Acridine-2,4-Dinitrophenyl Hydrazone Conjugated Silver Nanoparticles as an Efficient Sensor for Quantification of Mercury in Tap Water

H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 74200, Pakistan Department of Chemistry, Division of Science and Technology, University of Education, College Road, Lahore-, Pakistan Department of Chemistry, N.E.D. University of Engineering and Technology, Karachi 75270, Pakistan Department of Chemistry, Abdul Wali Khan University, Murdan, Pakistan Chemistry Department, Faculty of Science and Arts, King Abdulaziz University, Rabigh 21911, Saudi Arabia Department of Chemistry, University of Sahiwal, Sahiwal 57000, Pakistan


Introduction
The excretion of heavy metal from the industries in the environment has become a major global problem. Mercury (Hg 2+ ) is one of highly toxic pollutant which is found in soil, water, and air [1]. It is a highly dangerous metal and causes serious health problems to humans and animals. Its direct contact with eyes results in vision loss.
The human central nervous system is badly affected by action of Hg 2+ ions, if it presents in the blood. It also induces lung problem, the person feel breathing problem that leads to kidney failure and to ultimate death [2][3][4][5][6][7][8]. It has been estimated that the total amount of Hg added from industries into the environment is about 5,000-8,000 metric tons per year [9,10]. Various techniques have been employed for detection of Hg 2+ ions in the soil, water, and even in the air that include atomic absorption or emission spectroscopy (AAS/AES) [11,12], X-ray absorption spectroscopy (XAS) [13], inductively coupled plasma mass spectrometry (ICP-MS) [8], and atomic fluorescence spectrometry (AFS) [14]. Previous methods are difficult, costly, require expensive instruments, and also expertise of manpower to run these instruments. Therefore, it is necessary to develop an easy, economic, selective, and sensitive method for detection of mercury in the environmental samples. Recently, fluorescent and colorimetric sensors are found to be easily approachable, and facile method for the detection of environmentally pollutant metal ions (Hg 2+ ions) [15][16][17][18][19]. Due to presence of electronic configuration of d10 orbital of mercury, there is no spectral signature that inhibits its practical applications [20][21][22]. However, due to having this unique feature (Hg 2+ ions), the nanosensor has great optical response to detect Hg 2+ ions present in biological samples or in the environment [22][23][24]. The nanosensors have remarkable ability to detect mercury ions (Hg 2+ ) even if it is present in the very small amounts. In this context, silver nanoparticles of chemosensors were synthesized which possess strong optical response in the spectral range of 400 to 480 nm. The addition of external substances in the AgNPs results in the spectral shift (bathochromic, hyperchromic, hypsochromic, and hypochromic) due to interaction between external species with silver nanoparticles [25][26][27]. Moreover, nanoparticles of silver are cheap, nontoxic, and environment friendly. Furthermore, these have great capability to detect the heavy metal ions (Hg 2+ ions) and have high selectivity and sensitivity depending upon the nature of the used stabilizing agent. Various methods are present to synthesize AgNPs such as chemical reduction method, ion sputtering, and sol gel [28][29][30]. The abovementioned methods engage harmful chemicals and also need high energy which may cause decomposition of the chemosensors [31,32]. Due to the nontoxic nature and biological applications, the silver nanoparticles have great attention for the scientists, chemists, and pharmaceutics [33][34][35]. Moreover, silver nanoparticles provide remarkable opportunity to detect the heavy metal ions, dyes, pesticides, fertilizers, bacteria, fungi, and drugs in the environmental and biological systems.

Synthesis of ACR-AgNPs.
ACR-AgNPs were synthesized by chemical reduction method using sodium borohydride as a reducing agent. Stock solution of silver nitrate (1 mM) was prepared in deionized water which was further diluted to 0.1 mM. A 1.0 mM solution of ACR was prepared in ethanol and further diluted up to 0.1 mM. To synthesize ACR conjugated silver nanoparticles (ACR-AgNPs), equimolar solutions of synthesized ACR (0.1 mM) and silver nitrate (0.1 mM) were mixed in several ratios (1 : 5, 1 : 10, 1 : 15, and 1 : 20) and stirred at room temperature for10 minutes. After that, few drops (5-6) of NaBH 4 (4 mM) were added and resulted solution was stirred further. After 30 minutes of continuous stirring, yellow color appears in the initially colorless solution indicating the formation of colloidal silver nanoparticles. The synthesized ACR-AgNPs were centrifuged at 12000 rpm for 12 min until pallets were obtained, after discarding supernatant followed by freeze drying to obtain solid ACR-AgNPs. The obtained nanoparticles were stored at 4°C for further use.

Morphological Analysis by AFM.
For determination of particles size and surface morphology of the synthesized ACR-AgNPs. For the analysis of the complexation between ACR-AgNPs and Hg 2+ , both the solutions were mixed in 1 : 1 ratio (v/v). For AFM sample preparation, 10 μL freshly prepared solution of ACR-AgNPs was placed on a cleaned mica disc and dried in air. After complete drying, AFM analysis was performed in tapping mode.

FTIR Analysis.
FT-IR spectra in the range 400-4000 cm -1 were recorded by FT-IR 8900 Shimadzu using KBr disc. For FTIR analysis of ACR-AgNPs, 5 mg dried nanoparticles were mixed with a small amount of KBr powder and grounded to a fine disc. Similarly, 5 mg of ACR-AgNPs powder mixed with equal ratio with Hg 2+ was mixed with a small amount of KBr and grounded to a fine disc. Thereafter, analysis was done by FT-IR 8900 Shimadzu, Japan.
2.6. General Methods for Sensing Experiments. The photophysical potential of ACR-AgNPs towards metal ions was explored using UV-visible spectroscopy. For general screening experiments, 1.0 mL of freshly prepared ACR-AgNPs solution was mixed with various metal ions (100 μM), and changes in the absorption spectrum were recorded. For the determination of limit of detection for Hg 2+ , concentration-dependent experiments were performed, various concentration of Hg 2+ (5-100 μM) were treated with ACR-AgNPs. The limit of detection for Hg 2+ was calculated using standard deviation of blank and slope of straight-line equation using following formula LOD = 3:3 × S:D/Slope. For competitive analysis, several interfering metal ions were tested in presence of Hg 2+ , and obtained results were compared with that of Hg 2+ alone.

Spiking in Tap
Water. 100 μM solution of mercury was prepared in laboratory tap water taken from the University of Karachi. The freshly prepared ACR-AgNPs were mixed with mercury solution of equal concentration and equal quantity prepared in tap water. UV-visible spectrum was recorded and compared with spectrum recorded in deionized water.

Results and Discussion
3.1. Synthesis of ACR-AgNPs. UV-visible spectra were recorded to finalize the suitable ratio for optimum stabilization of ACR-AgNPs. Spectral analysis showed that ACR to AgNO 3 ratio of 1 : 10, 1 : 15, and 1 : 20 gave a sharp peak as compared to the ratio 1 : 5 at 410 nm ( Figure 2). Peak broad-ness may lead to incorrect results as the addition of any interfering substance especially low concentration may not induce a significant response. Thus, ratio 1 : 20 was used for the synthesis of ACR-AgNPs because in this ratio the more nanoparticles are formed and the peak is sharp and of high intensity.

3.2.
Characterization of ACR-AgNPs. The morphology and size of ACR-AgNPs were examined by two-dimensional and three-dimensional images obtained by atomic force microscopy (AFM), which showed spherical particles in the size range of 40 to 45 nm (Figure 3(a)). And also the SEM (scanning electron microscopy) analysis of the same revealed the spherical shape of the ACR-AgNPs having a size in similar range (Figure 3(c)).
Size distribution of ACR-AgNPs is examined through dynamic light scattering (DLS) in terms of percent intensity, ACR-AgNPs showed the average size of 40 nm and polydispersity index (PDI) of 0.556, Figure 3(b). FTIR spectra of ACR and ACR-AgNPs were recorded between 4000 and 500 cm -1 . Spectra showed prominent infrared absorbance peaks at 1618 cm -1 . Stretching vibration of carbon-carbon single and carbon-carbon double bond (aromatic) causes absorbance bands at 1585, 1618, 1477, and 1423 cm -1 . Asymmetric stretch of _ C _ O _ and CH 3 (bend) is observed at 1129 and 1253 cm -1 . Whereas peaks at 1511 and 1330 cm -1 indicate C-N and N-H stretching of aromatic amine. Stretching vibrations of secondary amine (N _ H) group of acridine showed a broadband at 3300 cm -1 while a sharp band appeared at 3644 cm -1 . In spectrum of acridine conjugated silver nanoparticles, the -N-H band shifted from 3300 cm -1 to 3443 cm -1 . These findings suggest that the secondary amine nitrogen of ACR takes part in the stabilization of silver nanoparticles (Figure 4) [41,42]. In order to determine the stability of newly synthesized ACR-AgNPs, different parameter was optimized such as pH of the medium and effect of temperature on the stability of nanoparticles. In order to determine the effect of temperature on the stability of nanoparticle, ACR-AgNPs were boiled to 100°C, cooled to room temperature, and spectra were recorded. As can be seen from Figure 5(a), ACR-AgNPs are found to be more stable after temperature treatment. The effect of pH on the stability of ACR-AgNPs was also optimized as shown in Figure 5(b). The newly prepared AgNPs stabilized by ACR were found to be highly stable in a wide range of pH (2-12).
3.4. ACR-AgNPs as Sensor. As a real target of this study, the effect of addition of commonly found ions/metal ions such as NH 4 + , Mn 2+ , Ni 2+ , Ba 2+ , Mg 2+ , Cr 3+ , Pb 2+ , Pd 2+ , Al 3+ , Sn 2+ , Fe 2+ , Co 2+ , Cu 2+ , Fe 3+ , Cd 2+ , and Hg 2+ in ACR-AgNPs is evaluated. UV-visible spectra of the ACR-AgNPs after addition of metal salt solutions (100 μM) in 1 : 1 ratio is recorded ( Figure 6). Addition of mercury solution in ACR-AgNPs resulted in decrease in the absorbance intensity that could be linked to the binding of Hg 2+ with the nitrogen of ACR-AgNPs by their lone pairs. As depicted in Figure 4 most probably, the Hg 2+ forms stable complex with Schiff base nitrogen atom of ACR-AgNPs. After the complex formation with Hg 2+ , the electronic environment of ACR-AgNPs is disrupted and consequently absorption intensity decreased significantly. All other tested metals did not have any pronounced effect on the SPR band of ACR-AgNPs. The interaction of Hg 2+-withACR-AgNPs can be monitored by several factors like surface modification, aggregate morphology, particle size, and shape [43].
Furthermore, sensitivity of ACR-AgNPs for Hg 2+ was checked in the presence of other metal ions viz. NH 4 + , Mn 2+ , Ni 2+ , Ba 2+ , Mg 2+ , Cr 3+ , Pb 2+ , Pd 2+ , Al 3+ , Sn 2+ , Fe 2+ , Co 2+ , Cu 2+ , Fe 3+ , Cd 2+ , and Hg 2+ by adding ACR-AgNPs and Hg 2+ along with all the aforementioned metals. The change in the absorption intensity of ACR-AgNPs+Hg 2+ upon addition of various other competing metal ions was recorded and compared with the intensity obtained of the same without interferent. The presence of other metal ions has no significant effect on the quenching of SPR band as obtained for the same amount of mercury without interferent, Figure 7. This indicates that the proposed sensor has efficiently detects the Hg 2+ ions in the real sample even in the presence of other metal ions.
In order to determine stoichiometry of ACR-AgNPs and Hg 2+ , Job's method was used in which total concentration was kept constant, while mole fraction of Hg 2+ was increased gradually from 0.1-1. Values of absorption intensities at 410 nm revealed that minimum absorbance was obtained at mole fraction value of 0.6, indicating 1 : 1 binding ratio of ACR-AgNPs with Hg 2+ (Figure 8).
In addition, sensing behavior of synthesized nanosensor at elevated concentration of Hg 2+ was checked between ranges of 5-100 μM Hg 2+ solutions ( Figure 9). An efficient and concentration-dependent decrease in the absorbance intensity of ACR-AgNPs is observed. A linear relation was observed with the increasing concentration of mercury with R 2 value of 0.9893. The limit of detection for mercury ion was found to be 1.65 μM.
3.5. Characterization of ACR-AgNP-Hg 2+ Complex. The morphology and size of ACR-AgNPs and Hg 2+ complex were observed through AFM and DLS and SEM ( Figure 10). Images obtained through AFM and SEM showed spherical and large size particles. Percent intensity of Hg 2+ complex of ACR-AgNPs indicated increment in particle size from 40 to 90 nm with a polydispersity index (PDI) of 0.577. Moreover, analysis through AFM, SEM, and DLS revealed increment in size which confirmed ACR-AgNPs complex formation with Hg 2+ .
The absorbance bands of ACR-AgNPs complex with Hg 2+ obtained in the range of 1000-4000 cm -1 are 3443, 1614, 1205, and 1138 cm -1 (Figure 11). Among these, broad absorbance band at 3443 cm -1 is due to the bending vibration of secondary amine (-NH). Whereas Hg 2+ binding to the nitrogen of aromatic amine and oxygen of hydroxyl results in shifting of this band from 3443 to  Journal of Chemistry 3580 cm -1 and also decreased broadness of the absorbance band appeared at 3580 cm -1 , because in the complex, both amine and hydroxyl groups of ACR-AgNPs are involved in chelation with Hg 2+ . The peak at 1687 cm -1 is due to C=N in ACR-AgNPs and in case of its complex with Hg 2+ , a sharp peak at 1614 cm -1 was observed. Moreover, C-N and C-O-C stretching bands were obtained at 1205 cm -1 and 1138 cm -1 (Figure 11). It is generally required to evaluate effect of pH on the host guest interaction in a complex in context of real sample applications. To determine an optimized pH for spectroscopic analysis, stability of ACR-AgNPs complex with Hg 2+ in the pH range of 2-13 is evaluated (Figure 12). To maintain the pH of solutions, the dilute sodium hydroxide and hydrochloric acid was added drop wise.
At pH range of 2-6, the decrease in intensity of the ACR-AgNPs complex with Hg 2+ might be associated with deprotonation of ACR-AgNPs. Conversely, the absorption intensity of ACR-AgNPs complex with Hg 2+ remained     Table 2. As can be seen in the Table that the limit of detection of the instrumental methods is lower compared to current method. However, these methods are based on the expensive instruments and timeconsuming analysis protocols. The method proposed in this study is based on UV-visible spectroscopy which is easy, economical without any need of an expert. Furthermore, the material used for the synthesis of ACR-AgNPs is cheaper compared. The method is swift, on-spot, and works well for real samples (water samples and biological system).

Conclusion
Acridine-based conjugated silver nanoparticles showed effective and sensitive method for the detection of Hg 2+ ions in the real water sample, and these nanoparticles also showed environmentally friendly behaviors. The addition of Hg 2+ ions prepared in the tap water with solution of silver nanoparticles of nanosensor (ACR-AgNPs) presents a significance decrease in the absorption intensity. The silver nanoparticles of acridine showed selectivity even in the presence of different metals ions and also exhibit significant stability in acidic and basic environment. The presence of electrolyte does not show any disturbance on the absorption intensity of the conjugated silver nanoparticles of acridine. Therefore, these results illustrate that derivative of acridine can be used as an efficient sensors for the detection of environmental pollutants. The limit of detection of proposed sensor obtained from experimental data is 1.65 μM. The percentage recovery of the spiked samples was in the range of 94.2-102.4%. All the above results show that the nanosensor could be used for the selective detection of mercury in the laboratory tap water and in biological samples.

Data Availability
All the data is presented in the manuscript.

Conflicts of Interest
The authors declare that they have no conflicts of interest.