Filling materials especially amalgam are the most frequently used restorative material for dental treatment and many concerns have been raised about them because of the existence of heavy metal ions like mercury, silver, copper, zinc and lead . It was demonstrated that amalgam filling is able to release these metal ions into the oral cavity causing very destructive effects on human body [2, 3]. For example, it was shown that Hg2+ release in saliva can represent a continuous source of oxidative damage to oral tissues . It also induces free radical production and changes the redox potential of cells. The release behavior of metal ions in the oral cavity is complex and involves several parameters, such as amalgam type, pH and oxygen variations in the oral cavity, presence of proteins, etc. Hence, certain trends might be outlined based on in vitro determination, and release measurement during laboratory tests should simulate the clinical situations. On the other hand, successful measurement of release through saliva sampling is difficult because of the fast flow of saliva in the oral cavity. In establishing in-vitro tests, synthetic environments are more frequently used than natural body fluids due to easy preparation to artificial saliva solutions. Saliva contains organic and inorganic substances suspended in an aqueous medium. Its pH may vary between 2 to 11 depending on the age, eating habits, and oral hygiene . Thus, metal release from materials used for dental applications has usually been studied in artificial saliva. Thus, artificial saliva solutions have been formulated to fulfilled different requirements. In all of these solutions, strong electrolytes usually NaCl and/or KCl, are present as the main constitute. Furthermore, there exists a buffer system such as HCO3-/CO32- or H2PO4-/HPO42- . In some cases, a complex anion like SCN- is also present which is actually present in human saliva at quite high concentration .
Recently, nanotechnology has been intensively applied in different research fields, such as separation science , drug delivery , and magnetic resonance imaging . Wide application of nanoparticles (NPs) has led to focusing on the use of these nanoparticles in many scientific areas because they have a high surface area which provides high interfacial area and therefore mass transfer is quicker, and equilibrium is achieved more rapidly. An important group of NPs in analytical chemistry have a permanent magnetism (ferromagnetism) or can be attracted by a magnetic field without retaining residual magnetism after the magnetic field is eliminated (superparamagnetism). Both types of magnetic NPs (MNPs) have been successfully used as solid-phase extraction (SPE) sorbents, because they aid the extraction procedure and save time in sample preparation. In conventional SPE, the sorbents are packed into cartridges, disks, or pipette tips, whereas in magnetic SPE (MSPE), which can be also classified as dispersive-SPE (d-SPE), the sorbent is added directly to the sample. After analyte adsorption, MNPs can retain on one side of the extraction recipient with an appropriate supermagnet. For elution, a suitable solvent is added, and the adsorbent is dispersed and retained with the supermagnet [10, 11]. This mechanism facilitates their application in different research areas [12-15]. High affinity to the analyte and reusability of nano-adsorbents provide significant cost reduction for any MSPE procedure. Furthermore, ability to remove adsorbed analytes from the adsorbent is a critical parameter when recovery of metal ions (and not removal) is desired for applications which recycling and resource recovery are needed. These desired features inspire searching a method which has capability of tuning analyte-affinity through direct adsorbent modification and to obtain an efficient adsorbent, particularly for applications in harsher matrices such as highly saline and biological fluids or processes where repetitive utilization is desired. Known properties of Fe3O4 nanoparticles (NPs) along with good adsorbent characteristics of MnO2 have suggested a hybrid nanomaterial of manganese (Mn) and Fe3O4 NPswhich yields a magnetically active adsorbent compare with or better than each precursor [16-19].
In this study, ethylene glycol bisthioglycolate modified gold nanoparticles grated Mn doped Fe3O4 nanoparticles (Au@Mn-Fe3O4 NPs) are used as a magnetic adsorbent for extraction and preconcentration of metal ions in artificial and natural saliva. The level of each target ion is determined by inductively coupled plasma-optical emission spectrometry (ICP-OES). The factors affecting extraction efficiency of the analytes are investigated and optimized. Finally, the proposed method is successfully applied to the extraction and preconcentration of target ions from natural saliva samples.
Chemicals and reagents
Lead nitrate, silver nitrate, ferrous chloride tetrahydrate (FeCl2.4H2O), ferric chloride hexahydrate (FeCl3.6H2O), manganese sulphate (MnSO4), hydrogen tetrachloroaurate (HAuCl4), sodium chloride, potassium chloride, calcium chloride (CaCl2.2H2O), sodium dihydrogen phosphate dihydrate (NaH2PO4.2H2O), sodium sulfide nine hydrate (Na2S.9H2O), urea (CO(NH2)2), lactic acid, sodium hydroxide, ammonia solution (25% w/w), hydrochloric acid, glycerol, and methanol were purchased from Merck company (Darmstadt, Germany). Ethylene glycol bisthioglycolate (EGBTG) was purchased from Sigma chemical (St Louis, MO, USA) and deionized water was used through the experiments. All chemicals and reagents were of analytical grade and used as supplied.
Determination of metal ions was carried out by a Varian Vista-Pro ICP-OES (Springvale, Australia). The instrument was operated at applied power of 1.2 kW and plasma and argon flow rates were 13.5 and 0.75 L min-1, respectively. Transmission electrom microscope (TEM) images of the adsorbent were obtained using a Philips CM200 transmission electron microscope (Amsterdam, Netherlands). The X-ray diffraction (XRD) analysis of modified nanoparticles was performed by an ItalStructures APD 2000 X-ray diffractometer (Riva Del Garda, Italy) using Cu Kα radiation source λ 1.540598 Aº with reflection scan mode. A Metrohm 827 pH/mV meter (Herisau, Switzerland) with a combined glass electrode was used for pH measurements.
Preparation of artificial saliva
The artificial saliva solution was prepared as described by Fusayama et al. [20, 21] in final volume of 1000 mL and its composition was as NaCl (0.40 mg mL-1), CaCl2.2H2O (0.79 mg mL-1), KCl (0.40 mg mL-1), NaH2PO4.2H2O (0.78 mg mL-1), Na2S.9H2O (0.005 mg mL-1), CO (NH2)2 (1.00 mg mL-1). The pH of the prepared artificial saliva was 5.5. In recommended procedure, 5 mL of the solution was diluted to 200 mL and lactic acid and ammonia solutions were used to adjust saliva pH in the recommended MSPE step.
Preparation of magnetic adsorbent
The Au@Mn-Fe3O4 NPs were prepared based on the reported method in the literature . Briefly, ferrous chloride tetrahydrate (2.15 g) and ferric chloride hexahydrate (5.84 g) were dissolved in 100 mL deionized water under nitrogen atmosphere with vigorous stirring at 90 ºC. Then, 30 mL of ammonia solution was added and the mixture was stirred for 15 min at the same condition. The prepared Fe3O4 NPs were cooled to the room temperature. The precipitate was isolated from the solution by applying an external magnet and the supernatant was removed. Then, the precipitate was washed with deionized water (3 ×250 mL) and 0.01 mol L-1 sodium chloride solution (2 ×100 mL). The later can accelerate magnetic separation particularly in alkaline solutions [23, 24]. Then, the suspension was transferred to a 250 mL round bottom flask and allowed to settle. The supernatant was removed and an aqueous solution of MnSO4 (10 mL, 0.1 M) was added to the mixture and stirred for 30 min, followed by the addition of KMnO4 (10 mL, 0.1 M) as oxidant. The mixture was then stirred for 1 h and the precipitate (Mn-Fe3O4 NPs) was washed with 200 mL of deionized water by decantation using supermagnet. After that, 5 mL of 5 mM HAuCl4 was added to the mixture and allowed to boil with constant stirring under reflux. The reaction was followed by addition of 10 mL of 25 mM trisodium citrate under continuous stirring. The mixture was remained at the same condition for 15 min and then rapidly cooled to the room temperature using an ice bath. In the next step, the supernatant was removed and an aqueous solution of EGBTB (1.0% v/v, 150 mL) was added followed by glycerol (60 mL). The mixture was stirred at 90 ºC for 2 h. After cooling down slowly to the room temperature, the precipitate was easily isolated from the supernatant using a supermagnet and the suspension was washed sequentially with deionized water (250 mL, three times), methanol (200 mL, twice), and deionized water (250 mL, once) before it was dried in vacuum oven at 50 ºC for 24 h.
Recommended MSPE procedure
A portion of artificial saliva solution containing 5 µg of target metal ion was transferred to a 250 mL beaker. The pH was adjusted by lactic acid or/and ammonia solution and diluted to the final volume of 200 mL. Then, 100 mg of Au@Mn-Fe3O4 NPs adsorbent was added and the solution was stirred for 5 min. The mixture was placed on a supermagnet and the supernatant was completely decanted. The magnet was removed; 2 mL of 0.1 M HCl solution was added and the mixture was stirred for 2 min. The solution was placed on a supermagnet again to hold the adsorbent in the bottom of the flask and the supernatant was transferred to a test tube for the subsequent ICP-OES analysis.
Linearity was evaluated by analyzing different concentrations of metal ions in the range of 5-250 ng mL-1 and calibration curves (five points) were constructed. Each point is the mean value of three-replicate analyses. The slope, intercept and correlation coefficient were calculated using the least square regression method. The limit of detection (LOD) and limit of quantitation (LOQ) were calculated based on the standard deviation of blank (Sb) and slope of calibration curve (m) determined according to the following equations:
LOD = 3.3Sb/m (1)
LOQ = 10Sb/m (2)
Precision of the proposed method was determined by five-replicate analyses of artificial saliva spiked with each analyte at 50 ng mL-1. Preconcentration factor (PF) of the method was calculated as PF = Vs/Ve× R%, where Vs is the sample volume, Ve is elution volume, and R% is percentage recovery of each analyte.
RESULT AND DISCUSSION
Soft coordination sites like SH groups are known to have a great tendency to soft metal ions, such as Ag+ and Pb2+. This work shows that combination of high adsorbent capacity of Mn-Fe3O4 NPs and modification with an S donor ligand significantly increase the quantitative retention of these ions as well as lowering their detection limits.
Characterization of adsorbent
Brunauer–Emmett–Teller (BET) analysis of Fe3O4 NPs before and after doping with Mn shows an increased surface area from 47 m2 g-1 to 73 m2 g-1, respectively, explaining the increase in analyte affinity of the adsorbent. Fig. 1 shows TEM image of the prepared adsorbent to determine its size and morphology. As can be seen , the prepared adsorbent has uniform size distribution with average diameter of 40±5 nm while most of the particles are quasi spherical in shape. The XRD patterns of the standard Fe3O4 particles and the prepared adsorbent before and after doping with Mn and modifying with Au NPs are presented in Fig. 2. This figure shows the Brag reflections indexed to the inverse cubic spinel phase of iron oxide for both samples which match well with standard magnetite obtained by JCPDS [25, 26]. There was no indication of additional phases and each sample contains identical peaks regardless of Mn content. There is no evidence of peaks corresponding to MnO2 or Mn3O4, confirming that Mn is incorporated into the ferrite structure rather than precipitating as oxides on the surface of Fe3O4 NPs. Further, peak positions of Mn-Fe3O4 NPs were shifted to lower peak position which also suggesting that the Fe3+ ions are effectively substituted by larger radius Mn2+ ions . For Au@Mn-Fe3O4 NPs two additional peaks corresponding to 111 and 200 plans of Au NPs were observed at peak positions about 37.7 and 44.5 . The saturation magnetization curves of Fe3O4 NPs, Mn-Fe3O4 NPs and Au@Mn-Fe3O4 NPs are shown in Fig. 3. As can be seen, Fe3O4 NPs has a saturation magnetization about 43 emu g-1. The saturation magnetization of Au@Mn-Fe3O4 NP adsorbents was decreased due to the thickening of the shells, however its magnetization still shows 24 emu g-1, which is more than 56% of Fe3O4 NPs’ magnetization. This appears to be due to the oxidative doping process removing Fe3+ ions from the lattice and incomplete replacement with the larger radius Mn ions, resulting in a more disordered structure and subsequent decrease in magnetic strength which confirms the results obtained by XRD analysis. However, the prepared adsorbent has enough magnetic responsiveness for applications which need magnetic manipulation.
Effect of pH
Optimum pH value which is required for quantitative extraction of the target ions was evaluated in the range of 2.0-7.0 for 5 µg mL-1 of each metal ion in artificial saliva solution. Higher pH was not considered due to probability of hydroxide precipitation, and the results were shown in Fig. 4. As can be seen, quantitative absorption was obtained in different pH values for each metal ion. Quantitative efficiency was obtained in pH 5.0 for Ag+, and it remains constant in higher pHs. Maximum adsorption of Pb2+ was obtained in the range of 4.5-7.0. Thus, optimum pH for simultaneous extraction of Ag+ and Pb2+ ions in artificial saliva ranged from 5.0-7.0. Considering the fact that normal pH of artificial saliva is 5.5 and -SH groups of EGBTG modifier can be oxidized in low pH, the value of 5.5 was selected for all our subsequent experiments including natural saliva samples.
Effect of Eluent
It was revealed from the pH studies that the adsorbent shows low tendency to target metal ions in high acidic media. Thus, the effect of desorbing solvent on the recovery of target ions was investigated by choosing some strong acids such as 0.1 M HNO3, 0.1 M HCl, and 0.1 M H2SO4 and some organic solvents such as ethanol and acetonitrile. The experimental results demonstrated that a solution of 0.1 M HCl has higher elution efficiency with respect to the other eluent. Therefore, it was selected for all subsequent experiments. On the other hand, eluent volume is a critical parameter affecting on the preconcentration factor and extraction kinetics. Thus, the effect of eluent volume for quantitative elution of target ions was investigated by changing it in the range of 0.5-10 mL. Based on the results, minimum volume for quantitative elution of the retained analytes was 2.0 mL. Thus, this value was selected for the following experiments.
Effect of the sample volume
Large sample volume and low eluent volume need to achieve higher preconcentration factor. The effect of sample volume on the extraction efficiency of targeted metal ions was studied by applying recommended MSPE procedure to different sample volumes of artificial saliva (25-250 mL) containing 5.0 µg of each metal ion. The results indicated that quantitative recovery of Ag+ and Pb2+ ions were achieved with maximum sample solution volume of 200 mL. Decreasing in the extraction recoveries can be explained by considering mass-balance equation. Theoretical recovery (R) can be calculated using R = 1/1+(β/Ks,w) , where, β is phase ratio (sample to adsorbent volume ratio) and Ks,w is the partition coefficient. From the equation, when the sample volume is too large, β increases and causes decreasing in recovery. Therefore, 200 mL was selected for sample volume in next experiments.
Adsorbent amount and adsorbent capacity
NPs have significantly higher surface area and short diffusion route compared to the traditional adsorbents which can result in high extraction efficiency and fast extraction dynamics. Hence, satisfactory results can be obtained with lower adsorbent amount. To obtain minimum adsorbent amount needed for quantitative extraction of target analytes, the amount of Au@Mn-Fe3O4 NPs was varied in the range of 10-200 mg. The results revealed that quantitative recoveries were obtained by using 100 mg of the adsorbent and this value was selected for the next experiments.
On the other hand, one of the most advantages of using modified MNPs is higher adsorption capacity which is due to their very high surface area. The capacity of an adsorbent determines how much adsorbent is requiring for quantitative extraction of certain amount of metal ions from the solution. In order to determine the adsorption capacity, 100 mg of Au@Mn-Fe3O4 NPs and 200 mL of various concentrations of metal ions were examined and the concentrations were increased until saturation occurred at equilibration time of 15 min. The maximum adsorption capacity was found to be 14.9 and 19.1 mg g-1 for Ag+ and Pb2+ ions, respectively. The results indicated that adsorption capacity of the prepared adsorbent was much higher than the required amount for determination of these ions in biological samples such as saliva.
Effect of extraction and desorption times
Efficient dispersion of the adsorbent can considerably increase its contact surface with the analyte leading to a higher adsorption efficiency and faster extraction time. Nanomaterials have higher surface area with respect to the traditional adsorbents and can be rapidly collected from the sample solution by using a magnetically assisted separation procedure. Hence, fast extraction and desorption times can be achieved with these materials. Fig. 5 shows the recovery values of metal ions as a function of extraction and desorption times. Based on the results, 5 min is sufficient for achieving an appropriate adsorption and 2 min was enough for a quantitative desorption, which are significantly shorter times compared to those in traditional column-passing SPE.
Reusability is one of the key parameters for evaluation of an adsorbent performance. In order to determine regeneration capability of Au@Mn-Fe3O4 NPs, the adsorbent which previously used in the general MSPE procedure was washed with desorbing solvent (10 mL, twice) and deionized water (10 mL, five times), then dried in oven at 50 ºC and reused. Experimental results revealed that the adsorbent can be reused at least up to ten times (RSD <3.9%) without sacrifice the analytical performance which makes it a suitable alternative to conventional SPE adsorbent.
Under the optimum conditions, the target ions were quantitatively extracted from spiked artificial saliva by applying the recommended MSPE procedure and the resulted analytical characteristics are summarized in Table 1. Linearity of the proposed method was obtained in the range of 5-250 ng mL-1 with correlation coefficients (R2) of 0.991. As can be seen from the table, LODs were obtained 0.09 and 0.11 ng mL-1 and the LOQs were obtained 0.27 and 0.32 ng mL-1 for Ag+ and Pb2+ ions, respectively. Relative standard deviation (RSD %) of the method for determination of the analytes was calculated 3.5 and 3.7 %, respectively. The enrichment factor for each metal ion was found to be 94 and 95 for Ag+ and Pb2+, respectively.
Application of the method
To investigate the accuracy and applicability of the optimized method in real biological samples, the concentration of Ag+ and Pb2+ ions was determined in saliva samples obtained from 9 volunteers (3 males and 6 females) with average of 19 years old and in the range of 15-29 years old that had no amalgam fillings. The informed consent was obtained from all volunteers at the beginning of the study and before fillings. Then, in 24, 72 and 96 hours after that one of their posterior decayed teeth was filled with amalgam, 2 mL of their saliva were collected by spitting in a special tube and kept at -18 ºC in a freezer immediately. Once all the samples were collected, they were transferred to the laboratory for analysis. Before testing, the samples were transferred into the three 200 mL flasks, put in three groups and labeled to saliva I, II and III. If needed, pH was adjusted and then diluted to the volume. Samples labeled saliva I were analyzed without any treatment and samples labeled saliva II and III were spiked with 10 and 100 ng mL-1 of each metal ion, respectively, for recovery tests, and then, were analyzed with the recommended MSPE procedure. The short-term release of the targeted metal ions was summarized in Table 2. As can be seen, the recoveries of target metal ions were in the range of 94 to 97% and the method has good merit for determination of these metal ions in biological high saline saliva.
An efficient modified adsorbent as Au NPs@Mn-Fe3O4 NPs was synthesized and successfully used for extraction and preconcentration of Ag+ and Pb2+ ions released to saliva of volunteers which were exposed to amalgam filling of their posterior decayed teeth. The method combines high adsorption capacity of the NPs and magnetic separation, and provides a fast, convenient, effective, and sensitive method for preconcentration and separation of these metal ions in high saline samples. In addition, reusability studies demonstrated the potential of the prepared adsorbent for recyclable usage compared to the conventional column-based SPE. The method is simple and inexpensive, and eliminates the need to use any auxiliary facilities to immobilize the adsorbent.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.