was continued for further 11 h to get dark red solid. It was filtered, washed with MeOH and chloroform to get pure compound and dried in air. Yield: 0.18 g, 56%. Elem. Anal. Calculated for C44H30N2O4Hg2+: C, 62.14; H, 3.33; N, 3.56. Found: C, 62.11; H, 3.29; N, 3.52. IR (KBr, cm−1) ν: 2969 (Ar–H), 1590 (br. C = N), 1513 (asym. OCO), 1236 (sym. OCO); ESI–MS: m/z 851.26 [(M + 1)+].

General Procedure for UV–Visible and Fluorescence Experiments

The stock solutions of sensor (1.0 × 10–5 M) and metal nitrate solutions (1.0 × 10–3 M) were prepared in aqueous acetonitrile (2:8). The UV–Visible and fluorescence response of fifteen metal ions (Cd2+, Co2+, Hg2+, Cu2+, Mg2+, Mn2+, Ni2+, K+, Na+, Zn2+, Sr2+, Pb2+, Al3+, Cr3+ and Fe3+) was investigated. Selectivity of the probe has been tested for all the metal ions using both UV–Visible and fluorescence experiments. To a 2 mL of sensor, 10 equivalents of each metal ion solutions were introduced and stirred for 1 min before carrying out absorption and fluorescence measurements. The fluorescence and UV–Visible titrations were achieved by using varied concentrations (0–2 eq.) of metal ion solution by maintaining the ligand concentration constant. The quantum yield (ϕ) of the sensor in presence and absence of the mercury(II) ions has been calculated according the formula [48].

Preparation of Solutions for 1H-NMR Titration

Stock solutions of ligand and mercuric nitrate were prepared in DMSO-d6 for 1H-NMR titrations.

Results and Discussion

UV–Visible and Fluorescence Spectral Studies of PAPM

The absorption and emission properties of the probe were measured using UV–Visible and fluorescence techniques in aqueous MeCN. At 275 and 355 nm, the probe showed two intense absorption bands, which can be assigned to pyrene based absorption bands. When the probe was excited at 355 nm, the pyrene based emission peaks (structured) were observed at 410 and 428 nm [41]. However, when the mercury(II) ions are added to the probe, a ratiometric shift was observed in the emission peak (non structured) at 456 nm with decrease in the fluorescence intensity. Furthermore, no significant changes in the emission peaks were noticed of probe after other metal ions were added to the sensor. The findings of fluorescence spectral studies of probe with various metal ions reveal that the receptor only responses to Hg2+ (Fig. 1).

Fig. 1
figure 1

Fluorescence intensity of PAPM with different metal ions (10 eq.) under irradiation at 365 nm. UV light

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Fluorescence Titration Studies

The stock solutions of receptor and mercury(II) ions were prepared in same concentration which is used for UV–Visible measurements. Among all the tested metal ions, the probe showed extreme sensitivity to Hg2+ (Fig. 2). Fluorescence titration has been carried out for thorough understanding of binding properties of sensor towards Hg2+. The probe exhibited substantially stronger fluorescence property (ϕ = 0.236) when irradiated at 365 nm. UV light, which can be perceived through naked eye. When mercury(II) ions were added to the probe solution, the ratiometric shift of emission band occurs at 456 nm with fluorescence quenching (ϕ = 0.024). Hence, PAPM functions as a potent “turn-off” sensor for determining mercury(II) ions. The quenching property of the probe has successively enhanced as the concentration of mercuric ions is increased in sequential order from 0–2 equivalent (0—40μL) (Fig. 3).

Fig. 2
figure 2

A absorption and B fluorescence spectral changes of PAPM (1.0 × 10–5 M) in the presence of 10 equivalent of diverse metal nitrates (1.0 × 10–3 M) in aqueous acetonitrile respectively

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Fig. 3
figure 3

A Fluorescence titration spectra of PAPM (1.0 × 10–5 M) with increase in concentration of Hg2+ with an excitation at 355 nm in aqueous acetonitrile. Inset: Change in the color of luminescence and quenching in photoluminescence intensity (under 365 nm. UV lamp) of probe after addition of mercury(II) ions (0–2 eq.) to PAPM. B Expansion graph of fluorescence titration of PAPM with increase in concentration of Hg2+

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Stoichiometry and Sensing Mechanism of PAPM with Hg2+

ESI–MS spectrometry, Job’s plot experimental data, IR spectral studies and 1H-NMR titration analysis confirm the formation of a complex between PAPM and mercury(II) ion in a 1:1 ratio. The fluorescence quenching of probe towards Hg2+ results from chelation enhanced quenching (CHEQ) which can be attributed to significant spin–orbit coupling constant of the heavy mercury(II) ion [49]. Additionally, the ligand structure lacks intramolecular hydrogen bonding and the presence of nitrogen donor sites aids in direct interaction with the mercury(II) ion as depicted in Scheme 2. The presence of rigid electron rich pyrene groups boosts the chelating ability of the probe through two nitrogen lone pairs resulting in fluorescence quenching [49].

1H-NMR titration studies were carried out in DMSO-d6 to assess the change in the chemical shift of imine protons of ligand upon binding to Hg2+. It is known fact that due to the electron donating nature of pyrene groups, the electron cloud is pulled towards both nitrogen atoms present in the fluorophore. The imine protons in the free ligand resonate at 9.50 ppm, after adding the 0.5 and 1 equivalent of mercury(II) ions to the free ligand, this peak shifted to 9.46 and 9.35 ppm respectively. This shift indicates the coordination of both nitrogen atoms to Hg2+ lead to decrease in electron density on nitrogen atoms [50] which is clearly indicated by upfield shift of imine protons by 0.15 ppm (Fig. 4). After addition of 1 eq. Hg2+ to the free ligand there is no appreciable change in chemical shifts of other protons.

Fig. 4
figure 4

1H-NMR titration of PAPM with Hg2+

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The appearance of a peak with maximum intensity at 851.26 m/z in the ESI–MS (Fig. S4) further supports to 1:1 complex formation between the probe and the mercury(II) ions.

The IR spectrum was recorded to ensure the complex formation between PAPM and Hg2+ and also to confirm the binding mode of acetate ion. PAPM showed a band of medium intensity at 1587 cm−1 (C = N) and 3041 cm−1 (Ar–H) which have been shifted to 1590 and 2969 cm−1 respectively upon complexation with Hg2+. The shift in the C = N stretching frequency clearly suggests the coordination of nitrogen atoms with mercuric ion. The presence of two new bands at 1513 and 1236 cm−1 in the complex are assigned to asymmetric and symmetric stretching (νOCO) vibrations of acetate ions respectively and the difference of 276 cm−1 among these two bands suggests the unidentate coordination mode of acetate [51].

The Job’s plot experiment [52] was carried out using stock solutions of PAPM and Hg2+ (1.0 × 10–4 M in aqueous acetonitrile) to determine the affinity of mercury(II) ion towards PAPM. Using the plot of photoluminescence intensity v/s mole fraction (XM) of the mercury(II) ions at 428 nm. (Fig. 5), the stoichiometry of PAPM and Hg2+ was established. The intersection point at 0.5 XM in the Job’s plot clearly reveals 1:1 stoichiometry between PAPM and Hg2+.

Fig. 5
figure 5

Job's plot showing 1:1 stoichiometry between PAPM and Hg2+. The molar ratio [Hg2+]/{[L] + [Hg2+]} was displayed as a function of fluorescence intensity at 428 nm

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Limit of Detection (LOD) and Association Constant (Ka)

The value of the LOD was determined using standard deviation (SD) and slope obtained by linear fittings based on fluorescence titration studies by using 3σ/slope [53] (where σ refers to standard deviation of probe). The emission data at 428 nm was used to plot the graph of LOD (Fig. 6). The graph of relative fluorescence intensity versus concentration of mercury(II) ion suggests the detection limit of PAPM for Hg2+ as 9.0 × 10–8 M. The obtained LOD value is comparable to other reported methods [27,28,29,30].

Fig. 6
figure 6

Limit of detection based on fluorescence titration of PAPM with change in concentrations of Hg2+

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The stability constant Ka was graphically evaluated using the Benesi-Hildebrand equation [54] by plotting (1/F-F0) versus 1/[Hg2+] (Fig. 7), where F0 denotes the fluorescence intensity of bare sensor and F denotes the maximum photoluminescent intensity as a function of Hg2+ concentration at 428 nm. The slope yielded the Ka value of 1.29 × 105 M−1 for PAPM towards Hg2+.

Fig. 7
figure 7

Benesi-Hildebrand curve for PAPM with Hg2+

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Competitive Study of Counter Metal Ions

To demonstrate the high selectivity of PAPM and to assess its practical application towards Hg2+, we also performed competitive studies in the presence of other fifteen metal ions (10 equivalent) at concentration of 10 μM Hg2+ (Fig. 8). These tests reveal that none of the specified ions significantly obstruct the detection of Hg2+ by PAPM.

Fig. 8
figure 8

Bar graph showing the relative photoluminescence intensity of PAPM (1.0 × 10–5 M) with Hg2+ in presence of 10 equivalent of other interfering metal ions (1.0 × 10–3 M) in MeCN:H2O (8:2)

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pH Studies

The fluorescence response of receptor towards Hg2+ was investigated at various pH levels as the effect of pH is vital for practical aspects of sensing. Buffer solutions ranging from 3.0 to 11.0 were prepared to test the pH sensitivity of the sensor (1.0 × 10–5 M) for the determination of mercury(II) ion (1.0 × 10–3 M) using phosphate buffer saline (PBS) environment. The mercury(II) ion was introduced to the buffered PAPM solution and the results showed that PAPM can effectively bind to Hg2+ in the pH range of 3.0–11.0 with no discernible changes in the fluorescence spectra (Fig. 9).

Fig. 9
figure 9

Fluorescence response of receptor to Hg2+ (10 eq.) with different ranges of pH (3.0–11.0) solutions of phosphate buffer saline

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Cytotoxicity by MTT Assay

HeLa cells purchased from National Center for Cell Science, Pune, India. The cells were seeded in a 96-well flat-bottom microplate and maintained at 37 °C in 95% humidity and 5% CO2 overnight. Different concentration (100, 50, 25, 12.5, 6.25 and 3.125 μg/ml) of PAPM in presence and absence of Hg2+ were treated. The cells were incubated further for 8 h. The wells were washed twice with PBS and 20 μL of the MTT staining solution was added to each well and plate was incubated at 37 °C. After 4 h, 100 μL of DMSO was added to each well to dissolve the formazan crystals and absorbance was recorded with a 570 nm using microplate reader. As a result of the MTT analysis, it was found that the PAPM was cytotoxic at higher concentrations but less toxic at lower concentrations. Cell viability was greater than 98% at a lower concentration of PAPM (3.125 μM), while it was 42.43% control at a higher concentration (100 μM). Despite of this, over 80% of the cells were continued to survive after PAPM concentration was increased to 12.5 μM. (Fig. S9).

Cell Imaging Studies

HeLa cells were sown separately on 16 mm coverslips and allowed to grow for 24 h in order to conduct bio-imaging experiment. Separate incubations of 10 μM of each compound were placed for 60 min at 37 °C in the dark. After removing the medium, the cells underwent three PBS washes before being fixed for 30 min with 4% para-formaldehyde. After that, a Zeiss LSM 710 confocal microscope was used to record the fluorescence of the cells. According to fluorescence imaging experimental results, PAPM exhibits high fluorescence, but when Hg2+ are added, the fluorescence intensity is quenched due to the combination of PAPM with Hg2+ in live HeLa cells (Fig. 10).

Fig. 10
figure 10

Fluorescence image of HeLa cells A incubated with PAPM (10 μM) for 1 h, B incubated with PAPM for 1 h and then Hg2+ (5 μM) for 1 h

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Sensing with Test Strips and TLC Plates

In order to explore the sensing properties of the probe towards mercury(II) ions in aqueous acetonitrile, we conducted the experiments on test strips as well as silica gel coated TLC plates for the practical utility. The four test strips (A to D; Fig. 11) were prepared by dipping test strips in PAPM solution (A), in PAPM and Hg2+ solution (B), in PAPM and various metal ion solution (C) and in solutions of PAPM, various metal ion and Hg2+ ion (D). Later these test strips were kept to dry at room temperature and exposed to UV lamp. This experimental results clearly illustrate that the color of the test strip changes from intense white luminescence (in the absence of Hg2+) to pale blue green color (minimum/almost no fluorescence) after irradiation at 365 nm.

Fig. 11
figure 11

A Test strip image after immersed in PAPM (high fluorescent). B Test strip image of (A) after immersed in 10 eq. of aqueous acetonitrile solution of Hg2+ (non-fluorescent). C Test strip image after immersed in PAPM in presence of other metal ions used in selectivity experiment (high fluorescent). D Test strip image of (C) after dipped in aqueous acetonitrile solution of Hg2+ (non-fluorescent). All test strips photographs were captured under irradiation at 365 nm

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The sensing application of receptor was also performed using a couple of TLC plates by immersing one of them in PAPM solution and other in the solutions of PAPM and mercury(II) ions sequentially. Further, both TLC plates were allowed to dry at room temperature for 20 min before being exposed to UV radiations at 365 nm. The TLC plate dipped in PAPM solution exhibited deep bluish white fluorescence (Fig. 12B), while another TLC plate displayed pale blue-green which is non-fluorescent in nature (Fig. 12C) (Fig. 12).

Fig. 12
figure 12

A Change in fluorescence intensity and its color. B Image of TLC plate dipped in PAPM solution showing intense fluorescence. C Image of TLC plate immersed in PAPM solution followed by immersion in Hg.2+ solution diminishing the fluorescence. (All images were captured under irradiation at 365 nm and solutions were prepared in aqueous MeCN)

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The successful usage of less expensive test strip and TLC plate techniques illustrates potential of the probe to recognize mercury(II) ions in future aspects.

Conclusion

The present study illustrates the synthesis of novel (N1E,N2E)-N1,N2-bis(pyrene-1-ylmethylene)benzene-1,2-diamine (PAPM) as a sensor to detect Hg2+ fluorometrically in aqueous MeCN. In presence of Hg2+, receptor experiences quenching in fluorescence intensity as well as a ratiometric redshift in the emission spectrum. The LOD and association constant (Ka) of sensor for Hg2+ were found to be 9.0 × 10–8 M and 1.29 × 105 M−1 respectively. The novel probe can be used for live cell imaging studies for detection of mercury(II) ions in HeLa cells. The present report also explored the sensing applications using low cost test strips and TLC plates for the detection of noxious mercuric ions. In summary, we conclude that the probe could serve as a potential sensor for the detection of mercury(II) ions.