Visual and colorimetric determination of mercury (II) based on lignosulfonate-capped silver nanoparticles

ABSTRACT
 Lignosulfonate /silver nanoparticles (L–AgNPs) were synthesized by a one-pot method. The structure of the prepared L–AgNPs was characterized by ultraviolet–visible spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The prepared L–AgNPs were spherical with a size of approximately 16–22 nm, whereas the structure of lignosulfonate did not change during the synthesis. The synthetic method is green, simple and fast. As a heavy metal, establishing a green and rapid detection method for mercury ion is very important. L–AgNPs exhibited high selectivity for mercury (II), a detection range of 0–68 µM, and a minimum detection limit of 7 nM. The detection method developed in this work was used for the determination of mercury (II) in actual water samples, and the results agreed well with those obtained by a colorimetric method. This study provides a new idea for the practice of green chemistry and a novel method for the detection of mercury (II). GRAPHICAL ABSTRACT


Introduction
Green reactants are very important in the preparation of nanomaterials (1).Lignin is the most abundant renewable and biodegradable natural resource worldwide, and it is a main research direction in the field of renewable resources.It has a large number of active groups, such as hydroxyl and aldehyde groups (2,3).The molecular structure of lignin components has a spatial configuration.The use of lignin as capping agents for the preparation of nanomaterials increases its utilization value and conforms to the concept of green chemistry (4,5).In addition, lignin has the advantages of renewability, biodegradability, and nontoxicity; these advantages could expand the application of nanomaterials in the field of medicine and biology.Lignin is mostly applied in the form of lignosulfonate.Sulfonation between the sulfite solution and the original lignin in wood flour upon the introduction of the sulfonic group during sulfite pulp production increases hydrophilicity (6).The further hydrolysis of lignosulfonate in the acid cooking liquid could depolymerize hemicellulose combined with lignin (7) to dissolve lignosulfonate; enable the separation of lignin, cellulose, and hemicellulose; and yield pulp.Moreover, the application of lignin is possible.Thus, lignosulfonate was used in the present work.
Silver nanoparticles (AgNPs) are precious metal nanomaterials with unique and excellent properties, such as electron, optics, catalysis, antibacterial, and surfacemodification properties; these properties largely depend on the sizes, shapes, and chemical environment of AgNPs (8,9).Therefore, the preparation and modification of AgNPs have great importance.The production technology of AgNPs could develop in the direction of outstanding material functions, low consumption of synthesis, reduced emissions of three types of wastes, and simple technical route (10).At present, chemical reduction is the most commonly used method for preparing AgNPs.However, the difficulty encountered in removing the chemical reduction reagent from the surfaces of AgNPs imposes considerable restrictions on the application of AgNPs in medicine and biology.Chemical reduction could also cause environmental pollution, which does not conform to the concept of green chemistry.The use of natural pollution-free biomass materials as reducing agents and stabilizers does not necessitate the separation of biomass from AgNPs; thus, this approach reduces environmental pollution and widens the application scope of AgNPs (11).Chandna et al (12) used lignin as a matrix to synthesize silver-gold bimetallic and monometallic nanocomplexes to explore the synergistic antioxidant and antimicrobial properties of ligninstabilized agents.Marulasiddeshwara et al (13) synthesized lignin-capped AgNPs and evaluated their antibacterial, antifungal, antioxidant, and antiplatelet properties.Hu et al (14) synthesized AgNPs with alkali lignin as a double reducing agent and capping agent and proved that its synthesis mechanism is highly related to pH.Aadil et al. synthesized Acacia ligninmediated AgNPs and detected hydrogen peroxide (15).
The harmful effects of mercury on the human body mainly involve the central nervous system, digestive system, and kidneys.The combination of mercury ions with numerous negatively charged groups, such as sulfhydryl groups in enzymes or proteins, in vivo affects cell function and growth by disrupting various metabolic pathways in cells.These pathways include energy generation and protein and nucleic acid synthesis.A large number of studies have reported that mercury (II) causes irreversible damage to many organs of the human body (16).Mercury (II) is difficult to detect during the early stage of poisoning because it is strongly concealed.Given that many cases of mercury poisoning are mainly due to the lack of rapid reaction detection methods, establishing a simple and rapid mercury (II) detection method has great research value.Mercury (II) detection methods include atomic absorption spectrometry (17), chromatography (18), fluorescence probe detection (19,20) and electrochemical method (21).Although these methods have the advantages of anti-interference and low limit of detection (LOD), they also have disadvantages, such as the use of expensive instruments and the conduct of complex sample pretreatment.Therefore, colorimetry has become the main research method for mercury ion detection in recent years because of its simplicity and rapidity (22,23).AgNPs were also used to detect mercury ions by colorimetry (Table 1); however, these methods do not use green materials to synthesize AgNPs (24)(25)(26).Zhan and Firdaus et al synthesized AgNPs from green materials, but the LOD was too high to be suitable for the detection of trace mercury ions (27,28).Some colorimetric methods have too small detection range (29,30), and some methods need other substances to detect mercury ions (24,31).Among them, detection using lignin-based AgNPs has the disadvantage of small detection range (32,33).
In this research, lignosulfonate/AgNPs (L-AgNPs) were prepared via one-step green synthesis.Lignin was used as an excellent dispersant and capping agent.The prepared L-AgNPs have good dispersibility, uniform morphology, and particle size.A rapid, low-cost, and pollution-free mercury ion detection method based on L-AgNPs was established.A naked-eye method for the detection of mercury ions based on the color change in L-AgNPs solution and color paper was also established, and its detection mechanism was studied.The following types of equipment were used: Ultraviolet-vis (UV-vis) 2550 ultraviolet spectrophotometer (Shimadzu, Japan), D8 Venture X-ray single crystal diffractometer (Bruker, Germany), and HT7800 transmission electron microscope (Hitachi, Japan).Fourier transform infrared spectra were obtained with a Spectrum Two spectrometer (PerkinElmer, USA).

Preparation of L-AgNPs
Lignosulfonate was prepared by sulfonation of lignin.Sodium lignosulfonate (0.30 g) was accurately weighed, placed in a round-bottomed flask, and dissolved in 30 mL of water.The solution was mixed under magnetic force at room temperature for 3 min.Next, the solution was mixed with 30 mL of 0.01 mol/L silver nitrate solution and 30 mL of 1 µM ascorbic acid aqueous solution, and then reacted for 5 h at 37 °C.
The color of the solution changed from brown to yellow.The solution was freeze-dried to obtain AgNPs in powder.

Standard curve
Mercury (II) standard solution was prepared as follows: 0.05 g of HgSO 4 was accurately weighed.The volume was adjusted to 100 mL by using 10% NaCl solution.
The mercury (II) standard solution was diluted and then added to0.5 mg/mL of L-AgNPs solution.Its absorbance was measured with a UV-vis spectrophotometer, and a standard curve was constructed.

Colored test paper
A filter paper was soaked in 0.5 mg/mL L-AgNPs was naturally dried and then processed into colored test paper.One drop of blank solution and one drop of mercury (II) solutions with six different concentrations were added.The amount of mercury ions was judged by observing the color change in the colored test paper.

Characterization of L-AgNPs
L-AgNPs were synthesized with lignosulfonate as capping agent, ascorbic acid as reducing agent, and AgNO 3 as raw material by heating in water bath.First, the reaction process was optimized, and the effects of reaction time, reaction temperature, and the amount of ascorbic acid were investigated.As shown in Figure 1-Sa, during L-AgNPs preparation, the absorbance at and specific wavelength of L-AgNPs increased with the increase in temperature.The absorbance intensity was the strongest when the temperature was 37 °C.With the increase in reaction time (Figure 1-Sb), the absorbance intensity of L-AgNPs became strong and stable after 5 h.Thus, the chosen reaction time was 5 h.The results showed that the reaction could not be carried out without adding ascorbic acid, and the optimal amount of ascorbic acid to add was 1 µM (Figure 1-Sc).Through the optimization of reaction conditions, the optimal reaction conditions were determined as follows: temperature of 37 °C, synthesis time of 5 h, and reductant dosage of 1 µM. Figure 1a and b are the SEM images of L-AgNPs.The prepared lignosulfonate-capped AgNPs appeared flake like, and AgNPs were uniformly dispersed in the lignosulfonate.The TEM image showed that L-AgNPs were spherical (Figure 1c and d) and that the diameter of the AgNPs was approximately 16-22 nm.Microscopy revealed the presence of a lattice plane with an interfering distance of 0.1192 nm for L-AgNPs.These results proved that lignosulfonate acted as a scaffold and good dispersant in the reaction.
On the basis of the infrared and X-ray diffraction spectra (Figure 2), lignosulfonate, L-AgNPs, and L-AgNPs added with mercury (II) were characterized to examine the structural changes in silver and lignosulfonate.The infrared characteristic peaks of lignosulfonate were observed.The peak at 3437 cm −1 was the expansion vibration peak of -OH; that at 2929 cm −1 was the antisymmetric stretching vibration of C-H bond in methylene; and the peaks at 1611, 1523, and 1388 cm −1 were the absorption peaks of the aromatic ring skeleton.The absorption peak became shallow after L-AgNPs were generated and mercury ions were added, so the small side-chain molecules on the benzene ring may be broken.The bending vibration peak of C-H bond in the methylene absorption peak was located at 1345 cm −1 ; the C-O-C stretching vibration peak was at 1137 cm −1 , and a slight change was observed in this absorption peak, probably due to the conversion of O-C-O in lignin into carboxyl during the reaction; the C-OH stretching vibration peak was situated at 1038 cm −1 ; the C-H stretching vibration peak was positioned at 2850 cm −1 ; and a strong S-O stretching vibration peak was observed at 620 cm −1 (34,35).Comparison revealed that the preparation of L-AgNPs and the addition of mercury (II) to L-AgNPs basically did not change the structure of lignosulfonate, except for minor changes in the S-O stretching peaks (Figure 2a).
Figure 2b shows the X-ray diffraction spectra of lignosulfonate, L-AgNPs, and L-AgNPs added with mercury (II).Sodium lignosulfonate is a kind of anionic surfactant, which is the reaction product of wood pulp with sulfur dichloride and sulfite, so the product contains many NaCl and Na 2 SO 4 .Through the match of NaCl (JCPDS No. 3050628) and Na 2 SO 4 (JCPDS No. 37-1465), the XRD of lignosulfonate showed that the crystal diffraction peaks mainly came from NaCl and Na 2 SO 4 (Figure 2-S) (36,37).The five peaks observed at 2 θ = 38.12°,44.2°, 64.37°, 77.50°, and 81.38°in the spectrum corresponded to (111), ( 200), ( 220), (311), and (222), respectively.These peaks were the same as the silver crystal structure in JCPDS No. 89-3722 and matched previously reported peaks (32,38,39).However, lignosulfonate did not contain these diffraction peaks.The appearance of these diffraction peaks after the synthesis of L-AgNPs indicated that high-purity AgNPs were prepared through the method used in this study.The XRD spectra obtained after the addition of mercury (II) ions were discussed in detail below.
The surface elemental analysis of lignosulfonate and L-AgNPs was performed via X-ray photoelectron spectroscopy.As shown in Figure 3a, the main elements in the product were O and C. Elemental Ag was also present in addition to these two elements.Figure 3b shows the Ag3d peak distribution in the reaction-  generated L-AgNPs and Ag 0 peaks at 368.2 and 374.2 eV (40).Combining these results with the results of the previous XRD analysis showed that the Ag + in the reactant was converted into Ag 0 to generate AgNPs (41).This phenomenon accounted for the formation of L-AgNPs.

Establishment of the method for the detection of mercury (II) by L-AgNPs
Figure 4a shows the UV-Vis spectrum of L-AgNPs after the addition of different concentrations of mercury (II).With the increase in mercury (II) concentration (from 0 µM to 68 µM), the absorbance of the L-AgNPs solution at the maximum absorption wavelength of 425 nm gradually decreased, a certain degree of blue shifting occurred, the color of the solution changed from yellow to colorless, and the freeze-dried sample changed from brown to yellow.A standard curve was constructed by taking the concentration of mercury (II) as the abscissa and the change value of the absorbance as the ordinate.Figure 4b presents a good linear relationship between mercury (II) concentration and the absorbance.The linear relationship between the absorbance of L-AgNPs and mercury (II) concentration after the addition of mercury (II) standard solution could be divided into two parts (y = 0.00956x + 0.68968, r = 0.99165; y = 0.00203x + 0.49987, r = 0.99698).
Mercury (II) standard solutions with different concentrations were added upon the dilution of the concentration of AgNPs to the signal-to-noise ratio of 3. The LOD for mercury (II) was calculated to be 7.0 nM by using 0.1 mg/mL L-AgNPs (42).The methods of detecting mercury (II) by AgNPs with other protective groups were compared, and the proposed method demonstrated wider detection range and lower LOD (Table 1).
As shown in Figure 3-Sa, the maximum absorption wavelength of L-AgNP changed under acidic conditions but basically did not change under neutral and alkaline conditions.L-AgNPs are suitable for the detection of mercury ions in neutral and alkaline conditions.If under acidic conditions, the pH must not exceed 5, because sodium lignosulfonate is an alkaline substance.Thus, it is easy to be affected by acidic conditions.However, the effect on L-AgNPs was not significant, because  lignosulfonate was only the template of L-AgNPs, and it did not affect the detection of mercury ions by L-AgNPs (Figure 3-Sb).Therefore, this method could be used for the detection of mercury ions under mild conditions.

Selectivity test
Under the same conditions for mercury (II), the possible interfering cations (Cu 2+ , Al 3+ , Ce 3+ , Cr 6+ , Ag +, Na + ,Ca 2+ , Pb 2+ , Ni + , Mn 2+ , K + , Fe 2+ , Zn 2+ , Cr 3+ , and Mg 2+ ) and anions (SO were added to the L-AgNPs solution.The test results are shown in Figure 5.The prepared L-AgNPs strongly responded to mercury (II), but did not respond to the other interfering ions that may exist in actual samples.This behavior proved that the prepared L-AgNPs are strongly selective for mercury (II) and suitable for the detection of mercury (II) in actual samples.

Colorimetry for the detection of mercury (II) by L-AgNPs
Mercury (II) solutions with different concentrations were successively added to the L-AgNPs solution.The color of the L-AgNPs solution lightened from bright yellow to light yellow with the increase in mercury (II) content in the system.The whole system became nearly colorless (as shown in Figure 6a) when its mercury (II) content reached 70 µM.Figure 6b shows filter papers soaked in the L-AgNPs solution.The filter papers were used as colored test paper after they had dried naturally.One drop of blank solution and one drop of mercury (II) solution with six different concentrations were added to the filter papers.The white area in the liquid circle of the colored test paper gradually expanded with the increase in mercury (II) content.Thus, the content of mercury (II) could be evaluated on the basis of the color change in the colored test paper.Therefore, colorimetric mercury detection and naked-eye observation were realized.

Mechanism of mercury (II) detection by L-AgNPs
The addition of mercury (II) could reduce the absorbance of L-AgNPs.This phenomenon enables the establishment of a quantitative method for mercury (II) determination.L-AgNPs were originally yellow but lightened upon the addition of mercury (II).Thus, colorimetry  could be used to detect mercury (II).Mercury (II) and L-AgNPs were speculated to interact.As shown in the infrared spectrum in Figure 2a, the structure of lignosulfonate, which acted as the L-AgNPs ligand, did not change after the addition of mercury (II), indicating that mercury (II) did not destroy the structure of lignosulfonate.The XRD spectrum (Figure 2b) showed that the diffraction position of silver in L-AgNPs changed drastically after the addition of mercury ions, indicating that mercury (II) and silver interacted (39).As shown in Figure 7a, L-AgNPs stabilized with lignosulfonate were well dispersed and defined.Figure 7b illustrates that L-AgNPs contained a high amount of silver, and lignosulfonate contained C and O elements.However, after mercury (II) was added, the L-AgNPs were destroyed, and their morphology became unclear (Figure 7c).Mercury (II) was present as seen in EDS, and the silver content was reduced (Figure 7d).Silver probably did not disappear but it was instead enveloped by mercury and destroyed (40).
Figure 8a and b show that the energy spectrum of Ag changed greatly after the addition of mercury (II).The peaks at 367.5 and 373.5 eV corresponded to Ag + and those at 368.2 and 374.2 eV were ascribed to Ag 0 .In contrast to the spectrum in Figure 3, Ag + was present in the spectra in Figure 8b. Figure 8c shows the peak distribution of Hg4f, in which the peak at 99.8 and 10 eV were Hg 0 (41).The results showed that Hg 2+ reacted  with Ag 0 in a redox reaction.This phenomenon may be the main reason for the reduction in the absorbance of L-AgNPs.
The changes in the absorption peak and solution color of AgNPs were analyzed in combination with the changes in the IR, XRD, and XPS spectra of L-AgNPs before and after the addition of mercury (II).The main reason is that a redox reaction occurs between mercury (II) and silver (25).The diffusion of newly generated mercury atoms to the surface of silver resulted in the intense blue shift and the reduction in the characteristic absorption band.The light-yellow discoloration was observable by the naked eye.The TEM images showed that the L-AgNPs changed irregularly and were in an aggregated state after the addition of mercury (II) (32).Nanoparticles are highly sensitive to their size and shape.The surrounding medium and the distance between particles correspond to the color change of scattered light (43).

Determination of mercury (II) in actual samples
Known concentrations of mercury (II) were added to Songhua River samples via standard addition method, and the mercury (II) content was detected through the established method to explore the application value of this method in the detection of mercury (II) in real samples.The calculation results are shown in Table 2.The relative standard deviation of mercury (II) detection via this method was between 2.36% and 2.61%, and the recovery was between 99.4% and 101.0%.These results showed that the prepared L-AgNPs are suitable for quantitative detection of mercury (II) in aqueous solutions.

Conclusions
L-AgNPs were synthesized using lignosulfonate as a stabilizer and ascorbic acid as a reducing agent.The synthesis of L-AgNPs did not require the use of other chemical reagents.Moreover, it did not change the structure of lignosulfonate and was environmental friendly.Given the interaction between the prepared L-AgNPs and mercury (II), mercury (II) could be quantitatively analyzed via UV-vis spectroscopy and objectively identified through colorimetry.This method has the advantages of fast response, high selectivity, and low cost.It provides an economical and efficient method for the detection of trace mercury (II), a novel method for the detection of heavy-metal ions, and a basis for the visual detection of heavy metals.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The

Figure 4 .
Figure 4. (a) UV-Vis spectrum of L-AgNPs with different concentrations of mercury (II).(b) relationship absorbance changes of L-AgNPs with different concentrations of mercury (II), (Inset: the standard curve).

Figure 5 .
Figure 5.The absorbance changes of L-AgNPs mixed with different interfering ions.

Figure 6 .
Figure 6.Colorimetric responses of L-AgNPs to mercury (II) with different concentrations.

Table 1 .
Comparison of mercury (II) detection methods using AgNPs with different protective groups

Table 2 .
present work was supported by the Scientific Research Projects of Colleges and Universities in Hainan Province [grant no Hnky-2022ZD-19] and supported by Hainan Provincial Natural Science Foundation of China [grant no 222QN333] Determination of mercury (II) content in water samples using the proposed method (n = 3).