A Highly Sensitive and Selective Colorimetric Hg2+ Ion Probe Using Gold Nanoparticles Functionalized with Polyethyleneimine

A highly sensitive and selective colorimetric assay for the detection of Hg2+ ions was developed using gold nanoparticles (AuNPs) conjugated with polyethyleneimine (PEI). The Hg2+ ion coordinates with PEI, decreasing the interparticle distance and inducing aggregation. Time-of-flight secondary ion mass spectrometry showed that the Hg2+ ion was bound to the nitrogen atoms of the PEI in a bidentate manner (N–Hg2+–N), which resulted in a significant color change from light red to violet due to aggregation. Using this PEI-AuNP probe, determination of Hg2+ ion can be achieved by the naked eye and spectrophotometric methods. Pronounced color change of the PEI-AuNPs in the presence of Hg2+ was optimized at pH 7.0, 50°C, and 300 mM·NaCl concentration. The absorption intensity ratio (A700/A514) was correlated with the Hg2+ concentration in the linear range of 0.003–5.0 μM. The limits of detection were measured to be 1.72, 1.80, 2.00, and 1.95 nM for tap water, pond water, tuna fish, and bovine serum, respectively. Owing to its facile and sensitive nature, this assay method for Hg2+ ions can be applied to the analysis of water and biological samples.


Introduction
Mercury ion (Hg 2+ ) is ubiquitously distributed in the environment, and it is considered to be one of the major environmental pollutants to be widely used in industry, agriculture, and medicine. It is nonessential and toxic to the human body. Hg 2+ is considered to be one of the major environmental pollutants to be widely used in industry, agriculture, and medicine. is mercury ion exists in inorganic and organic mercury ions. Upon entering the body, inorganic mercury ions are accumulated mainly in the kidneys and give rise to vomiting and diarrhea, followed by hypovolemic shock, oliguric renal failure, and possibly death [1][2][3][4]. Organic mercury ions such as methylmercuric (MeHg + ), ethylmercuric (EtHg + ), and phenylmercuric (PhHg + ) ions can also cause injuries at the central nervous system and lead to paresthesias, headaches, ataxia, dysarthria, visual eld constriction, blindness, and hearing impairment [5][6][7]. erefore, detection of mercury ion in various sample matrices has been an urgent issue.
Several analytical techniques, such as direct mercury analyzer (DMA) [8], ion chromatography (IC) [9], and high performance liquid chromatography (HPLC) [10,11], have been utilized to detect mercury ions. However, these ways generally require complicated sample pretreatment process, skillful technicians, and sophisticated instrumentations. erefore, the low-cost and facile analytical method for selective detection of mercury ions remains to be a challenge for analytical chemists.
Polyethyleneimine (PEI) was applied to use a chemical functionalizer as a harmless gene delivery mediator, templates, stabilizers, and molecular gum to arrange metal nanoparticles [19][20][21][22]. Various amine groups could give su cient active sites for strong combining capability, and these characteristics of PEI can be useful to control the selectivity of di erent ions. For its application, AuNPs conjugated with PEI (PEI-AuNPs) have been utilized as delivery of drug and gene in breast cancer therapy [23].
is study showed that PEI-AuNPs were aggregated with inorganic and organic mercury ions, and these ions induced the de nite color change of AuNPs selectively among other diverse ions. Dispersed and aggregated AuNPs were characterized by ultraviolet-visible spectroscopy (UV-Vis), highresolution transmission electron microscopy (HR-TEM), and dynamic light scattering (DLS) upon addition of Hg 2+ . Hg 2+ ion binding sites on the surface of PEI-AuNPs were elucidated by 13 C nuclear magnetic spectroscopy ( 13 C NMR), X-ray photoelectron spectroscopy (XPS), and time-of-ight secondary ion mass spectrometry (TOF-SIMS) [24,25]. e    interference e ects were tested in the presence of other metal ions and anions. Also, PEI-AuNP assay method for detection of Hg 2+ ion was optimized in terms of pH, temperature, and salt condition.

Preparation of PEI-AuNPs.
PEI-AuNPs were synthesized as following literature procedures by mixing aqueous solutions of HAuCl 4 ·3H 2 O (1 mL, 0.025 M) and PEI (7.8 mL, 9.74 mM), with subsequent reduction of HAuCl 4 at ∼pH 7.0 [26]. e mixture was kept to react for three days at ambient temperature, and ∼10 nm PEI-AuNPs were produced.

Sample Preparation and Hg 2+ Sensing Test Using PEI-AuNPs.
e suspended particles in all water samples were removed by a syringe lter (0.20 μm pore size) prior to analysis, and sample aliquots (9 mL) were mixed with a 500 μM·Hg 2+ solution (1 mL) to produce a 50 μM·Hg 2+ stock solution.
Tuna sh was obtained from a local supermarket. Its muscle tissues were crushed and dried on petri dishes overnight at 85°C. A small portion (ca. 0.3 g) was incubated in 2 mL HNO 3 for 1 h before addition of 0.5 mL HClO 4 (70%). en, the samples were irradiated under a UV lamp for 3 h to convert all possibly contained organic mercury to inorganic mercury. Finally, the acid extracts were transferred to 50 mL volumetric asks, and the volume was adjusted to 50 mL with Milli-Q water [27]. ese samples were spiked with 100 μg·mL −1 of Hg 2+ .
Stock solution of bovine serum was made to be 0.1% concentration in water. e suspensions were stirred and centrifuged alternately for 30 min and the solutions (9 mL) were blended with 1 mL of a 100 μg·mL −1 Hg 2+ solution.
0.9 g of citrus leaf was added to 5 mL concentrated HNO 3 and heated for 2 h in the boiling water bath. After being cooled to room temperature, the samples were added with 2 mL of 30% H 2 O 2 , followed by heating for 1 h in the boiling water bath. Finally, the sample volume was made to 25 mL with double-distilled water. Before conducting experiments, the pH value of samples solution was neutralized by solid NaOH [28].
To evaluate the utility of our proposed method, Hg 2+ concentrations added in tap, pond water, tuna sh, and bovine serum were measured with 1 mL of the PEI-AuNP solution, and followed by UV-Vis spectrophotometry. ose analytical results were con rmed with DMA.

Instrumentation.
e absorption spectra were recorded by UV-Vis spectrophotometer (S-3100, Sinco, Seoul, Republic of Korea). UV-Vis spectra were acquired in the range  Journal of Analytical Methods in Chemistry of 300-800 nm by using 4 mm path length quartz cells. e pH measurements were conducted with an HI 2210 pH meter (Hanna Instruments, Woonsocket, RI, USA). e concentrations of Hg 2+ ions in various samples were measured by DMA (DMA 80, Milestone, Italy). 13 C NMR spectra were measured on an Avance III 400 MHz 1 H NMR spectrometer (Bruker, Billerica, MA, USA). XPS analysis was performed using a PHI 5000 VersaProbe III instrument (ULVAC-PHI, Chigasaki, Japan). Mass spectra were measured using TOF-SIMS (TOF-SIMS 5, ION-TOF, Mȕnster, Germany). e size distributions of nanoparticles were recorded by a Zetasizer (Malvern Instruments Ltd., Worcestershire, UK). e images and sizes of PEI-AuNPs and their Hg 2+ -induced aggregates were measured on a micrograph using transmission electron microscope (TEM; CM30, Philips, NC, USA). TEM samples were obtained by settling the scattered AuNPs and evaporating the solvent.

Characterization of PEI-AuNPs and eir Complexes with
Hg 2+ . PEI-AuNPs were prepared as ∼10 nm size as reducing HAuCl 4 with amine group of PEI. e AuNPs were usually synthesized by the citrate reduction of HAuCl 4 , and their sizes were ∼33 nm as described in earlier studies [29], but AuNPs conjugated with PEI (PEI-AuNPs) under these conditions became much smaller. As a result, the mean AuNP size depended on the quantity and type of reducing agents, pH, temperature, and reaction time [30,31]. A strong localized surface plasmon resonance (LSPR) peak of these label-free AuNPs appeared at ca. 514 nm in their UV-Vis spectrum, resulting in the red color of the corresponding solution. e size of AuNPs in uenced to change their surface plasmon absorption maxima at 514 nm [32]. e sizes of PEI-AuNPs and Hg 2+ -PEI-AuNPs were distributed to be ∼15 and ∼75 nm, respectively, according to TEM  images and Zetasizer measurements (Figures 1(b) and 1(c)). e color of these PEI-AuNPs was similar to those of label-free AuNPs, but distinct color change occurred from red to dark violet upon addition of Hg 2+ ions. UV-Vis absorption spectra for AuNP, PEI-AuNP, and Hg 2+ -PEI-AuNP solutions are demonstrated in Figure 1(a). Upon addition of Hg 2+ ions, the strong absorption band of PEI-AuNPs at 514 nm was gradually shifted to 700 nm, and a new absorbance band concomitantly increased in intensity upon addition of Hg 2+ ions (Figure 1(a)). When PEI-AuNPs are aggregated, the conduction electrons near their surfaces become delocalized and are shared amongst neighboring particles. As a result, the surface plasmon resonance (SPR) shifts to lower energies, causing the shift of absorption and scattering peaks to longer wavelengths.

Selectivity of PEI-AuNPs for Hg 2+ Ions and Related Interference E ects.
e selectivity for PEI-AuNP assay method was tested in 50 μM·Hg 2+ and various 500 μM metal cations (Zn 2+ , Cd 2+ , Cu 2+ , Cr 3+ , Pb 2+ , As 3+ , Al 3+ , Mg 2+ , Co 2+ , Mn 2+ , Sn 2+ , Fe 3+ , Ge 4+ , Ni 2+ , Ga 3+ , Li + , Ti 4+ , K + , Ba 2+ , and Ca 2+ ions) and anions (F − , Cl − , Br − , SO 4 2− , PO 4 3− , NO 2 − , and NO 3 − ions). e interference by other 500 μM numerous anions and cations for the selectivity of Hg 2+ was further examined. Any metal cations and anions did not induce any color changes except Hg 2+ , as shown in Figure 2(a). UV-Vis absorption spectra of PEI-AuNPs solutions at pH 7, 50°C, and 300 mM·NaCl concentration were recorded in the presence of various metal cations and anions (Figure 2(b)). e strong absorption band at 700 nm distinctly appeared for Hg 2+ , enabling to discriminate easily from di erent metal cations and anions. e absorbance ratios (A 700 /A 514 ) of the PEI-AuNPs solution upon addition of each cation and anion were measured to test the selectivity for Hg 2+ ion (Figure 2  (c)). e absorbance ratio of PEI-AuNPs solution in the presence of Hg 2+ was ∼11 times greater than those in the presence of other ions. A high absorbance ratio of Hg 2+ -PEI-AuNPs was attributed to the aggregation of PEI-AuNPs, whereas a low absorbance ratio of PEI-AuNPs in the presence of other ions indicated to keep well-dispersed forms of PEI-AuNPs. erefore, Hg 2+ ion must be selectively coordinated with a speci c site of PEI-AuNPs. e interference e ect by other ions in the selectivity of Hg 2+ toward PEI-AuNPs was tested in PEI-AuNP solutions upon addition of Hg 2+ ions mixed with other ions. Other ions did not interfere with determination of Hg 2+ , even though their concentrations were ten times greater than that of Hg 2+ . No metal cations and anions except Hg 2+ perturbed absorption bands at 514 and 700 nm (Figure 2(d)).

Binding Sites of PEI to AuNPs and Hg 2+ to PEI-
AuNPs. XPS spectra for PEI-AuNPs and Hg 2+ -PEI-AuNPs were measured to con rm the binding site of Hg 2+ ion to PEI-AuNPs (not shown) [33]. e high-resolution N 1 s signal in Hg 2+ -PEI-AuNPs at 406.2 eV showed the binding energy of Hg 2+ -N bonds [34]. us, it was found that Hg 2+ ion must be bound to nitrogen atom of PEI. e binding site of Hg 2+ to PEI was further examined with 13 C NMR spectra for free PEI and PEI bound to Hg 2+ (Hg 2+ -PEI) as a model of PEI-AuNPs and Hg 2+ -PEI-AuNPs ( Figure 3). 13 C NMR spectra showed that two CH 2 peaks (peaks 1 and 3) resonating 56.5 ppm and 51.0 ppm in free PEI shifted signi cantly to 54.5 and 49.8 ppm, respectively, in comparison to those of Hg 2+ -PEI, as shown in Figure 3. e chemical shifts of other peaks changed a little, which indicated that Hg 2+ ions must be coordinated to nitrogen atoms of tertiary amine in PEI [35,36].

Optimum Conditions for PEI-AuNP Probe.
To optimize the sensitivity of the PEI-AuNP probe for Hg 2+ , the probe was tested as functions of pH, temperature, salt concentration, PEI concentration, and reaction time. e absorbance ratios changed as a function of pH, and it was the highest at pH 7 ( Figure 5(a)). is optimum pH of the PEI-AuNP probe must be something to do with pKa of tertiary amine and the conformation of PEI [38]. us, Hg 2+ must be optimally coordinated to nitrogen elements of PEI in its N-tetrahedral form at pH 7, leading to the highest sensitivity of the probe [39]. e sensitivity of the PEI-AuNP probe for Hg 2+ ions was examined as a function of temperature in the range of 30-100°C, and its sensitivity was optimized at 50°C ( Figure 5(b)). Also, the sensitivity of PEI-AuNP probe was monitored as a function of NaCl concentration, and it was optimized at 300 mM·NaCl concentration ( Figure 5(c)). e optimum concentration of PEI conjugated to AuNPs was examined in the presence of 0.4 μg·mL −1 Hg 2+ solution, and the absorbance ratio of UV-Vis spectra as a function of PEI concentration revealed that optimum concentration of PEI was ∼33 μM (data not shown). 2+ , EtHg + , MeHg + , and PhHg + Using the PEI-AuNP Assay Method.

Quantitation of Hg
e change for color, UV-Vis spectra, and TEM image of the PEI-AuNP probe upon addition of inorganic (Hg 2+ ) and organic mercury ions (MeHg + , EtHg + , and PhHg + ) were monitored. e color of PEI-AuNPs changed gradually from red to dark violet as the concentration of mercuric ions increased ( Figure 6). Also, the absorbance increases at 700 nm and decreases concomitantly at 514 nm, as the concentration of mercuric ions increases (0.05, 0.15, 0.25, 0.50, 1.5, 2.5, 3.5, and 5.0 μM) in PEI-AuNPs solutions. e absorbance ratios for concentrations of each   Paper-type sensor was fabricated, and the present probe solution was dropped to the Whatman paper, and dried it. e color of the Whatman paper turned red (Figure 7(a)), and the functionality of the sensor was tested on water sample containing Hg 2+ ion. When water sample of 0.1 ppm Hg 2+ was added onto the Whatman paper disc, its color turned dark purple (Figure 7(b)). is fact showed this paper disc coated with PEI-AuNP solution can be utilized as a paper-type Hg 2+ sensor.

Application of the PEI-AuNP Probe in the Analyses of Real Samples.
To validate the present assay method, the colorimetric responses in real water samples were tested. e tap water, pond water, bovine serum, and tuna sh samples spiked with 0.6 and 1.8 μM·Hg 2+ were analyzed using the PEI-AuNP probe and DMA. As shown in Table 1, the analytical results of the proposed probe are nearly identical to those obtained using DMA. Hg 2+ ions in real citrus leaf samples were also determined using both the colorimetric AuNP probe and DMA, as shown in Table 2, and their analytical results are almost the same. us, present AuNPbased probe in determination of Hg 2+ ions seemed to be more advantageous than instrumental methods in terms of simplicity, sensitivity, cost, and time. e previously reported instrumental methods and nanoparticle assay methods for the detection of Hg 2+ ions are compared in Table 3, showing that the colorimetric PEI-AuNP probe o ers the lowest LOD for the determination of Hg 2+ ions in aqueous samples.

Conclusions
A highly sensitive and selective colorimetric probe to determine Hg 2+ ions was developed using AuNPs conjugated with branched polyethyleneimine. e sensing mechanism of this colorimetric probe was originated from the aggregation of PEI-AuNPs in the presence of Hg 2+ , and the Hg 2+ ion was found to be selectively coordinated by nitrogen element of PEI conjugated with AuNPs. is method o ers simple, highly sensitive, highly selective, and cost-e cient on-site monitoring of the Hg 2+ ion, allowing the detection of concentrations as low as 1.72 nM to be visually achieved within 40 min.