Molecular Imprinted Based Quartz Crystal Microbalance Nanosensors for Mercury Detection

Abstract Mercury(II) ions are emerging as a result of more human activity, especially coal‐fired power plants, industrial processes, waste incineration plants, and mining. The mercury found in different forms after spreading around diffuses the nature of other living things. Although the damage to health is not yet clear, it is obvious that it is the cause of many diseases. This work detects the problem of mercury(II) ions, one of the active pollutants in wastewater. For this purpose, it is possible to detect the smallest amount of mercury(II) ions by means of the mercury(II) ions suppressed quartz crystal microbalance nanosensor developed. Zinc(II) and cadmium(II) ions are chosen as competitor elements. Developed nanosensor technology is known as the ideal method in the laboratory environment to detect mercury(II) ions from wastewater because of its low cost and precise result orientation. The range of linearity and the limit of detection are measured as 0.25 × 10−9–50 × 10−9 m. The detection limit is found to be 0.21 × 10−9 m. The mercury(II) ions imprinted nanosensors prepared according to the obtained experimental findings show high selectivity and sensitivity to detect mercury(II) ions from wastewater.


Introduction
As a result of rapid population growth, and industrialization, wastewater has exceeded than that nature can cope with, and the receiving environment faces the risk of pollution. [1][2][3][4][5] The need to purify the wastewaters from hazardous metals has arisen in order to prevent this situation that could affect the ecological balance. [6][7][8] Contaminants present in wastewater can be dissolved in either water or solid matter. [9,10] Mercury is one of the persistent pollutants in wastewater, and when it reaches surface waters or soils, microorganisms may transform it into methyl mercury, a substance that is rapidly absorbed by most bodies and is known to cause nerve damage. [11][12][13][14] Acid surface waters can contain significant amounts of mercury. [15] When the pH is between five and seven, the concentration of mercury in the water increases. Because of this, there is a severe environmental and public health problem, and it has become necessary to detect it in the aqueous environment. [16][17][18][19] recommended method. For repeatability, four samples from the same concentration were prepared and measured. This day-to-day study was carried out at three different times a day at the same concentration. The same procedure was observed for three different days. The Hg(II) ions imprinted pHEMAC nanofilm we obtain is very attractive due to its high biocompatibility, selectivity, surface modification, reusability, and relatively low cost. [30,31]

Preparation and Characterization of Hg(II) Ions Imprinted QCM Nanosensors
Surface morphology of the Hg(II) ions imprinted pHEMAC nanosensors were characterized with FTIR, contact angle, AFM, and ellipsometry dimensions and performed to calculate the thickness of polymeric film onto the gold surface of QCM nanosensor chips. FTIR spectrum of MAC has the characteristic stretching vibration amide I adsorption bands at 1606 and 1387 cm −1 . For the characteristic determination of pHEMAC polymer, the characteristic strong -SH stretching vibration bands at 3021 cm −1 slips to the higher frequency field at 2910 cm −1 as a result of decreasing the electron density of sulfhydryl group of MAC monomer. The FTIR spectrum of MAC has showed the characteristic -SH absorbance peak at 3021 cm −1 shifted to 2910 cm −1 due to MAC incorporation into the HEMA monomer and was confirmed by carbonyl stretching bands at 1713 cm −1 . Hydrophilicity of nanofilm was determined by contact angle measurements. The contact angle of the unmodified QCM nanosensor decreased from 81.4° to 67.2° when the Hg(II) ions imprinted pHEMAC nanofilm was attached onto the modified gold surface (Figure 1A,B). Decrease in the contact angle shows the increased hydrophilic property of nanosensor chip surface. The surface morphologies of Hg(II) ions imprinted pHEMAC nanosensors were investigated with AFM measurements ( Figure 1C,D). The surface depths of Hg(II) ions imprinted QCM nanosensors were determined with AFM as 8.03 and 93.59 nm, respectively. AFM images indicate clearly that a polymeric film was synthesized on the nanosensor surfaces. The ellipsometric thicknesses were measured as 92.5 ± 0.8 nm for gold QCM surface ( Figure 1E) and 113 ± 0.7 nm for Hg(II) ions imprinted QCM nanosensor ( Figure 1F). For the results of ellipsometry, we can say the chip surfaces are homogeneous. MAC mono mer contains carboxylic acid group and has hydrophilic structure. These results are consistent with results of AFM.

Kinetic Analyses with Hg(II) Ions Imprinted QCM Nanosensors
The detection of Hg(II) ions from an aqueous solution was performed using Hg(II) ions imprinted and non-imprinted pHEMAC nanosensors. First, Hg(II) ions imprinted pHEMAC nanosensor was equilibrated with 1% HNO 3 solution. After, a series of various concentration solutions of Hg(II) ions ranging from 0.25 × 10 −9 to 50.0 × 10 −9 m were applied to QCM system in Figure 2A. As seen in the figure, the increase in Hg(II) ions concentration caused an enhancement in QCM nanosensor response.
The plateau was reached after 35 min and 1.0% HNO 3 solution was injected onto the QCM nanosensor for washing of unbounding molecules. Initially, the nanosensor's response increases linearly, and then it reaches its plateau at a relatively high concentration of Hg(II) ions (10.0 ng mL −1 ) as saturation is the accessible imprinted voids. The whole cycle containing adsorption, desorption, and regeneration was completed in about 50 min. Figure 2B shows the linear range of Hg(II) ions imprinted pHEMAC nanosensor (0.25 × 10 −9 -7.5 × 10 −9 m).
The data were obtained from this concentration range, which was used to determine the limit of detection (LOD) and limit of quantitation (LOQ) values of the Hg(II) ions imprinted pHEMAC nanosensor.
Limit of detection was calculated by the parity Limit of quantification was conjectured by the parity where S is the standard deviation of the intercept and m is the slope of the regression line. [32][33][34] LOD and LOQ values were calculated to be 0.21 × 10 −9 and 0.73 × 10 −9 m, respectively. A summary of the different detection methods for Hg(II) ions is given in Table 1.
To evaluate the applicability of the QCM nanosensor, water sample (sampled from Beytepe, Ankara, Turkey) was spiked with Hg(II) ions and tested in the QCM nanosensor. To further demonstrate the applicability of our QCM nanosensor in practical applications, we performed recovery experiments using spiked water sample with 1.0 × 10 −9 m of Hg(II) ions. We observed an average recovery value of 92%, indicating that the QCM nanosensor can be used for the detection of Hg(II) ions in water sample even at concentrations below the allowed Hg(II) ions concentration (10 × 10 −9 m) defined by the U.S. Environmental Protection Agency. High recovery percentages even at very low Hg(II) ions concentration and low standard deviation in the experiments indicate the high accuracy of our QCM nanosensor.  Kinetic analysis of Hg(II) ions imprinted and non-imprinted pHEMAC nanosensors was performed in aqueous solution in real time. Freundlich, Langmuir, and Langmuir-Freundlich adsorption isotherm models were calculated by using experimental results. The adsorption parameters are given Table 2.
According to the obtained data, this results are in good agreement with the Langmuir model (R 2 : 0.999), indicating that the prepared binding sites for Hg(II) ions on the nanosensor surface are monolayer, co-energy, homogeneously distributed, and minimal lateral interaction (Figure 3A,B). The Δm max value calculated from Langmuir model is very close to the value obtained experimentally (Δm max : 1.66 ng mL −1 ). The Scatchard plot analysis indicated that the polymer binds a single molecule to each binding  site (R 2 : 0.986), which confirms the good fit of the Langmuir model.

Selectivity of Hg(II) Ions Imprinted QCM Nanosensors
The selectivity of Hg(II) ions imprinted pHEMAC nanosensor was examined using Cd(II) and Zn(II) ions. Selective recognition of Hg(II) ions with Hg(II) ions imprinted and non-imprinted pHEMAC nanosensors was examined with 25.0 × 10 −9 m of each Hg(II), Cd(II), and Zn(II) ions solutions. The selectivity coefficients (k) and relative selectivity coefficients (k′) valuation are dedicated in Figure 4. Hg(II) ions imprinted QCM nanosensors were 10.48 and 9.35 times more picky for Hg(II) ions whence Cd(II) and Zn(II) ions, seriatim. Fastening capacities of Hg(II) ions imprinted and non-imprinted pHEMAC nanosensor were compared. As seen in Figure 4, fastening capacity of Hg(II) ions imprinted pHEMAC nanosensor is higher than non-imprinted pHEMAC nanosensor. It shows that the Hg(II) ions imprinted pHEMAC nanosensor recognizes Hg(II) ions with good selectivity because of the Hg(II) ions imprinting methods that chemical recognition cavity and create shape.

Reproducibility and Stability
The equilibration-adsorption-regeneration cycles were repeated for four times using aqueous Hg(II) ions solution with concentration of 50 × 10 −9 m in Figure 5. As can be seen from Figure 6, Hg(II) ions imprinted pHEMAC nanosensor has displayed reproducible mass shift during the cycles and Hg(II) ions imprinted pHEMAC nanosensor shows that there is no decrease in adsorption capacity during four cycles. Four samples were prepared and measured at the same concentration to perform the repeatability study.
For the intraday study, the Hg(II) ions solution was prepared at the same concentration. The sample was prepared at three different times of the day. Intraday sensitivity was also determined by the same procedure. Interday application was observed for three different days. The result was recorded as %RSD. Average %RSD was 1.119. The consequences of the studies are shown in Tables 3 and 4.

Conclusion
As a result, we have developed a new method of quartz crystal microbalance nanosensor to detect Hg(II) ions from wastewater. In this method, we first converted the MAC and Hg(II) ions into the precomplex and then modified the nanosensor chips to obtain pHEMAC-Hg(II) ions and pHEMAC polymers. Characterization analyses were made with high precision, and the obtained polymers were compared with previous studies. The range of linearity and the limit of detection were measured as 0.25-50 × 10 −9 m. The detection limit was found 0.21 × 10 −9 m. Compared to other methods in literature, our method is known to be as cheap and fast as Global Challenges 2019, 3, 1800071  it is sensitive. Moreover, it is also favored in terms of time and timing. Rapid increase in population, urbanization, and industrialization, excessive and unconscious use of fossil fuels such as coal, natural gas, and oil, and increase in consumption per capita are the main factors accelerating the pollution and deterioration process. This study contributes to the detection of heavy metal ions from wastewaters resulting from environmental pollution.
Global Challenges 2019, 3, 1800071   After this process, the gold-coated surface of the QCM chips was connected with allyl mercaptane (CH 2 CHCH 2 SH). After modification procedure allyl groups were cleaned from the gold-plated QCM chip surface with ethyl alcohol and desiccated with nitrogen gas. At the completion of surface modification QCM chips were rinsed with ethyl alcohol to remove unbounded allyl mercaptan and desiccated with nitrogen gas.
Surface Modification of the QCM Chips-Preparation of Hg(II) Ions Imprinted and Non-Imprinted pHEMAC Nanosensors: For the preparation of Hg(II) ion imprinted and non-imprinted pHEMAC QCM nanosensors, MAC:Hg(II) ions pre-complex as template molecule Hg(II) ions was prepared by using Hg(II) ions and MAC monomer (Figure 6).
The stoichiometric molar ratio of MAC:Hg(II) ions pre-complex was determined by preparing in different molar ratio as 1:1, 2:1, 3: Thereafter, 5.0 µL was pulled of the stock monomer solution with pipette and modified with allylmercaptan on QCM chip using the spin coating method. Polymerization was carried out by means of UV-irradiation (100 W, 365 nm) for 45 min at room temperature under a nitrogen atmosphere. The QCM chip was washed four times with ethanol and desiccated in a vacuum oven. 0.05 m EDTA and 0.05 m HCl solutions were utilized as the desorption agent to detect Hg(II) ions from the QCM nanosensor. The non-imprinted pHEMAC nanosensor was prepared by applying the same procedure without Hg(II) ions.
Surface Modification of the QCM Chips-Instrumentation: Characterization studies of Hg(II) ion imprinted and non-imprinted pHEMAC nanosensors were completed with FTIR, contact angle, AFM, and ellipsometer measurements. The characterization of pHEMAC and Hg(II) ion imprinted pHEMAC nanosensor was performed using FTIR spectrophotometer (Thermo Fisher Scientific, Nicolet iS10, Waltham, MA, USA) in the wavenumber range of 700-4000 cm −1 . For the contact angle of the chips surface, KRUSS DSA100 (Hamburg, Figure 6. The molecular formula of MAC-Hg(II) ion complex monomer.